TY - JOUR
AB - Sunday CEP, Comet Assay: Talk 1/5 How to perform a high quality in vivo comet assay that is acceptable to the regulators? Ulla Plappert-Helbig Novartis Institutes for BioMedical Research, Basel, Swizerland The revised ICH S2 ‘Guideline for genotoxicity testing and data interpretation for medicines intended for human use’ recommends the in vivo comet assay as a second in vivo genotoxicity endpoint. Furthermore the guideline includes also the new option in the genetic toxicology testing strategy ‘battery without in vitro mammalian cell assay but two in vivo endpoints.’ The combined micronucleus/comet assay test fits perfectly into that new strategy. Therefore, the in vivo rodent alkaline Comet assay has increasingly been used for regulatory genotoxicity testing in recent years. The assay has now been validated formally with a standardized study protocol in the international validation study led by the Japanese Center for the Validation of Alternative Methods (JaCVAM). The result of the validation study was submitted to the OECD for the establishment of the OECD test guideline. The experience from the international JaCVAM validation study as well as further analysis and discussions in the meetings of the IWGT (International Workshop on Genotoxicity Testing) revealed that a crucial part is the proper experimental performance of the assay. Examples will be shown how experimental conditions can influence data variability and assay sensitivity. Recommendation will be given how positive and vehicle control data as well as historical control data should be used to show the validity of the assay and the proficiency of the laboratory. Furthermore the influence of experimental design, cytotoxicity and selection of organs on data interpretation and the study conclusion will be discussed. Sunday CEP, Comet Assay: Talk 2/5 Interpretation of comet data - what does it all mean? Carol Beevers Covance Laboratories Ltd, UK The alkaline comet assay is frequently described as a simple and quick technique for examining genotoxicity effects in a wide range of animal tissues. However, interpretation of the results may not always be so straight forward and a simple and quick approach to data evaluation could lead to an incorrect conclusion. DNA strand breaks are the endpoint measurement in the assay but are not formed solely by direct chemical interaction with the DNA. A variety of other mechanisms such as indirect secondary effects following target tissue toxicity, oxidative stress, pharmacological activity of the test chemical and mechanical shearing during tissue processing can produce comets identical in appearance to those formed following genotoxic damage. Interpretation of all comet data should include an assessment of other supportive information and may trigger further experimentation in order to reach a final conclusion on the genotoxicity of the test compound. Sunday CEP, Comet Assay: Talk 3/5 Combination and Integration of the Comet assay in vivo: Experiences with repeated dosing of methyl methanesulfonate, temozolomide and dibenzopyrene Melanie Guerard, Juergen Funk, Andreas Zeller Pharma Research and Early Development, Basel, Switzerland The comet assay is frequently used as a second in vivo endpoint to determine the genotoxic potential of new drug candidates. Due to a testing design comparable with the micronucleus test (MNT) in vivo, both assays can be combined within one experiment. While the MNT is often integrated in repeat dose toxicology studies, a survey among pharmaceutical companies demonstrated that this is not regularly done for the comet assay. The main reasons for not combining or integrating theassay are; the complexity of tissue sampling during necropsy, the risk of cytotoxicity impacting the comet read-out after repeated dosing and the potential impact of an additional administration required before necropsy on other toxicological endpoints. By using different genotoxic test compounds, i.e. methyl methanesulfonate, temozolomide and dibenzopyrene, we assessed whether an integration of the comet assay in a repeat dose study is feasible. We used a Pig-a assay with a dosing period of 5 to 28 days and a treatment free period of 28 days. In addition, MNT in bone marrow and yH2AX staining as well as histopathological examinations in liver were performed. In order to assess DNA damage by the comet assay, animals were also treated following the recovery period over 2 or 3 days, just as in an acute stand-alone comet experiment. The data obtained demonstrated that the comet assay can be combined with a variety of genotoxicity endpoints and even in a repeat-dosing regimen, without compromising the comet read-out. The results obtained therefore are in favor for combining the comet assay with additional genotoxicity endpoints and further its integration into repeat dose experiments. Further data is needed as during our exploratory studies, a considerably lower amount of tissue samples was obtained compared to a regulatory repeat dose toxicology study. Sunday CEP, Comet Assay: Talk 4/5 Results of the EFPIA survey on the in vivo comet assay Bas-jan van der Leede Janssen Research & Development, Belgium In the 2012 annual meeting of the EFPIA Preclinical Development Committee (PDC) and the EMA Safety Working Party regulators, one of the European regulatory agencies mentioned the perception that a number of pharmaceutical companies had been encountering difficulties with the interpretation of some findings in the in vivo comet assay. On request of the EMA Safety Working Party, the PDC organized a survey to investigate among the European representatives of pharmaceutical companies their experience with the in vivo comet assay focused on issues/problems with interpretation of study results and regulatory acceptance of 2 negative in vivo (comet and micronucleus assay) to mitigate a positive in vitro mammalian cell assay following the current ICH S2 guidance. A group of comet assay experts was identified by the PCD to collect and analyze the survey responses, to review the studies with less interpretable findings in more detail, and to report the survey results. The survey reports on the incidence of studies with positive, negative, equivocal or inconclusive results, the rationale for conducting the studies, further exploration of the equivocal studies (i.e. not clearly interpretable as positive or negative or leading to debate with the regulatory authorities) and the studies showing positive results, and the regulatory acceptance of the assay. More than 140 studies have been conducted (in-house or outsourced) by the pharmaceutical companies responding to the survey. The final outcome of the survey and guidance on interpretation of the in vivo comet assay results will be discussed. Sunday CEP, Comet Assay: Talk 5/5 Experience with (in vivo) comet assays in pharmaceutical testing: A regulator’s view Roland Froetschl BfArM Federal Institute for Drugs and Medical Devices, Germany The in vivo comet assay has become a standard test in regulatory genotoxicity testing with the revision of the ICH S2 guidance. In ICH S2 (R1) the in vivo comet assay is the recommended second in vivo assay in the option 2 standard battery. In genotoxicity testing for regulatory purposes the in vivo comet assay was already used before implementation of ICH S2 (R1) as a follow up test to further investigate the relevance of positive results in in vitro genotoxicity tests. Regulatory testing for genotoxicity is done early in drug development and therefore submission of comet assay reports as part of the standard battery testing is still rare and mostly limited to scientific advice procedures during early development. Most of the studies submitted so far are studies with acute dosing whereas studies integrated into repeated dose studies are still an exception. Advantages from a regulators point are the flexibility of the assay with respect to target tissues other than bone marrow and the endpoint compared to the limitations of the UDS test used most frequently for follow up testing in the past. This improves the assessment of the potential of a substance to cause DNA damage under in vivo condition at target sides of toxicity of the drug and in the main metabolic organ the liver. Furthermore the potential for integration into repeated dose studies is considered a pro as more toxicological data (e.g. toxicokinetic, histopathology) are available from the same study but experience is still very limited with this respect. However there are currently still difficulties in interpretation of results when variability between animals or dose groups is high and results are equivocal or borderline. It is expected however that these problems will be solved with increase of use and data available on background and historical control levels. Monday, Plenary Lecture: 0905 The genetic basis of how mammals protect against genotoxic metabolites Ketan Patel MRC Laboratory for Molecular Biology, Cambridge, UK What does the manner by which your body clears your last night’s gin and tonic have in common with epigenetic regulation? Both processes generate highly reactive aldehydes (acetaldehyde and formaldehyde); these simple molecules can cause considerable damage and so the body has to restrain them. My talk will put forward an explanation of how we protect ourselves from these molecules. My story will begin with a devastating human genetic illness called Fanconi’s anaemia – children with this condition have developmental defects, blood stem cell attrition and an enormous cancer risk. The basic defect in this disease is an inability to repair DNA crosslinks (these are caused when cells are exposed to chemotherapy agents such as Cisplatin). The experiments I will discuss will show that these individuals are unable to protect themselves from the two endogenously generated aldehydes. Lastly the take home message may just alter the way you interact with your cocktail later today! Langevin F, Crossan GP, Rosado IV, Arends MJ, Patel KJ (2011) ‘FANCD2 counteracts the toxic effects of naturally produced aldehydes in mice.’ Nature. 475:53–58. Rosado IV, Langevin F, Crossan GP, Takata M, Patel KJ (2011) ‘Formaldehyde catabolism is essential in cells deficient for the Fanconi anemia DNA-repair pathway.’ Nature Structural and Molecular Biology. 2011 Nov 13. Garaycoechea JI, Crossan GP, Langevin F, Daly M, Arends MJ, Patel KJ (2012) ‘ Genotoxic consequences of endogenous aldehydes on mouse haematopoietic stem cell function.’ Nature 2012 489(7417):571–5. Monday, Symposium 1: Talk 1/5 Polymerase theta-mediated end joining (TMEJ) of replication-associated DNA breaks Marcel Tijsterman Leiden University Medical Center, Netherlands DNA lesions that block replication fork progression are drivers of cancer-associated genome alterations, but the error-prone DNA repair mechanisms acting on collapsed replication are incompletely understood, and their contribution to genome evolution largely unexplored. To investigate the consequences of blocked DNA replication on genome stability, we sequenced the genomes of animals that are mutated for the Y family translesion synthesis (TLS) polymerases pol η, pol κ and/or REV1. We found that the inability to replicate across DNA damage, even under non-challenged conditions, leads to non-typical DNA deletions, which are characterized by an extremely narrow size distribution, minimal homology of exactly one nucleotide at the junctions, and by the occasional presence of templated insertions. Although found randomly spread out throughout the genome, these deletions are identical to deletions observed near G4 DNA motifs in animals lacking the DNA helicase DOG-1/FANCJ, together arguing that replication blocks at spontaneous DNA damage as well as at stable secondary DNA structures (i.e. G-quadruplexes) are processed by a non-canonical error prone repair pathway. We found this pathway to critically depend on the A-family polymerase theta, which protects the genome against gross chromosomal rearrangements. Pol Theta-Mediated End Joining (TMEJ) prevails over non-homologous end joining and homologous recombination and prevents genomic havoc at replication fork barriers at the expense of small deletions. Strikingly, by comparing the genomes of isolates of C. elegans from different geographical regions, we found that in fact most spontaneously evolving structural variations match the signature of TMEJ, arguing that this pathway is an important source of genetic diversification. Monday, Symposium 1: Talk 2/5 Signatures of mutational processes in human cancer Ludmil Alexandrov Wellcome Trust Sanger Institute, UK All cancers are caused by somatic mutations. These may be the consequence of the intrinsic slight infidelity of the DNA replication machinery, exogenous or endogenous mutagen exposures, enzymatic modification of DNA or defective DNA repair. Different mutational processes often generate different combinations of mutation types, termed ‘signatures.’ Notably, C:G>A:T transversions predominate in smoking-associated lung cancer, while C:G>T:A transitions occurring mainly at dipyrimidines and CC:GG>TT:AA double nucleotide substitutions are common in ultraviolet-light associated skin cancers. Our understanding of the mutational processes that cause somatic mutations in most cancer classes is remarkably limited. We recently developed an algorithm to extract mutational signatures from cancer genomics data and applied it to 21 breast cancer whole genome sequences. Both novel and known signatures were revealed. Global sequencing projects are generating catalogues of somatic mutations from tens of thousands of cancers. Here, we report the results from a large-scale analysis that revealed the signatures of mutational processes in human cancer. We compiled 4,938,362 somatic mutations from the mutational catalogues of 7,042 primary cancers of 30 different classes (507 from whole genome and 6,535 from exome sequences). In all cases, normal DNA from the same individuals had been sequenced to establish the somatic origin of variants. Our analysis revealed more than 20 distinct mutational signatures. For most of these processes we do not know the underlying biological mechanism. However, we show that one such signature, which we have attributed to abnormal activity of APOBEC enzymes, is present in 16/30 cancer types and is therefore one of the most common mutational mechanisms operative in human cancer. We relate other mutational signatures to age of cancer diagnosis, smoking, UV light, and anticancer drug exposure. Our results reveal the diversity of mutational processes underlying the development of human cancer. Monday, Symposium 1: Talk 3/5 Mapping ‘Breakomes’ by Next Generation Sequencing: Methods and Perspectives Nicola Crosetto1, Ivan Dikic2. 1Hubrecht Institute–KNAW, Royal Utrecht Medical Centre;2Goethe University Medical School, Germany DNA double-strand breaks (DSBs) can be caused by numerous physical and chemical agents – including ionizing radiation and radio mimicking chemotherapy drugs – posing a serious threat to genomic stability, and potentially leading to the formation of oncogenic mutations, including translocations, deletions, and amplifications. Here, I present a genome-wide method to map DNA double-strand breaks (DSBs) at nucleotide resolution by direct in situ breaks labeling, enrichment on streptavidin, and next-generation sequencing (BLESS)1. We comprehensively validated and tested BLESS using different mammalian cell models, DSBs-inducing agents, and sequencing platforms. BLESS was able to detect telomere ends, Sce endonuclease-induced DSBs, and complex genome-wide DSBs landscapes. As a proof of principle, we characterized the genomic landscape of sensitivity to replication stress in human cells, and identified over 1,000 non-uniformly distributed aphidicolin-sensitive regions (ASRs) overrepresented in genes and enriched in satellite repeats. ASRs were also enriched in regions rearranged in human cancers, with many cancer-associated genes exhibiting high sensitivity to replication stress. Recently, we developed a modified BLESS labeling and amplification method based on nanoliter dispensing devices (roboBLESS), which is suitable for single-cell DSBs mapping as well as high-throughput drug screening. Our methods can be applied to various cells and experimental conditions with a specificity and resolution unachievable by other techniques. 1. Crosetto, N. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013). Monday, Symposium 1: Talk 4/5 Mutational landscape of yeast mutator strains Alain Nicolas, Alexandre Serero, Claire Jubin, Sophie Loeillet, Patricia Legoix-Né Institut Curie, UMR3244 CNRS, UPMC, Paris, France The acquisition of mutations is relevant to every aspect of genetics, including cancer and evolution of species on Darwinian selection. Genome variations arise from rare stochastic imperfections of cellular metabolism and deficiencies in maintenance genes. I will present the genome-wide spectrum of mutations that accumulate in a WT and in nine S. cerevisiae mutator strains deficient for distinct genome maintenance processes: pol32∆ and rad27∆ (replication), msh2∆ (mismatch repair), tsa1∆ (oxidative stress), mre11∆ (recombination), mec1∆ tel1∆ (DNA damage/S-phase checkpoints), pif1∆ (maintenance of mitochondrial genome and telomere length, unwinding of G-quadruplexes), cac1∆ cac3∆ (nucleosome deposition), and clb5∆ (cell cycle progression). This approach revealed the diversity, complexity, and ultimate unique nature of each mutational spectrum, composed of punctual mutations, chromosomal structural variations, and/or aneuploidies. The mutations produced in clb5∆/CCNB1, mec1∆/ATR, tel1∆/ATM, and rad27∆/FEN1 strains extensively reshape the genome, following a trajectory dependent on previous events. It comprises the transmission of unstable genomes that lead to colony mosaicisms. This comprehensive analytical approach of mutator defects provides a model to understand how genome variations might accumulate during clonal evolution of somatic cell populations, including tumor cells. Serero A., Jubin C., Loeillet S., Legoix-Né P. & Nicolas A. (2014) Mutational landscape of yeast mutator strains. PNAS, 111, 1897–1902. Jubin C., Serero A., Loeillet S., Barillot E. & Nicolas A. (2014) Sequence profiling of the Saccharomyces cerevisiae genome permits deconvolution of unique and multi-aligned reads for variant detection. Genes/Genomes/Genetics (3G) 4:707–715 Monday, Symposium 1: Talk 5/5 Genetic Toxicology by whole genome sequencing of C. elegans Anton Gartner1, Bettina Meier1, Susanna L Cooke2, Joerg Weiss1, Aymeric P Bailly1,3, Ludmil Alexandrov2, John Marshall2, Keiran Raine2, Mark Maddison2, Elizabeth Anderson2, Michael R Stratton2, Peter J Campbell2,4,5 1Centre for Gene Regulation and Expression, University of Dundee;2Cancer Genome Project, Wellcome Trust Sanger Institute;3CRBM/CNRS UMR5237, University of Montpellier, France;4University of Cambridge, UK;5Addenbrooke’s Hospital, Cambridge, UK Mutation is associated with developmental and hereditary disorders, ageing and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We developed C. elegans whole genome sequencing to model mutational signatures, analysing 183 worm populations across 17 DNA repair- deficient backgrounds, propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was ~1/genome/generation, stable across many DNA repair deficiencies. Telomere erosion led to complex chromosomal rearrangements initiated by breakage-fusion-bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin-B1 induced substitutions of guanines in GpC context, more pronounced in worms lacking nucleotide excision repair, matching patterns observed in aflatoxin-induced liver cancers. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions and rearrangements. Strikingly, both agents induced clustered rearrangements resembling ‘chromoanasynthesis’, a replication-based mutational signature seen in constitutional genomic disorders, suggesting interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease. We are confident to suggest that NGS sequencing will be routinely used for genotoxicology testing in the future, providing a ‘whole genome’ Ames assay. Monday, Symposium 2: Talk 1/6 Characterizing the epigenome and exposome in evaluating environmental exposures and cancer risk Zdenko Herceg International Agency for Research on Cancer, France Recent advances in laboratory sciences offer much in the challenge to unravel the aetiology of complex human diseases, notably cancer, potentially providing an evidence-base for prevention. For example, remarkable advances in epigenomics have an important impact on our understanding of biological phenomena and importance of environmental stressors in complex diseases. Environmental and lifestyle factors are thought to be implicated in the development of a wide range of human cancers by eliciting changes in the epigenome. These changes thus represent attractive targets for biomarker discovery intended for the improvement of exposure and risk assessment, diagnosis and prognosis, and provision of short-term outcomes in intervention studies. The epigenome can be viewed as an interface between the genome and environment, therefore aberrant epigenetic events associated with environmental exposures are likely to play an important role in the onset and progression of different human diseases. Remarkable advances in epigenomics and the advent of powerful technologies for analyzing epigenetic patterns in both cancer tissues and normal cells indicate that the next few years will be fundamental for the identification of critical cancer-associated and exposure-associated epigenetic changes and for their evaluation as new generation of biomarkers. Here I will discuss recent progress in our understanding of whether epigenetic changes in cancer and surrogate tissues can be used as biomarkers for exposure assessment, early detection and an intermediate biomarker for different health outcomes. Monday, Symposium 2: Talk 2/6 Signatures of mutagenesis in mammalian cells Serena Nik-Zainal1,2, Ludmil Alexandrov1, David Wedge1, Peter Campbell1, Mike Stratton1 1Wellcome Trust Sanger Institute & Cambridge University Hospitals NHS Trust, UK;2East Anglian Medical Genetics Service, Cambridge University Hospitals NHS Foundation Trust, UK Cancer is the ultimate disorder of the genome, characterised by not one or two substitutions, indels or copy number aberrations, but hundreds to thousands of acquired mutations that have been accrued through the development of a tumour. The set of mutations observed in a cancer genome is not simply a random accumulation of variants. It is the aggregate outcome of several biological mutational processes comprising an underlying mechanism of DNA damage mitigated by the DNA repair pathways that exist in human cells. Each mutational process will leave its distinctive mark or mutational signature on the cancer genome. The recent increase in the speed of sequencing, offered by modern sequencing technologies (1), permits unprecedented access to the entire tumour genome of a cancer patient. Utilising this surge in scale, we set out to extract the mutational signatures that have been operative in 21 whole-genome sequenced breast cancers (2, 3). Our mathematical methods (4) reveal known signatures of mutagenesis as well as novel signatures. Delving deep into the detailed architecture of these signatures, we uncover new biological insights (2, 3). We find from further interrogation of nearly 5 million mutations sourced from over 7000 cancers sequenced worldwide, that more than 20 different signatures exist; imprints determined by the underlying sources of endogenous and exogenous DNA mutagenesis and of DNA repair (5). Furthermore, using whole-genome sequencing approaches in model systems, the biology of mutagenesis can be explored at an unprecedented scale. Monday, Symposium 2: Talk 3/6 The role of metabolomics in characterising the human exposome Toby Athersuch Imperial College London, UK The complement of low molecular weight compounds that comprise the human metabolome are a critical subset of the internal chemical milieu and a key component of the human exposome. They perform a vast array of functions in the body including acting as osmolytes, signaling molecules, energy substrates, and subunits of cellular macromolecules. The human metabolome is intimately linked to gene-environment interactions, as changes observed in the metabolome across multiple levels of biological complexity (e.g. cellular, tissue, organ, and system levels) result from the interaction of numerous micro- and macro-scale processes including nutrition, environmental exposure, biological regulation, and physiological response. Furthermore, the concept of a ‘continuum of metabolism’, has previously been described, and articulates the limitations of the rigid designations of compounds as ‘endogenous’, ‘exogenous’ or ‘xenobiotic’, which do not reflect the true relationship of the internal and external chemical environments. Metabolic phenotyping can therefore provide a rich source of individual-level biochemical information that can be used to help contextualise and unite environmental exposure data, other biochemical measures, and disease endpoints, and has found utility population-based studies of chronic disease. In this contribution, the rationale for applying metabolic profiling in the context of environmental health studies will be discussed, with reference to current examples of large-scale exposome initiatives. The recent developments in the profiling platforms used to generate metabolic profiles (principally nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry) will be discussed as these have greatly expanded both the coverage and accessibility of metabolic profiling methods in the research community. Monday, Symposium 2: Talk 4/6 Internal exposome: ‘omics’ approaches for developing novel biomarkers of exposure Theo de Kok Maastricht University, Netherlands With the introduction of genomics-based technologies, providing large scale data on biological responses to dietary or environmental factors, our understanding of the molecular mechanisms behind increased cancer risk as well as the preventive action of specific dietary factors has started to grow. This understanding strengthens the biological plausibility of the observed links between exposures and cancer risk in epidemiological studies. Results from the NewGeneris project (http://cordis.europa.eu/publication/rcn/13390_en.html) have shown that by building gene networks, exposures can be linked to biological effects (e.g. induction of micronuclei) at the level of gene expression, and that gene network analysis can be used to establish potential gene expression biomarkers. Furthermore, linking transcriptomics data to specific exposures has shown gender specific responses. This was recently confirmed in the Flemish Environment and Health Survey (FLEHS) in which also new approaches were applied to study combined exposures using Z-scores as well as differences in genetic susceptibility to environmental exposures using an at-risk value based on the sum of risk alleles. The analysis of genomics responses to dietary factors can also be used to identify molecular mechanisms behind disease prevention. In a recent study with 168 participants receiving a fruit juice containing various phytochemicals, we demonstrated that lymphocytes can be used for biomonitoring of chemopreventive effects. Analysis of whole-genome transcriptomic responses shows clear associations with phenotypic markers of antioxidant and antigenotoxic effects, and provides information on specific molecular processes involved in these beneficial effects. The preventive effects and mechanisms involved are strongly associated with polymorphisms of genes involved in phase I and II metabolism and cellular antioxidant defence. These findings demonstrate that genomics techniques provide valuable tools to establish mechanistic pathways involved in both initiation and prevention of disease induced by dietary and environmental factors. Monday, Symposium 2: Talk 5/6, Poster 009 Effects of particulate air pollution on pulmonary alterations in promoter DNA methylation in healthy Sprague-Dawley rats Tsun-Jen Cheng1, Yuan-Horng Yan2, Cheng-Da Hsu2, Michael Chan3, Cheng-Huang Shen4 1National Taiwan University;2Department of Medical Research, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chiayi, Tai;3Graduate Institute of Molecular Biology, National Chung Cheng Univer;4Department of Urology, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chiayi, Taiwan Particulate air pollution showed a serious risk to human health. However, the underlying mechanisms are not completely understood. Epidemiological studies have indicated that exposure to particulate matter (PM) was associated with DNA methylation, but toxicological evidence is lacking. We investigated pulmonary alterations in promoter DNA methylation in Sprague-Dawley (SD) rats following subchronic exposure to ambient PM2.5 in a traffic/ urban environment. We constructed a real world PM2.5 exposure system, the Taipei Air Pollution Exposure System for Health Effects (TAPES), to continuously deliver non-concentrated PM. SD rats were exposed to PM using TAPES in Taipei, Taiwan, 24 hours/day, 7 days/week, for a total of 16 weeks between December 22, 2009, and April 9, 2010. Control rats were housed in the same environment, but received only high-efficiency particle filtered air (HEPA-FA). Whole lung parenchyma tissues were collected from rats exposed for 16 weeks. To further explore methylation changes in response to PM exposure, we used a methylated-CpG island recovery assay-assisted microarray method. An Agilent 105k CpG microarray, Gene Pix 4000A Scanner, and GenePix Pro 4.0 software were used to identify methylated target genes. The Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics tools was used to identify the specific biological processes, functional categories, or pathways associated with the targeted genes containing hyper-methylated promoters. The average concentrations of PM2.5 in the PM and FA chamber of TAPES during the study period was 13.30 and 0.13µg/m3, respectively. Microarray results revealed hyper-methylation of promoter in genes similar to ribosomal protein (Rps10), ATP synthase (Atp5h) and hypothetical protein (Ehd2), and in genes related to the TGF-β signaling pathway (Inha gene), Cadherin signaling pathway (Pcdhga9 and Pcdhgb7 genes), and Wnt signaling pathway (Pcdhga9 and Pcdhgb7 genes) in rats exposed to PM. Monday, Symposium 2: Talk 6/6, Poster 001 Adductomics – measurement of DNA and protein adducts in human tissues as an approach to defining the exposome David H Phillips, George Preston, Osman Sozeri Centre for Environment and Health, King’s College London, UK Characterising the exposome involves profiling prominent disease causing toxicants, including reactive electrophiles, endocrine disruptors, modulators of immune response, metals and agents that bind to cellular receptors. Adductomics involves the identification of endogenous and exogenous electrophiles, by characterisation of protein and DNA adducts, in a ‘top-down” approach. Measurement of DNA adducts in human tissues is a well established biomonitoring approach in molecular epidemiology studies, with a number of different methods, including postlabelling, immunoassay and mass spectrometry, in widespread use. While the amount of DNA obtainable from biobank samples can be limited, there is usually a more abundant supply of human serum available, from which larger amounts of albumin can be extracted. This circulating protein accumulates modifications at its nucleophilic cysteine residue (Cys-34). As described by Rappaport and co-workers [1], following high-pressure tryptic digestion of HSA, these modifications can be found in a 21-residue peptide termed T3. Liquid chromatography is used to purify T3, and the resulting liquid fraction is infused directly into a triple quadrupole mass spectrometer. Using a specialised method based around selected reaction monitoring, the amount of added mass at the Cys-34 site can be defined. The method has potential as a high-throughput approach to detecting events and exposures that may shed light on the aetiology of chronic diseases including cancer. Thus the application of adductomics to the exposome concept involves both an untargeted investigation of the internal exposome based on the measurement of complete categories of features (e.g. protein adducts), and a targeted approach based on the investigation of defined external sources of exposure. Reference: 1. Li, H., Grigoryan, H., Funk, W.E., Lu, S.S., Rose, S., Williams, E.R. and Rappaport, S.M. (2011) Profiling Cys34 adducts of human serum albumin by fixed-step selected reaction monitoring. Mol. Cell. Proteomics, 10, M110.004606. Symposium 2: Poster 002 Analytical Approach for Determination of 2’Deoxynucleoside Adducts with Electrophiles: Investigation of Reactivity and Selectivity of para-Benzoquinone Maria Schmied-Tobies UFZ, Germany The reaction of an electrophile with DNA is considered one type of molecular initiating events that can lead to genotoxic effects1. The electrophile may enter the organism or be formed from a precursor within the organism by bio-activation. Relatively hard oxygen, and nitrogen containing moieties of DNA may be attacked by Michael addition, SN2 reactions, Schiff base formation, and acylation2. In consequence a reactive toxicity arises which leads to adducts (addition products)3,4. Typically LC-MS analysis takes advantage of the labile glycosidic bond within the deoxynucleosides. Here, the loss of the sugar moiety (116Da) can be monitored in the positive ionisation mode as a constant neutral loss (CNL) by using a triple quadrupole MS5. Within our study an LC-MS method using constant neutral loss was established to investigate reactivity and selectivity of 2’deoxycytidine, 2’deoxyadenosine, 2’deoxyguanosine and 2’deoxythymidine towards para-benzoquinone as a model electrophile. 1. Farmer P. B., Toxicol. Lett. 2004, 149, 3. 2. Schwöbel J. A. H., Koleva Y. K., Enoch S. J., Bajot F., Hewitt M., Madden J. C. Roberts D. W., Schultz T. W., Cronin M. T. D.: Chem. Rev. 2011, 111, 2562. 3. Rappaport S. M., He L., Grigoryan H.Funk W. E., Williams E. R.: Toxicol. Lett, 2012, 213, 83. 4. Jacobs A. T., Marnett L. J.: Acc. Chem. Res. 2010, 43, 673. 5. Balbo S., Turesky R. J., Villalta P. W.: Chem Res Toxicol. 2014, 27, 356. Symposium 2: Poster 003 Association between Glutathione S-transferases GSTM1 and GSTT1 polymorphisms and Risk of Coal Workers’ Pneumoconiosis Diana M. Narvaez1, Diana Carolina Polania-Villanueva1, Carlos Torres2, Leonardo Briceño2, Gloria Morgan3, Ruth Marien Palma4, Marcela Varona2, Helena Groot1 1Laboratorio de Genética Humana, Universidad de los Andes.;2Laboratorio de Genética Humana, Universidad de los Andes.;3Universidad del Rosario.;4Universidad del Rosario.;5ARP Positiva;6Instituto Nacional de Salud, Colombia.;7Universidad del Rosario.;8Laboratorio de Genética Humana, Universidad de los Andes Coal workers’ pneumoconiosis (CWP) is one of the most widespread occupational lung diseases caused by inhalation and accumulation of organic or mineral particles like silica, asbestos, and airborne coal mining dust. The deletion polymorphism of glutathione S-transferases GSTM1 and GSTT1 represents a risk factor for the development of lung diseases. The aim of this study was to investigate the relationship between these polymorphisms and the risk of CWP. In total 128 underground coal miners from Colombia were selected, 49 with pneumoconiosis and 79 controls. Blood samples were taken and an occupational health surveillance including physical examination, spirometry and chest radiograph were taken. Genomic DNA was extracted from peripheral blood lymphocytes and GSTM1 and GSTT1-null alleles were genotyped. The associations between genotypes and CWP were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for possible confounders. We found no significant association of CWP and GSTT1 (OR = 1.423, 95%CI= 0.463–4.376, p>0,05) and GSTM1 (OR = 1.615, 95%CI= 0.750–3.476, p>0,05) variants, although it seems to be a risk factor. Symposium 2: Poster 004 Can the Pig-A gene mutation assay be used as a biomarker to predict the risk of developing Oesophageal Adenocarcinoma? Hasan Haboubi, Benjamin Rees, Lisa Williams, James Manson, Cathy Thornton, George Johnson, Gareth Jenkins Swansea University Background: Barrett’s Oesophagus (BO) is a pre-malignant condition affecting the lower oesophageal mucosa, caused by chronic Gastro-Oeosophageal Reflux Disease (GORD). Noxious refluxate constituents in patients with GORD can induce reactive oxygen species and subsequent DNA damage, driving the progression of Barrett’s Oesophagus to Oesophageal Adenocarcinoma (OA). The incidence of OA is on the rise, culminating in intensified efforts to better stratify patients at risk of progression. Regular endoscopic surveillance allows for such monitoring to take place but this is costly and has a potential for physical harm. The Pig-A gene mutation assay is a fast and reproducible method of assessing genomic instability through the measurement of fluorescently labeled antibodies to specific membrane proteins by flow-cytometric methodology. We postulate that blood cells circulating through the inflamed oesophageal mucosa, exposed to mutagenic chemicals such as bile, accumulate mutations that can be investigated using this assay. Methods: Blood based cell lines were exposed to physiological carcinogens such as bile and the Pig-A mutant frequency measured. Subsequent ex-vivo analysis of blood was undertaken in patients attending endoscopy with symptoms of GORD. Pig-A analysis of erythrocytes and leucocytes was performed and results correlated with histopathological analysis of oesophageal biopsies as well as a detailed lifestyle questionnaire. Results: In-vitro investigations confirmed the carcinogenicity of bile acids to blood based cell lines, with increased mutant frequencies detected through the Pig-A gene mutation assay (p<0.05). Subsequent ex-vivo erythrocyte analysis demonstrated no effect from patient age or gender on mutant-frequency. Higher mutant frequencies were observed in OA patients compared to both normal GORD patients and those with Barrett’s (p<0.01) but there was no significant difference between BO and normal controls. Conclusions: The application of this simple, non-invasive blood-based mutation assay to patients with GORD suggests OA patients have higher mutational events than patients with Barrett’s or normal oesophageal mucosa. Symposium 2: Poster 005 Development of Stem Cell Therapies for the Treatment of Ocular Surface Stem Cell Disease Eleni Chrysostomou, Nigel J Fullwood, Francis L Martin Lancaster University The cornea is located at the front of the eye protecting it and its contents, a fundamental element for the maintenance of ocular surface. Alterations in its structure can lead to vision impairment. Its renewal is governed by a limbal stem cell (LSC) population, which resides at the limbal epithelium. Transplantation of ex-vivo cultured corneal epithelium has been successfully used through culturing limbal epithelium stem cells (LESCs) on amniotic membrane [1]. In this study we investigated the potential of non-corneal stem cells for the treatment of ocular surface disease. The cell types investigated are nasal epithelial cells and periodontal ligament (PDL) cells, both cultured in an ex-vivo system on amniotic membrane [2]. We employed scanning electron microscopy, image analysis and transmission electron microscopy (TEM). Our results show that the cultivated nasal epithelial cells become much more corneal-like as a result of ex-vivo expansion and could potentially be used as an alternative source for ocular reconstruction in patients with Limbal Stem Cell Deficiency (LSCD). The PDL cells do not change during culture and would thus be unsuitable for ocular surface reconstruction. Future experiments will include more TEM and Raman spectroscopy studies [3]. Cultivated nasal epithelium can also be used as a base for toxicity tests to investigate the potential cytotoxic effects of the exposure of human nasal epithelium to hazardous urban atmospheric and chemical agents [4]. The presence of goblet cells in the cultivated nasal epithelium was observed, but not the presence of ciliated cells. Alterations of the culture techniques can be performed in order to promote the differentiation of ciliated cells in the future. Symposium 2: Poster 006 Diagnostic Potential Of Near-infrared Raman Spectroscopy In Plasma: An Exploratory Study For Detection Of Endometrial Cancer Alana Mitchell1, George Theophilou1, Ketan Gajjar2, Helen Stringfellow1, Pierre Martin-Hirsch1, Francis Martin3 1Lancashire Teaching Hospitals NHS Trust;2Cambridge Universities NHS Trust;3Centre for Biophotonics, Lancaster Environment Centre, Lancaster University Background: Endometrial cancer is the most common gynaecological malignancy in the Western world, with a rising incidence due to increasing rates of obesity and an ageing population. Although there has been some research into potential biomarkers such as apolipoprotein A-1, transferrin and transthyretin, a biomarker with the required sensitivity and specificity for a screening test has not yet been developed. These limitations have led to an interest in vibrational spectroscopy as an applicable tool in the diagnosis of gynaecological malignancies [1] Raman spectroscopy is one such tool, based on the principle of exploiting inelastic light scattering from a laser source. This results in a shift in wavelength that provides chemical and structural information for the sample under analysis -- a biochemical-cell ‘fingerprint’ Methods: A total of n = 60 plasma samples (30 from patients with endometrial cancer and 30 controls) were obtained from the Lancashire Hospitals genitourinary tissue bank. Ten Raman spectra were acquired from 200µl of each plasma sample deposited and dried on a Low-E glass slide using 10% laser power over 15 seconds of acquisition time. Spectra were wavelet de-noised and vector normalized using MATLAB software. Results: To test the capability of plasma spectra to distinguish between cancer versus control, principal component analysis (PCA) was performed with the first principal component (PC) accounting for 86% of the variance. Paired t-tests showed significant differences between the mean spectra (P <0.001) with notable spectral alterations in the peak at 1338 cm-1, which is characteristic of the C--H vibration of nucleic acid bases. This may indicate that the cancerous patient’s blood plasma may be associated with an increase in the relative amounts of nucleic acids. Conclusion: These results build on previous findings from our group [2]. They demonstrate tremendous promise for the development of blood plasma analysis into a clinical tool for non-invasive detection and screening of endometrial cancers. References: 1) Mitchell, A.L.et al. (2014) J. Biophotonics doi: 10.1002/jbio.201400018. 2) Gajjar, K et al (2013) Analyst 138:3917–3926. Symposium 2: Poster 007 DNA methylation patterns in respiratory allergy cases: comparability of saliva vs. blood Sabine Langie1, Sophie Traen1, Patrick de Boever1,2, Katarzyna Szarc vel Szic3, Ken Declerck3, Gudrun Koppen1, Anne Schepers4, Guy Van Camp4, Wim Vanden Berghe3, Greet Schoeters1,3 1Environmental Risk and Health unit, Flemish Institute of Technological Research (VITO), Mol, Belgium;2Centre for Environmental Sciences, Hasselt University, Belgium;3Department of Biomedical Sciences, University of Antwerp, Wilrijk, Belgium;4Laboratory of Cancer Research and Clinical Oncology, Center for Medical Genetics, University of Antwerp, Belgium Environmental exposures during fetal and early life may contribute to complex disease later in life such as allergy, neurodegenerative diseases and cancer. The underlying mechanisms are still undiscovered, but changes in the epigenetic regulation of gene expression (such as DNA methylation) is a prominent candidate mechanism. Longitudinal (birth)cohorts are instrumental to study the relation between early-life environmental factors and the development of complex diseases, but investigations are hampered since blood sampling in children is kept minimal for practical and ethical reasons. Saliva is suggested as an alternative DNA source because samples can be obtained in a decentralized manner with less constraints. It is of interest to know how saliva DNA methylation patterns compare with the blood patterns, with the latter being considered as a reflection of systemic effects. The aim of the current pilot study was to set up an analysis pipeline for whole-genome DNA methylation analysis using the Illumina BeadChip platform and to investigate the comparability of DNA methylation patterns in mononuclear cells (MNC) versus saliva samples. Furthermore, we hypothesized that differential methylation regions can be detected in respiratory allergy (RA) cases (n=5) when compared to healthy controls (n=5). R-based software was used for data normalization and to identify DMR. The methylation status of the majority of the probes was comparable between MNC and saliva with about 10% of the probes having a differential methylation pattern (p<0.05 and β-value>10%). When cases were compared with healthy controls, 216 differentially methylated probes were identified in common between MNC and saliva samples. All the probes showed the same methylation polarity. Some probes were linked to RA, but bioinformatics analysis did not identify enriched pathways. We selected a list of 12 differentially methylated regions for further biological and technical validation with bisulfite pyrosequencing and those results will be presented. Symposium 2: Poster 008 Does mobile phone radiation induce changes in cells of the hematopoietic system in vitro? Katharina Taichrib1, Henning Hintzsche1, Marina Rohland2, Kai Baaske2, Thomas Kleine-Ostmann2, Thorsten Schrader2, Helga Stopper1 1Institute of Pharmacology and Toxicology, University of Würzburg, Germany;2Physikalisch-Technische Bundesanstalt, Braunschweig, Germany Mobile phones are used worldwide to a huge amount and the number is still increasing. Therefore questions concerning electromagnetic radiation and its potential to affect biological systems at low intensity levels are of great and current interest. Although many studies have been performed to investigate this issue no complete consensus has been reached so far. Most of the investigations do not indicate a harmful potential of this radiation. Two main questions remain open, concerning long-term effects and specific effects on children. It has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. These absorptions might occasionally exceed the recommended safety limits. According to these questions, the aim of this study is to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. As biological system, two cell types are investigated, HL-60 cells as an established cell line, and hematopoietic stem cells. Cells are irradiated at frequencies of the major technologies, GSM (900 MHz), UMTS (1.950 MHz), and LTE (2.535 MHz). LTE modulation is now commonly applied but has not been studied sufficiently so far. The exposure takes place for a short and a long period and with different intensities ranging from 0 to 4W/kg. Studied endpoints include apoptosis, cell cycle, differentiation, DNA damage, DNA repair, epigenetics, and oxidative stress. Results of irradiated HL-60 cells did not reveal any changes in the examined endpoints, neither after short-term nor after long-term exposure. Investigations with hematopoietic stem cells are still in progress. These results will also be presented and discussed. Symposium 2: Poster 010 Environmental pollutants and DNA damage in adolescents of the 2nd Flemish Environment and Health Study (FLEHSII) Carmen Franken1-2, Eva Govarts1, Nathalie Lambrechts1, Gudrun Koppen1, Elly Den Hond1, Ann Colles1, Liesbeth Bruckers3, Ilse Loots4, Bert Morrens4, Vera Nelen5, Isabelle Sioen6, Kim Croes7, Willy Baeyens7, Nicolas Van Larebeke8, Greet Schoeters1-2–9 1Environmental Risk and Health, Flemish Institute for Technological Research (VITO), Mol, Belgium;2Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium;3Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Has;4Political and Social Sciences, University of Antwerp, Antwerp, Belgium;5Provincial Institute for Hygiene, Antwerp, Belgium;6Department of Public Health, Ghent University, Ghent, Belgium;7Department of Analytical and Environmental Chemistry, Free University of Brussels, Brussels, Belgium;8Department of Radiotherapy and Nuclear Medicine, Ghent University, Ghent, Belgium;9University of Southern Denmark, Institute of Public Health, Odense, Denmark. A total of 606 adolescents (14-15y) were recruited from the industrial hotspot areas Genk-Zuid and Menen, and from the general population of Flanders as part of the second Flemish Environment and Health Study (FLEHSII, 2007–2011). DNA damage was measured using the alkaline comet assay (with and without formamidopyrimidine DNA glycosylase to detect oxidized bases) in whole blood samples, and by an enzyme-linked immunosorbent assay (ELISA, Genaur) quantifying 8-hydroxy-2-deoxyguanosine in urine. Exposure-effect associations were analyzed via stepwise multiple regression analyses adjusted for fixed confounders and statistically significant covariates (p<0.20 in single regression models). For this analysis the exposure biomarkers were divided into tertiles, as the relationship with the DNA damage markers possibly might be non-linear. (Borderline) significant exposure-effect associations with DNA breaks in the comet assay were observed for urinary chromium (p=0.03), blood thallium (p=0.09), the urinary concentration of the polycyclic aromatic hydrocarbon metabolite 1-hydroxypyrene (p=0.08) and the urinary benzene metabolite t,t’-muconic acid (p=0.07). Additionally, increased urinary concentrations of arsenic (p=0.002), toxic relevant arsenic (p=0.0004) and thallium (p=<0.0001) were significantly associated with decreased concentrations of 8-hydroxy-2-deoxyguanosine. In conclusion, various environmental pollutants present in the life environment of youngsters were associated with short term DNA damage. Via gene expression analysis, the pathways behind the observed DNA damage will be further explored. This study was commissioned, financed and steered by the Ministry of the Flemish Community (Department of Economics, Science and Innovation; Flemish Agency for Care and Health; and Department of Environment, Nature and Energy). Symposium 2: Poster 011 Formation of DNA base adducts of the food contaminant patulin with and without involvement of glutathione Carolin Pfenning, Harald L Esch, Leane Lehmann University Wuerzburg, Section of food chemistry, Wuerzburg, Germany The mycotoxin patulin (PAT), an α,β,γ,δ-unsaturated lactone, is a frequent contaminant in fruits and products thereof. At submicromolar concentrations, PAT has shown mutagenic potential in the hprt locus of mammalian cells, most sensitive to base substitutions caused by e.g. DNA-adducts. It also induced DNA-DNA-cross-links, displaying its potential to react directly with DNA. In contrast, small electrophiles such as PAT are assumed to be inactivated by intracellular glutathione (GSH). Thus, the reactivity of PAT towards DNA bases was investigated by incubation of PAT with nucleobases in the presence or absence of GSH under cell-free conditions followed by LC-MS/MS analysis. Structure of reaction products was elucidated by i) interpreting their fragmentation pattern, ii) exact mass measurements, and iii) characterizing the nucleophilic nitrogen of the nucleobase involved in adduct formation. In this way, two different structures of DNA base-adducts were identified: i) adducts substituted with one or two DNA bases, even occurring in the presence of GSH, and ii) adducts with one molecule of DNA bases and GSH each, revealing mixed GSH-PAT-DNA base adducts which have neither been described for PAT nor for any other α,β-unsaturated carbonyl compound before. Adduct formation was observed for all nucleobases, while the reactivity of PAT towards purine bases was preferred. Thus, the formation of DNA base adducts under cell-free conditions despite the presence of GSH could contribute to the PAT-induced mutations observed at the cellular level. Interestingly, conjugation to GSH not abolishes the reactivity of PAT against DNA bases, but rather forms a reactive intermediate, which is able to react with nucleophilic centers of DNA bases. This reaction mechanism could be a mode of action to consider for other reactive α,β-unsaturated carbonyl compounds relevant for the human exposure. Symposium 2: Poster 012 Genotoxic and epigenetic effects of Lead in recycling automotive battery workers Diana M Narvaez1, Andres Hernandez-Gil1, Valeriano Lopez1, Sonia Diaz2, Ruth Marien Palma2, Marcela Varona3, Helena Groot1 1Laboratorio de Genética Humana, Universidad de los Andes.;2Laboratorio de Genética Humana, Universidad de los Andes.;3Laboratorio de Genética Humana, Universidad de los Andes.;4Instituto Nacional de Salud, Colombia.;5Instituto Nacional de Salud, Colombia.;6Universidad del Rosario;7Laboratorio de Genética Humana, Universidad de los Andes The oxidative damage generated by Lead (Pb) through Fenton-like reactions has been indicated by years as a source of genotoxic damage, it transforms nucleotide structures to its oxidized forms like 8-oxoguanine and other molecules that indirectly may produce 5-hydroxymethylcytocine (5hmC). This DNA damage is recognized by the base excision repair system (BER) in standard conditions. When high Pb exposure occurs, this system has been described as non functional by the inhibition of Apurinic Endonuclease 1 (APE1).When the oxidized nucleotides are recognized by the non functional BER system it generates an apurinic site that could be detected by the Comet assay. On the other hand, the effect of the oxidative stress could produce a loss of melthylation marks trough synthesis of 5hmC. The aim of this study was to correlate the blood Pb levels with the DNA damage in a population occupationally exposed, and additionally to scan the behavior of the global epigenetic markers 5-methylcytocine (5mC) and 5hmC. To achieve this, a population of 60 participants were selected and classified in two equally sized groups by age and gender. Lifestyle habits and symptoms were included among the variables to be analyzed. Blood samples were taken to measure DNA damage by the alkaline Comet assay. Blood Pb concentration, 5mC and 5hmC was quantified by Anodic Stripping Voltammetry and ELISA Assay respectively. We found differences between the exposed and non exposed groups regarding DNA damage, but it indicates a negative relation among damage and Pb levels. Moreover, dose-dependent correlation were not found between Pb levels and DNA damage. Finally, the epigenetic markers 5hmC and 5mC seems not to change among the exposed and non-exposed individuals but interestingly, it shows significant differences when controlling by alcohol intake and drinking rate. Symposium 2: Poster 013 Hematopoietic Stem Cell Mutation is Indicated by the Extreme Persistence of Cisplatin-Induced Pig-a Mutant Phenotype Cells Stephen Dertinger1, Svetlana Avlasevich1, Dorothea Torous1, Jeffrey Bemis1, Souk Phonethepswath1, Carson Labash1, Kristine Carlson1, Jared Mereness1, John Cottom1, James Palis2, James MacGregor3 1Litron Laboratories, Rochester, NY, USA;2Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA;3Toxicology Consulting Services, Bonita Springs, FL, USA Cisplatin is a cytostatic agent used in the treatment of many types of cancer, but its use is associated with increased incidences of secondary leukemia. We evaluated cisplatin’s in vivo genotoxic potential by analyzing peripheral blood for Pig-a mutant phenotype erythrocytes and for chromosomal damage in the form of micronuclei. Mutant phenotype reticulcoyte and erythrocyte frequencies, based on anti-CD59 antibody labeling and flow cytometric analysis, were determined in male Sprague Dawley rats treated for 28 consecutive days (Days 1–28) with up to 0.4mg cisplatin/kg/day, and sampled on Days -4, 15, 29, and 56. Vehicle and highest dose groups were evaluated at additional time points post-treatment up to 6 months. Day 4 and 29 blood samples were also analyzed for micronucleated reticulocyte frequency using flow cytometry and anti-CD71-based labeling. Mutant phenotype reticulocytes were significantly elevated at doses ≥ 0.1mg/kg/day, and mutant phenotype erythrocytes were elevated at doses ≥ 0.05mg/kg/day. In the 0.4mg/kg/day group, these effects persisted for the 6 month observation period. Cisplatin also induced a modest but statistically significant increase in micronucleus frequency at the highest dose tested. The prolonged persistence in the production of mutant erythrocytes following cisplatin exposure suggests that this drug mutates hematopoietic stem cells and that this damage may ultimately contribute to the increased incidence of secondary leukemias seen in patients cured of primary malignancies with platinum-based regimens. Studies with cancer patients undergoing cisplatin-based therapies are currently being planned to test the translational potential of the Pig-a gene mutation assay. Symposium 2: Poster 014 Comparative analysis of the DNA damage response elicited by UVA and UVB radiation H Steel1, C O’Connor2, M Brown2, M Bell2, TJ McMillan1,3, SL Allinson1 1Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University,UK;2Alliance Boots, Nottingham, UK;3Vice-Chancellor’s Office, University of Keele, UK Ultraviolet (UV) radiation causes skin cancer and photoaging as a result of the damage it is able to cause to a range of biomolecules. Among these it is well established as being capable of causing both direct and indirect damage to DNA. Shorter wavelength UVB (280-315nm) can be directly absorbed by DNA bases, causing cross-linked mutagenic adducts i.e. cyclobutane pyrimidine dimers (CPDs) and 6-4-photoproducts (6-4PP). By contrast, for longer wavelength (UVA; 315–400nm) radiation photosensitiser-mediated processes are thought to be primarily responsible for genotoxic damage, which may include directly induced double strand breaks although there are conflicting reports of this latter. Here we compare the kinetics of activation of the DNA damage response (DDR), as measured by γH2AX foci formation, in normal human epidermal keratinocytes by varying equitoxic doses of UVA and UVB radiation. We demonstrate that UVA elicits an earlier response of shorter duration than the equivalent dose of UVB and that this response is independent of cell cycle stage. We have also characterised the activation of downstream components such as p53 and find substantial differences in both the intensity and duration of the responses to UVA and UVB radiation. Symposium 2: Poster 015 Molecular Dynamics shows similar DNA structure distortion when BPDE is bound to major mutation hotspots in TP53 and KRAS Georgina Menzies1, Andrea Brancale2, Ian Prior3, Paul Lewis 1Centre for NanoHealth and College of Medicine, Swansea University;2Cardiff School of Pharmacy, Cardiff University;3Institute of Translational Medicine, University of Liverpool A group of well studied and highly mutable genes are TP53 and the RAS isoforms of HRAS, KRAS and NRAS. A mutation hotspot in TP53 at codon 157, is predominantly found in lung cancer. The RAS genes, are also known to mutate in many sub types of cancer, including in the lung, where an unusual mutation occurs. In this gene set there is a highly mutable region in the K-RAS isoform at codon 12. Studies have shown that sites, including these, in TP53 and the RAS isoforms correspond to adduct sites for the cigarette carcinogen benzo[a]pyrene diol epoxide (BPDE). Using our molecular dynamics and statistical analysis pipeline we have investigated the 3D DNA structure similarity between codons 12, 13 and 14 in the RAS isoforms and codon 157 in TP53 when BPDE is bound to guanine forming an adduct. We have shown that DNA sequences involving these two codons – although distinct and from different genes having different functions – have similar distortion after BPDE is bound. Structural changes involve the helicoidal parameters, stagger, tilt, incline, propeller, roll, rise and shift. We propose this similarity in structural distortion between 157 and K12 is due to the similarity between thymine and methylated cytosine, which, in the two sequences, are the bases proceeding the mutating guanine, other adjacent sequences also had an effect on distortion. Multivariate statistical analysis for the RAS and TP53 gene sequences showed K12 and 157 cluster separately from the other adducted RAS isoform sequences. However, they are most similar by structure to the control group. This is a similar result to previously presented 10S BPDE studies. We suggest that these results show that codons 157 in TP53 and 12 in KRAS have little 3D structural distortion after BPDE forms an adduct relative to other adducted sequences. Monday, Workshop 1: Talk 1/6 Interpreting in vitro micronucleus positive results: Simple biomarker matrix discriminates clastogens, aneugens, and irrelevant positive agents Stephen Dertinger1, Steven Bryce1, Jeffrey Bemis1, Jared Mereness1, Kristine Carlson1, Richard Spellman2, Jocelyn Moss2, Donna Dickinson2, Maik Schuler2 1Litron Laboratories, Rochester NY;2Pfizer Worldwide Research and Development, Genetic Toxicology Center of Emphasis, Groton, CT The specificity of in vitro mammalian cell genotoxicity assays is considered relatively low, as they yield a high incidence of positive results that are not expected to have in vivo relevance. We set out to develop a rapid, effective follow-up testing strategy that predicts whether apparent in vitro micronucleus (MN)-inducing effects are due to a clastogenic, aneugenic, or secondary irrelevant mode(s) of action. Priority was given to biomarkers that could be multiplexed onto flow cytometric acquisition of MN frequencies, or could be accomplished in parallel using a homogeneous-type assay. A training set of 30 chemicals comprised of clastogens, aneugens, and misleading positives was studied in human TK6 cells over a range of closely spaced concentrations in a continuous exposure design. In addition to MN frequency, the following endpoints were investigated: cleaved PARP-positive chromatin, cleaved caspase 3-positive chromatin, EMA positive chromatin, polyploid nuclei, phospho-histone H3-positive cells, TMRE negative cells, cellular ATP levels, cell cycle perturbation, and shift in γ-H2AX fluorescence relative to solvent control. Logistic regression identified endpoints that effectively predicted chemicals’ a priori classification. Cross validation indicated that a promising base model includes γ-H2AX shift and change in phospho-histone H3-positive events (25/30 correct calls). Improvements were realized when one or two additional endpoints were included (26 - 30/30 correct calls). These models were further evaluated with a test set of 10 chemicals, and by evaluating 3 chemicals at a collaborating laboratory. The resulting data support the hypothesis that a matrix of high throughput-compatible biomarkers can effectively delineate two important modes of genotoxic action, as well as identify cytotoxicity that can lead to irrelevant positive results. More recently, we have explored configurations of select endpoints into multiplexed, add-and-read assays that may be suitable for early compound screening programs that require miniaturized, high-throughput methodologies. Monday, Workshop 1: Talk 2/6 The ICH M7 guideline: industrial feedback and case studies Jim Harvey GlaxoSmithKline The EMA and FDA genotoxic impurities guidelines are expected to be superseded in 2014 by the ICH M7 guideline on the ‘Assessment and Control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk.’ While ICH M7 retains many of the central tenants of the EMA and FDA guidelines, there are some important additions regarding the assessment and control of mutagenic impurities in drug products. Two case examples based on published synthetic drug substance schemes will be used to illustrate the major changes from a safety perspective (1) the focus on ‘mutagenic” rather than ‘genotoxic” impurities (2) the assessment of any potential impurities in both expert and statistical rule based SAR tools and the subsequent impact on impurity control limits in final drug product (3) the testing of impurities in OECD compliant in vitro or in vivo safety tests and (4) the control of mutagenic impurities to the staged threshold of toxicological concern or mutagenic carcinogens to compound specific ‘acceptable intakes” in final drug product. Given the downstream consequences of the (Q)SAR predictions regarding impurity assessment, it is important that in silico assessments are accompanied by ‘expert knowledge” to ensure their robustness. Examples of where sharing of this ‘expert knowledge” across industry has led to improvements the performance of these (Q)SAR models will also be discussed. Monday, Workshop 1: Talk 3/6 Genetic toxicology testing of therapeutic biologicals: Current discussions on best practices Peter Kasper Federal Institute for Drugs and Medical Devices (BfArM), Germany Evolving therapeutic biologicals which are still less visible on the market include oligonucleotides (ONs) and antibody drug conjugates (ADCs). Both product groups fall under the ICHS6 guideline for non-clinical safety assessment of bio-therapeutics, which states that genetic toxicology testing is not needed unless there is a ‘cause for concern.’ ON-based drugs such as antisense ONs, siRNAs, miRNAs, or aptamers usually contain chemical modifications to improve their stability and/or affinity. A cause for concern with respect to genotoxicity could arise from the chemically modified mononucleotide ‘metabolites” which could be incorporated into nuclear DNA leading to either chain termination during replication (strand breakage) or mis-pairing and mutation. These effects would be detected by standard battery tests. Another possible mechanism of genotoxicity is related to the formation of triple helices when an ON binds in a sequence-specific manner with genomic double-stranded DNA which can result in site-specific mutations. Such effects would not be detected by standard (reporter gene specific) mutagenicity tests. However, stringent sequence and biochemical conditions required for triplex interaction are well known and provide an approach to evaluate the potential of ONs for triplex-mediated mutagenesis. ADCs consist of small molecule drugs that are conjugated to antibodies by a linker molecule to provide preferential cell targeting capabilities for the delivery of the drug. The moieties of potential genotoxic concern are primarily the (free) linker and/or the drug. The possible need for genotoxicity testing depends on a number of factors, including the cleavage mechanism and physio-chemical characteristics (e.g. stability or reactivity) of the test article. This presentation summarizes proposals for genotoxicity assessment of ONs and ADCs as currently discussed in the Genotoxicity Subcommittee of the Oligonucleotide Safety Working Group and the ‘Biologics Subgroup’ of the ILSI HESI Genetic Toxicology Testing Committee. Monday, Workshop 1: Talk 4/6 Genotoxicity testing of peptides: folate deprivation as a marker of exaggerated pharmacology Melanie Guerard, Andreas Zeller, Matthias Festag, Christine Schubert, Thomas Singer, Lutz Mueller Pharmaceutical Sciences, pRED, F. Hoffmann-La Roche Ltd., Basel, Switzerland The incidence of micronucleated-cells is considered to be a marker of a genotoxic event and can be caused by direct- or indirect-DNA reactive mechanisms. In particular, small increases in the incidence of micronuclei, which are not associated with toxicity in the target tissue or any structurally altering properties of the compound, trigger the suspicion that an indirect mechanism could be at play. In a bone marrow micronucleus test (MNT) of a synthetic peptide (a dual agonist of the GLP-1 and GIP receptors) that had been integrated into a regulatory 13-weeks repeat-dose toxicity study in the rat, small increases in the incidence of micronuclei had been observed, together with pronounced reductions in food intake and body weight gain. Because it is well established that folate plays a crucial role in maintaining genomic integrity and pronounced reductions in food intake and body weight gain were observed, folate levels were determined from plasma samples initially collected for toxicokinetic analytics. A dose-dependent decrease in plasma folate levels was evident after 4 weeks of treatment at the mid and high dose levels, persisted until the end of the treatment duration of 13-weeks and returned to baseline levels during the recovery period of 4 weeks. Based on these properties, and the fact that the compound tested (peptide) per se is not expected to reach the nucleus and cause DNA damage, the rationale is supported, that the elevated incidence of micronucleated polychromatic erythrocytes is directly linked to the exaggerated pharmacology of the compound resulting in a decreased folate level. Monday, Workshop 1: Talk 5/6 A high throughput genotoxicity screen using γH2AX Marc Audebert INRA TOXALIM, Toulouse, France In vitro genotoxicity tests used in regulatory toxicology studies are sensitive, but the occurrence of irrelevant positive results is high compared to in vivo studies. The development of fast and specific high-throughput in vitro genotoxic assays for predicting in vivo genotoxicity is a major priority. The present study was undertaken to validate an in vitro genotoxic assay in a 96 well plate format allowing the simultaneous examination of cytotoxicity and genotoxicity. The test is based on the quantification of the phosphorylation of the histone H2AX (‘γH2AX”), which reflects a global genotoxic insult, by the In Cell Western technique. The assay was evaluated on four human cell lines with different metabolic capabilities (HepG2, Hep3B, LS-174T and ACHN). We selected 61 compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM) and 24 other chemicals with different mode of mutagenic action (aneugenicity, DNA adduct formation, DNA alkylating, induction of oxidative stress, topoisomerases inhibitors,…), including some known to require specific metabolic activation. We tested also 25 cytotoxic compounds with different apoptotic properties. The γH2AX ICW assay demonstrated a very high sensitivity and specificity. Moreover, results in the different cell lines were complementary as they enabled differentiation between clastogen and aneugen compounds, and directly from bioactivated genotoxins, providing key information about the genotoxic mode of action of the compounds. Overall, our results confirm the high predictivity of the γH2AX ICW assay for genotoxic and carcinogenic compounds without the addition of an exogenous metabolic activation system. The assay is expected to provide its usefulness in the frame of in vitro genotoxicity testing, which in turn is expected to lead to a reduction of the number of animals used for genotoxicity assessment. Monday, Workshop 1: Talk 6/6, Poster 079 Investigation of the effect of compensatory erythropoiesis on the outcome of the in vivo Pig-a Mutation Assay M Kenyon, S Coffing, J Ackerman, W Gunther, K Dobo Pfizer Global Research and Development, Genetic Toxicology Center of Emphasis, Groton, CT, USA The in vivo Pig-a assay, which identifies cells with a mutant Pig-a phenotype based on absence of glycosylphosphatidyl inositol anchored cell surface markers, was first introduced for use in rodents in 2008. It has rapidly developed and gained interest as an assay useful for assessing the mutagenic potential of compounds in vivo, as evidenced by its adoption for development by the International Life Sciences Institute Health and Environmental Science Institute in 2012 and as a topic of the 2013 International Workshop on Genotoxicity Testing. Although a large number of compounds, including both mutagens and non-mutagens, have been tested in the rat Pig-a assay in hematopoietic cells, there is limited understanding of how perturbations in hematopoiesis (e.g. red blood cell lysis followed by erthyropoietic regeneration) affect assay performance. It is possible that jackpot clonal expansion of spontaneous mutants could result in an elevated Pig-a mutant frequency after treatment with an agent that perturbs hematopoiesis, especially as large numbers of reticulocytes are generated after hemolysis. However, Pig-a mutations are thought to be growth neutral so selective expansion of mutants is unlikely. Therefore, the average mutant frequency from treated animals should not be affected. To test this hypothesis, Wistar-Han rats were dosed by oral gavage with concentrations of a non-genotoxic hemolytic agent, 2-butoxyethanol, which were expected to induce levels of intravascular lysis and compensatory erthyropoiesis ranging from marked to little or none. Both single dose and 28-day treatment regimens were used. Hematology parameters were assessed at minimum within the first 24 hours of dosing and 8 days after the final dose. Pig-a mutant frequencies were assessed on days 15 and approximately 30 for both treatment protocols, and also on days 43 and 57 for the sub-chronic protocol. Even at doses of 2-butoxyethanol that induced marked intravascular lysis and strong compensatory erythropoiesis, the average Pig-a mutant phenotype red blood cell and reticulocyte frequencies were within the historical range. Workshop 1: Poster 060 A Collaborative Approach towards Risk Assessment for Mutagenic Impurities Susanne Abigail Stalford1, Elizabeth Covey-Crump1, Simon Gayton1, Nicole McSweeney1, Martin Arien Ott1, Andrew Teasdale2 1Lhasa Limited, UK;2AstraZeneca It is expected that ICH M7 guidance on mutagenic impurities (MIs) will support the control of potential MIs based on a sufficient understanding of the manufacturing process. This strategy would reduce the need to perform genotoxicity or analytical testing on MIs predicted to be purged during synthesis of the final active ingredient. A concept was brought forward by A Teasdale at AstraZeneca, in which semi-quantitative ‘purge factors” are calculated to give confidence that a MI is likely to be absent in an end-product. This approach, directly aligned to the ICH M7 guideline, is used by several organisations within the pharmaceutical industry and has been used to support regulatory submission. The goal though is to expand its use more widely; therefore a consortium has been set up to establish a framework which would aid in the generation of ‘purge factors” and support inclusion of these arguments within regulatory submissions. The key aims are to 1) standardise how calculations are performed throughout industry with engagement of regulators, 2) collate existing data and promote cross-industry data sharing to facilitate supported and accurate decision making, and 3) aim to provide an automated in silico system based on experimental data and expert knowledge. A successful international collaboration has been established, with a number of pharmaceutical companies and regulators guiding the development of the software and establishing principles and a framework for data-sharing. This work has the potential to save both time and money in regards to regulatory submission and also to ensure effort is focussed correctly on those impurities that present a substantive risk; however this relies on data-sharing driving the need for continued engagement across industry. Workshop 1: Poster 061 Analysis of early genotoxicity screening of pharmaceutical candidates – assay performance and adaptive approaches to screening Paul Hastwell, Amy Lane, Dean Thomas, Kate Simpson, Benjamin Moore, Angela White, Jim Harvey, Anthony Lynch, Sharon Robinson GSK, UK Genotoxicity of drug candidate molecules is rarely a source of drug attrition during clinical development due to the availability of a robust battery of in vitro and in vivo assays, a long tradition of pre-clinical genotoxicity testing and a well developed regulatory environment. Based on historical data we estimate that if the assessment of genotoxicity was first assessed in GLP studies performed in support of first time in human clinical trials Genotoxicity might account for ~15% of non-clinical safety related attrition. To avoid this at GlaxoSmithKline (GSK) assessment of genotoxicity is started as early as target validation, using a screening cascade that utilises literature review of the target pharmacology, in silico SAR tools to assess the chemical structures and in vitro genotoxicity assays as appropriate. Here we describe the analysis of the performance of the core assays of the genotoxicity screening cascade (in silico, BlueScreen, Ames and MLA) from 2009 to present. We have compiled the available genotoxicity data for >4000 proprietary compounds in this period. Analysis of compounds where a battery of in silico and in vitro genotoxicity tests have been performed (n>250) reveals that for example, ~75% of BlueScreen Positive compounds tested are also positive in the Ames and/or MLA. Conversely 75% of negative BlueScreen GSK compounds tested are negative both the Ames and MLA. Based on the findings from our analysis we describe how we use data from in silico and the BlueScreen assay to rank compounds in terms of the probability of success. The aim is to tip the balance towards working in chemical space that has the least chance of having genotoxic liabilities. Using case studies we will illustrate how the adaptive use of in silico tools, BlueScreen and the Ames test can reduce development cycle times when investigating genotoxic liabilities. Workshop 1: Poster 062 Automated determination of PCE and NCE sub-populations in mammalian erythrocyte micronucleus test samples from animals treated with azidothymidine Christian Schunck1, Melanie Guerard2, Christine Marchand2 1MetaSystems, Basel, Switzerland;2F. Hoffmann La Roche The mammalian erythrocyte micronucleus (MN) test using rodent haematopoietic cells from bone marrow or peripheral blood is a widely used test system to assess clastogenicity and aneugenicity of chemicals. The test is based on the measurement of micronucleated immature erythrocytes (polychromatic erythrocytes; PCE). PCE are distinguished from mature erythrocytes (normochromatic erythrocytes; NCE) by their hue values in standardized samples. Like for other microscope based toxicology tests reproducibility and documentation of results are crucial, which makes automation of imaging highly desirable. Though automated evaluation of MN is well established, a specific challenge of the erythrocyte MN assay is the automated determination of PCE and NCE sub-populations. We have identified the following obstacles for this automation step that arise even in well standardized experimental setups: a. Variability of staining quality between different samples, b. Cells with equivocal hue values, c. Variations in the cardinal numbers of PCE and NCE populations. Azidothymidine (3′-azido-3′-deoxythymidine) is known to induce micronuclei in vivo and at the same time is haematotoxic, i.e. impacting the PCE to NCE ratio. AZT was therefore used to validate an automated imaging system (MetaSystems Metafer). Here we present a new algorithm (‘Classifier’) for automated differentiation of PCE and NCE based on hue values. Results obtained with the new algorithm have been compared to data based on half-manual differentiation of sub-populations. Our results indicate that automated discrimination of PCE and NCE is possible, and that the system copes with the above mentioned obstacles. Workshop 1: Poster 063 Automated In vitro Micronucleus Scoring: Flow Cytometry vs Image Analysis Jatin Verma1, Benjamin Rees1, Gareth Jenkins1, Steve Bryce2, George Johnson1 1Swansea Unviersity, UK;2Litron Laboratories, USA The in vitro micronucleus assay (MN) assay is an internationally accepted test for detecting genotoxic agents, within the environment and in pharmaceuticals. The major drawback with the conventional MN assay is its subjectivity and time spent manually scoring cells. Automation of the MN assay is starting to reduce this subjectivity, providing higher statistical power and reducing overall sampling time. The MicroFlow technique is an automated flow cytometry-based scoring approach, which enables analysis of cell-cycle and apoptosis, in addition to dose response. This project aims to evaluate the sensitivity of the flow cytometry based MN assay (MicroFlow kit, Litron Laboratories USA) for dose response analysis, and compare it to an automated image analysis (Metafer) method. Human lymphoblastoid TK6 cells were treated with methyl methane sulfonate (Clastogen), Carbendazim (aneugen) and Ochratoxin A (weak genotoxic carcinogen). Different treatment strategies included a 4 hrs or 30 hrs treatment time, with or without recovery and in presence or absence of Cytochalasin-B. A clear dose dependent induction in MN was detected by both MicroFlow and Metafer for MMS and Ochratoxin A. However, an increase in MN was not detected in presence of Cytochalasin-B, following 30 hrs treatment with Carbendazim, no recovery. Furthermore, cell cycle analysis with MicroFlow revealed a dose dependent accumulation of cells in G2/M cell cycle stage for MMS and Ochratoxin, whereas Carbendazim caused a significant G1 block. The MicroFlow approach overestimated the MN frequency at the top dose for all three chemicals as compared to Metafer following 30 hrs treatment. Factors such as early apoptotic bodies, MN within bi, tri and multinucleated cells can influence the MN scoring with MicroFlow. Similar issues occurred with Metafer where irregular shaped micro-nucleated cells and co-treatment with Cytochalasin-B can result in underestimation of MN following treatment with Carbendazim. Both MicroFlow and Metafer techniques are rapid, high content and automated scoring platform suitable for scoring MN in the in vitro MN assay. Workshop 1: Poster 064 Avoid Misleading Positives And Identify Genotoxic Mechanisms In The Flow Cytometric In Vitro Micronucleus Assay In TK6 Cells R Spellman, E Rubitski, Z Sobol, M Homiski, D Dickinson, J Moss, K Dobo, M Schuler Pfizer Global Research & Development, Genetic Toxicology Center of Emphasis, Groton, CT, USA. Toxicity and apoptosis are well known confounding factors in the vitro micronucleus (MN) assay. This is especially true using flow cytometric methods with lysed cells, which makes the distinction of micronuclei from apoptotic bodies and nuclear debris very difficult. We have employed two strategies to overcome this problem. First, we evaluated a BCL-xL over expressing cell line derived from the TK6 cell line by lentiviral transfection which renders it resistant to apoptosis induction. Second, we followed up positive MN responses with aneugen marker phosphorylated histone 3ser10 (H3) and double strand breakage marker γH2AXser139 (γH2AXDSB, γH2AXAPO) to identify the MN inducing mechanism. Both TK6 and TK6 BCL-xL cell lines were dosed in 96-well plates with clastogens (mitomycin C, etoposide, cytosine arabinoside), aneugens (diethylstilbestrol, vinblastine, noscapine, colchicine) and cytotoxicants (CCCP, tunicamycin, anisomycin C). Replicate plates were examined for MN induction using the Litron in vitro MN kit as well as for the presence of H3 and γH2AXDSB / γH2AXAPO on ETOH fixed samples. As expected, all compounds induced a positive MN response in TK6 cells but only the aneugens and clastogens increased MN frequencies in TK6 BCL-xL cells within accepted cytotoxicity parameters. All compounds produced lower responses for apoptosis markers EMA, γH2AXapo and annexin V in TK6 BCL-xL compared to TK6 cells. While absolute micronucleus frequencies were lower in TK6 BCL-xl including the negative control, fold increases in micronuclei were similar. Aneugens showed similar increases in H3 positive cells and clastogens identical increases in γH2AXDSB in both cell lines. No increases in H3 or γH2AXDSB were seen with the cytotoxicants in either cell line. Our results show significant enhancements to the in vitro MN flow cytometric assay could be developed by either using apoptosis resistant TK6 BCL-xl cells and/or by the addition of the aneugen and clastogen specific markers H3 and γH2AXDSB. Workshop 1: Poster 065 Differences in the genotoxic responses of two tobacco specific nitrosamines: NNK and NNN Fiona Cunningham1, Damien Breheny1, Sarah McGarry2, Mark Ballantyne2, Debbie Dillon1, Clive Meredith1 1British American Tobacco, United Lingdom;2Covance Laboratories Ltd, United Kingdom Tobacco smoke contains over 6,000 constituents, some with well-established toxicological properties. Tobacco-specific nitrosamines (TSNAs) are related to nicotine and other tobacco alkaloids and many are carcinogenic in laboratory studies. Here, we compared the genotoxicity of two TSNAs (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) andN-Nitrosonornicotine (NNN)) in a range of in vitro assays, in the presence and absence of rat liver S9. NNK and NNN were assayed for bacterial reverse mutation in Salmonella typhimurium strains from the standard regulatory battery. NNK caused dose-dependent mutations in TA100 and TA1535 with S9 when tested up to 5000 µg/plate. NNN gave no conclusive results, in TA100, TA1535 or in the additional strain of YG7108 with and without S9, even when supplemented with individual human Cytochrome P450 enzymes CYP1A1, CYP1A2, CYP2A6 and CYP2A13. NNK and NNN were analysed for the ability to induce mutation at the tk locus in L5178Y mouse lymphoma cells. NNK induced mutation in L5178Y cells following 3 hour treatment with S9 when tested up to 10mM; however, NNN did not induce mutations with or without S9 when tested up to 10mM. NNK and NNN were assessed in the in vitro micronucleus assay using Chinese hamster V79 cells. NNK induced micronuclei in V79 cells when tested for 3+21 hours with S9 up to 10mM. NNN did induce micronuclei in V79 cells when tested for 3+21 hours with S9, but this result is questionable with no dose-related increase in MNBN cell frequency. Both NNK and NNN are on the list of toxicants identified by the WHO Study Group on Tobacco Product Regulation (TobReg) for mandatory lowering. Based on these results, we conclude that there is a clear difference between NNK and NNN in terms of their genotoxic responses. Workshop 1: Poster 066 Effects of In-life Blood Sampling During Repeat Dose Rat Toxicity Studies on Bone Marrow and Peripheral Blood Micronucleus Frequencies Julia Kenny, Dean Thomas, Joanne Collins, Jonathan Howe, Mark Burman GlaxoSmithKline, UK Integration of the micronucleus (MN) endpoint into non-GLP rodent toxicity studies offers the opportunity for an in vivo assessment of genotoxicity early in drug discovery. At GSK, toxicokinetic assessment on non-GLP rodent studies is routinely performed on main study animals. As published literature indicated that in-life bleeding of rodents can increase background MN frequency, we assessed the impact of various blood sampling regimes (e.g. micro-sampling and dried blood spot) on bone marrow and peripheral blood MN frequencies. Seven-day repeat dose studies using three different strains of rats were conducted where MN samples were taken from bled and un-bled vehicle control and cyclophosphamide-treated rats; the bleed timepoints were typical of those used on a toxicity study on Days 1 and 7, but with reduced blood volumes (80–100 mcL/sample). Subsequently, MN samples were taken from vehicle control animals on 4 - 14 day repeat dose studies using smaller volume blood sampling methods (<100 mcL/sample). The data generally indicated in-life bleeding increased the baseline MN frequency when assessed by both manual bone marrow and flow peripheral blood analyses. There was no direct correlation between the % circulating blood volume sampled and MN frequency and there was an inconsistent correlation between the increases in MN frequency in rats with a lower body weight. The CPA-bled rats showed further increases in MN frequency when compared to the un-bled CPA-treated rats. Furthermore, whilst the degree of cytotoxicity (measured as %PCE or %RET) was also increased in both vehicle control and CPA-treated bled rats compared to un-bled rats, the magnitude of effect was more pronounced when measured in the blood compared to the bone marrow. Standardisation of bleeding regimes and/or microsampling techniques, together with generation of new historical control ranges, may allow integration of the MN endpoint in to repeat dose toxicity studies with in-life bleeding protocols. Workshop 1: Poster 067 Evaluating the genotoxicity of tobacco smoke-derived aerosols using the flow cytometry-based in vitro micronucleus assay Blandine Pouleau, Daniel Smart, Damian McHugh Genetic and Cellular Toxicology, Biological Systems Research, Switzerland The in vitro micronucleus (MN) assay is used to assess the genotoxic potential of test substances by measuring the induction of micronuclei in mammalian cells. The flow cytometry-based assay (in vitro MicroFlow®, Litron Laboratories) coupled with a high-throughput sampler permits the analysis of thousands of nuclei rapidly. Furthermore, clastogenic or aneugenic mechanistic signatures can be discerned using this high content approach. Using this assay version, the genotoxicity of the total particulate matter fraction and the saline-soluble portion of the gas-vapour phase (GVP) from 3R4F reference cigarettes (University of Kentucky) along with several prototypical genotoxins was evaluated using a study design compliant with OECD guideline n°487 and recommendations from Litron Laboratories. To maximise the possibility of detecting a genotoxic response, three different treatment conditions were deployed in CHO-K1 cells: extended (24h), short-term without metabolic activation (4h -S9) and with S9 metabolic activation system (4h +S9). In order to understand cytotoxicity vis-à-vis genotoxicity in the assay, three different cytotoxicity indices were explored, namely relative population doubling (RPD), relative increase in cell counts (RICC) and relative cell counts (RCC. Known clastogens, e.g. methyl methanesulphonate, and aneugens, e.g. colchicine, were shown to induce marked genotoxicity as well as clear clastogenic and aneugenic mechanistic signatures, while several non-genotoxic cytotoxins, e.g. phthalic anhydride, were confirmed as non-genotoxic in the assay. 3R4F-derived TPM and GVP induced clastogenic responses but there was no detectable evidence of aneugenicity. Cytotoxicity was underestimated by RCC for most of the substances tested, whereas RICC appeared to be the most sensitive index and RPD typically yielded levels of cytotoxicity midway between RCC and RICC. In conclusion, the flow cytometry-based in vitro MN assay is appropriate to evaluate the genotoxic potential of tobacco smoke-derived aerosols. Workshop 1: Poster 068 Evaluation of the mutagenicity induced by tobacco smoke-derived total particulate matter using the mouse lymphoma assay Anne-Laure Demoute, Oirda Al-Jobori, Blandine Pouleau, Daniel Smart, Damian Mc Hugh Genetic and Cellular Toxicology, Biological Systems Research, Switzerland The mouse lymphoma assay (MLA) is a validated, regulatory in vitro test used to evaluate the mutagenicity of test substances in L5178Y mouse lymphoma cells. The goal of the study was to evaluate the testing laboratory’s proficiency by performing a series of ten experiments with reference positive controls, i.e. methyl methanesulfonate (MMS), 7,12-dimethylbenz(a)anthracene (DMBA) and total particulate matter (TPM) generated using 3R4F reference cigarettes (University of Kentucky) plus solvent controls. The chosen study design was compliant with the Organisation for Economic Co-operation and Development (OECD) guideline 476 and recommendations of the International Workshop on Genotoxicity Testing (IWGT) MLA workgroup. L5178Y tk+/-clone 3.7.2C IVGT (Public Health England) cells were exposed to the test substances for 4 hours ± S9 and 24 hours (-S9). The 3R4F TPM fraction was generated following the Health Canada Intense smoking regimen. On nine out of ten independent test occasions, the 3R4F TPM was found to produce biologically-relevant and concentration-related increases in mutagenicity following the 4h +S9 treatment condition. The lowest observed genotoxic effect level (LOGEL) for this treatment condition occurred at 30–60 µg/ml with relative total growth values of 20–40%. 3R4F TPM was also found to induce concentration-related and biologically-relevant mutagenicity in the absence of S9 with LOGELs between 12–30 µg/ml. The responses observed under these treatment conditions were both less pronounced and frequent than in the presence of S9, and were observed exclusively at the assay’s cytotoxic limit. MMS, DMBA and solvent controls induced responses that were within IWGT recommended acceptability limits. In conclusion, these data demonstrate the proficiency of the laboratory at performing the MLA and at determining the mutagenicity induced by 3R4F TPM. Workshop 1: Poster 069 Evaluation of thioacetamide genotoxicity in rat liver and stomach as measured by the in vivo alkaline Comet assay Carol Beevers1, Zena Keig-Shevlin1, Robin Neft2 1Covance Laboratories Ltd, UK;2Covance Laboratories Inc. Thioacetamide is a known hepatotoxic agent and dietary administration caused hepatocellular carcinoma in mice and rats and tumors of the bile duct in rats. It is negative in Ames and in vitro chromosome aberration tests, however, there are conflicting reports regarding its genotoxicity in vivo. It induced micronuclei in the bone marrow of two strains of mice, albeit mostly at lethal dose levels, but similar effects were not seen in rats. No micronuclei were induced in the liver or peripheral blood of thioacetamide treated rats. In the Comet assay, thioacetamide was positive in stomach, colon and urinary bladder, but not in bone marrow or liver of mice treated by intraperitoneal injection. We examined thioacetamide activity (up to a maximum tolerated dose) in a rat alkaline comet assay and measured DNA migration in liver and stomach after 3 exposures (21–24 hours apart), sampling 3 hours after the final administration. Thioacetamide caused an increase in DNA migration in the stomach of rats treated at 38 and 75mg/kg/day as well as an increase in hedgehogs, a Comet morphology indicative of highly fragmented DNA that is excluded from Comet analysis. Thioacetamide did not cause any increase in DNA migration in the liver, despite evidence of marked hepatotoxicity. These findings confirm that toxicity does not always confound assessment of genotoxicity-related DNA migration in the Comet assay. Workshop 1: Poster 070 Functional analysis of the ToxTracker reporter assay for highly predictive in vitro genotoxicity testing Alice Bolner, Bruno Morolli, Fabienne Calléja, Harry Vrieling, Giel Hendriks Leiden University Medical Center, Netherlands Industry continuously develops new chemicals for several applications. In order to consider compounds safe for introduction on the market, rigorous testing for potentially toxic properties is required. To reliably identify the potential hazards for human health, mechanistic understanding of the often complex toxicological properties of chemicals is needed. We previously developed the ToxTracker assay that allows identification of the mechanism of toxicity by visualizing the specific signaling pathways that are elicited upon chemical exposure. Toxtracker is a murine stem (mES) cell-based in vitro assay that uses selective activation of fluorescent reporters by different cellular signaling pathways associated with (geno)toxicity. The ToxTracker can discriminate between compounds that induce DNA damage, oxidative stress and protein damage. To identify the pathways associated with activation of the different ToxTracker reporters and thereby gain further insights in the mechanisms of toxicity of chemicals, we performed functional analysis of the different ToxTracker reporter genes. We exposed the reporter cell lines to various reference compounds with a known mode of action in combination with specific inhibitors of key regulators of different cellular damage signaling pathways. Furthermore, key (geno)toxicity response pathways were blocked by siRNA knockdown or CRISPR/Cas-dependent gene knockout. We found that the ATR/Chk1 DNA damage and the cytokine-associated NF-kB transcription regulator signaling pathways play an essential role in activation of the DNA damage reporters. Cellular oxidative stress is detected by a reporter gene that depends on the Nrf2-dependent antioxidant response and a Nrf2-independent reporter. Activation of the protein damage reporter has been linked to the unfolded protein response and detection of cellular stress depends on the p53 tumor suppressor. Taken together, this study provides further insights in the cellular damage responses detected by the ToxTracker assay and will contribute to elucidate the mechanisms of toxicity of compounds. It will thereby improve in vitro health hazard predictions. Workshop 1: Poster 071 Further Developments of a Mammalian Cell in-vitro Pig-A gene mutation assay Benjamin Rees1, Cathy Thornton1, Gareth Jenkins1, David Dow2, Nalini Mehta2, Lucy Field2, George Johnson1 1Swansea University, UK;2GlaxoSmithKline Current developments within the field of Genetic Toxicology, has led to an ever increasing need for sensitive, cost effective and robust in-vitro test systems for use in therapeutic compound Safety Assessment. Recently, the development of the in-vitro Pig-A gene mutation assay was identified as one of the emerging technologies (Schuler et al., 2011), utilising an analogous basic concept to the current flow cytometry (FCM) based in-vivo Pig-a assay (Dobrovolsky et al., 2010). Presently, increasing reviewer demand for specific in-depth appraisal of the Pig-a mechanism has led to the requirement of use of the in-vivo human lymphocyte or in-vitro Pig-A test systems. Preliminary dose response analysis showed qualitative assessment following acute exposure to the model alkylating agent, Ethyl Methanesulfonate (EMS), in human lymphoblastoid cell lines. Therefore, the present study endeavoured to meet two separate goals; initially to further design and validate the in-vitro equivalent assay in order to assess the feasibility of quantitative dose response modelling, which then therefore, could potentially supersede current in-vitro gene mutation assays (HPRT and MLA). Secondly, by means of the in-vitro test system, begin to discover the true identity of presumptive Pig-A mutants; observed phenotypic GPI-AP negative events. Human lymphoblastoid cells used within the in-vitro Pig-A assay were shown to demonstrate p53 functionality, generating dose dependent p21 (WAF1/CIP1) expression when exposed to MMC (4hr treatment). Bench mark dose (BMD) metrics were also generated that were in-line with those presented for EMS in different in vitro gene mutation endpoints (Gollapudi et al., 2013). Novel flow cytometry approaches provided data beneficial in broadening understanding between the flow cytometry defined Pig-A mutant phenotype with functional insight into the true identity of such events. BB. Gollapudi et al., (2013) Quantitative Approaches for Assessing Dose Response Relationships in Genetic Toxicology Studies. Environmental and Molecular Mutagenesis, 54, 8–18 Dobrovolsky, V. N., Miura, D., Heflich, R. H., and Dertinger, S.D. (2010) The In-vivo Pig-A Gene Mutation Assay, A Potential Tool for Regulatory Safety Assessment. Environ Mol Mutagen. 51, 825–835 Schuler, M., Gollapudi, B. B., Thybaud, V., & Kim, J. H. (2011). Need and potential value of the Pig-a in vivo mutation assay-a HESI perspective. Environ Mol Mutagen, 52(9), 685–689. doi: 10.1002/em.20687 Workshop 1: Poster 072 Genotoxic effect of polycyclic aromatic hydrocarbons alone or in mixture Laure Khoury1, Vanessa Graillot1, Cleo Tebby2, Florence Zeman2, Alexandre Péry2, Jean-Pierre Cravedi1, Marc Audebert1 1INRA, Toulouse, France;2INERIS, Verneuil-en-Halatte, France Polycyclic Aromatic Hydrocarbons (PAHs) are a family of more than one hundred compounds and are potentially carcinogenic to humans. Hazard characterization of PAH complex mixtures is currently based on Toxicity Equivalent Factors (TEFs). However, this method present some limits and it is relevant to define new TEFs for a large number of PAH in order to improve cancer risk assessment for humans. In this study, we used a new genotoxic assay (based on gH2AX quantification) to analyze the genotoxicity of 27 PAHs on three human cell lines representative of major target organs of PAHs: liver (Hep3B), colon (LS-174T) and lung (NCI-H358). Mathematical models were used to determine new TEFs values for PAHs. Dose-genotoxic response modelling was performed using a Hill model, with a focus on effects equivalency between PAHs at low dose. We observed a significant dose-response of genotoxicity in the three cell lines tested for 16 tested compounds and developed new TEFs that we named GEF for Genotoxic Equivalent Factor. Calculated GEFs for the tested PAHs were generally higher than usually utilized TEFs. We further explored the effect of PAH mixtures by testing 16 binary mixtures on LS-174T cells. In some cases, antagonistic, potentiation or synergic actions, or a variation of the maximum response were observed in relation to concentration addition. To confirm these interactions, DNA adducts quantification and metabolic studies are under investigation. Finally, more complex mixtures of PAHs based on 4, 8 or 16 compounds were tested and the genotoxic effect of these mixtures was predicted with concentration addition. In this study, we proposed new TEFs for PAHs based on their genotoxic effects on human cells investigated at low doses. Moreover, we developed mathematical models to predict PAH mixtures genotoxic effect. This method will be useful to improve cancer risk assessment for humans exposed to PAHs. Workshop 1: Poster 073 Genotoxic potential of phosphorothioate mononucleotide analogue in human TK6 lymphoblastoid cells Amer Saleh1, Mick Fellows1, Nigel Gooderham2, Catherine Priestley1 1AstraZeneca R&D, UK;2Imperial College, London, UK Phosphorothioate oligonucleotide (S-ODNs), in which one of the non-bridging oxygen atoms is replaced with a sulphur atom, are widely employed in antisense therapeutics. Despite their enhanced nuclease resistance, S-ODNs could slowly degrade and release monophosphorothioate nucleotides, which may imbalance nucleotide pools or become a substrate for DNA polymerase with potential incorporation into newly synthesised DNA. In this work, the effect of deoxyadenosine monophosphorothioate (dAMPαS) on the thymidine kinase (TK) locus was investigated. Treatment of human TK6 lymphoblastoid cells with 0.5mM dAMPαS gave a significant increase in mutation at the TK locus and increasing dAMPαs concentration up to 1.5mM gave a highly significant response. The increase in TK mutant frequency was associated with a decrease in cell survival. However, dAMPαs failed to induce HPRT mutation and only a weak increase was seen in induced micronuclei. Accordingly the phenotypic stability of the trifluorothymidine (TFT) resistant colonies was assessed. When the TFT resistant colonies from high concentrations of dAMPαS were rechallenged with TFT, the TFT resistance phenotype was lost. These data suggest that dAMPαs treatment in some way interferes with the TFT selection system and may lead to unstable TFT resistant colonies (not true TK / mutants) via a mechanism which is yet to be identified. It is clear that caution should be taken with interpretation of mammalian cell gene mutation data when assessing modified nucleotides. Workshop 1: Poster 074 Genotoxicity Testing Requirements for Cosmetics, Medical Devices and Pharmaceuticals – a Missed Opportunity for Alignment of Regulations? Philip Clay Chorley Consulting Ltd, UK The regulations which describe the process and requirements to assess the genotoxicity of products which are classified as cosmetics (EC1223/2009), medical devices (ISO 10993) or pharmaceuticals (ICH S2(R1)) have all recently been, or are currently being, reviewed and updated. The primary intention of all these regulations is to protect the public from exposure to potentially hazardous materials. Many personal care products may fall into different regulatory classifications based on subtle differences in ingredients, claims or mode of action but the requirements for the assessment of the genotoxicity of the products can be varied and confusing. Here, we describe the various approaches to genotoxicity assessment and discuss if the recent and proposed updates may be missing an opportunity to better align across regulatory classifications. Workshop 1: Poster 075 Historical Data for SHE and Bhas Cell Transformation Assays Kamala Pant1, Shannon Bruce1, Michelle Klug-Laforce1, Jamie Sly1, Timothy Lawlor1, Marilyn Aardema2 1BioReliance by SAFC;2Marilyn Aardema Consulting Two of the cell transformation assays (CTA) are going through the OECD guideline process for use in predicting carcinogenicity. These are the assays used to assess a test article’s potential to cause morphological transformation of the cell cultures. The Syrian Hamster Embryo (SHE) assay assesses the transforming activity of the test article in primary SHE cells as measured by induction of morphologically transformed (MT) colonies consisting of randomly oriented, three dimensional, stacked growth pattern, with criss-crossing of cells. The Bhas 42 cell transformation assay uses the Bhas 42 cells and measures induction of MT foci that show invasive growth into the monolayer of surrounding contact-inhibited cells. Bhas 42 cells were established by transfection of v-Ha-ras gene into BALB/c 3T3 cell line, therefore are considered initiated in a two-stage carcinogenesis theory. The assay protocol consists of an assay for initiation (Sasaki, et.al. 1988, 1990, Asada, et.al. 2005) and promotion (Ohmori, et.al.2004, 2005). BioReliance has been performing these CTAs for more than a decade and over the years has accumulated an extensive historical database for the SHE and Bhas 42 CTAs. The positive control used for the SHE CTA is benzo(a)pyrene at 5.0 µg/mL, for the Bhas 42 initiator assay it is 3-methylcholanthrene (MCA) at 1.0 µg/mL and for the Bhas promoter assay it is 12-O-tetradecanoylphorbol-13- acetate (TPA) at 50ng/mL. The mean frequency of MT colonies in the SHE assay is 0.45 and 2.1 for the vehicle and positive controls, respectively. In the Bhas Initiator assay the frequency of MT foci is 5.8 and 40.71 for the vehicle and positive controls, respectively. In the Bhas Promoter assay the mean values are 6.51 and 33.87 for the same. These data are for up to year 2013. In this poster we will present the vehicle and positive control data over the years for both CTAs. Workshop 1: Poster 076 Idiosyncrasies of in vitro approaches to toxicity testing: Lessons from Genetic Toxicology Martha Moore1, Julie Clements2, B Bhaskar Gollapudi3, Matthew LeBaron4, Elisabeth Lorge5, Stefan Pfuhler6, Paul White7 1ENVIRON International Corporation, Little Rock, AR, USA (Little Rock, AR, US);2Covance, Harrogate, UK;3Exponent, Midland, MI, USA;4Dow Chemical, Midland, MI, USA;5Servier Group, Orleans-Gidy, France;6Proctor and Gamble, Mason, OH, USA;7Health Canada, Ottawa, ON, Canada. There is an international focus on developing in vitro predictive toxicology assays for chemical hazard assessment, driven by a desire to reduce laboratory animal use. This, coupled with the new molecular ‘omics” technologies has provided the major impetus behind the Tox21, ToxCast and other efforts to move toward primary reliance on an in vitro hazard test battery. The discipline of genetic toxicology which began more than four decades ago can offer many valuable lessons to this new toxicity assessment paradigm. Early on, the possibility that the in vitro tests (bacterial and/or cell culture-based) would be good predictors of chemicals carcinogens was proposed and demonstrated with a limited set of chemicals, prompting the hope that these tests would prove to be inexpensive and reliable predictors of chemical carcinogens. Over the decades there has been substantial knowledge gained concerning the strengths, weaknesses and issues involved in using in vitro assays for hazard assessment. In addition, major improvements to the genetic toxicology assays and data interpretation have been accomplished. There are many parallels between the emerging field of in vitro toxicology testing and the evolution of in vitro genetic toxicology tests. A number of major issues were identified and tackled that are just now being recognized as important for 21st Century in vitro assays. Currently, there is a multi-sector international effort with a focus on better understanding such issues like cell stewardship, cell line characteristics and the appropriate strategies for providing/assuring the required metabolic activation for in vitro systems. This effort, coupled with other genetic toxicology community experience, including the use of 3-D vs 2-D cell cultures, and the selection of endpoints and correlations with apical health outcomes, provide extensive information that should inform the current efforts to establish all in vitro approaches for chemical hazard assessment. Workshop 1: Poster 077 Influence of receptor fluid on dermal absorption of caffeine Kristina Trenz, Sabine Bruszies, Hana Hofman-Huether BSL Bioservice Laboratories Dermal absorption describes the transport of chemicals from the surface of the skin into the skin and into the systemic circulation. An in vitro method, based on Franz cells, using preferably human skin is widely used for the investigation of penetration properties of substances. To ensure a correct interpretation of results it is important to investigate factors influencing this assay. In the current study the influence of different receptor fluids on the penetration properties of caffeine was investigated. The study was performed in a dynamic flow through system using human skin. Caffeine was applied at the start of the study. At various time points receptor fluid was collected. At the end of the study all matrices (skin, strips, skin wash and receptor fluid) were analysed for the content of caffeine. The receptor fluids used were water with 50% Ethanol and DPBS supplemented with BSA (5%), DMSO (5%) or SDS (5%). The results confirmed, that the receptor fluid used for dermal absorption studies in vitro has an influence on the penetration properties. The total absorption of Caffeine varied depending on the receptor fluid composition between 45% (DMSO and BSA) and 15% (50% Ethanol and SDS). Workshop 1: Poster 078 Integration of cII, Pig-a and Micronucleus Endpoints into Big Blue® Transgenic Mutation Assay Using Benzo(a)pyrene and Ethylnitrosourea Robert R Young1, Harshini Dinesdurage1, David Bruning1, Leon F Stankowski1, Rohan Kulkarni1, Timothy E Lawlor1, Marie McKeon1, Yong Xu1, Kamala Pant1, Svetlana Avlasevich2, Dorothea K Torus2, Steven D Dertinger2, Marilyn J Aardema1,3 1BioReliance by SAFC, USA;2Litron Laboratories, USA;3Marilyn Aardema Consulting, USA Recent trends in genetic toxicology have led to the integration of multiple endpoints into repeat dose in vivo studies to investigate multiple modes of DNA damage and to reduce the number of animals used in testing. As part of qualification of the Lambda LIZ/lacI C57BL/6 homozygous (Big Blue®) transgenic mouse mutation assay system to OECD TG488 standards, we examined induction of cII mutations in liver and bone marrow, micronuclei in blood reticulocytes and mature erythrocytes (mnRET and mnNCE, respectively), and Pig-a mutant phenotype reticulocytes and erythrocytes (RETCD24- and RBCCD24-, respectively). Animals were treated with olive oil (5mL/kg/day for 28 days), BaP (50mg/kg/day for 28 days), or ENU (40mg/kg/day on Days 1–3), with sacrifice on Day 31. Background cII mutant frequencies in liver and bone marrow were 43.6±6.20x10^-6 and 38.1±14.2^-6 respectively. Significant increases (p<0.001) in cII mutant frequencies were observed for BaP in liver (4.89-fold) and bone marrow (4.15-fold), and for ENU in liver (17.7-fold) and bone marrow (10.6-fold). Peripheral blood was collected ~3 hours before sacrifice and analyzed for Pig-a mutant phenotype and micronucleus frequencies by flow cytometry using MutaFlow®and MicroFlow® kits, respectively (Litron Laboratories). Background micronucleus frequencies were 0.139±0.0419% and 0.106±0.131% for mnRET, mnNCE respectively and for Pig-a mutant phenotype were 0.327±0.0160x10^-6 and 0.565±0.0552x10^-6 for RETCD24- and RBCCD24- respectively. BaP induced significant increases (p<0.001) in mnRET, mnNCE, RETCD24- and RBCCD24- (1.56-, 2.11-, 616, and 101-fold, respectively). ENU induced significant increases (p<0.001) in mnNCE, RETCD24- and RBCCD24- (1.40-, 1050-, and 208-fold, respectively). As expected, mnRET were not elevated by ENU (p>0.05) due to the extended sampling time after dosing with ENU. This study demonstrates the robustness of the Big Blue® Mouse Mutation Assay to detect induced mutagenicity by both direct and indirect acting mutagens and demonstrated the ability to integrate additional endpoints for mutation and clastogenicity. Workshop 1: Poster 080 Multiplexed flow cytometric DNA Damage response assay provides high throughput, comprehensive profiling of genotoxicity and cytotoxicity Steven Bryce, Ariel Berg, Jeffrey Bemis, Stephen Dertinger Litron Labs, Rochester NY Characterization of relevant endpoints, e.g. γH2AX, cleaved PARP, as part of early screening prior to genotoxicity testing could yield valuable information in advance and enable better prediction and design of required tests such as the in vitro micronucleus assay. However, in order to effectively operate in early screening environments, assays must be exceptionally simple and miniaturized. We developed a multiplexed, homogeneous assay that simultaneously liberates cells’ nuclei and labels specific nuclear epitopes with fluorescent antibodies. These samples are compatible with 96 and 384 well plate formats and can be rapidly analyzed via flow cytometry. Initial studies involved exposing both human TK6 and mouse L5178Y cells to a 22-member reference set of compounds consisting of known genotoxicants and irrelevant positive agents. Cells were treated for 24 continuous hours and sampled at 4 and 24 hours for assessment of γH2AX, phospho-H3, cleaved PARP, cell cycle and cell proliferation. Processing involved addition of cells to labeling solution followed by a 10min incubation and analysis. Information on the respective endpoints was obtained from about 2000 cell equivalents analyzed in approximately 10sec. Qualitative examination of the multitude of endpoints across both cells types revealed responses and patterns consistent with the expected activities of the reference compounds. Clastogens elicited increases in γH2AX, reductions in phospho-H3, and characteristic G2/M cell cycle block. Aneugens induced no change in γH2AX, accumulation of mitotic cells marked by phospho-H3 positivity, and increased polyploidy. While positive indications of cytotoxicity were observed for certain concentrations of the genotoxic agents, the lack of response in γH2AX and phospho-H3 served to differentiate non-genotoxic cytotoxic chemicals. Future investigations involving this one-step, multiplexed screening approach will involve examination of additional chemicals, identification of other useful markers and application of statistical modeling programs to further automate the classification process. Workshop 1: Poster 081 News from the pre-validation of the Hen’s Egg Test for Micronucleus-Induction (HET-MN) Katrin Maul1, Dagmar Fieblinger1, Andreas Heppenheimer2, Juergen Kreutz3, Manfred Liebsch1, Andreas Luch1, Ralph Pirow1, Albrecht Poth2, Kerstin Reisinger3, Pamela Strauch2, Thorsten Wolf4 1Federal Institute for Risk Assessment, Berlin, Germany;2Harlan Cytotest Cell Research, Rossdorf, Germany;3Henkel AG & Co KgaA, Duesseldorf, Germany;4University of Osnabrueck, Osnabrueck, Germany The HET-MN assay (Hen’s Egg Test for Micronucleus-Induction) is different from other in vitro genotoxicity assays because it includes toxicologically important features such as absorption, distribution, metabolic activation and excretion of test compounds. The approach combines a regulatory accepted read-out parameter (the evaluation of micronuclei) with a widely used test system (the hen’s egg). As a promising follow-up approach to supplement existing in vitro test batteries for genotoxicity, the HET-MN has recently undergone a formal pre-validation. In 2010 the transfer of the methodology from the originator laboratory, the University of Osnabrueck, to three other laboratories was finalized (Federal Institute for Risk Assessment, Harlan Cytotest Cell Research and Henkel) with testing Cyclophosphamide and 7,12-Dimethylbenzanthracene. In the second phase three compounds (Ampicillin, Carbendazim, Acrylamide) were tested in a blind study in the laboratories of the BfR, Harlan and Henkel. These data showed a good intra- and inter-laboratory reproducibility. In the final pre-validation 20 chemicals comprising different chemical classes and mode of actions were tested blinded. The chemicals comprised 10 known clastogens and aneugens as well as 10 compounds with an expected negative outcome. The later group comprised the so called true negatives (negative in both historical in vitro and in vivo experiments) and compounds which were reported positive in regulatory accepted in vitro assays but were shown to be negative in in vivo experiments (the so called misleading positives). The data will be presented concerning the reproducibility and predictivity. The work was funded by the German Ministry for Research and Education. Workshop 1: Poster 082 p53 may be important for avoidance of misleading positives in in vitro genotoxicity tests Katherine E Chapman, Anna L Seager, Katja Brüsehafer, Ume-Kulsoom Shah, Eleanor Wilde, Jane Mikhail, George E Johnson, Shareen H Doak, Gareth JS Jenkins DNA Damage Research Group, Institute of Life Science, Swansea University, UK Current in vitro genotoxicity tests display low sensitivity, as illustrated by the high frequency of misleading positive results. Therefore, identification of a biomarker that allows genotoxic agents to be distinguished from non-genotoxic agents could be advantageous. A possible candidate for such a biomarker is p53, a protein that is crucial in coordinating DNA repair, cell cycle kinetics and apoptosis in response to genotoxicity. Here, the In Vitro Micronucleus Assay, Western blotting, analysis of mutation spectra and comparison of human lymphoblastoid cell lines were used to establish whether p53 expression and status were linked to genotoxicity. Protein analysis demonstrated that p53 was up-regulated in response to doses of hydrogen peroxide between 10µM and 20µM, coinciding with the LOGEL of 12µM for micronucleus frequency. In contrast, di(ethylhexyl)phthalate, which produced a negative micronucleus response, did not produce any changes in p53 expression. Comparisons between micronucleus induction in p53-proficient cell line TK6, and a p53-deficient cell line isogenic to TK6, NH32, demonstrated that p53 could be responsible for an adaptive (hormetic) dose-response following N-methyl-N-nitrosourea treatment at 0.009µg/ml. Further, greater micronucleus induction was observed in NH32 compared to TK6 following mitomycin-C or cytosine arabinoside, compared to TK6. Mutation spectra analysis confirmed that cell lines MCL-5 and AHH-1 both contain a partial CCGG to TTGG transition mutation at codon 282 in exon 8 of the TP53 gene, which encodes p53. Such a mutation might confer heightened sensitivity of these cells to genotoxins, relative to cells with wild-type p53 status. Together, these data demonstrate that p53 shows promise as a candidate for distinguishing between genotoxins and non-genotoxins. Further, the data support the use of p53-competent cells in avoidance of misleading positives. This knowledge might lead to more effective initial assessment of the genotoxic potential of chemicals, therefore reducing the number of unnecessary animal follow-up tests currently performed. Workshop 1: Poster 083 Reconstructed Skin Comet assay: Update on the ongoing validation Stefan Pfuhler1, Marc Bartel2, Veronika Blatz2, Joep Brinkmann3, Thomas R Downs1, Ursula Engels4, Anja Fischer4, Frank Henkler3, Sebastian Hoffmann5, Stefanie Jeschke3, Cyrille Krul6, Manfred Liebsch7, Ralph Pirow3, Astrid Reus8, Markus Schulz2, Kerstin Reisinger4 1The Procter & Gamble Co;2BASF SE, Ludwigshafen, Germany;3Federal Institute for Risk Assessment, Germany;4Henkel AG & Co KGaA, Düsseldorf, Germany;5seh consulting + services, Germany;6TNO, Zeist, The Netherlands;7Federal Institute for Risk Assessment, Berlin, Germany;8TNO Triskelion, Zeist, The Netherlands In vitro cytogenetic assays have been associated with a high rate of irrelevant positive results. Therefore skin tissues which take into account the exposure route and potential metabolism of ingredients were combined with the Comet assay approach with the aim to provide a new approach to be used as a follow-up for positive results from the current in vitro genotoxicity test battery for compounds that are in direct contact with the skin. Here, we provide an update on the progress of the validation of the Reconstructed Skin (RS) Comet assay. In 2012 Cosmetics Europe (CE) joined forces with the German Federal Ministry of Education and Research (BMBF). Whereas the CE project had focused on an epidermal model, the project funded by the BMBF worked on a number of full thickness (FT) models. While each skin model requires a different method for cell isolation, both projects used the same protocol for the Comet assay procedure, allowing for a comparison between the models. As a result, the Phenion® and EpiDermTM Full-Thickness Skin Models were selected to enter the validation phase. This decision was made after statistical evaluation of results from the method transferability phase using, e.g., data obtained with the direct-acting mutagen and taking into account inter- and intra-laboratory variability, as well as the number of valid experiments. The validation includes testing of 30 chemicals, selected by external experts, in an incomplete block-design. The initial testing phase is now complete and focused on inter- and intra-laboratory reproducibility, such that 8 coded chemicals were each evaluated in three laboratories comprising (1) true positives (cadmium chloride, mitomycin C, 7,12-dimethylbenz(a)anthracene, N-ethyl-N-nitrosourea), (2) misleading positives (eugenol, propyl gallate) and (3) true negatives (cyclohexanone, di-(2-ethylhexyl)phthalate). The results of this phase showing good predictivity will be presented. Workshop 1: Poster 084 Reconstructed Skin Micronucleus Assay: Update on the ongoing validation Stefan Pfuhler1, Marilyn Aardema2,6, Brenda Barnett1, Emilia Costin3, Sebastian Hoffmann4, Greg Mun3, Gladys Ouedraogo5, Shambu Roy6, Rodger Curren3 1The Procter & Gamble Co;2Marilyn Aardema Consulting, LLC;3IVS, Inc;4seh consulting + services;5L’Oreal Life Sciences Research;6Bioreliance, USA Cosmetics Europe has funded projects to establish and evaluate models for genotoxicity using 3D reconstructed skin tissues. These provide a more realistic model for dermally applied chemicals/products, such as cosmetics, and are expected to be used to follow-up on positive results from the in vitro genotoxicity battery. Phase 1 and 2 studies validating the RSMN using EpiDerm™ tissues showed good transferability, inter- and intra-laboratory reproducibility. In Phase 3, the number of chemicals was extended from 5 to 28. Results demonstrated excellent specificity (90%) but the number of true positives tested so far (8) was considered too few to draw a final conclusion about the sensitivity of this assay. Therefore, an additional 12 compounds selected by external experts are being tested with a focus on genotoxic carcinogens. These chemicals for the next testing phase include 9 expected positive and 3 chemicals that were showing precipitation, two of which led to unexpected positive results in previous Phase 3 testing and are now being retested under conditions that specify handling of precipitating chemicals in more detail. Overall, the data generated to date support the use of the 3D skin EpiDerm™ model for genotoxicity testing of dermally applied chemicals. This current study on 12 additional chemicals is expected to provide a conclusive estimation of the sensitivity and specificity of the RSMN. Workshop 1: Poster 085 Response of p53 competent human cell lines – Differences after treatment with genotoxins and non-genotoxins Florian Engel1, Prashant Singh2, Jürgen Borlak2, Sandra Weickhardt1, Gabriele Kaufmann1, Roland Frötschl1 1BfArM, Bonn, Germany;2MH-Hannover, Germany For the risk evaluation of pharmaceuticals the in vitro genotoxicity testing plays an essential role. However cells frequently used and mentioned in guidelines as those with greatest experimental experience are diverse of origin and genetic background. IWGT recommended the use of p53 competent cells for testing, however also between those unlike sensitivities were described for the same substances. We tested 10 different non-genotoxic and genotoxic substances in two p53 competent human cell lines, HepG2 and TK6, and compared the treatment response of these cells widely used in genotoxicity testing. Substances used were N-Nitroso-N-methylurea, 7,12-Dimethylbenzanthracene (DMBA), Benzpyrendioepoxid (BPDE), Streptonigrin, Etoposid, Paclitaxel, Lamivudin, Phorbol-12-myristate-13-acetate, Phenobarbital, Chloroquine and Nicotine. Treatment was done for 24h (48h Lamivudin) with a concentration associated with 20 % cytotoxicity in the particular cell line and whole genome microarray analysis was performed. Results show that the master regulators (MR) for the same substance are different in the two cell systems. The overlap of the corresponding networks is little (1 - 29 %) between the cell systems. Because of the differences between the MR networks for the same substance in the two cell systems it is obvious that the response to the substance treatment is dissimilar. This is confirmed by the biochemical role of the MR`s. With DMBA/BPDE in the HepG2 cells for example the MR is involved in the metabolism of the substance whereas in TK6 cells, with low metabolic competence, the MR is involved in DNA mismatch repair and recombination. The different response of the two cell systems clearly recommends that the choice of test system has to be done carefully with consideration of the target organ exposed. Further investigation in the relevance of master regulators for organ specific differences in genotoxic potency of chemicals are needed. Workshop 1: Poster 086 Revision of OECD guidelines for genotoxicity: current status and next steps Veronique Thybaud1, Jan Van benthem2, Nathalie Delrue3, George R Douglas4, Elisabeth Lorge5, David P Lovell6, Martha M Moore7, Rita Schoeny8, Tim Singer4 1Sanofi, Vitry-sur-Seine, France;2National Institute for Public Health and the Environment, Bilthoven, Netherlands;3OECD, Paris, France;4Health Canada, Ottawa, Canada;5Servier Group, Orleans-Gidy, France;6St George’s, University of London, UK;7ENVIRON International Corporation, Little Rock, AR, USA;8US EPA, Washington, USA Over the past 30 years, in vitro and in vivo assays have been developed to evaluate genotoxic properties of chemicals, and OECD Test Guidelines (TG) for genotoxicity were released to describe the assay procedures to be used in regulatory safety testing. Since last revision (1997), tremendous scientific and technological progress has been made on assay performance and understanding of underlying mechanisms. In order to improve regulatory assay performance and data interpretation, a project for updating the existing OECD Test Guidelines on Genotoxicity was launched in March 2010. Canada, Netherlands, France and USA were identified as lead countries, and an Expert Group (EG) comprised of international experts was established. EG has recommended the deletion of several TGs, including the assays conducted in yeast and Drosophila, considering that assays conducted in mammalian cells are more appropriate. The purpose of the revision process is 1) to improve the clarity and consistency between TGs, 2) to improve the statistical power, 3) to refine the methodology to improve the relevance of the results, and 4) to provide more up to date recommendations for acceptability criteria and data interpretation. The OECD Working group of National coordinators of the Test Guidelines Programme approved in April 2014 the revised TGs for the in vitro and in vivo TGs for the micronucleus (TG487, TG474), and chromosomal aberration tests (TG473, TG475), and a new TG for in vivo Comet assay. The next steps will consist of 1) the revision of TGs for in vivo germ cell, and in vitro mammalian gene mutation tests, 2) the update of the Introduction Document to provide an overview of the TGs and background for the recent updates, and 3) the development of a Guidance Document dealing with assay strengths and weaknesses, weight of evidence approaches and data interpretation. Workshop 1: Poster 087 Statistical Analysis of the Pig-a In Vivo Gene Mutation Assay Bernd-Wolfgang Igl1, Andreas Sutter1, Stephen Dertinger2 1Bayer Pharma AG, Berlin, Germany;2Litron Laboratories, Rochester, NY. The peripheral blood Pig-a in vivo gene mutation assay is a novel method for the detection of somatic gene mutations. Mutation of the Pig-a gene renders glycosylphosphatidylinositol (GPI) anchors non-functional. Without these anchors, specific markers on the exterior of cells such as CD59 will not be present. The Pig-a assay measures the frequency of cells not exposing these markers on their surface, also known as ‘Pig-a mutant phenotype cells”. This assay is currently under validation; however, it has already achieved regulatory relevance as it has been suggested as in vivo follow-up for directly acting Ames positives in the recent ICH M7 step 3 document. Presently there is no recommended standard for the statistical analysis of this assay. Thus, we propose a statistical strategy involving a down-turn protected Poisson-regression to determine a dose-dependency of rare mutant phenotype erythrocytes. We demonstrate appropriateness of this strategy using example data sets obtained with the current processing protocol. Workshop 1: Poster 088 Study towards the genotoxic potential of migration products from plastic food contact materials Birgit Mertens1, Roel Anthonissen1, Coraline Simon2, Heidi Demaegdt3, Tara Vandermarken4, Kersten Vanlangenhove4, Marc Elskens4, Karin Vandermeiren3, Marie-Louise Scippo2, Luc Verschaeve1 1Toxicology Unit, Dep. Public Health & Surveillance, Scientific Institute for Public Health, Belgium;2Laboratory of Food Analysis, Dep. of Food Sciences, University of Liège, Belgium;3Environment, Health and Safety of the Food Chain Unit, CODA-CERVA, Belgium;4Analytical and Environmental Chemistry, Vrije Universiteit Brussel, Belgium Bisphenol A (BPA) is often used as a monomer for the manufacture of plastics such as polycarbonate. This transparent thermoplastic has many applications including the fabrication of several food containers like baby bottles, cups, etc. Reports on the migration of BPA from polycarbonate into food together with studies identifying BPA as an endocrine disruptor have resulted in a worldwide concern about the use of BPA. In January 2011, the European Commission decided to ban the use of polycarbonate in baby feeding bottles. In a recent opinion, the Belgian Superior Health Council highlighted however that at present little is known on the currently used alternatives for polycarbonate. The ALTPOLCARB project aims to study the migration of compounds from non-polycarbonate plastic material and their genotoxic and endocrine disruptor activities. In the current part of the study, the genotoxic potential of pure substances for which the migration from plastic baby bottles has been reported in literature, was investigated. The screening was performed with the Vitotox test, an indicator test for genotoxicity. The Vitotox test is based on two recombinant Salmonella typhimurium reporter strains and detects activation of the SOS operon by an integrated lux operon. None of the forty compounds tested fulfilled all criteria for a positive result in the Vitotox test. Although more tests are required to complete the full genotoxic profile of the compounds, these results already provide a first indication of the lack of genotoxicity associated with the different migration products. Workshop 1: Poster 089 The combination of two tobacco blend technologies to reduce tobacco smoke toxicant yields: Assessment in the Ames test Ian Crooks, Ken Scott, Annette Dalrymple, Debbie Dillon, Clive Meredith British American Tobacco, UK Tobacco smoke from a combustible cigarette contains more than 6000 constituents, approximately 150 of these are identified as toxicants. Technologies that modify the tobacco blend to reduce toxicant emissions have been developed, including tobacco sheet substitute (TSS) to dilute toxicants in smoke, and blend treated tobacco (BT) to reduce the levels of nitrogenous precursors and some polyphenols. In this study, BT and TSS technologies were combined into an experimental cigarette (EC) with filter additives to further modify smoke constituents. A control cigarette was included for comparative purposes; both cigarettes had a tar pack yield of 7mg. The EC and control cigarette were smoked to ISO (35mL puff volume of 2 s duration, every 60 s, 0% vent blocking) and HCI (55mL puff volume of 2 s duration, every 30 s, 100% vent blocking) puffing parameters. The resulting particulate matter (PM) from both cigarettes were subjected to an Ames test with 5 Salmonella typhimurium strains in the presence and absence of S9. The results indicate that some statistically significant (p ≤ 0.05) reductions in bacterial mutagenicity were observed in TA98 and TA100 relative to the control cigarette, regardless of smoking regime. With TA1537, the EC HCI PM was less mutagenic (p ≤ 0.05) than the control however, the ISO EC PM was equivalent to the control, overall. Furthermore, these reductions in mutagenicity are more consistent than ECs that contained single blend technologies. In conclusion, the data demonstrates that cigarettes with reduced toxicant emissions may be developed without introducing any additional mutagenic hazards as assessed by the Ames test, with the tester strains specified. In addition, significant reductions in Ames mutagenicity of the EC relative to the control cigarette were observed. Workshop 1: Poster 090 The development of a DNA adductomic liquid chromatography-tandem mass spectrometry genotoxicity screening system Laura Turnham1, Rajinder Singh1, Michael D Fellows2, Karen Brown1, Peter B Farmer1 1University of Leicester, Department of Cancer Studies and Molecular Medicine, UK;2AstraZeneca, R&D, UK In the EU, US and Japan, legislation requires the evaluation of drug genotoxicity prior to commencing clinical trials. Agents that can react directly with DNA and do not display threshold toxicity are of particular concern. Liquid chromatography- tandem mass spectrometry (LC-MS/MS) has become one of the most sensitive and specific methods for DNA adduct detection. The aim of this project is to develop an assay using tandem mass spectrometry to screen for 2’-deoxynucleoside DNA adducts, utilising a common fragmentation pattern, the neutral loss of the 2’-deoxyribose sugar (116u). Both 2’-deoxyguanosine (dG) and calf thymus (CT) DNA were incubated with different model genotoxic agents at low and high pH to identify conditions for maximal adduct formation and minimise decomposition. CT DNA was subjected to enzymatic hydrolysis to 2’-deoxynucleosides prior to analysis. Samples were analysed with LC-MS/MS, scanning for the constant neutral loss of 116u. Scans were performed over the range 200–650 m/z, or alternatively each sample was injected five times with the scan range limited to 100 m/z increments. For dG treated with malondialdehyde (MDA) two adducts were detected, one of which was confirmed to be 3-(2’-deoxyribosyl)pyrimido[1,2-a]purine-10(3H)-one (M1dG). Treatment of dG with glycidamide at pH 9.0 resulted in two adducts with [M+H]+ ions at 355 and 356 m/z. Both M1dG and 3-(2’-deoxyribo-N6-adenosinyl)propenal (M1dA) adducts were detected following the treatment of CT DNA with MDA. No DNA adducts were observed for the glycidamide treated CT DNA samples, possibly due to adduct loss via depurination in the enzymatic hydrolysis stage. In conclusion, limiting the LC-MS/MS CNL scan range to 100 m/z increments enhanced sensitivity allowing the detection of DNA adducts in both CT and dG treated samples. The results of this study indicate the potential of the developed method for use in screening compounds for DNA adduct formation. Workshop 1: Poster 091 The difference in response of aneugenic compounds in TK6 and L5178Y cells Julie Hayes, Katie Wood, Ann Doherty 1Discovery Safety, Drug Safety & Metabolism, AstraZeneca, UK A compound series in development at AstraZeneca, with a mode of action of an epigenetic reader has been investigated in the in vitro (MNvit) and in vivo micronucleus assays. The chemical series has been shown to induce aneugenic damage. The MNvit assay is utilised to screen compounds and predict for the in vivo micronucleus outcome. In this project we have been able to obtain in vivo bone marrow samples from early discovery studies, and therefore we have a more thorough in vitro to in vivo data set than we would normally have for a compound series, which has shown some interesting findings. Of the 6 compounds which gave a positive signal in the in vivo micronucleus test, 4 were from the same chemical scaffold and 2 were from a different chemical scaffold, but all hit the same target with similar potency. However, the results from the in vivo assay for these 6 compounds differed from the results of the in vitro micronucleus assay using TK6 cells, thus warranting further investigations. The results of the MNvit assay using TK6 cells showed that only 2 of the 6 compounds were positive, however all 6 compounds showed a positive induction of micronuclei when using L5178Y cells. Thus, the aneugenic effect seen with all 6 compounds does not fulfil the criteria for a ‘positive’ in the MNvit in the TK6 cells but does in the L5178Y cells. When the micronuclei in the toxic range (cytotoxicity as measured by relative population doublings) were examined in the TK6 cells, the aneugenicity was observable as centromeric positive micronuclei. Therefore, the difference in the results with these cell lines is considered to be a factor of the toxicity (or cell blockage). Workshop 1: Poster 092 The in vitro genotoxicity of topoisomerase II poisons is driven by their activity against the alpha isoform of the enzyme Mick Fellows AstraZeneca, UK Both gemifloxacin and etoposide are potent mammalian cell in vitro and in vivo mutagens and etoposide is a known carcinogen. Their genotoxic mode of action is driven by their ability to stabilise topoisomerase II / DNA cleavable complexes, thus interfering with replicative machinery and inducing permanent DNA strand breaks. Several recent publications have suggested that the carcinogenic mode of action of etoposide is via it’s interaction with the topoisomerase II β isoform1 (1,2). However, after in vitro treatment with etoposide and gemifloxacin, measuring DNA bound topoisomerase II using isoform specific antibodies demonstrated that topoisomerase II α cleavable complex formation was far more prevalent than β. Furthermore, following electroporesis transfection of mammalian cells with a specific topoisomerase II α targeting siRNA, the in vitro genotoxicity of both etoposide and gemifloxacin was effectively mitigated. These data suggest either different isoforms of topoisomerase II drive genotoxicity and carcinogenicity or the suspected mechanism of topoisomerase II induced carcinogenesis is yet to be fully elucidated. Gaining this increased understanding of the isoform specific genotoxicity of topoisomerase II poisons will help with the risk assessment of both topoisomerase II targeting antibiotics and compounds with off-target topoisomerase II inhibition. Reference(s) 1. Cowell IG, Austin CA. (2012) Cell Cycle. 11(17), 3143–31444. 2. Cowell IG, et al. (2012). Proc Natl Acad Sci U S A. 109(23), 8989–8994 Workshop 1: Poster 093 The use of quartz crystal microbalances to assess delivery and deposition of e-cigarette aerosols in vitro Bethany Samways, Jason Adamson, David Thorne, Debbie Dillon, Clive Meredith British American Tobacco Electronic cigarettes (e-cigarette) are relatively new nicotine delivery products and are postulated to be less harmful to the consumer than conventional combustible cigarettes, based on their respective ingredients and emissions. Toxicological and biological assessment of combustible cigarette smoke constituents in vitro have been conducted for decades; however e-cigarettes are yet to be characterised fully in vitro. To aid future e-cigarettes in vitro research, the Vitrocell VC10 smoke engine with QCMs within a cell exposure module, were used to assess the durability and delivery of four commercially available e-cigarettes. E- cigarettes tested were: Product A, Product A Menthol, Product B, Product B Menthol which contained 4.5, 3.0, 4.5 and 3.0% nicotine respectively and allowed comparison between brands (A/B) and nicotine contents (4.5%/3.0%). The VC10 was set at 0.25L/min constant dilution. Ten repeats were tested for each e-cigarette, until battery exhaustion, thus identifying the lowest common puff denominator. Each product showed variation in exhaustion time and deposited aerosol mass between repeats (standard deviations of deposited aerosol mass ± 14.49, 15.48, 14.27 and 29.61µg/cm2 for Product A, Product A Menthol, Product B, Product B Menthol, respectively). Data were plotted with deposited aerosol mass against exhaustion time and show four distinct groups of data representing the four products. Therefore this method could distinguish differences between different brands as well as e-cigarettes with varied nicotine content. These data suggest that there is inherent product variability, which needs to be taken into consideration for future in vitro and possibly in vivo testing. Indeed, this study demonstrates the need for further testing to understand the variability of the products and the differences between products, to facilitate protocol development. This study also highlights the importance of dosimetry tools to quantify the dose of aerosol reaching the cells within in vitro systems. Workshop 1: Poster 094 ToxTracker: a cell-based reporter assay that provides mechanistic insight into the genotoxic properties of chemicals Giel Hendriks, Fabienne Calléja, Bruno Morolli, Branislav Misovic, Gonzalo Congrains Sotomayor, Harry Vrieling Leiden University Medical Center. The ever-increasing number of chemical compounds that are developed by industry for a wide range of applications pose a potential threat for human health. These compounds may react with various biomolecules and disrupt proper cellular functioning. Depending on the nature of the chemical insult, cells activate specialised defence mechanisms that provide protection against the toxic, mutagenic and possibly oncogenic consequences of exposure. Monitoring the activation of specific cellular signalling pathways upon exposure may therefore allow reliable and mechanism-based assessment of potential (geno)toxic properties of chemicals, while providing insight into their primary mode of toxicity. We therefore developed the ToxTracker assay, a panel of mouse embryonic stem (mES) cells that contain different GFP reporter genes. To identify biomarker genes that are predictive for exposure to different classes of genotoxic chemicals, we performed extensive whole-genome transcription profiling. The differential responsiveness of these biomarker-based GFP reporters enables disclosure of the primary reactivity of chemicals. Genotoxic potential of compounds is assessed by a panel of 6 different reporter cell lines that can discriminate between induction of DNA damage, oxidative stress, protein damage and general cellular stress. For automated analysis of ToxTracker reporter activation we have developed a dedicated software tool, ToxPlot. For each reporter, GFP induction is calculated for a fixed level of cytotoxicity, the induction profile for each compound is compared to a panel of reference chemicals and finally, the ToxTracker results are summarised in an easy-to-interpret heat map. Extensive validation of the ToxTracker assay using different reference compound libraries reveals an outstanding sensitivity and specificity of the test compared to the conventional in vitro genotoxicity assays. Together, the integrative approach of ToxTracker provides a powerful tool for identification of potentially carcinogenic properties of chemicals by unveiling the cellular signalling pathways that are activated upon exposure. Workshop 1: Poster 095 Validation of Litron MicroFlow® In Vitro Micronucleus Test Cameron Bain, Dawn Innes, Clive Roper Charles River The aim of this study was to validate flow cytometry analysis of micronuclei obtained from TK6 cells following exposure to a panel of 8 test items with known or predicted genotoxic effects. The test items included negative controls, clastogenic and aneugenic materials and an apoptogen. Test item details are summarised below. TK6 cells were tested in the presence and absence of exogeneous metabolism (rat S9) over a range of test item concentrations intended to assess any dose response to the test item. Samples were analysed by flow cytometry using the Litron MicroFlow® kit. The results generated are summarised below. Cytosine arabinoside (clastogen) correct assignment (positive). Mitomycin C (clastogen) correct assignment (positive). Cyclophosphamide with S9 (clastogen) correct assignment (positive). Colcemid (aneugen) correct assignment (positive). Vinblastine (aneugen) correct assignment (positive). Diethylhexylphthalate without S9 (negative) correct assignment (negative). Diethylhexylphthalate with S9 (negative) correct assignment (negative). Pyrene (negative) correct assignment (negative). Dexamethazone (apoptogen) was assigned as suspicious. For Dexamethazone, a small non dose-dependent response was observed and, therefore, this was assigned as suspicious. The assay correctly predicted the genotoxic classification for all test items investigated. Measurable inter-operator and inter-cytometer differences were observed. However, correct assignment of genotoxic classification was generated irrespective of operator or cytometer. Therefore, Charles River will perform this test by a single lead operator using only a single flow cytometer to analyse samples. In conclusion, the Litron MicroFlow®in vitro micronucleus test was shown to be a valid system for quantifying the micronucleus inducing potential of test items in the in vitro micronucleus TK6 test system. Monday Lunchtime Lecture (supported by ECVAM): 1315 Mammalian cell results with Ames-positive chemicals – correlations with presence or absence of in vivo genotoxic or carcinogenic activity David Kirkland1, Errol Zeiger2, Federica Madia3, Raffaella Corvi3 1Kirkland Consulting, Tadcaster, UK;2E Zeiger Consulting, USA;3EURL ECVAM, Ispra, Italy Positive results in the Ames test are accepted to correlate well with carcinogenic potential, at least in rodents. However, situations can be envisaged where this may not be the case. Such situations might involve bacterial-specific metabolism, exceeding a detoxification threshold or the induction of oxidative damage to which bacteria may be more sensitive than mammalian cells in vitro or tissues in vivo. Since most chemicals are tested in mammalian cells as well as in the Ames test, the pattern of mammalian cell results (positive, negative) may be informative regarding potential for in vivo genotoxic or carcinogenic activity. A database of >750 Ames-positive chemicals that have been tested in vivo has been compiled from multiple sources, and the results in mammalian cell tests for gene mutation (tk or hprt) and clastogenicity/aneugenicity (CAvit; MNvit) have been analysed. Although high proportions (>85%) of carcinogens and in vivo genotoxins gave positive results in both gene mutation and CAvit/MNvit tests, large numbers of chemicals that were not carcinogenic or genotoxic in vivo (>47%) also gave the same pattern of mammalian cell results. Although the different frequencies were statistically significant, positive results in 2 mammalian cell tests, with an Ames-positive chemical, cannot be considered definitively predictive of either carcinogenic or in vivo genotoxic potential. By contrast, the occurrence of negative results in both in vitro mammalian cell tests with Ames-positive carcinogens and in vivo genotoxins was rare. However, such in vitro negative results were significantly more frequent for chemicals that are not carcinogenic or genotoxic in vivo. Thus, with an Ames-positive chemical, negative results in 2 mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered an indicator of the absence of in vivo genotoxic or carcinogenic potential. Monday, Symposium 3: Talk 1/6 Structure and catalytic mechanism of adp-ribosyl glycohydrolases Ivan Ahel University of Oxford, Sir William Dunn School of Pathology, Oxford, UK Poly(ADP-ribosyl)ation is a reversible post-translational protein modification employed in several important nuclear processes including the response to DNA damage and the regulation of chromatin structure. Poly(ADP-ribose) (PAR) is a highly negatively charged polymer that is formed from repeating ADP-ribose nucleotide units linked via glycosidic ribose-ribose bonds, and is synthesised by the poly(ADP-ribose) polymerase (PARP) family of enzymes. The persistence of PAR signalling after a specific pathway has been completed would be highly detrimental, and thus the cells have to use the poly(ADP-ribose) glycohydrolase (PARG) enzyme to degrade PAR chains in a timely manner, by cleaving the unique glycosydic bonds between the ADP-ribose monomers. PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the modified protein and this last step of PAR removal is catalysed by several ADP-ribosylglycohydrolases related to PARG, such as TARG1, MACROD1 and MACROD2. Here we present our data describing the structure and the catalytic mechanism of PARG and TARG1 enzymes, which provide a better understanding of the cellular mechanisms for reversal of PARP signalling. Monday, Symposium 3: Talk 2/6 Dissection of DNA damage responses using genetic interaction maps Haico van Attikum Leiden University Medical Center, Netherlands To protect the genome, cells have evolved a diverse set of pathways designed to sense, signal and repair multiple types of DNA damage. To assess the degree of coordination and crosstalk among these pathways, we systematically mapped changes in the cell’s genetic network across a panel of mechanistically distinct DNA-damaging agents. Each agent was associated with a distinct interaction pattern, which, unlike single mutant phenotypes or gene expression data, has high statistical power to pinpoint the specific repair mechanisms at work. Our networks revealed novel roles for the histone acetyltranferase Rtt109, the neddylation machinery and the RSC chromatin remodeling complex in the DNA damage response. In particular, we observed several links between RSC and the nucleotide excision repair (NER) machinery and found that RSC contributes to efficient removal of UV lesions throughout the genome by two NER pathways: global genome repair and transcription-coupled repair. Thus, our conditional genetic interaction map provides a unique resource that identifies novel factors and pathways involved in the DNA damage response. Monday, Symposium 3: Talk 3/6 Nucleotide Excision Repair Factors Directly Regulate dNTP Synthesis in Response to UV Damage in Yeast Simon Reed1, Zheng Zhou1,2, Neil Humphryes1,3, Patrick van Eijk4, Raymond Waters4, Shirong Yu4, Julia Smirnova4, Rolf Kraehenbuehl4, Edgar Hartsuiker4 1Cardiff University, UK;2College of Biology, Hunan University, Changsha, China;3New York University Department of Biology, USA;4North West Cancer Research Institute, Bangor University, UK The DNA damage checkpoint is a protein kinase cascade that maintains replication fork integrity, promotes DNA repair and regulates cell-cycle progression in response to DNA damage. Regulating gene expression programs is a central facet of this pathway. The Dun1 kinase controls expression of DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Here we demonstrate that the yeast Rad4-Rad23 nucleotide excision repair complex binds directly to the promoters of DNA damage response genes including DUN1, to regulate their expression. Ubiquitination of Rad4 by the GG-NER E3 ligase in response to UV radiation determines the occupancy of Rad4-Rad23 at the promoters of these genes, thus controlling their expression. Our study shows how core NER factors regulate the production of dNTPs, the building blocks required for DNA synthesis and reveal how the nucleotide excision repair pathway integrates with the DNA damage checkpoint. Monday, Symposium 3: Talk 4/6 Coordination of DNA damage signalling and repair Svetlana Khoronenkova, Grigory Dianov Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Oxford, UK Base excision repair (BER) is the major cellular system involved in the repair of DNA damage caused by endogenous and exogenous mutagens. BER therefore is responsible for maintaining genome integrity, and its deficiency has been implicated in many human diseases, including neurodegeneration, premature aging and cancer. Endogenous DNA damage load depends on cellular metabolism and exogenous mutagens, and it is not clear how the levels of BER enzymes are controlled in response to the changing environment. Moreover, to identify the specific mechanisms of response of normal and cancer cells to DNA damage stress, a better understanding of signal transduction pathways upstream of base excision repair is required. I will therefore discuss our recent findings on mechanisms involved in the regulation of proteins and signalling pathways involved in the coordination of base excision repair. Monday, Symposium 3: Talk 5/6, Poster 016: Withdrawn Monday, Symposium 3: Talk 6/6, Poster 020 Influence of SIRT6 on the repair of oxidatively generated DNA modifications Elena Robeska, Waldemar Hess, Bernd Epe Johannes Gutenberg University Mainz Essential cellular functions such as oxidative respiration and lipid peroxidation create reactive oxygen species that can damage DNA. The DNA modifications are repaired by various pathways, depending on the type of lesion. Sirtuins, which are NAD+-dependent deacetylases of histones and other proteins, are known to modulate the accessibility of DNA repair proteins to DNA. In this study, we investigated the influence of sirtuin-6 (SIRT6), on the repair of single-strand breaks (SSB) and of oxidatively generated purines in the DNA. SSB were induced in primary Sirt6-/- mouse embryonic fibroblasts (MEFs) and wild-type comparisons by incubation with H2O2 (50 µM; 15min; 37°C), while oxidised purines (8-oxoG) were generated by exposure to the photosensitizer Ro19-8022 (50nM) plus light. The repair kinetics od the induced lesions were followed by an alkaline elution assay, using the repair glycosylase Fpg as a probe for oxidized purines. We found that Sirt6-/- MEFs showed no retardation of the repair of SSB. Also the Fpg-sensitive DNA base modifications were romoved with the same rate as in the wild-type MEFs. Moreover, the frequency of micronuclei in erythrocytes, which is a sensitive marker for genotoxicity, was not elevated in the Sirt6-/- mice. It has to be concluded that SIRT6 has no influence on the global repair rate of oxidatively induced DNA damage. Symposium 3: Poster 017 Epigenetic modifications by DNA methyltransferase inhibitors in TK6 cells: effects on methylation status and GADD45a expression Mark Tate1, Adam Djouani2, Ioanna Myrtziou-Kanaki1, Nick Billinton1, Richard Walmsley1,2 1Gentronix Ltd, BioHub at Alderley Park, Cheshire;2University of Manchester, UK Knowledge of the mechanisms of epigenetic regulation of gene expression is increasing and a role for methylation and histone modifications in the onset of carcinogenesis is becoming apparent. Current in vitro safety assessment approaches for cancer hazard are primarily focused on the mutagenicity of novel chemistries, though the relationship between mutagenicity test results and epigenetic changes is currently not understood. The in vitro expression of a DNA damage response gene, GADD45a, has previously been shown to be repressed (in an osteoblast cell line) by methylation of a CpG island [1]. Resistance to cytotoxic drug therapy, as well as GADD45a down-regulation have been shown to be reversible by treatment with a DNA methyltransferase inhibitor (DMTi) Decitabine. The GADD45a gene is exploited in the ‘GreenScreen HC” GFP reporter assay. It produces positive results for all classes of genotoxins, with both high sensitivity and specificity [2,3]. Little is currently known about its sensitivity to epigenetic effectors, though validation studies produced a positive result for the demethylating agent 5-azacytidine [1]. Data will be presented from experiments designed to detect epigenetic signatures following treatment of GADD45a-GFP reporter transfected TK6 cells with 4 DMTi’s. Phenotypic effects were measured by GFP fluorescence, with bisulphite genomic sequencing conducted to investigate changes to the TK6 methylome. Increases in GADD45a-GFP expression following exposure to 3 DMTi’s, were reversed following passage without DMTi. For the 4th, there was not a full reverse. Bisulfite sequencing approaches revealed an increase in methylation status changes following DMTi treatment against untreated controls. qPCR is now being used to assess transcriptional effects of DMTi exposure and differences between the genomic and plasmid-borne GADD45a genes will be reported. References: [1] Al-Romaih et al., (2008) Neoplasia 10(5):471–480. [2] Hastwell et al., (2006) Mutat. Res. 607:160–175. [3] Birrell et al., (2010) Mutat. Res. 695:87–95. Symposium 3: Poster 018 Impact of p53 on DNA damage and metabolic activation of PhIP in Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice Annette M. Krais1, Joost P.M. Melis2,3, Ewoud N. Speksnijder3, Marie Stiborova4, Rajinder Singh5, Goncalo Gamboa da Costa6, Mirjam Luijten2, David H. Phillips1, Volker M. Arlt1 1King’s College London, UK;2National Institute for Public Health and the Environment (RIVM);3Leiden University Medical Center;4Charles University Prague;5University of Leicester;6National Center for Toxicological Research (NCTR) 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of foods and is regarded as a potential human carcinogen. PhIP is metabolically activated by cytochrome P450 (CYP) enzymes, mainly CYP1A2, but also CYP1A1. Recent findings in human cells have indicated that the cellular TP53 status can influence the metabolism of environmental carcinogens such as benzo[a]pyrene, suggesting a new role for TP53 in CYP1A1-mediated carcinogen bioactivation. In order to investigate the potential role of p53 in the in vivo metabolism of PhIP, Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice were treated with a single oral dose of 50mg/kg body weight PhIP and tissues were collected after 24 hours. DNA adduct formation by PhIP (dG-C8-PhIP) was analysed by liquid chromatography-tandem mass spectrometry incorporating an isotopically [13C10]-labelled dG-C8-PhIP internal standard. In the liver 65.9±27.6, 42.9±19.7 and 31.0±4.4 adducts per 108 nucleotides were detected in Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice, respectively. Similarly, a ~2-fold lower DNA binding was also observed in the colon of Trp53(-/-) relative to Trp53(+/+) mice. Cyp1a1 and Cyp1a2 protein levels, determined by Western blotting, were lower in the livers of PhIP-treated Trp53(-/-) than in Trp53(+/+) mice. Hepatic microsomes were also analysed for specific Cyp1a1 and Cyp1a2 activities by monitoring ethoxyresorufin O-deethylation (EROD) [Cyp1a1/2] and methoxyresorufin O-deethylation (MROD) [Cyp1a2] which were both ~2.5 times lower in Trp53(-/-) than in Trp53(+/+) mice. Cyp1a protein levels/activities in the livers correlated with the DNA adduct formation by PhIP in this tissue in each mouse line. Collectively, our results indicate that p53 can affect the expression of Cyp1a in vivo after PhIP treatment. Symposium 3: Poster 019 Impact of p53 on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its human metabolite 3-aminobenzanthrone Ann-Christin Baranski1, Annette M. Krais1, Laura U.E. Wohak1, Osman Sozeri1, Heinz H. Schmeiser2, David H Phillips1, Volker M Arlt1 1King’s College London, MRC-PHE Centre for Environment & Health, London, UK;2German Cancer Research Center (DKFZ), Heidelberg, Germany.. 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen linked to diesel exposure. We previously found that the cellular TP53 status can influence the metabolism of environmental carcinogens such as benzo[a]pyrene. To further investigate the role of TP53 in the bioactivation of 3-NBA and its human metabolite 3-aminobenzanthrone (3-ABA) a panel of isogenic colorectal HCT116 cells was used, differing only with respect to their endogenous TP53 status. HCT116 cells having TP53(+/+), TP53(+/-), TP53(-/-), TP53(R248W/+) or TP53(R248W/-) were treated with 1 µM 3-NBA or 10 µM 3-ABA up to 48 hrs. DNA adduct levels, as analysed by 32P-postlabelling, were ~1.8-fold higher in TP53(R248W/-) compared to TP53(+/+) cells treated with 3-NBA; adduct levels were not significantly different in the other cell lines. This result suggests that 3-NBA metabolism can be influenced by mutant p53. In contrast, DNA adduct levels induced by 3-ABA were up to 15 times lower in TP53(+/-) and TP53(-/-) relative to TP53(+/+) cells indicating that 3-ABA metabolism depends on wild-type p53 function; no significant difference in adduct levels was found in TP53(R248W/+) or TP53(R248W/-) cells. CYP1A1 mRNA expression, determined by qRT-PCR, was ~2-fold higher in 3-NBA-treated TP53(R248W/-) compared to TP53(+/+) cells, while NQO1 mRNA expression remained unchanged in all cell lines. These results are in line with previous findings indicating that, besides NQO1, recombinant CYP1A1 is capable of activating 3-NBA by nitroreduction and suggest that enhanced CYP1A1 expression in TP53(R248W/-) cells leads to higher 3-NBA-DNA adduct formation. After 3-ABA treatment CYP1A1 mRNA expression was ~3-5-fold lower in all cell lines relative to TP53(+/+) cells, indicating that loss of wild-type p53 function but surprisingly not mutant p53 is linked to lower CYP1A1-mediated 3-ABA-DNA adduct formation. Collectively, this study demonstrates a link between p53 and the expression of CYP1A1 by environmental pollutants and reveals a role for p53 in xenobiotic metabolism. Symposium 3: Poster 021 Investigating the mechanisms of HDAC inhibitor-induced DNA damage Wenbin Wang1, Anthony Lynch2, Simon Reed1 1Cardiff University, UK;2GlaxoSmithKline Histone deacetylase inhibitors (HDACi) are a class of compounds targeting the epigenome considered to be safe from a genetic toxicology point of view and non-DNA reactive, instead they have been shown to cause DNA damage in healthy cells, posing significant risks in safety assessment. Therefore understanding the mechanisms of HDACi-induced DNA damage is important for future safety assessment of compounds targeting the epigenome. Based on the effective application of these drugs in the treatment of lymphomas, the lymphoblastoid TK6 cell line, harboring wild-type p53, was selected to represent cells with normal DNA damage signaling. To confirm this, we compared the response of TK6 (p53 WT), MDA-MB-231 (p53 mutant); PC-3 (p53 null) to treatment with nutlin-3, a p53-dependent cell death activator. The results showed a significant dose-dependent increase in apoptosis only in TK6 cells at 48h (40% at 10µM), measured using the Annexin-V/PI flow assay, validating the p53 proficiency of these cells. Next we showed using western blots that 1µM Trichostatin A (TSA), a first generation pan-HDAC inhibitor, exhibits up to a 7-fold increase in global acetylation at H3K9, a key target of these compounds with implications in gene expression. Since γH2AX has been suggested as a marker of DNA damage, we performed flow analysis of samples treated with varying doses of TSA (150nM – 2µM) at 24h. In our experiments we did not observe an increase in the γH2AX response for this particular compound despite the findings of previous studies. Additionally, the same treatments showed an increase in cell death (40% at 2µM) and a 43% increase in γH2AX in response to 20µM H2O2 as a positive control. It is possible that the decrease in survival may be due to factors other than DNA damage alone. Future studies, using ChIP-Chip, will focus on examining how epigenetic changes induced by HDAC inhibitors affect DNA repair (marker of damage) and chromatin structure at a genome-wide level in order to better understand the underlying mechanisms. Symposium 3: Poster 022 The fine tuning of autophagy and differentiation in the skeletal muscle Paola Fortini1, Chiara Ferretti1, Egidio Iorio1, Lorenza Garribba1, Sara Baccarini1, Mario Falchi1, Monica Cagnin2, Ciro Isidoro2, Eugenia Dogliotti1 1Istituto Superiore di Sanità-Rome-Italy;2Università del Piemonte-Novara-Italy Post-mitotic cells are expected to be provided of a tight mechanism of genome integrity control to guarantee the maintenance of their correct functioning along the life span. By using an in vitro skeletal muscle cell differentiation system we have previously shown that DNA repair is down-regulated in terminally differentiated muscle cells (myotubes). Despite the accumulation of damage, myotubes are extremely resistant to the cytotoxic effects of DNA damaging agents such as ionizing radiation, alkylating agents and campthotecin. Conversely, an extreme sensitivity to the cytotoxic effects induced by doxorubicin and menadione was observed in myotubes indicating that a cell death pathway is still functional in terminally differentiated cells. Preliminary data indicate that autophagy, a cell mechanism with pro-death and pro-survival functions, is activated during myogenesis as testified by immunofluorescence staining and western blotting analysis of LC3, a hallmark of the autophagosome formation. In addition, myoblasts with silencing of beclin 1, a key player of autophagy, show a block of the autophagic flux associated to a clear reduction of the fusion index indicating a strict connection between autophagy and the maturation of myocytes into multinucleated myotubes. Interestingly, an attenuation of the basal autophagic response was observed in p53 deficient muscle cells in parallel to a reduced level of muscle specific proteins, further supporting a crucial role of autophagy in the accomplishment of the muscle differentiation program. Monday, Symposium 4: Talk 1/6 Less is more for cancer chemoprevention: evidence of a non-linear dose response for the protective effects of resveratrol Karen Brown University of Leicester, UK Diet-derived compounds are attractive candidates for the prevention of cancer. However, despite preclinical data indicating that phytochemicals/micronutrients can protect against cancer, these findings have failed to translate into successful clinical outcomes; this may be due to failure to identify the optimal dose for clinical assessment. This important issue has received little attention and it has generally been assumed that in terms of dosage, more is better. The fact that diet-derived compounds are often identified based on epidemiological observations suggesting activity at low, chronic intake, seems to have been overlooked. Consequently, little is known about the pharmacokinetics or activity of dietary attainable concentrations for any of the commonly investigated agents. This study challenges the current developmental paradigm using resveratrol as a model chemopreventive phytochemical. Although resveratrol has been touted as worthy of clinical evaluation, knowledge gaps, including identification of the optimum dose and key molecular targets, preclude the rational design of chemopreventive efficacy trials. To address these deficiencies we compared the pharmacokinetics and activity of a low dietary relevant dose, equivalent to the amount contained in a glass of red wine with an intake 200-fold higher. Using accelerator mass spectrometry the dose-response relationship and metabolite profile of [14C]-resveratrol was determined in plasma of healthy volunteers and colorectal tissue of cancer patients. Subsequently, an equivalent dietary dose was shown to inhibit intestinal adenoma development in ApcMin mice more effectively than the higher dose. Efficacy correlated with increased activation of AMP-activated protein kinase (AMPK) and further in vitro examination revealed a bell-shaped dose response for AMPK signalling, leading to autophagy and senescence. Finally, using human explant cultures and samples from resveratrol-treated patients we found that low dietary exposures not only elicit biological changes relevant to chemoprevention, they may have superior activity compared to high doses, and should be included in preclinical testing strategies. Monday, Symposium 4: Talk 2/6 Cyclooxygenase inhibitors for the prevention of cancer Ann Williams University of Bristol, UK Colorectal cancer remains the second most common cause of cancer deaths in the UK and incidence is set to rise with an ageing population: primary and secondary prevention represents one of the greatest opportunities to impact significantly on colorectal cancer-associated mortality. The pathology of colorectal cancer can been explained by a combination of genetic and epigenetic changes as well as environmental influences; chronic inflammation has long been associated with increased risk of developing colorectal carcinogenesis. Whilst activation of the WNT-signalling pathway is believed to account for the initiation of colorectal tumours, aberrant expression of cyclooxygenase-2 [COX-2, a major pro-inflammatory mediator] occurs in the majority of colorectal tumours and plays a crucial role during colorectal cancer development. Deregulation of COX-2 expression leads to increased prostaglandin E2 (PGE2) production, the pleiotropic effects of which appear to promote most, if not all, of the hallmarks of cancer. The ability of the COX/PGE2 pathway to affect multiple aspects of tumour development may also offer an explanation for the effectiveness of COX inhibitors at reducing both the incidence and progression of intestinal tumours in animal models of cancer, and more importantly in human cancer patients. NSAIDs such as aspirin have been traditionally used as analgesics and anti-inflammatories and are some of the world’s most widely used drugs. Recent epidemiological evidence shows that daily aspirin or acetylsalicyclic acid (ASA) consumption can reduce the risk of colorectal cancer, although most studies specify the need for long term aspirin usage before effective inhibition is evident. However due to associated toxicities, there remains a need to better understand how NSAIDs such as aspirin work, not only to allow the delivery of effective chemoprevention but also to discover new ways in which the anti-cancer effects of these important drugs can be fully exploited in the clinic. Monday, Symposium 4: Talk 3/6 Epigenetics and cancer chemoprevention Clarissa Gerhauser German Cancer Research Center, Epigenomics and Cancer Risk Factors, Heidelberg, Germany Within the past decade, epigenetic mechanisms and their modulation by natural products have gained major interest in the chemopreventive community. The term ‘epigenetics’ refers to modifications in gene expression caused by heritable, but potentially reversible, changes in DNA methylation and chromatin structure. Major epigenetic mechanisms include DNA hyper- and hypomethylation, histone acetylation and methylation (among others), and non-coding (micro) RNAs. Given the fact that epigenetic modifications occur early in carcinogenesis and represent potentially initiating events in cancer development, they have been identified as promising new targets for prevention strategies. Chemopreventive agents from various dietary sources, including green tea, soy, fruit and berries, cruciferous vegetables, turmeric and others have been shown to directly target enzymatic activities or modulate expression of enzymes involved in epigenetic gene regulation, including DNA methyltransferases, histone acetyltransferases, deacetylases and demethylases, as well as sirtuins. Also, many cancer preventive compounds were shown to alter miRNA expression. Research over the last decade is accumulating that these activities might contribute to their chemopreventive efficacy by effects on signal transduction mediated by nuclear receptors and transcription factors such as NF-kappakB, cell proliferation and cell cycle progression, cellular differentiation, DNA repair, apoptosis induction, cell motility, metastasis formation, and cellular senescence So far, evidence of in vivo epigenetic activities in animal models and human pilot studies is still limited (literature reviews in Huang et al., 2011; Gerhauser, 2013; Gerhauser, 2014; reprints at http://www.dkfz.de/en/tox/download/gerh/clarissa_gerhauser-publ-list.html). Outlook: Current epigenetic research is highly dynamic field. With the available technologies for quantitative methylome profiling (e.g. Illumina 450k bead arrays for human samples, reduced representation and whole genome bisulfite sequencing (RRBS, WGBS)), future studies will have to focus on genome-wide analyses, integration of effects on various epigenomic mechanisms with gene expression, and the link to chemopreventive outcome to identify best strategies for chemopreventive intervention targeting the epigenome. Monday, Symposium 4: Talk 4/6 Drug repurposing: From Cancer treatment to prevention Farhat Khanim, Mark Drayson, Christopher Bunce University of Birmingham Irrespective of cancer type, age and fitness impact significantly upon the therapeutic options that are available to patients. If we consider that many cancers occur at increasing frequency with age, that modern lifestyle choices are increasing the frequencies of some cancers, and changing demographics are giving rise to an ageing population then we need to reconsider our strategies for therapy development. These therapies not only need to deliver effective anti-cancer activity, but also come with acceptable side-effect profiles tolerable for our older patients and ideally with affordable costs. Indeed in the developed world, the added pressure of fiscal restraints imposes even greater restrictions upon therapeutic choices resulting in worse patient outcomes. We have developed a translational research program in haematological malignancy based upon the principle of drug redeployment of drug combinations. Drug redeployment, the use of drugs for a new clinical indication, is an efficient effective drug development pipeline that has changed the clinical landscape in some cancers. Our initial studies identified BaP (bezafibrate and Provera) for the treatment of acute myeloid leukaemia (AML), Burkitt’s Lymphoma (BL) and chronic lymphocytic leukaemia (CLL). In parallel with ongoing mechanism of action (MOA) laboratory studies, we have performed clinical trials in the UK in AML and Malawi in paediatric BL which have demonstrated clinical activity of BaP. Using a screening approach we have identified 2 further drug combinations, valproate and niclosamide (VaN) for myeloma, and zinc and colchicine (ZAC) for BL, which are soon to enter clinical trial phase. These agents are associated with low-acceptable toxicity profiles suitable for these difficult patient cohorts, as well as being affordable. Although our studies are primarily based around drug redeployment for treating cancers, there is an ever growing literature around the potential of some drugs in cancer chemoprevention which will be discussed. Monday, Symposium 4: Talk 5/6 Current state of the use of SERMS for women unaffected with breast cancer: Uptake of tamoxifen in young women D Gareth Evans1,2, Louise Donnelly2, Julia Wiseman2, Rosemary Greenhalgh2, Jill Fox2, Paula Stavrinos2, Sarah Dawe2, Anthony Howell2 1Manchester University, UK;2The Genesis Breast Cancer Prevention Centre, University Hospitals of South Manchester, UK Introduction: An overview of the four randomised trials of tamoxifen versus placebo indicates that tamoxifen reduces breast cancer risk by approximately 33–40%, and tamoxifen along with raloxifene has recently been approved by NICE to be offered to women at high risk of breast cancer in the UK. However, the uptake in women at increased risk is reported to be low. Historically approximately 10% of women in our clinic entered the IBIS-I tamoxifen prevention trial. In this mixed methods paper, we assess the uptake of tamoxifen in a consecutive series of premenopausal women not in a trial and use qualitative interviews to explore the reasons for uptake. Methods: All eligible women between the ages of 33 and 46 at ≥17% lifetime risk of breast cancer undergoing annual mammography in our service were invited to take a five year course of tamoxifen. The risk benefit ratio was explained in a specially designed decision aid (DA). Reasons for accepting (n=15) or declining (n=15) were explored using semi-structured interviews. Results: Of the 1,279 eligible women, 136 (10.6%) decided to take tamoxifen. Women >40 were more likely to accept tamoxifen treatment (74/553 [13.4%]). Interviews highlighted four themes surrounding decision making: perceived impact of side effects, socially constructed knowledge of tamoxifen, tamoxifen as a ‘cancer drug’ and daily reminder of cancer risk. Concerns were similar in women who did or did not accept tamoxifen and decision making appears to be embedded in the experience of significant others. The decision aid was welcomed as a source of information without affecting the outcome decision. Conclusions: Tamoxifen uptake was similar in this study to previously ascertained uptake in a randomised placebo controlled trial (IBIS-I). It is possible that raloxifene which has fewer side effects and may be viewed more positively will have higher uptake in post-menopausal women. Monday, Symposium 4: Talk 6/6, Poster 024 Assessing molecular mechanisms of curcumin and its in vivo efficacy in targeting colorectal cancer stem-like cells Ankur Karmokar1, Lynne Howells1, Leonie Norris1, Hong Cai1, Baljit Singh2, Muhammad Imran Aslam2, Kevin West2, Angus Mcgreggor2, Richard Bayliss2, Anja Winter1, Sameena Khan1, William Steward1, Andreas Gescher1, Karen Brown1 1University of Leicester;2Leicester Royal Infirmary The turmeric constituent curcumin has recently been shown to target cancer stem-like cells (CSCs) in cancer cell line populations, and we have observed similar activity in primary colorectal adenoma and cancer cells derived from patients. Here, we aim to elucidate the mechanisms responsible for this activity and determine whether the inhibitory effects of curcumin on CSCs translate to efficacy in vivo. To investigate the molecular effects of curcumin in CSCs, Caco2 adenocarcinoma cells were exposed to curcumin (0.1 and 1µM) for 7 days. Cells were sorted based on aldehyde dehydrogenase (ALDH) activity to isolate CSCs. Curcumin significantly down regulated (p<0.05) various stem cell proteins in the ALDHhigh population. Use of an affinity pull-down assay revealed a direct interaction between curcumin and one of these target proteins. Curcumin had no effect on the expression of the target gene, but caused a significant (p<0.01) decrease in phosphorylation of the target protein, specifically in the CSCs, which may destabilise the protein leading to reduced levels. Direct interaction of curcumin and its target protein was further verified by a tryptophan fluorescence binding assay that indicated a two-site binding model. To examine the in vivo efficacy of curcumin against tumours arising from stem-like populations, 2000 ALDHhigh primary colorectal cancer cells were injected subcutaneously into the flank of NOD-SCID mice (5 per group) maintained on a diet containing curcumin (0.2%) or a control diet. Consumption of curcumin was associated with delay in time to palpable tumours, increase in murine survival and reduced rate of tumour growth. There was also a ~60% reduction in the ALDHhigh cell fraction in tumours from curcumin treated mice compared to controls. These results indicate that clinically achievable concentrations of curcumin may inhibit the growth and expansion of CSCs by interacting with a specific target protein, and this may contribute to anti-cancer efficacy in human tissues. Symposium 4: Poster 023 Allele-specific Mutation Detection qPCR: A Sensitive Method for Detection of Early Colorectal Cancer Mutations Using Circulating Cell-free DNA Ni Ni Moe Myint1, Karen Brown1, J. Howard Pringle1, Jacqui Shaw1, Ricky Trigg1, Alessandro Rufini1 University of Leicester, UK Colorectal cancer (CRC) is the third most common cancer in the UK, with a five-year survival rate estimated at ~55%. To reduce mortality and morbidity, an effective screening method is needed for detection of early preneoplastic adenomas. Circulating cell-free DNA (cfDNA) offers enormous potential as a biomarker of tumour burden and for monitoring response to preventive interventions and treatments. CfDNA are thought to be primarily released from dying cells and exist as small fragments in blood (~70-200bp). Whilst low levels of cfDNA (~1-10ng/ml) are detected in healthy individuals, the amount can increase significantly to >100ng/ml in cancers. This project aims to develop a sensitive assay for genetic profiling of adenoma-derived cfDNA in humans, as a relatively non-invasive diagnostic tool to circumvent the sampling bias associated with biopsying mutationally heterogeneous solid tumours, for use in future chemoprevention trials. A database search on Catalogue Of Somatic Mutations In Cancer (COSMIC) revealed that mutations in APC, KRAS, GNAS and TP53 are frequently associated with non-serrated colorectal adenomas, whereas BRAF and KRAS mutations are predominantly found in serrated adenomas. Using real-time quantitative PCR technology, allele-specific mutation detection assays using peptide nucleic acid ‘clamp’ biochemistry have been designed and optimised for early colorectal mutations. Using DNA extracted from cancer cell lines, the limits of sensitivity have been determined at ≈0.2% for single base substitutions in KRAS (G12, G13) and BRAF (V600), corresponding to detection of as few as six mutant copies amongst ~3,000 copies of wild-type alleles. This approach will be expanded to include APC, TP53 and GNAS mutations that are associated with a high risk of CRC development. Future work also includes detection of mutations in adenoma tissues and corresponding plasma cfDNA samples from patients to validate the potential of cfDNA as a biomarker for early detection. Symposium 4: Poster 025 Auranofin induced genotoxicity and cytotoxicity correlates with membrane fluidity in ovarian cancer cells Deepu Oommen1, Nicholas Dodd1, Dennis Yiannakis2, Awadhesh Jha1 1School of Biomedical and Biological Sciences, University of Plymouth;2Plymouth Oncology Centre, Derriford Hospital, Plymouth, UK Auranofin (Riduara) is a US federal food and drug administration approved small molecule drug used widely in the treatment of rheumatoid arthritis. Auranofin inhibits thioredoxin reductase component of thioredoxin system and perturbs the cellular redox balance. However, recently, it has been reported that auranofin has anticancer properties and in fact being tested in clinical trials for re-purposing to treat a wide range of cancers. Membrane fluidity is probably the most important physicochemical property of cell membranes and changes in fluidity can affect the functional properties of the cell. Furthermore, increased membrane fluidity has been reported in irradiated and cisplatin treated cancer cells. In this study, we investigated whether membrane fluidity modulates the sensitivity of ovarian cancer cell lines (OVCAR5, OVCAR4 & IGROV1) to auranofin. Membrane fluidity measured by electron spin resonance (ESR) using the spin label 5-doxyl stearic acid (5-DS) revealed a more fluidic state of membrane in IGROV1 compared to OVCAR4 and OVCAR5 (OVCARs). In addition, all three cell lines exhibited increased membrane fluidity upon auranofin treatment. Interestingly, IGROV1 was found to be more sensitive to auranofin than OVCARs as cytotoxic and genotoxic effects of auraniofin were profoundly high in IGROV1. Genotoxicity in auranofin treated cells was assessed by the extent of double strand breaks as determined by immunostaining of radiation induced foci (RIF) using antibodies against p53 binding protein (53BP) and phosphorylated histone 2X (ƔH2AX). Cell survival was measured by MTT assay. Taken together, our study demonstrated that membrane fluidity might be a significant factor in determining the sensitivity of ovarian cancer cells to auranofin. Symposium 4: Poster 026 Cytogenetic damage in buccal epithelium of gastrointestinal cancer patients Maria Byakhova, LP Sycheva Russian Academy of Medical Science, Moscow The aim of this study is to determine a cytogenetic status of oral mucous in patients with cancer of the gastrointestinal tract (CGT). This study was based on examination of a group of people (31 pers) with malignant tumors of the gastrointestinal tract II and IIIa, b stades. The control group included 30 apparently healthy people. The main result was a statistically significant (p <0.01) increase in the buccal epithelium in patients with cytogenetic indices CGT: proportion of cells with micronuclei in 9 times, 3,3 times protrusions, the total index 6 times compared with healthy ones. Indicators apoptosis also were significantly increased in patients (the proportion of cells with karyolysis 10 times, apoptotic index by 4,4 times) compared with healthy ones. In 6 months after surgery the same patients were examined repeatedly. It was found that a statistically significant reduction of cells ammount with micronuclei in 2,5 times, with micronuclei and protrusions in 1,6 times in buccal epithelium in patients after radical treatment in comparison with a primary case. The rate of the cells with karyorrhexis was decreased in 2,7 times, the cells with karyolysis in 2,1 times, the apoptotic index by 2,1 times in the buccal epithelium in patients after radical treatment in comparison with a primary case. The conclusion of this study is that the proportion of cells with cytogenetic abnormalities in buccal and nasal epithelium in patients with cancer of the gastrointestinal tract is significantly higher than in healthy ones. It should be emphasized that the identified cytogenetic irregularities were of the same character in both men and women. The results indicate the systematic nature of the changes. It is also shown the reduction of karyological parameters after radical treatment, which indicates its prognostic significance. Symposium 4: Poster 027 Does Obesity Induce Genomic Damage Due to Elevated Oxidative Stress? Ezgi Eyluel Bankoglu1, Florian Seyfried2, Laura Rotzinger2, Arno Nordbeck2, Christoph Thomas Germer2, Christoph Otto2, Helga Stopper1 1Institute of Pharmacology and Toxicology, University of Wuerzburg, Germany;2Department of General-, Visceral-, Vascular- and Paediatric Surgery, University Hospital of Wuerzbur Obesity is defined as excessive fat accumulation that presents a health risk, contributing to the development of diabetes, cardiovascular diseases and cancer. In this ongoing study, we investigate the association between obesity, oxidative stress and genomic damage. Furthermore, whether caloric restriction can reduce genomic damage and therefore cancer risk. Zuc-Lrpfa male rats were used as obesity model in addition to Lean control (fa/+) and divided into two groups: obese (Zuc-Lrfpa ad libitum fed) and caloric restriction (Zuc-Lrfpa). Rats were observed for 6 weeks before caloric restriction and 4 weeks after. During these 10 weeks daily food intake and body weight were monitored and urine was collected at 4 different time points (before caloric restriction and after 1st, 2nd, 3rd week of caloric restriction). Finally, rats were sacrificed and colon, liver, kidney were harvested for further analysis. At the time of sacrifice, body weight was significantly different between the obese rats and the lean control group, while the caloric restriction rats did not differ from the lean control. Evaluation of DNA damage was performed with freshly isolated primary cells by comet assay. DNA damage was higher in the obese group compared to the other groups. In order to detect oxidative stress dihydroethidium (DHE) staining was performed. Results of DHE staining revealed elevated oxidative stress in liver, kidney and colon of obese rats. Malondialdehyde content was measured in order to detect lipid peroxidation products. To quantify the oxidation of DNA, RNA and the repair rate the urinary oxidized DNA base 8-oxodG, the oxidized RNA base 8-oxoGuo and the oxidation of nucleotide precursors 8-oxoGua were quantified by LC/MS/MS. Our data show different values between obese and lean animals, which need to be stratified further as additional data become available in the course of the project. Symposium 4: Poster 028 Effects of Bone Marrow – Mesenchymal Stem Cells on genotoxic sensitivity of Myeloma cells to Melphalan in vitro Simon Andrews, Mohammad RezaSalehan, Ruth Morse UWE Multiple myeloma (MM) remains incurable due to resistance to chemotherapeutics (1,2), which may result from protection to in vivo exposure by interaction of bone marrow stromal/mesenchymal cells (BM—MSC) with MM (3). Patients with a haematological malignancy also sustain ‘damage” to their BM, however it is currently unknown if this ‘damage’ is related to altruistic protection of MM by the BM-MSC. Furthermore, whilst ‘damage’ is demonstrated as compromised functionality, little is known of genotoxicity to both MM and BM-MSC post-exposure to treatment. The myeloma cell line U266 and BM-MSC were exposed to a clinically relevant dose of melphalan (32.8µM) for 1hr, both alone and in co-culture (both treated or one compartment treated, then co-cultured). Genotoxic damage was assessed using the in vitro comet assay. Tail intensity was recorded in U266 cells. Decreased tail intensity for all MM controls and treatments during co-culture with MSC inferred MM genome stabilisation, perhaps via reduction of apoptosis/proliferative index of MM cells (4). At 16hr, all directly exposed U266 cells showed tail retardation, suggesting crosslink formation, however untreated U266 co-cultured with treated BM-MSC (indirectly exposed) had increased tail intensity (p<0.05) suggesting a bystander effect, unrelated to cellular efflux of melphalan. After 48hr tail intensities returned to baseline levels (p>0.05), suggesting repair of damage. Treated U266 co-cultured with untreated BM-MSC had reduced tail intensity at 16hr (p<0.05) and 48hr (p<0.001); tail retardation at 48hr supports reduced proliferation and therefore damage repair. Tail retardation at 16hr from crosslinks for U266 alone was greater than U266 in co-culture (~50% vs 33%); suggesting that BM-MSC interaction may reduce crosslink formation. These results suggest MSC interaction with MM may protect from genotoxicity, which supports the observed functional protection by previous studies. Further confirmation of crosslink formation and repair kinetics may improve understanding of resistance mechanisms in myeloma therapy. 1. Lonial, S. & Cavenagh, J. (2009). Br J Haem. 145: 681–708. 2. Allegra, A., et al. (2011). Eur J Haem. 86: 93–110. 3. Kemp K, et al. (2011). Ann Hematol 90:777–789. 4. Paraguassú-Braga et al (2003) Cell Death & Diff 10: 1101–1108. Symposium 4: Poster 029 Folic acid receptor mediated cellular uptake of albumin-ß-carboline-conjugates Kathrin Butzbach1, Federico A.O. Rasse Suriani2, M. Micaela Gonzales2, Franco M Cabrerizo2, Bernd Epe1 1Johannes Gutenberg-University, Mainz, GERMANY;2IIB-INTECH CONICET UNSAM, Chascomús, Argentina In photodynamic therapy (PDT) cells are killed by irradiation in presence of a photosensitizer, which produces cytotoxic reactive oxygen species (ROS). This therapeutical concept would be more powerful, if it was possible to accumulate the photosensitizer specifically in malignant cells, thereby protecting surrounding tissue. Many solid tumors overexpress folic acid receptor a (FRa), to ensure the supply of folic acid (FA). FRa is an endocytotic receptor with a high FA affinity and a fast turnover rate which makes it an attractive vehicle for cancer cell specific drug delivery. We wanted to find out whether albumin-folate-conjugates are useful to transport photosensitizers into FRa overexpressing cancer cells. As a diagnostic tool we used a highly fluorescent ß-carboline derivate, which we coupled covalently to BSA. Afterwards we coupled FA to this albumin-ß-carboline-conjugate (AßC) to produce an albumin-ß-carboline-FA-conjugate (AßCF). These two different conjugates were characterized in size, conjugation pattern and photophysical properties. The fluorescence quantum yield of the fluorophor was not diminished significantly by albumin therefore this conjugate is a suitable molecule to visualize AßCF under the microscope. For experiments in cells we used KB cells (human nasopharyngal carcinoma), which are FRa overexpressing. We could show that AßCF accumulates in the lysosomes of the cells after 90min while under the same conditions AßC is not entering the cell at all. This proves that the uptake of AßCF is mainly FA mediated. By using a ß-carboline derivate with a higher triplet quantum yield and a comparable coupling behavior it should be possible to kill only FRa positive cells by irradiation. Symposium 4: Poster 030 Regulation of the alternative splicing of genes involved in leukaemia through the splice factor kinases CLK1 and SRPK1 Hanan Alabouh1, David Corry 1, Sebastian Oltean2, Masatoshi Hagiwara3, Ruth Morse1, Michael Ladomery1 1Faculty of Health & Applied Sciences, University of the West of England, Bristol UK;2School of Physiology and Pharmacology, University of Bristol, UK;3Graduate School of Medicine Kyoto University, Kyoto, Japan In humans, over 94% of multi-exonic genes are alternatively spliced1. Alternative splicing provides cells with many protein isoforms with often opposing functions derived from the same gene2. Malignancies take advantage of this crucial aspect of gene expression producing, for example, higher levels of anti-apoptotic proteins3. Alternative splicing is regulated by splice factors and protein kinases and phosphatases. The activity and intracellular localization of the oncogenic splice factor SRSF1 is regulated by the splice factor kinases SRPK1 and CLK1. We have previously shown that the alternative splicing of vascular endothelial growth factor (VEGF-A) is regulated through SRPK1 and SRSF14. However the role of SRPK1 and CLK1 in the regulation of the alternative splicing of key genes in leukaemia has not yet been investigated. The aim of this project is to understand the effect of inhibiting CLK1 and SRPK1 on alternative splicing of key genes associated with leukaemia. CLK1 was inhibited in K562 cells (CML) with the compound TG003 at 150 µM, and SRPK1 was inhibited with SRPIN340 at 10–20 µM for 24 hours. Standard RT-PCR was performed to investigate the effect of both CLK1 and SRPK1 inhibition on the alternative splicing of key genes. These included the tyrosine kinase RON, in which exon 11 skipping in cancer produces a constitutively active isoform5; the apoptotic gene caspase 9; and the transcription factor RUNX1 (AML1). The relative intensity of PCR amplicons was quantified using ImagJ software. Inhibition of the splice factor kinase CLK1 has a significant effect on the alternative splicing of RON, reducing the amount of oncogenic exon 11 skipping. This result is consistent with the finding reported elsewhere that CLK1 is involved in the regulation of cancer hallmarks6. We therefore propose that targeting CLK1 has the potential to provide new treatment options in leukaemia. Monday, Workshop 2: Talk 1/6 Consideration of Relevant Investigations Providing a Picture of Genotoxic Potential of Nanomaterials Alan Poole ECETOC Potential adverse effects of nanomaterials to human health has been a topic of investigation for a number of years and some interim conclusions have been drawn. The results of genotoxicity testing of nanomaterials in different in vitro cell systems have produced mixed results requiring the introduction of clear guidelines and harmonisation of testing conditions including consideration of exposure conditions, cell harvesting and analysis. In vivo investigations have also provided mixed results. For example following intratracheal instillation of high doses doses of selected nanomaterials cells in the brovnchiolar lavage fluid exhibited signs of DNA damaging effects suggesting an inflammatory mode of action. Following gavage, negative results have been detected on bone marrow while some effects were detected at low doses in colon cells. The Nanogenotox EU project concluded: Importance of new in vivo assays apart from Micronucleus on bone marrow especially in organs of contact sites for the investigation of genotoxicity Any genotoxic TG should be amended to include some toxicokinetic testing Organs accumulating materials like lung … should be investigated for adverse effects especially in long-term low dose studies Long-term health effects of nanomaterials is still an ongoing research area in particular the mode of action by which nanomaterials might induce lung tumours after inhalation. For risk assessment and regulation consideration must be given to the relevance of high dose effects to humans as well as potential thresholds of effects. This workshop goes beyond the question if nano-materials are primary genotoxicants and which methods are appropriate. It brings together the right experts to present a comprehensive picture of biological pathways leading to long-term effects and potential tumour induction by nanomaterials Monday, Workshop 2: Talk 2/6 Caveats to investigating the genotoxicity of nanomaterials Shareen Doak Swansea University, UK There is increasing pressure to define a hazard identification and risk management strategy for nanomaterials to prevent stifling innovation within the nanotechnology industry, which is developing an increasing array of nano-containing consumer products. Consequently, a battery of appropriate in vitro assays assessing several genotoxicity endpoints is required to minimise extensive and costly in vivo testing. However, the validity of using the established protocols in current OECD recognised genotoxicity assays is currently being questioned for nanomaterials because their unique physico-chemical properties can result in unexpected interactions with experimental components that generate misleading data-sets. Several caveats to investigating the genotoxicity of nanomaterials have now become evident and thus represent important factors that need to be considered in order to ensure that in vitro genotoxicity assays report true biological impacts in response to nanomaterial exposure. Additionally, our blossoming understanding of such limitations in the current testing regime presents the need for a systematic approach to answer the remaining questions with regards to the genotoxic in vitro testing strategy required for nanomaterial safety assessment and regulation. Monday, Workshop 2: Talk 3/6 Novel insights in nano-genotoxicology: from 2009 till now Laetitia Gonzalez Vrije Universiteit Brussel, Belgium At the 10th International Conference on Environmental Mutagens (ICEM, 2009) some conclusions were drawn regarding genotoxicity testing of nanomaterials. Since then the number of research studies on the genotoxicological effects induced by nanomaterials has steadily increased, leading to some novel insights in the field. An overview of the recent research efforts will be given with special consideration to the issues identified in 2009, which include nanomaterial agglomeration/aggregation, protein corona formation, solubility and the lack of mechanistic information. In recent years, research efforts have focused on the suitability/adequacy of the methodologies used for nanomaterials testing. Furthermore, nano- genotoxicology research is evolving from mere assessment of induced effects towards a better understanding of their in vitro and in vivo action. Although the induction of reactive oxygen species was (and still is) regarded as the main mode of action, assessing indirect genotoxicity, as a result of nanomaterial-protein interactions and/or physical interference of nanomaterials with intra-cellular components, is gaining importance. These aspects will be discussed highlighting the recent insights and the remaining questions. Monday, Workshop 2: Talk 4/6 The role of oxidative stress in nanomaterial related genotoxicity in vitro Martin Clift University of Fribourg, Adolphe Merkle Institute, Switzerland The oxidative stress paradigm is the most notable theory concerning the onset of adverse biological effects associated with exposure to nanomaterials. Whilst the impact of oxidative stress can primarily affect cellular homeostasis by disrupting intracellular signaling (i.e. calcium signaling has been shown to be affected by both accidental (e.g. ultrafine carbon black) and manufactured (e.g. quantum dots) nanoparticles), the loss in antioxidant status by a cell, as caused by nanomaterials, can initiate subsequent intrinsic and extrinsic detrimental effects which include the production of (pro)-inflammatory cytokines and chemokines. Such processes, either individually or combined, have been shown to further elucidate a genotoxic response (i.e. DNA damage, nuclear deformation and cell proliferation) in both in vivo and in vitro systems following exposure to a variety of different engineered nanoparticle types. The elicited genotoxic response in this manner has been described as a secondary effect, due to the uptake and intracellular localisation of the nanoparticle type with the biological system of interest. The ability for nanoparticles to induce secondary genotoxicity has been the most commonly reported mechanism in this regard. Despite published research suggestive of nanomaterials predominantly causing secondary genotoxicity, there remain heightened discrepancies as to the precise role of oxidative stress as a driving factor for nanoparticle genotoxicity. As, potentially, the genotoxic responses reported could be simply a subsequent effect of the (pro)-inflammatory response only. Furthermore, increased discussion surrounds the precise, responsible physical characteristics of the nanoparticles that contribute to this response. The aim of this presentation is to provide an overview of the methodological approach towards understanding of the mechanisms in which oxidative stress can be determined as mediating nanomaterial induced genotoxicity in vitro, with a particular focus upon carbon nanotubes. Monday, Workshop 2: Talk 5/6 Genotoxicity of nano and non-nano materials: Lessons from different dimensions Roel Schins IUF - Leibniz Research Institute for Environmental Medicine, Germany Genotoxicity research on nanomaterials has expanded tremendously in recent years. Various modes of action have been proposed to be involved, while the validity of specific tests and assay protocols is being questioned. Such aspects have also been observed and discussed with non-nanosize materials including asbestos and man-made fibers, crystalline silica dust and low toxicity poorly soluble particles. The focus of this presentation will be on the established roles of particle size, shape and solubility as well as on the key relevance of inflammation induced mutagenesis. Bridging studies with nanoparticles and their larger counterparts continue to be important for risk assessment. Such investigations help to clarify whether established as well as potential newly identified modes of action are nano-specific or whether the genotoxic effects of nano versus non-nano materials merely differ in a quantitative rather than a qualitative manner. Monday, Workshop 2: Talk 6/6 A life-time inhalation carcinogenicity study with two nano materials Lan Ma-Hock, Jana Keller, Sibylle Groeters, Volker Strauss, Bennard van Ravenzwaay, Robert Landsiedel Experimental Toxicology and Ecology, BASF SE, Germany Many nanomaterials form poorly soluble biopersistent particles (PSP). A life-time inhalation carcinogenicity study with PSP nanomaterials according to OECD guidelines is not available, nor are there study to investigate the role of lung (over)load, inflammation and potential genotoxicity on lung tumor formation. A long-term study has been started in June 2013 and is testing CeO2 and BaSO4 according to OECD guideline no. 453. The animals are exposed (whole-body) to the dusts for 6 hours/day, 5 days/week for 24 months. Tumor incidence is determined after 24 and 30 months. To examine the relationship between inflammation and lung tumor formation, interim sacrificing after 3 and 12 months are performed. Exposure of selected organs including lungs, lung-draining lymph nodes and olfactory bulbs are assessed, genotoxicity in peripheral blood is determined. BaSO4 (NM220) and CeO2 (NM212) are substances of the OECD WPMN sponsorship program (http://ihcp.jrc.ec.europa.eu/our_activities/nanotechnology/nanomaterials-repository). Concentrations are selected based on short-term inhalation studies: CeO2 at 3mg/m3 to cause inflammation and (slight) overload, at 1 and 0.3mg/m3 to cause inflammation, but no overload and at 0.1mg/m3 to cause neither overload nor inflammation. BaSO4 at 50mg/m3 is expected to cause lung overload but no or little inflammation. The first interim sacrifice showed lung burdens of 1.39 and 0.012mg CeO2 per lung at 3 and 0.1mg/m3, respectively, while, 1.73mg per lung was found at 50mg/m3 BaSO4. Analyses of lavage fluid revealed concentration-related increases of neutrophils, lymphocytes and enzyme activities in CeO2-exposed animals, while only a slight inflammation was detected in BaSO4-exposed animals. This project is a collaboration of the German Environmental Ministry with the Federal Institute for Occupational Safety and Health, the Federal Institute for Risk Assessment, the Federal Environmental Agency and BASF SE. As part of the European NanoREG project, it is supervised by an external expert committee. Workshop 2: Poster 096 An in vitro coculture method for detection of secondary genotoxic effects in bronchial epithelial cells by carbon nanotube exposed macrophages Saila Pesonen1, Sampsa Matikainen1, Hannu Norppa1, Julia Catalán1,2 1Finnish Institute of Occupational Health, Helsinki, Finland;2University of Zaragoza, Zaragoza, Spain Clastogenic factors (CF) such as lipid peroxidation products (4-hydroxynonenal aldehyde), inosine nucleotides (ITP and IDP), and cytokines (TNF-α) released by phagocytizing alveolar macrophages after exposure to particles may induce DNA damage in lung tissue such as alveolar epithelial cells in vivo. Such a secondary genotoxic effect, mediated by immune cells, cannot be detected in traditional assays based on ordinary cultures of a single cell type. To investigate these effects, we set up an in vitro co-culture system where human bronchial epithelial BEAS 2B cells were cultured with human monocyte-derived macrophages, using polycarbonate transwell inserts (pore size 3 µm) to separate the cell layers. Peripheral blood mononucleated cells were isolated from donor blood buffy coats, and magnetic bead labelling was used for further isolation of CD14 positive cells to achieve a pure monocyte population. Monocytes were differentiated into mature macrophages in the presence of human recombinant granulocyte macrophage colony stimulating factor during a 7-day culture. We tested several schedules and culture media for combining the two cell types into one co-culture. The most successful procedure consisted of culturing each cell type separately in their own culture media prior to exposure. For exposure of the macrophages, the inserts were placed on top of the BEAS 2B cells on the transwell plate. This culture method will enable us to investigate the possible genotoxic effects of clastogenic factors released by macrophages exposed to carbon nanotubes in vitro. We are presently searching for a suitable positive control. After a 24-h exposure, crocidolite asbestos did not induce secondary DNA damage (Funded by the Finnish Work Environment Fund). Workshop 2: Poster 097 Assessment of SiO2 nanoparticle genotoxicity in blood cells of rats after sub-chronic exposure Eugenia Cordelli1, Patrizia Eleuteri1, Paola Villani1, Francesca Maranghi2, Roberta Tassinari2, Laura Narciso2, Francesco Cubadda2, Federica Aureli2, Marilena D’Amato2, Andrea Martinelli3, Antonio Di Virgilio3, Francesca Pacchierotti1 1Laboratory of Toxicology, ENEA, Italy;2Dept. Food Safety and Veterinary Public Health, Istituto Superiore di Sanità;3Service for Biotechnology and Animal Welfare, Istituto Superiore di Sanità In the framework of the European funded Project ‘NANoREG: A common European approach to the regulatory testing of nanomaterials”, a 90-day oral toxicity study of synthetic amorphous silica nanoparticles (NM-203) is being carried out at the Italian National Institute of Health. The study includes the assessment of genotoxicity in blood cells at the DNA, gene and chromosome level, by the comet, pig-a and micronucleus assay, respectively, carried out at ENEA laboratories. Sprague Dawley rats are exposed daily by gavage to 0, 2, 5, 10, 20, or 50mg/kg b.w. SiO2 nanoparticles for a total of 90 days. After 45 days of treatment, a sample of blood was withdrawn from the tail vein of 5 male rats per group, to investigate possible induction of genotoxicity at an intermediate exposure timepoint. A second sampling is planned at the end of the 90-day treatment time to evaluate possible cumulative effects. Pig-a mutation and micronucleus induction were assessed at 2, 20 and 50mg/kg, whereas comet assay was performed in all dose groups. A group of rats, orally treated with 3 daily doses of 20mg/kg ethylnitrosourea (ENU), from which blood was sampled at the appropriate assay-specific timepoints, served as positive control. As expected, ENU induced statistically significant increases of comet parameters and percentage of micronucleated reticulocytes at 4 days, and of pig-a mutant reticulocyte and erythrocyte frequencies at 15 and 30 days after the beginning of treatment, respectively. Conversely, in spite of a good homogeneity of individual data around their group mean, no significant differences between control and SiO2 nanoparticles treated groups were detected for any of the analysed endpoints. This study is partially financed by EU FP7 project NANoREG, grant 310584 Workshop 2: Poster 099 Cytotoxicity of poli lactic-co-glycolic acid nanoparticles and their localization in hepatic and kidney cells. Patricia Ramírez, Leonardo Erick Vázquez, Giovanna Ramírez, Roberto Díaz UNAM, FES-Cuautitlán Nanotechnology offer expectant and new opportunities to improve the quality of life of individuals, but scientific knowledge on risks associated with these materials is limited, especially for the diversity of nanomaterials and their potential applications. Among the polyesters used in the development of nanoparticles (NP) include aliphatic polyesters as monomer and dimer units of lactic acid or lactic-co-glycolic acid. These biopolymers have been approved by the FDA for development of drug delivery systems and other biomedical applications. PLGA NP have been used in controlled release of several molecules. Design of experiments provides the statistical framework for varying factors influencing the production of NP systems simultaneously and obtains reliable information with a small number of experiments with maximum efficiency. In this work we define the major factors involved in obtaining PLGA NP. The results of the exploratory design, showed the variables which most affect the size of the NP systems. Beside, the NP characterization the nanostructures were localized in liver and kidney cells by microscopy confocal fluorescent assays. The results showed differential sensitivity to the NP income in hepatic and kidney cell lines. NP of PLGA with different physicochemical properties were tested. We estimated in epithelial cells exposed to different NP doses, cell viability and quantification of intracellular GSH and thiobarbituric reactive species as cytotoxic responses associated with short term exposure of NP. Some physicochemical properties of NP studied were correlated with the cytotoxic effects. The results showed a dose-response effect of NP on intracellular GSH and time exposure is a significant factor affecting redox modulation in cells exposed. Workshop 2: Poster 100: Withdrawn Workshop 2: Poster 101: Withdrawn Workshop 2: Poster 102 Mid-infrared spectroscopic assessment of nanotoxicity in Gram-negative vs. Gram-positive bacteria Kelly A Heys1,2, Matthew J Riding1, Richard F Shore2, M Gloria Pereira2, Kevin C Jones1, Kirk T Semple1, Francis L Martin1 1Lancaster Environment Centre, Lancaster University;2Centre of Ecology and Hydrology, Lancaster, UK Nanoparticles appear to induce toxic effects through a variety of mechanisms including generation of reactive oxygen species (ROS), physical contact with the cell membrane and indirect catalysis due to remnants from manufacture. The development and subsequent increasing usage of nanomaterials has highlighted a growing need to characterize and assess the toxicity of nanoparticles, particularly those that may have detrimental health effects such as carbon-based nanomaterials (CBNs). Due to interactions of nanoparticles with some reagents, many traditional toxicity tests are unsuitable for use with CBNs. Infrared (IR) spectroscopy is a non-destructive, high throughput technique, which is unhindered by such problems. We explored the application of IR spectroscopy to investigate the effects of CBNs on Gram-negative (Pseudomonas fluorescens) and Gram-positive (Mycobacterium vanbaalenii PYR-1) bacteria. Two types of IR spectroscopy were compared: attenuated total reflection Fourier transform infrared (ATR-FTIR) and synchrotron radiation-based FTIR (SR-FTIR) spectroscopy. This showed that Gram-positive and Gram-negative bacteria exhibit differing alterations when exposed to CBNs. Gram-positive bacteria appear more resistant to these agents and this may be due to the protection afforded by their sturdier cell wall. Markers of exposure also vary according to Gram status; Amide II was consistently altered in Gram-negative bacteria and carbohydrate altered in Gram-positive bacteria. ATR-FTIR and SR-FTIR spectroscopy could both be applied to extract biochemical alterations induced by each CBN that were consistent across the two bacterial species; these may represent potential biomarkers of nanoparticle-induced alterations. Vibrational spectroscopy approaches may provide a novel means of fingerprinting the effects of CBNs in target cells. Workshop 2: Poster 103 Silicon Dioxide Nanoparticles Alter Fibrin Clot Structure in vitro Xiaoxi Pan1, Robert AS Ariëns1, Yun Yun Gong2, Michael N Routledge1 1Leeds Institute for Genetics, Health and Therapeutics, School of Medicine, University of Leeds, UK;2Institute for Global Food Security, Queens’s University Belfast, UK Silicon dioxide nanoparticles (SiO2 NP) are manufactured on an industrial scale as additives to cosmetics, paints and printer toners. These particles have been found to have genotoxicity, but there has been little research on possible effects on coagulation. The aim of this study is to investigate the possible influence of SiO2 NP on fibrin clot structure. Nanopowder SiO2 (Sigma), with sizes between 10 and 20nm were added to both human normal pooled plasma and fibrinogen purified from human plasma and clot formation induced. Clot structure and function was assessed using turbidity assay, laser scanning confocal microscopy (LSCM) and permeability assay. In plasma, turbidity assay revealed significant changes in fibre thickness at concentrations of 0.1ng/ml and above (p<0.05). At these concentrations the clot was also less permeable than in controls (p<0.05). LSCM results demonstrated that more fibres formed in the clots after treatment with 10ng/ml of SiO2 (p<0.05). Dose dependent effects were seen in all assays. These effects were not seen in clots formed from purified fibrinogen in the presence of the SiO2. In conclusion, the SiO2 NP generated denser fibrin clot structures compared to control in normal pooled plasma samples. Unlike purified fibrinogen, plasma contains several coagulation factors such as Factor XII which could enhance the clot formation when in contact with negatively charged surface area, such as that provided by SiO2 NP. Exposure to such NP, or NP with similar surface charge could alter clot structure and increase risk of cardiovascular events. Tuesday, Symposium 5: Talk 1/5 Genome instability following mitotic transmission of unreplicated DNA lesions Piya Temviriyanukul, Sandrine van Hees, Anastasia Tsaalbi-Shtylik, Jacob Jansen, Niels De Wind Leiden University Medical Center, The Netherlands Replication stress can lead to the generation of double stranded DNA breaks (DSBs) that are correlated with loss of genetic information, structural chromosomal aberrations and tumorigenesis. We have investigated the mechanistic basis of the induction of DSBs using mammalian cells defective in nucleotide excision repair and post-replicative translesion synthesis as models. We show that, upon treatment with a low dose of ultraviolet (UV) light, cells accumulate patches of ssDNA containing (6-4)pyrimidine-pyrimidine photoproducts ((6-4)PPs) in genomic DNA. Rather than rapidly collapsing into DSBs, these ssDNA gaps are transmitted through mitosis into the subsequent cell cycle. During the ensuing S phase the ssDNA gaps are converted into DSBs, leading to genomic instability. Tuesday, Symposium 5: Talk 2/5 Regulation of canonical and non canonical functions of specialized DNA polymerase eta in response to DNA damage Emmanuelle Despras, Natasha Zlatanou, Noémie Delrieu, Charlène Garandeau, Sana Ahmed-Seghir, Patricia L Kannouche Institut de cancérologie, France The faithful transmission of genetic material through cell generations relies on DNA replication by high-fidelity and high-processivity polymerases. However, the presence of DNA damage during S phase can stall these replicative polymerases, increasing the risk of mutagenesis, chromosomal rearrangements and segregation defects. Translesion synthesis (TLS), a mechanism conserved from bacteria to mammals, insures the direct replication of blocking DNA lesions through the transient recruitment of specialized low-fidelity DNA polymerases. As TLS polymerases are highly error-prone on undamaged templates, their access to DNA is tightly regulated. The post-translational modification of PCNA by mono-ubiquitination (Ub-PCNA) induced at stalled forks is considered a key event in this process. The identification of both PCNA- and ubiquitin-interacting motifs in most TLS polymerases provided an attractive molecular mechanism for the preferential interaction of TLS polymerases with Ub-PCNA. However, increasing evidence plaid for complex layers of regulation. The importance of TLS for genome stability is highlighted by the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, caused by hereditary mutations in the gene coding for specialized polymerase eta (polη). Polη is able to accurately bypassed the most abundant UV-induced DNA lesion and we showed that this TLS activity promotes replication fork progression after UV irradiation. Recently, we have identified a non canonical role of polη in the repair of oxidative damage. This pathway also requires Ub-PCNA but is independent of DNA replication. We will discuss the relative contribution of polη PCNA- and ubiquitin-interacting motifs on the regulation of polη during these processes. Tuesday, Symposium 5: Talk 3/5 The role of exonucleases in replication-coupled DNA interstrand crosslink repair Peter McHugh University of Oxford, UK DNA interstrand cross-links (ICLs) are an extremely toxic form of DNA damage that inhibit basic cellular processes such as DNA replication and transcription. Defects in ICL repair are associated with cancer pre-disposition, as exemplified by Fanconi anemia. Moreover, many important cancer chemotherapeutics exert their cytotoxic effects by ICL formation. Therefore, understanding ICL repair is important both for preventing and treating cancer. In yeast, nucleotide excision repair (NER) initiates the major repair response to ICLs throughout the cell cycle. However, the major repair response to ICLs in mammalian cells is triggered by the collision of replication forks with ICLs. Replication-coupled ICL repair involves several structure-specific nucleases including XPF-ERCC1, and Mus81-Eme1, although the details and sequence of events in this pathway remain poorly understood. A highly conserved family of 5’-3’ exonuclease, the founder member of which is yeast Pso2/Snm1 is also critical for ICL repair in all eukaryotes. We have recently found that both purified yeast Pso2 and its human homologue SNM1A can digest past an ICL on their substrate strand. This leaves a residual tethered mononucleotide, a preferred substrate for downstream processing steps such as translesion synthesis. We have discovered that yeast Pso2 acts downstream of the NER apparatus to process incised ICL repair intermediates. By contrast, failure of the mammalian SNM1A repair pathway leads to fork cleavage by Mus81, producing double-strand break intermediates. Recent genetic, biochemical and structural studies that are beginning to reveal how these replication-associated ICL repair events are controlled and coordinated in both yeast and mammalian cells will be presented. Tuesday, Symposium 5: Talk 4/5 Discovery of novel Fanconi anemia genes: fundamental research and clinical implications Jordi Surralles Universitat Autonoma Barcelona, Spain Fanconi anemia (FA) is genome instability disorder characterized by bone marrow failure, malformations, chromosome fragility, hypersensitivity to DNA interstrand cross-linking (ICL) mutagens and a high predisposition to cancer. The only cure of the hematological disease, which is the major cause of early death, is bone marrow transplantation using an HLA compatible donor. Those families with unavailable donor rely on the future implementation of advanced gene and cell therapies. These therapeutic approached are further complicated by the fact that at least 16 genes, from FANCA to FANCQ, are involved in this disease and their products interact in a complex genome stability and tumor suppression network. Notably, 4 out of 16 FA genes (FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1 and FANCO/Rad51C) are breast cancer susceptibility genes in otherwise unaffected mutation carriers. Therefore, the discovery of novel Fanconi anemia genes is important not only for the affected patients but also for the general population. In this context, I will present data on the discovery of a novel Fanconi anemia gene by whole exome sequencing. Interestingly, this gene (ERCC4 o FANCQ) is involved not only in FA but also in xeroderma pigmentosum type F (XPF), a melanoma susceptibility disorder with defective nucleotide excision repair (NER), and in XFE-type progeria. Our functional genetic and biochemical studies indicate that depending on the balance between ICL repair and NER, mutations in the same gene lead to three clinically distinct disorders highlighting the multitask nature of the XPF nuclease in human health. The therapeutic implications of the discovery of FA genes will be shown in the frame of gene and cell therapy. Tuesday, Symposium 5: Talk 5/5 NEDD4 and Cul3 dependent RNA pol II degradation in the presence of DSBs Evi Soutoglou, Tibor Paknotai, Audrey Furst Université de Strasbourg, INSERM, France DNA double-strand break (DSB) repair interferes with ongoing cellular processes, including replication and transcription. We showed before that infliction of a single DSB at a human RNAPII transcribed gene leads to inhibition of transcription elongation and reinitiation in a DNAPKcs dependent manner. We also demonstrated that the mechanism of DNAPK-mediated transcription inhibition involves the proteasome-dependent pathway. Here we have evidence that as it was shown for UV lesions, DSBs trigger the recruitment of the E3 ubiquitin ligase NEDD4 and the Cul3 complex that leads to the break –dependent ubiquitination and subsequent degradation of RNA polII at damaged genes. The results point to the pivotal role of protein degradation in the eviction of RNAPII from the DNA upon encountering a DNA lesion. Symposium 5: Poster 031 5-aza-2`deoxycytidine induces DNA lesions that are repaired via Transcription-Coupled Repair involving Cockayne syndrome protein B Manuel Luis Orta Vázquez1-2, Ángel Ruíz-Castizo1, Nuria Pastor Carrillo1, Inmaculada Domínguez García1, Mª del Prado Vargas Duarte1, Estefanía Burgos-Morón3, Javier Manzano-López1, Jose Manuel Calderón-Montaño2-3, Andreas Höglund2, Manuel Muñiz Guinea1, Miguel López-Lázaro3, Thomas Helleday2, Santiago Mateos1 1Dep of Cell Biology, University of Seville, Spain;2Science for Life Lab. Dep. of Medical Biochemistry and Biophysics, Karolinska Institute, Sweden;3Dep. of Pharmacology, University of Seville, Spain It is well known that the reason by which actively transcribed genes are better repaired than those that are silenced is the transcription coupled repair pathway (TCR). A deficiency in key proteins of this route, such as CSA and CSB, generates the still poorly understood Cockayne syndrome. In this pathway, transcription blocks of RNA polymerase II (RNApol II) are stabilized by CSB, which also recruits DNA repair proteins such as those involved in Nucleotide Excision Repair or Base Excision Repair. On the other hand, 5-aza-2′deoxycytidine (decitabine, hereinafter named 5-azadC) is a chemotherapeutic drug recently approved to treat myelodysplasic syndromes and acute myeloid leukemia. This cytidine analogue is very efficient trapping covalently DNA methyltransferases (DNMTs) onto the DNA, which induce replication associated Double Strand Breaks. In this study we show that 5-azadC induced chromosome breaks are prevented by transcription inhibition which point that trapped DNMTs are an obstacle to the progression of RNApol II. Also we show that CSB null hamster cells are hypersensitive to this drug, displaying reduced survival and increased apoptosis rates. Using γ-H2AX and 53BP1 foci as markers of the DNA damage response and a 6 hour experimental schedule, we demonstrate that 5-azadC is able to trap DNMT1 onto DNA and that a DNA damage response is activated in wild type cells but not in CSB mutant cells. Furthermore, we also present data where CSB null cells have defects in the repair of DNMT1 foci. In order to investigate more about this phenotype, we discover an epistatic effect between transcription progression and CSB using DRB as transcription inhibitor. Together, the data point to a model in which transcription is arrested when collides with trapped DNMT1, which generates DSBs and the subsequent repair of DNMT1 adducts. Symposium 5: Poster 032 Effect of damaged ribonucleoside triphosphates on the activity of NTP-dependent enzymes Antonina Dovgerd, Dmitry Zarkov Institute of Chemical Biology and Fundamental Medicine Aerobic organisms cannot avoid oxidative stress because reactive oxidative species (ROS) are generated by the normal cellular metabolism. Oxidative stress has been implicated in the onset and development of several pathological processes, including cancer and age-related neurodegenerative diseases such as Alzheimer disease and Parkinson disease. One of the most common DNA lesions generated by ROS is an oxidized guanine (8-oxoguanine; 8-oxoG), which can pair with adenine in DNA and cause transversion mutation. However, a considerable amount of 8-oxoG is generated in the cellular nucleotide pool as well as in DNA by ROS because the nucleotide pool is mainly located in the cytoplasm, and the cytoplasmic nucleotides can be more easily attacked by ROS than DNA in the nucleus. Furthermore, the pool size of cellular ribonucleotides is hundreds of times larger than that of deoxyribonucleotides, and therefore, significant amounts of 8-oxoG-containing ribonucleotides like 8-oxoGTP are probably produced in the cytoplasm. The same situation is observed for 8-oxoadenine ribonucleotide triphosphate (8-oxoATP). In fact, GTP and ATP is involved in many intracellular processes as well as in RNA synthesis; these include energy metabolism, microtubule assembly, protein synthesis, and cell signaling. Accordingly, the conversion of GTP into 8-oxoGTP and ATP into 8-oxoATP by oxidative stress may affect the above cellular processes. Therefore, the present study was undertaken to examine the interaction between 8-oxoATP and NTP-dependent enzymes, such as DNA ligase and T4 polynucleotide kinase. The results obtained demonstrate that the presence of 8-oxoATP in reaction mix significantly decreased activity of the enzymes. The study of this problem is an interesting and promising, since the mechanisms of elimination of 8-oxoATP from the cellular nucleotide pool are unknown. Symposium 5: Poster 033: Withdrawn Symposium 5: Poster 034 Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by Homologous Recombination. Manuel Luis Orta Vázquez1, Nuria Pastor Carrillo1, Inmaculada Domínguez García1, Estefanía Burgos-Morón3, Jose Manuel Calderón-Montaño2, Jose Antonio Bejarano García1, Irene García-Domínguez1-3, Ángel Ruíz Castizo1, Mª del Prado Vargas Duarte1, Miguel López-Lázaro3, Thomas Helleday2, Santiago Mateos Cordero1 1Dep. of Cell Biology, University of Seville, Spain;2Science for Life Lab. Dep. of Medical Biochemistry and Biophysics, Karolinska Institute, Sweden;3Dep. of Pharmacology, University of Seville, Spain Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially targets cancer cells. It acts primarily trapping DNA methyl transferases (DNMTs) protein by forming tight covalent complexes between DNMT protein and zebularine-substrate DNA. They represent endogenous fork obstacles and it’s well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSBs are nonhomologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested fork. Here we investigate in more detail the importance of the lesions induced by Zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferential repaired by HR. Also we show that the production of DSBs is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. We found a decreased amount of chromatid breaks in agreement with this hypothesis. Here we report that cells deficient in single-strand break repair (SSB) are hypersensitive to zebularine. We show more zebularine-induced DSBs in XRCC1 deficient cells, likely to be the result of conversion of a SSBs into a toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR. Tuesday, Symposium 6: Talk 1/6 Using Adverse Outcome Pathways to support mechanistic based safety assessment Maurice Whelan European Commission Joint Research Centre, Systems Toxicology Unit and EURL ECVAM, Italy Regulatory toxicology is undergoing a steady transformation driven by the push towards more systematic use of mechanistic knowledge and reasoning in decision making. But an uncomfortable paradox has emerged in that the more toxicological knowledge we generate, the more difficult it is to exploit for practical purposes. Although there is already a phenomenal amount of mechanistic information available in the literature, scientists continue to dig deeper and deeper. But with a gain in resolution, the trade-offs are usually a loss in coverage and coherence, which ultimately renders the knowledge unusable by decision makers. The Adverse Outcome Pathway (AOP) Development Programme at the OECD aims to address the fundamental challenge of making mechanistic knowledge available in a form that users can understand and exploit for chemical safety assessment. An AOP is essentially a conceptual construct that is reductionist at the process level, describing a sequential chain of causally linked Key Events (KE) that lead to an adverse health or ecotoxicological effect. KEs occur at different levels of biological organisation and are related through Key Event Relationships (KER). A fundamental principle behind AOP thinking is that AOPs assemble into a causality network through the sharing of common KEs and KERs. AOPs can vary in resolution and expanse and can be tailored for multiple uses such as providing a blueprint for computational prediction models, designing integrated testing strategies, or structuring information for weight-of-evidence assessments. Moreover, the AOP framework is fast becoming a tool for international crowdsourcing and sharing of mechanistic knowledge for the purposes of advancing chemical safety assessment. Tuesday, Symposium 6: Talk 2/6 Applying the TT21C principles to safety assessments of new chemicals including DNA damaging agents Rebecca Clewell The Hamner Institutes for Health Sciences As part of a larger effort to provide proof-of-concept in vitro only risk assessments, we have developed a suite of high throughput assays for key readouts in the p53 DNA damage response toxicity pathway: DSB DNA damage (p-H2AX), permanent chromosomal damage (micronuclei; MN), p53 activation, p53 transcriptional activity, and cell fate (cell cycle arrest, apoptosis, MN). Dose-response studies were performed with these protein and cell fate assays, together with whole genome transcriptomics, for nine prototype chemicals: including double strand break agents, and chemicals causing DNA alkylation or oxidative damage. Data were collected in human cell lines expressing wild-type p53 (HT1080, HCT 116). Dose-response data for each of the chemicals indicates that the p53 transcriptional response does not prevent MN induction at low concentrations. In fact, the no observed effect levels (NOELs) and benchmark doses (BMDs) for MN induction were less than or equal to those for p53-mediated gene transcription regardless of the test chemical, indicating that p53’s post-translational responses may be more important than transcriptional activation in the response to low dose DNA damage. p53 acts as a cofactor in complexes of repair proteins at DSBs (DNA repair centers; DRCs). We are currently developing models for the DRC dose- and time-response with these DNA damaging chemicals to evaluate how p53’s post-translational activity affects the dose-response curves for MN. This presentation describes the process of defining key assays required for a pathway-based, in vitro-only risk assessment, using the p53-mediated DNA damage response pathway as a prototype. Tuesday, Symposium 6: Talk 3/6 Towards a High Throughput Microscopy Pathway in Toxicity Reporter Platform for Chemical Safety Assessment Bob van de Water Leiden Academic Centre for Drug Research, Leiden University, The Netherlands Adaptive cellular stress responses are paramount in the healthy control of cell and tissue homeostasis after cell injury during hypoxia, oxidative stress or unanticipated side-effect of medications and other chemical exposures. To increase our understanding of chemically-induced adaptive stress response pathway activation and its contribution to safety assessment a time-resolved, sensitive and multiplex readout of chemical-induced toxicological relevant cellular stress responses is essential. For this we develop a platform containing a panel of distinct adaptive stress response fluorescent protein reporter cell lines. These are used for automated high content live cell imaging and quantitative multi-parameter image analysis to elucidate critical adaptive stress response pathway activation that can contribute to human chemical safety assessment. To conserve the endogenous gene regulatory programs, we tag selected reporter target genes with GFP using BAC-transgenomics approaches. Here we demonstrate the functionality of individual BAC-GFP pathway in toxicity reporter cell lines to their respective specific model compounds. The application of these reporters in chemical safety assessment in relation to drug-induced liver injury will be discussed. We anticipate that ultimately a phenotypic adaptive stress response profiling platform will allow a high throughput and time-resolved classification of chemical-induced stress responses assisting in the safety assessment of chemicals. This work is part of the MIP-DILI project supported by the Innovative Medicines Initiative (grant agreement n° 115336), and the FP7 SEURAT-1 DETECTIVE project (grant agreement 266838). Tuesday, Symposium 6: Talk 4/6 Implementing Toxicity Testing in the 21st Century (TT21C) using Quercetin as a Case Study for Consumer Safety Risk Assessment Yeyejide Adeleye Unilever The US National Academy of Sciences/National Research Council report on TT21C (1) proposes an approach to toxicity testing that could ultimately negate the requirement to generate hazard data in animals. At the core of this vision is the use of in vitro approaches to investigate perturbations in the critical cellular processes that lead to adverse events (toxicity pathways), and a safety assessment approach that ensures human exposure is kept below the level that is expected to cause adverse effects. Using a case study chemical (quercetin), a defined exposure scenario (0.5% in skin lotion) and a prototype pathway (p53) we have examined key elements of the TT21C strategy to determine the applicability of pathways based risk assessment of consumer products. Doses determined from in vitro assays (measuring key components of the p53 pathway) were compared to the output from biokinetic modelling and the potential for in vitro to in vivo extrapolation explored. A first tier risk assessment was performed comparing the total quercetin concentration in the in vitro systems with the predicted total quercetin concentration in plasma and tissues (2). We aim to demonstrate the practical applicability of using pathway-based risk assessment for consumer safety and highlight the challenges. 1. Krewski et al 2007 Toxicity Testing in the 21st Century: a Vision and a Strategy. US National Academies of Science. The National Academies Press. 2. Adeleye et al, 2014; Implementing Toxicity Testing in the 21st Century (TT21C): Making safety decisions using toxicity pathways, and progress in a prototype risk assessment. Toxicology (Epub) http://www.ncbi.nlm.nih.gov/pubmed/24582757 Tuesday, Symposium 6: Talk 5/6, Poster 039 MutAIT, a genetic toxicology portal: Structure and Applications in health risk assessment Daniele Avancini1, George Johnson2, Georgina Menzies1, Nick Sullivan2, Andrea Brancale3, Paul Lewis1 1Centre for NanoHealth, College of Medicine, Swansea University, Swansea, UK;2College of Medicine, Swansea University, Swansea, UK;3Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK Gene mutation data are frequently analysed by industry, academia and regulatory bodies for human health risk assessment. A number of databases housing genetic toxicology mutation data have been created and curated at different institutions, with the aim of providing public access to facilitate further research and assessment. We have developed a genetic toxicology portal to provide easy access to two of the largest developed mutation databases. The Mammalian Gene Mutation Database (MGMD) has over 50,000 records on mutation events for over 150 chemical and physical mutagens. Health Canada has also developed a transgenic gene mutation database (TransGenicDB) that has been seamlessly integrated with transgenic model data from MGMD. We have developed a user-friendly online platform to allow access to this powerful resource to industry and academia and statistical tools for mutation spectra and point of departure analysis on results retrieved from the databases or users own data. Called MutAIT (Mutation Assay Informatics Tools), the development of a website front end allows users to search the databases via custom querying. We provide an example of how this platform can be used to study the carcinogen, Benzo(a)Pyrene Diol Epoxide (BPDE). BPDE mutation spectra and corresponding spontaneous spectra were retrieved for the cII gene in transgenic animals and analysed to identify differences in frequencies and distributions of mutations. The spectra were then analysed using different statistical including relative dinucleotide mutability (RDM) calculations to find the likelihood of mutation within specific sequences. Coupled with analysis of BPDE dose response data, which were used to derive point of departure metrics such as NOGEL, BPD and STD, the platform provided a full report of BPDE target specificity under varying conditions. A further example of the benefits of such analysis shows molecular dynamic simulations of cII mutation hotspot sequences for BPDE, selected from the above analysis, to determine structural changes to DNA sequence when a BPDE adduct forms and correlation to mutability. As shown, MutAIT provides resources and tools to perform a comprehensive analysis in the wide field of genetic toxicology. Tuesday, Symposium 6: Talk 6/6, Poster 037 Developing an ‘in vitro’ carcinogenicity tool Eleanor Wilde, Anna Seager, George Johnson, Shareen Doak, Gareth Jenkins Swansea University, UK The National Centre for the Replacement, Refinement and Reduction of Animals in Research aims to minimise animal use in crucial carcinogenicity tests. One cause of increased animal use is the high frequency of misleading positive results shown by genotoxicity assays, which are vital in drug development. This project therefore aims to improve in vitro safety assessment by combining the analysis of cell genotoxicity, signalling and phenotype caused by known carcinogens, identifying effects specific to potent carcinogens, non-genotoxic carcinogens and misleading positives. The cytokinesis-block micronucleus assay was used to quantify the genotoxicity of Methyl-Methanesulfonate (MMS), N-Nitroso-N-Methylurea (MNU), Methyl Carbamate and 2,4-Dichlorophenol, treating TK6 cells for 1.5 cell-cycles with 1.5 cell-cycles recovery. Western blotting was also used to analyse the effects of chemicals on expression of tumour suppressor proteins, p53 and phospho-p53. Finally cells were treated for 1.5 cell-cycles, fixed and imaged using the InCell Analyzer 2000. These images were analysed using Matlab to monitor changes in cell-cycle and cell morphology. The micronucleus assay indicated that significant genotoxicity was initiated by MMS, MNU and 2,4-Dichlorophenol at 0.7, 0.3 and 30 µg/mL (Dunnett’s t-test, p < 0.05). Also Western Blotting showed dose-dependent increases in p53 and phospho-p53 expression in response to MMS and MNU. Finally a dose-dependent G2/M cell-cycle block and an increase in average nuclear area and perimeter were observed using the InCell Analyzer in response to both MMS and MNU. These results tie in with the genotoxicity shown by these chemicals and also the increase in p53 and phospho-p53 since these proteins are known to cause G2 cell-cycle arrest in order to initiate DNA repair or apoptosis. Linking these data-sets will indicate how the effects of specific compounds relate to their mechanisms of action, improving the identification of potential carcinogens in vitro. Symposium 6: Poster 035 Cell Cycle Modifies Susceptibility of MCF-7 Cells to Benzo[a]pyrene Kirsten Snijders Lancaster Environment Centre Benzo[a]pyrene is an environmental contaminant mainly found in tobacco smoke, car exhaust fumes and industrial wastes. It has pro-mutagenic and pro-carcinogenic properties and damages DNA through the formation of DNA adducts. B[a]P is an exogenous ligand for the aryl hydrocarbon receptor (AhR) responsible for the regulation of transcriptional responses. To investigate the susceptibility of MCF-7 cells to B[a]P at different stages of the cell cycle the cells were cultured then concentrated in S-phase or G0/G1-phase and subsequently treated with 10-6M and 10-9M B[a]P, along with a vehicle control of DMSO. Cell cycle progression was examined through flow cytometry. The cells were fixed onto Low-E slides with 70% ethanol and bio-spectroscopy analysis, ATR-FTIR and RAMAN, was performed. Changes in gene expression levels for CYP1A1 and CYP1B1, cytochrome P450 genes involved in the xenobiotic metabolism of B[a]P, were detected through RT-PCR and western blotting. Thus far this study demonstrates that B[a]P is capable of inducing alterations in exposed cell populations even at very low doses of 10-9M. B[a]P gradually reduces cell survival after 7days exposure and induces long term inhibition of cell growth. High dose exposure of 10-6M B[a]P causes short term evasion of G1 arrest and accumulation in S-phase. These findings indicate that growth kinetics in vitro are important pre-determinants of MCF-7 cell susceptibility to genotoxic agents. Bio-spectroscopy and computational analysis will produce biochemical signatures characterising dose and cell cycle related effects of B[a]P. CYP isoform expression will indicate the gene response and level of DNA damage following B[a]P exposure. Hamouchene, H. (2011). Influence of cell cycle on responses of MCF-7 cells to benzo[a]pyrene. BMC Genomics. 12 Jiao, HY. (2007). Growth kinetics in MCF-7 cells modulate benzo[a]pyrene-induced CYP1A1 up-regulation. Mutagenisis. Pang, W. (2012). Identification of benzo[a]pyrene-induced cell cycle-associated alterations in MCF-7 cells using infrared spectroscopy with computational analysis. Toxicology. 298 Symposium 6: Poster 036 Characterisation of the stem cell population in human endometrium Marina Ellie Afami, Francis L Martin, Nigel J Fullwood, Pierre L Martin-Hirsch Lancaster University The human endometrium is a highly regenerative tissue that experiences functional and structural changes during each menstrual cycle in order to provide a favourable environment for implantation of the embryo. Underpinning this cycle must be a stem cell population. The aim of this project was to characterise this stem cell population in situ in normal tissue. To this end, scanning electron microscopy and image analysis was employed. Different stages during the menstrual cycle were investigated; in particular, the process of epithelial-cell shedding and regeneration in order to determine their role in proliferation and migration from the endometrial crypts. Our results demonstrate a detailed and complex cycle of events, which includes desquamation of the superficial layer of endometrium, the appearance of cone-shaped endometrial glands, formation of fibrin mesh which is later replaced by rapid proliferation of epithelial cells, ciliogenesis, re-establishment of vasculature, participation of white blood cells to clear away cellular debris and finally remodelling of the endometrium. The functionalis is the layer that is sloughed off during each menstrual cycle whilst the basalis, which contains the potential for endometrial regeneration remains intact. The current evidence supports the hypothesis that the endometrial stem cell population is located in the endometrial crypts, which probably reaches the basal layer. However, the process is more complex than previously thought. For example, the factors triggering the production of uterodomes and their precise function remains unclear. Further insights into biochemical differences in these different cell types will be obtained using Raman spectroscopy. Symposium 6: Poster 038 In vitro assessment of genotoxicity and cell cycle abnormalities to better understand carcinogenic risk Leanne Stannard1, Shareen Doak1, Ann Doherty2, Gareth Jenkins1 1Swansea University, UK;2AstraZeneca Safety assessment in the 21st Century requires more sophisticated in-vitro tools to obtain richer datasets. We aim here to couple micronucleus data to other aspects of cell biology to obtain more predictive data for carcinogenicity. Firstly, automated micronucleus detection is utilised to describe dose responses for compounds using TK6 cells (to define the thresholds). To determine the mechanisms allowing the cells to tolerate low doses, many key cellular processes are being investigated, including cell cycle analysis (flow cytometry), cell signalling analysis (real-time PCR and Western blotting) and morphological alterations in cellular and subcellular organisation (InCell analyser). We aim to discriminate potent carcinogens from weak carcinogens and non-genotoxic carcinogens. The chemicals studied so far are Cadmium Chloride (CdCl2), which has been classed as a potent carcinogen, and Quercetin, a suspected misleading positive. The micronucleus data for Quercetin shows that micronuclei are only induced in the toxic dose range, (based on 50% RPD), of 110–150µM. After solving problems with the dissolution of CdCl2, the RPD data shows that the toxic dose region is between 0.2-0.4mg/ml. Current work is focussing on the cell cycle, cell signalling and cell phenotype, to identify their mechanism of action and confirm previous findings. As well as determining underlying biological processes impacting on the genotoxic responses to mutagenic chemicals, this project aims to develop state of the art cell biology tools within the mechanistic studies to investigate pathways and processes important in carcinogenesis. Tuesday, Workshop 3: Talk 1/6 Applications of biomarkers in aquatic organisms: linking human and environmental health Awadhesh Jha Plymouth University, UK Historically, a large number of landmark studies using aquatic organisms have provided a wealth of information for basic biomedical research and to elucidate the underlying mechanisms of human diseases. Given that a large number of stress related genes are highly conserved, advancement in ‘omics’ approaches are now also realising the potential of these organisms to correlate human and environmental health. Furthermore, despite the fact that similar biological responses (including genotoxicity) are observed in natural biota and in humans, relatively little importance has been given to applications of these biomarkers in environmental risk assessment (ERA) until recently. New legislations around the world are however emphasizing the need for biological effects of contaminants as the criteria for ERA. We have attempted to develop and implement a range of genotoxicological and other biological responses at different levels of biological organisation in several ecologically relevant species. These include transcriptional expression of key genes, direct damage to DNA / chromosomes, histopathology and physiological responses. The broader aims have been to determine the relative sensitivity of the species to a range of contaminants and compliment the observed responses with bioavailability and body burden data, from analytical tools. The synthesized information could provide added value when obtaining information through traditional bioassays, chemical and ecological measures, in order to adopt a preventive approach for human health and environmental sustainability. It is however to be remembered that biota occupy a range of habitats with many confounding factors influencing the responses, often simultaneously and differentially in different tissues, species and life stages. Use of these model species will therefore deliver little if the fundamental understanding of how they respond under complex environmental scenarios is not understood at molecular and cellular levels in an integrated manner following proper optimisation and validation of the applied tools. Tuesday, Workshop 3: Talk 2/6 Mussel micronucleus cytome assay, an approach to evaluate cytotoxic and genotoxic environmental pollution: examples of field application Claudia Bolognesi Environmental Carcinogenesis Unit, IRCSS IRCCS AOU San Martino-IST, Italy The micronucleus (MN) assay is one of the most widely used genotoxicity biomarkers in aquatic organisms, providing an efficient measure of chromosomal DNA damage occurring as a result of either chromosome breakage or chromosome mis-segregation during mitosis. The collected experimental evidence on the sensitivity of the MN assay allowed it to be recommended as a test in the battery of biomarkers in the Mussel Watch programs. The MN assay is applied in laboratory and field studies using hemocytes and gill cells from bivalves, mainly from the genera Mytilus. The experimental protocol of the MN assay was standardized to include also the evaluation of other nuclear alterations, such as nuclear buds, apoptotic and necrotic cell, following the cytome approach already applied in mammalian cells. The experimental design involving the use of wild and caged mussels was defined. The sample size, the role of main confounding factors and the data analysis and interpretation were also outlined. The mussel micronucleus cytome (MUMMNcyt) assay was applied between 2007–2013 in a biomonitoring program using caged mussels to assess the effects of a decontamination processes in a remediation site highly contaminated by chromium in the west part of the Liguria coast (Italy). Significant increases in frequency of nuclear anomalies, MN and nuclear buds, as well as of cytotoxicity biomarkers were observed in hemocytes and gill cells of mussels from the remediation site compared with the reference area, related to the accumulation of Cr and polycyclic aromatic hydrocarbons in mussel tissues. Differences in biomarker responses observed in hemocytes or gill cells suggest to apply the MUMNcyt assay to both target tissues from the same animals to have a more complete picture of the genotoxic and cytotoxic effects induced by the cumulative exposure to a mixture of contaminants in polluted areas. Tuesday, Workshop 3: Talk 3/6 Impact of environmental exposures on metabolic pathways and behavior in fish Rebecca Van Beneden, Patrick Carlson, Lindsay Grumbach, Torey Bowser, Marissa Giroux University of Maine, USA Arsenic exposure is a concern worldwide and has been linked to cancer, reproductive disorders, cardiovascular disease, diabetes and metabolic disease. The prevalence of type II diabetes has risen dramatically in many parts of the world in the last two decades. Like many other diseases, its etiology is a complex interaction of genetic, behavioral and environmental factors. An emerging hypothesis suggests that exposure to environmental contaminants may play a larger role than previously believed. Notably, arsenic exposure has been linked to diabetes and metabolic disease, although the mechanism of toxicity is still poorly understood. The adult zebrafish (Danio rerio) was used to investigate liver toxicity following arsenic exposure. A proteomic approach was employed to investigate arsenic-induced alteration in the zebrafish liver proteome following a 7-day exposure to 50 ppb sodium arsenite. These studies revealed altered lipid transport and metabolic pathways, as well as alterations in expression of cell cycle regulatory genes. The proteomics studies were expanded to further investigate the effect of arsenic on protein kinase B (AKT1) which plays a critical role in insulin signaling and glycogen synthesis and regulation of many of the downstream effects we observed in our proteomic studies. Studies are also underway on the effect of arsenic exposure on fish behavior. Initial results suggest that arsenic exposure may impact the phosphorylation state, and thus activity, of AKT. Arsenic also appears to impact behavior of zebrafish, exposed either as adults or embryos, in a number of behavioral tests. The combined results suggest that arsenic may affect organisms at multiple levels. Tuesday, Workshop 3: Talk 4/6 Reflection of exposure of aquatic organisms to environmental pollutants via the transcriptome and epigenome Kevin Chipman University of Birmingham, UK Carson’s ‘Silent Spring” approximately 50 years ago raised awareness of the need to protect the environment from chemicals that have the potential to cause ecological and human harm. However the methods for assessing the impact of pollutants on organisms in which there is little genomic information has been limited to crude, insensitive end points. The rapid expansion of knowledge about gene sequence and function, coupled with ‘omic’ technologies has provided an unprecedented opportunity to transform environmental monitoring into mechanism-based, sensitive early-warning alerts based on ‘adverse outcome pathways.’ Transcriptomic analyses, and associated bioinformatic interrogation, can reveal the complex responses to chemicals with potential to cause cancer and other disorders. Models of chemical-induced liver carcinogenesis in zebrafish have revealed remarkable similarities in gene expression and epigenetic marks to those associated with human liver cancers. However, although the incidence of liver tumours in fish can reach levels up to 20% incidence, little is known about the relative importance of genetic and epigenetic causes. A sub-group of stress-related genes (identified from toxicogenomic studies in fish under laboratory conditions) can be predictive of the pollution exposure in fish taken from different environments in the wild (Williams et. al., PLoS Comput Biol. 2011). Gene expression networks can reveal associations between key nodes of the network with health-related parameters. Moreover epigenetic changes, mediated by disturbance of the 1-carbon cycle, can also inform on environmental conditions associated with tumour formation in wild fish. An epigenetic progenitor cell mechanism may be important in carcinogenesis (Mirbahai et. al. J. Proteome Res., 2013). Thus transciptome and methylome profiles have potential utilisation as complex monitoring biomarkers. Tuesday, Workshop 3: Talk 5/6, Poster 106 Comparative DNA Damage and Repair in Echinoderm Coelomocytes Exposed to Genotoxicants Ameena H El-Bibany, Andrea G Bodnar, Helena C Reinardy Bermuda Institute of Ocean Sciences The capacity to withstand and repair DNA damage differs among species and plays a role in determining an organism’s resistance to genotoxic stress, life history, and susceptibility to disease. Environmental stressors that affect organisms at the genetic level are of particular concern in ecotoxicology due to the potential for chronic effects and trans-generational impacts on populations. Echinoderms are valuable organisms to study the relationship between DNA repair and resistance to genotoxic stress due to their history as ecotoxicological models, large variation in lifespans between species (interspecific differences), little evidence of senescence, and few reported cases of neoplasia. In this study, DNA damage was assessed (by Fast Micromethod) in coelomocytes of four echinoderm species (sea urchins Lytechinus variegatus, Echinometra lucunter lucunter, and Tripneustes ventricosus, and a sea cucumber Isostichopus badionotus) after acute exposure to H2O2 (0 - 100mM) and UV-C (0 - 9999 J/m2), and DNA repair was analyzed over a 24-h period of recovery. Results show no difference in DNA damage and repair capacity between small (young) and large (old) L. variegatus, E. l. lucunter, and I. badionotus (GLM, p > 0.05), indicating maintenance of DNA repair with age. There was a general correlation between DNA repair capacity and lifespan of different species, with longer-lived species showing higher levels of repair than shorter-lived species 24 hours following exposure to the highest concentration of H2O2 (100mM) and UV-C (9999 J/m2). Interspecific differences in genotoxic susceptibility and capacity for DNA repair are important to consider when evaluating ecogenotoxicological model organisms and assessing overall impacts of genotoxicants in the environment. Tuesday, Workshop 3: Talk 6/6, Poster 105 Biospectroscopy as a tool to detect the effects of environmentally-relevant levels of two fungicides in an amphibian cell line Rebecca J Strong1, Crispin J Halsall1, Kevin C Jones1, Richard F Shore2, Francis L Martin1 1Lancaster University;2Lancaster Environment Centre, UK Amphibians are regarded as sensitive sentinels of environmental pollution due to their highly permeable skin and complex life cycle comprising both aquatic and terrestrial phases. In particular, environmental contamination from agriculture has been associated with detrimental effects in amphibian populations. Fungicides are widely applied in temperate climates and have been associated with genotoxicity and developmental effects in aquatic animals. Therefore it is important to determine the effects of environmentally-relevant concentrations of such contaminants in target cells. Infrared (IR) spectroscopy with multivariate analysis has previously been utilized to signature the biological effects of different contaminants found in environmental systems at low levels(1). Using this approach, the Xenopus laevis kidney epithelial cell line was exposed to low concentrations of the fungicides carbendazim and flusilazole (0.5 x 10-10 - 0.5 x 10–8M) for 24 hours. Cells were then examined using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy and the resulting spectra processed with multivariate analysis. Initial results show differences in regions associated with glycogen, protein and collagen for cells exposed to carbendazim, with the lowest dose, 0.5 x 10-10M clustering furthest away from the control. Cells treated with flusilazole also showed differences in regions associated with glycogen and protein, with the higher doses clustering further away from the control. Future work will assess the combination of these two agents, as amphibians are exposed to multiple stressors simultaneously. References 1. Llabjani, V., Crosse, J. D., Ahmadzai, A. A., Patel, I. I., Pang, W., Trevisan, J., Jones, K. C., Shore, R. F., and Martin, F. L. (2011) Differential Effects in Mammalian Cells Induced by Chemical Mixtures in Environmental Biota As Profiled Using Infrared Spectroscopy, Environ. Sci. Technol. 45, 10706–10712. Workshop 3: Poster 104 A new Ames tester strain for specific detection of the genotoxicity of polycyclic aromatic hydrocarbons Masami Yamada, Keiko Matsui, Takehiko Nohmi National Institute of Health Sciences, Tokyo, Japan The pollution of air, water and soil with polycyclic aromatic hydrocarbons (PAHs) is a serious problem in many countries all over the world. To sensitively detect the genotoxicity of PAHs including benzo[a]pyrene in complex mixtures extracted from various polluted environmental sources, Salmonella enterica serovar Typhimurium (S. typhimurium) strain YG5161 has been used in several countries. The strain was developed with the idea that pYG768 carries the dinB gene encoding Escherichia coli DNA polymerase IV (pol IV), that can efficiently bypass the DNA adduct caused by PAHs and frequently generate mutation, is introduced into a conventional Ames tester strain TA1538. Strain YG5161 exhibits higher sensitivity to the genotoxicity of PAHs than do conventional strains TA1538 and TA98. There has been a problem, however, the genotoxic PAHs can be veiled in complex mixtures extracted from various polluted environmental sources if genotoxic nitroaromatics or aromatic amines are contaminated. This is because strain YG5161 also detects the mutagenicity of aromatic amines and nitroaromatics with high sensitivity as the conventional Ames tester strains do. To solve the problem, we disrupted the nfsB and oat genes encoding the activation enzymes in strain TA1538 to reduce the cross sensitivity, and introduced plasmid pYG768 again into the delta-nfsB/oat strain. The resulting strain YG5185 retained similar high mutability to PAHs as did strain YG5161, and substantially decreased the sensitivity to nitroarenes, 1-nitropyrene, 1,8-dnitropyrene etc. We propose that the novel tester strain YG5185 can contribute to the environmental mutagen study with its specific and sensitive ability to detect the genotoxic PAHs in complex mixtures from polluted environmental sources. Workshop 3: Poster 107 DNA Double-Strand Breaks In Relation To Persistent Organic Pollutants In Fasting Common Eiders (Somateria Molissima) Anette A Fenstad1, Bjørn Munro Jenssen1, Børge Moe2, Sveinn A Hanssen2, Chris Bingham1, Dorte Herzke3, Jan O Bustnes2, Åse Krøkje1 1Department of Biology, Norwegian University of Science and Technology, Norway;2Norwegian Institute for Nature Research, Framsenteret, Norway;3Norwegian Institute for Air Research, Framsenteret, Norway Lipophilic persistent organic pollutants (POPs) are released from fat reserves during fasting, causing increased blood concentrations. Thus, POPs represent a potential anthropogenic stressor during fasting periods. Blood of female common eiders (Somateria mollissima) was analysed by using agarose gel electrophoresis and image data analysis to quantify the DNA-fraction, of total DNA, that migrated into the gel (DNA-FTM) as a relative measure of DNA double strand-breaks (DSBs) during the fasting incubation period in the high arctic. In 2008 and in 2009 blood samples were obtained for analysis of 9 POPs and DNA-FTM at day 5 of the incubation period, and then in the same individuals at day 20. This unique study design gave us the opportunity to analyse the same individuals throughout two points in time, with low and high stress burdens. During the incubation period the body mass (BM) decreased by more than 20%, whereas the POP levels increased by at least 148%. The DNA-FTM increased by more than 60% (being proportional to the increase in DSBs). At day 5, but not day 20, DNA-FTM was positively correlated with most analysed POPs. The increase in DNA-FTM was positively correlated with the decrease in BM (g) during incubation. Thus, we suggest that fasting stress (BM loss) decreases DNA integrity and that stress caused by fasting on BM loss appeared to override the additional stress caused by concurrent increase in levels of the analysed POPs in the eiders. Workshop 3: Poster 108 Fish cell lines in 3 dimensional (3D) cultures in ecotoxicological studies: is there oxygen in the middle? Laura Langan1,2, Nicholas Dodd1, Stewart F Owen, Simon Jackson1, Wendy M Purcell1, Awadhesh Jha1 1Plymouth University, UK;2AstraZeneca, UK In mammalian research, three-dimensional (3D) in vitro culture is a well-established model exhibiting micro environments which are close to that of the in vivo conditions in terms of gene expression, cell-cell interaction and cellular function. In parallel with mammalian studies, the fish liver spheroid model is now being considered for environmental applications in line with the 3Rs approach (reduction, replacement, and refinement). The limitations of fish spheroids originating from cells of other organs have not yet been assessed. A stumbling block to the widespread use of this technique in fish ecotoxicology is the lack of information regarding the morphology and physical micro-environment of spheroids. It is not known whether the spheroids, formed from fish cells, form the same micro-environment as tumour models or whether they are more representative of the in vivo tissue they are formed from. Oxygen gradients across the micro-structure are believed to play an important role in driving xenobiotic metabolism. It is therefore important to know whether the growing spheroids have a homogenous supply of oxygen, which would have a considerable impact on the metabolism of xenobiotics, and provide key information as to the applicability of the model. For the first time, the oxygen saturation level within spheroids formed from the rainbow trout gonad cell line RTG-2 was measured using Electron Paramagnetic Resonance (EPR). Spheroids were grown in a range of sizes from 100µm to 800 µm, with the EPR oxygen sensitive probe lithium phthalocyanine embedded at the core. Initial findings indicate that for smaller spheroids, percentage oxygen saturation was approximately 88% in the 100–300µm size range up to 7 days post formation. Interestingly over a 14 day period, oxygen saturation levels halved from day 7 to day 14 in these smaller spheroids which was not replicated in the larger spheroids. Overall, the oxygen measurements within this spheroid model suggest that fish spheroids have a homogenous supply of oxygen at smaller size ranges for the first week. As they mature, it appears that an oxygen gradient is formed that may mimic the conditions identified in mammalian tissue. Workshop 3: Poster 109 Induction of Genotoxicity and an Inflammatory Response by Ultrafine Superparamagnetic Iron Oxide Nanoparticles (USPION) Stephen Evans, Neenu Singh, Thomas Wilkinson, Gareth Jenkins, Shareen Doak Swansea University, UK Ultrafine superparamagnetic iron oxide nanoparticles (USPION) hold great potential for a number of uses in the medical field such as in the enhancement of imaging techniques, tissue repair and targeted drug delivery. If its usage increases so will its rate of manufacture and therefore, its inhalation. This study aimed to assess the potential of USPION to promote genotoxic damage or inflammatory responses in relation to their physico-chemical properties, focusing on the impact of redox status by considering both magnetite (Fe3O4) and maghemite (Fe2O3). Genotoxicity was assessed in the bronchial cell line 16HBE140- using the automated micronucleus assay, while the ability of the test nanoparticles to promote an inflammatory signalling response was examined in the monocyte like THP-1 cell line utilising ELISAs for TNF-α and IL-8. Both undifferentiated and cells differentiated to a macrophage like state via treatment with PMA were investigated. Cellular uptake was assessed using the ferrozine assay and full particle characterisation was carried out by dynamic light scattering, differential centrifugal sedimentation and XPS analysis in addition to hard protein corona characterisation. Interestingly, this study demonstrated that maghemite was capable of promoting a greater inflammatory response in THP-1 cells than magnetite which could subsequently be responsible for genotoxicity induced by exposure to this nanomaterial. Maghemite displayed a greater degree of particle agglomeration following DLS analysis than magnetite suggesting a possible explanation of different degrees of immuno/genotoxicity between redox state. Iron oxide redox status therefore plays an important role in influencing the biological impact of nanomaterial exposure in addition to the importance of agglomeration state. The inflammatory response of the THP-1 cell line demonstrates the importance of considering the interaction of different cell types when testing nanomaterial toxicity in vitro. Tuesday, Young Scientist Session & Awards: EEMS Award Winner, Talk 1/6 Using Genetic Toxicity Data for Human Health Risk Assessment, the Quantitative Way… George E Johnson Institute of Life Science, College of Medicine, Swansea University, UK There is a growing body of evidence that supports using genetic toxicity data in a more quantitative manner for human health risk assessment. This is causing a paradigm shift away from only using these data in a qualitative binary fashion (i.e., positive or negative), and places more emphasis on the point of departure (PoD) metrics. It is also becoming clear that genetic toxicity could be considered as an apical endpoint in itself, and if this were the case there would another paradigm shift away from only using genetic toxicity data to inform cancer risk assessment. In support of this, there is growing realisation that the PoDs derived from genetic toxicity endpoints are more accurate and well defined in terms of informing human risk assessment. Due to this changing landscape, it is important that the assays used in genetic toxicology can provide suitable statistical power to define accurate PoDs, while maintaining high sensitivity and specificity. This applies to both in vitro and in vivo assays for gene mutation and chromosome damage endpoints, and here we present PoD metrics from many of these high content assays. Furthermore, in order to have confidence in a PoD defined using one of these endpoints, the mode of action must be assessed and clearly linked to the dose response. This has been done using gene expression analysis and knockdown technologies following treatment with mono-functional alkylating agents, and here we show that DNA repair has been clearly linked to the PoDs. The next step is to utilise in vitro data in a more quantitative manner, and eventually carry out human health risk assessments based on in vitro data alone. Our initial investigations into in vitro to in vivo extrapolations (IVIVE) suggests that this is a possibility, and our EEMS 2014 satellite workshop with the ILSI-HESI GTTC brings in global experts on this topic to indicate best practise and what should be done next. Tuesday, Young Scientist Session & Awards: Talk 2/6, Poster 113 Adverse outcome pathways for metal and radionuclide exposure: an ecotoxicological perspective using mussels as a sentinel organism Lorna J Dallas1, Yanan Di1, Tim P Bean2, Brett P Lyons2, Andrew Turner3, Awadhesh N Jha1 1School of Biological Sciences, Plymouth University, UK;2Cefas Weymouth Laboratory, UK;3School of Geography, Earth and Environmental Sciences, Plymouth University, UK In ecotoxicology, the primary unit of interest is the ecosystem, community or population (i.e. at an ecological rather than individual level). It is, however, usually necessary to measure biological effects of contaminants at lower levels (e.g. molecular, biochemical etc). One solution to this problem is to explore the mechanistic links between effects at differing levels of biological organisation, and therefore be able to predict the consequences at the ecosystem level (i.e. an adverse outcome pathway [AOP] approach). In this context, a suite of biomarkers at different levels of biological organisation were investigated in an ecologically relevant bivalve species, Mytilus galloprovincialis, after exposure to nickel (a metal), zinc pyrithione (an organometal) and tritiated water (a radionuclide). These contaminants were chosen based on their differing properties, and hence, mechanisms of action. All three contaminants produced genotoxicity (DNA strand breaks, as measured by the comet assay, and induction of micronuclei [MN]). For nickel (> 1800 µg L−1) and tritiated water (15 MBq L−1), biomarkers at lower levels of biological organisation (i.e. DNA strand breaks, MN, changes in the expression of key stress response genes) were more sensitive than those at higher levels (i.e. clearance rate, attachment, tolerance of anoxia). In particular, exposure to tritiated water for 14 days resulted in DNA damage and molecular alterations without affecting higher level responses. In contrast to nickel and tritiated water, exposure to both 0.2 and 2.0 µM zinc pyrithione caused significant deviation from concurrent controls for every biomarker examined, suggesting that further investigation of the environmental impacts of this contaminant is particularly necessary. Variation in biological responses induced by these three different contaminants suggests that potential links between levels of organisation are contaminant-specific, i.e. that there are several AOPs. Tuesday, Young Scientist Session & Awards: Talk 3/6, Poster 110 Epigenetic alterations induced by nanoparticles Ali M Tabish1, Katrien Poels1, Andrea Baccarelli3, Peter Hoet1, Katrien Luyts1, Dorota Napierska1, Lode Godderis1,2 1Katholieke Universiteit Leuven, Belgium;2IDEWE, Belgium;3Bldg 1, Boston, MA, USA Humans are at risk of chronic exposure to nanoparticles (NPs) due to the manufacturing of NPs and/or NPs occurring naturally. The possible toxic health effects of NPs associated with human exposure are largely unknown. In this study, we assessed epigenetic effects of NPs exposure in vivo. We exposed BALB/C mice to well-characterized gold NPs (Au NPs) of 5nm, 60nm and 250nm diameter; single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) at two dose levels (high dose: 1mg/ml; low dose: 100 µg/ml) for 48 hrs. Control mice received either vehicle (0.2% saline) or air (sham-control). Mice were sacrificed after 48 hrs, and lung and blood cells were analyzed for epigenetic endpoints i.e., global and gene specific methylation, and DNA hydroxymethylation. NPs exposure showed alterations in global DNA hydroxymethylation in mice lung DNA while no alterations were observed for global DNA methylation. NPs exposure in mice also altered methylation level of number of genes in lung and blood tissues. The epigenetically altered genes were involved in DNA repair pathway, cell cycle control pathway, oxidative stress response pathway and immune related pathways. We are further translating in vivo findings in humans who are exposed to NPs. In conclusion our results indicated the involvement of epigenetic factors in response to NPs exposure in vivo. Tuesday, Young Scientist Session & Awards: Talk 4/6, Poster 111 New insights into activity of the formamidopyrimidine-DNA glycosylase from E.coli Anton Endutkin, Zharkov Dmitry SB RAS Institute of Chemical Biology and Fundamental Medicine, Russia Formamidopyrimidine DNA glycosylase (Fpg) from Escherichia coli is one of the main tools protecting the bacterial genome from lesions caused by environmental and internal insults. Fpg initiates base excision repair in DNA by removing oxidized nucleobases. A major DNA oxidation product is 8-oxoguanine (8-oxoG), a base with a high mutagenic potential. We have used kinetic and thermodynamic approaches to analyse 8-oxoG recognition and excision by Fpg. First, we have found that the energy of stacking interactions of 8-oxoG was in strict dependence on nucleotide environment, which may affect lesion recognition and the efficiency of eversion of 8-oxoG from the double helix by DNA glycosylases. In addition, we observed that the presence of the lesion affects the kinetics of association of oligonucleotides into a duplex. Using site-directed mutagenesis, we have addressed the functions of many previously unstudied amino acid residues that were predicted to be important for Fpg activity and structure maintenance. Of note, many substitutions abolished the excision of 8-oxoG but did not affect the cleavage efficiency of abasic substrates. Finally, we investigated the contribution of separated structural domains of Fpg to specific enzyme-substrate interactions. Surprisingly, despite the absence of the catalytic domain, C-terminal domain of Fpg possessed a low residual ability to recognize and cleave abasic substrates. This is can shed light on unknown functions of truncated Fpg homologs from Mycobacterium tuberculosis. Our study uncovers new mechanistic details of Fpg activity, allowing us to consider Fpg as a promising scaffold for applied nanotechnology and molecular medicine. This work was supported by RAS Presidium (Molecular and Cellular Biology Program), RFBR. Tuesday, Young Scientist Session & Awards: Talk 5/6, Poster 112 The PARP inhibitor Olaparib disrupts Base Excision Repair of 5-aza-2′-deoxycytidine lesions. Manuel Luis Orta Vázquez1-2, Andreas Höglund2, Jose Manuel Calderón Montaño2-3, Nuria Pastor1, Inmanculada Domínguez1, Cecilia Ström2, Estefanía Burgos Morón3, Miguel López-Lázaro3, Thomas Helleday2 1Dep. of Cell Biology. Fac. of Biology. University of Seville, Spain;2Science for Life Lab. Dep. of Medical Biochemistry and Biophysics. Karolinska Institute, Sweden;3Dep. of Pharmacology. Fac. of Pharmacy, Univ. of Seville, Spain Decitabine (5-Aza-2’-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks (DSBs). The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) Polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage and disrupts BER. Here we show that after 5-azadC treatment, PARP inhibition prevents XRCC1 and DNMT1 foci co-localization, thereby disrupting BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML. Tuesday, Young Scientist Session & Awards: UKEMS Award Winner, Talk 6/6 Using the comet assay to study DNA repair: progress in the past decade Sabine AS Langie Environmental Risk and Health unit, Flemish Institute of Technological Research (VITO), Mol, Belgium The comet assay is a versatile and sensitive method for measuring strand breaks in DNA. The first paper on this single-cell gel electrophoresis assay was published in 1984 by Ostling and Johanson (Biochem. Biophys. Res. Commun., 1984), meaning that this year we are celebrating 30 years of the comet assay. This assay (with and without inclusion of lesion-specific enzymes) is widely used as a biomarker assay in human population studies - primarily to measure DNA damage, but increasingly also to assess the capacity of cells for DNA repair. Ostling and Johanson were also the first to report experiments to measure DNA repair, by simply following the decrease of DNA damage over time after challenging cells with ionising radiation. However, this approach is time-consuming and laborious, and therefore not ideal for biomonitoring studies, which typically require high-throughput processing of many samples. As an alternative approach, the in vitro comet-based repair assay was developed: a cell extract is incubated with a DNA substrate containing specific lesions, and DNA incisions accumulate. The in vitro comet-based repair assay was first devised to measure base excision repair in lymphocytes (Collins et al., Mutagenesis, 2001). However, over the past decade it has been modified and improved to study incision of other lesions and thus other repair pathways, as well as being applied to tissue samples in addition to cell suspensions. In my presentation I will give a short overview of the history of the in vitro comet-based repair assay and the contributions I have made in this area of research. The application of the in vitro repair assay in dietary intervention, environmental biomonitoring and animal studies will be discussed. In addition, I will also highlight the importance of an international network and long-lasting collaborations, the opportunities I was given and the challenges a young scientist might face, all of which have allowed me to grow and become a more mature scientist. Wednesday, Symposium 7: Talk 1/5 DNA Strand Breaks and Neurological Disease Keith Caldecott Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, UK DNA strand breaks arise in the nervous system from a variety of sources, including endogenous abortive topoisomerase activity and oxidative stress. Both single- and double-strand breaks arise endogenously, and most likely can impact on critical cellular processes such as neural cell transcription and differentiation. Here, I will discuss recent published and unpublished developments in my laboratory that address underlying mechanisms by which DNA strand breaks impact on neural cell function and human neurological disease. Wednesday, Symposium 7: Talk 2/5 DNA breaks and chromosome pulverisation from errors in mitosis Karen Crasta Nanyang Technological University, SIngapore This talk will focus on a mechanism by which DNA breaks are generated as a consequence of whole chromosome missegregation via micronuclei. Whole-chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and often extensive fragmentation of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for ‘chromothripsis’ in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement. Wednesday, Symposium 7: Talk 3/5 Unexpected sequelae of endogenous DNA damage Laura Niedernhofer1, Tania Rozgaja1, Xuesen Li1, Jin Wang2, Ismene Karakasilioti3, Amanda Beck1, Sara McGowan1, Salony Maniar4, Nadiezhda Cantu-Medellin4, Donna Beer Stolz4, Eric Kelley4, Claudette St. Croix, Yinsheng Wang2, Paul Robbins1, George Garinis3 1Scripps Florida, USA;2U.C. Riverside, USA;3IMBB;4University of Pittburgh, USA XFE progeroid syndrome is a disease of accelerated aging. It is caused by mutations in XPF, which encodes the catalytic subunit of the DNA repair endonuclease XPF-ERCC1. The nuclease plays a key role in nucleotide excision repair of helix-distorting lesions, interstrand crosslink repair, and the end-joining of some double-strand breaks. Accelerated aging is presumed to arise as a consequence of failure to repair spontaneous, endogenous DNA damage. Mutations in mErcc1 have been created to make murine models of XFE progeroid syndrome with the goal of identifying the mechanism by which nuclear DNA damage drives aging. Ercc1-/D mice express ~5% of the normal complement of XPF-ERCC1 and spontaneously develop multiple progressive age-related degenerative diseases early in adulthood including cerebral atrophy, emphysema, glomerulosclerosis, sarcopenia, osteoporosis, and cataracts. They live only 7 months. As anticipated, these mice accumulate oxidative DNA lesions more rapidly than wild-type mice. Careful analysis of these mice, as well as tissue-specific mutant strains, reveals both cell autonomous and non-autonomous mechanisms contributing to aging. Recent findings of quite unexpected downstream consequences of unrepaired endogenous DNA damage in vivo will be discussed. Wednesday, Symposium 7: Talk 4/5 Reprogramming in vivo: The power of manipulating cell plasticity Maria Abad Spanish National Cancer Research Centre The ability to reprogram differentiated cells into induced pluripotent stem cells (iPS cells) has considerably improved our current understanding of cellular plasticity and has also helped to paving the way towards regenerative medicine. However, little was known about whether or not in vivo reprogramming is feasible and if so, what type of cells are generated in vivo and what are their implications in the organism. We have generated a ‘reprogrammable” transgenic mouse strain that ubiquitously express the so-called Yamanaka factors upon treatment with doxycycline. We have shown that transitory induction of the four Yamanaka factors in mice, for just one week, is able to induce dedifferentiation and pluripotency in a variety of tissues and to various degrees. Mice that had activated the reprogramming factors developed multiple teratomas, which is indicative of complete reprogramming events in vivo. Indeed, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptomic level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage and generate embryo-like structures that express embryonic and extraembryonic markers, suggesting that they achieve a more plastic or primitive state than ES cells. Our results could be relevant for the development of future applications of reprogramming in regenerative medicine. Wednesday, Symposium 7: Talk 5/5 High-throughput genetic screens to identify novel regulators of mammalian genome maintenance Rebecca McIntyre, David Adams Wellcome Trust Sanger Institute, Cambridge, UK We are performing two landmark screens for genes that regulate genome stability in mice using a high-throughput, flow cytometric micronucleus assay. So far, we have screened over 800 loss-of-function mouse mutants as part of the International Mouse Phenotyping Consortium. We have discovered many novel models of genome instability disorders including mouse models of CENPJ-Seckel syndrome1, SLX4-Fanconi anaemia2, SMC3-Cornelia de Lange3. Unbiased screening has identified novel regulators of vertebrate genome maintenance, including Mysm1, and we are working to identify mechanism. We have also assessed the susceptibility of genome instability mutants to tumour formation and by sequencing tumour genomes from these mutants, we aim to understand the mutational signatures at play. In a parallel effort we have screened over 1,800 outbred mice and have performed a genotype-wide association study to identify loci that contribute to genome maintenance. We have identified five hotspots in the mouse genome that have the greatest impact on genome maintenance. Several of the SNPs identified were orthologous to those found in the human population and so we are currently assessing whether they are functional using Cas-9/CRISPR technology. 1. McIntyre RE et al., (2012). Disruption of Mouse Cenpj, a Regulator of Centriole Biogenesis, Phenocopies Seckel Syndrome. PLoS Genet 8: e1003022. 2. Crossan GP, van der Weyden L, Rosado IV, Langevin F, Gaillard PH, McIntyre RE, Gallagher F, Kettunen MI, Lewis DY, Brindle K, Arends MJ, Adams DJ, Patel KJ (2011). Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia. Nature genetics 43: 147–152. 3. White JK et al., (2103). Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes. Cell 154 (2): 452–464. Symposium 7: Poster 040 Fanconi anemia human cells deficient in FANCD2 are hypersensitive to the cytotoxic effects of coffee and caffeic acid Estefanía Burgos-Morón1, José Manuel Calderón-Montaño1-3, Manuel Luis Orta Vázquez2-3, Santiago Mateos Cordero2, Miguel López-Lázaro1 1Department of Pharmacology, University of Seville, Spain;2Department of Cell Biology, University of Seville, Spain;3Science for Life Lab. Department of Medical Biochemistry and Biophysics, Karolinska Institute, Sweden Background: Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by genetic instability and hypersentivity to DNA damage agents. Previous findings indicate that coffee and caffeic acid may induce DNA damage. The aim of this work is to evaluate if Fanconi anemia cells deficient in FANCD2 are hypersensitive to the cytotoxic and DNA-damaging effects of coffee and caffeic acid. Methods: FANCD2-deficient human cells (PD20-/-) and FANCD2-complemented PD20-/- cells were used. Cytotoxicity activity of coffee and caffeic acid was evaluated by clonogenic survival assay. DNA damage was studied by an immunofluorescence assay in which γH2AX and 53BP1 foci was measured. The t-test (paired, two-tailed) was used for statistical analysis. Results and Discussion: The cytotoxicity activity of coffee and caffeic acid in PD20-/- cells was significantly higher than in PD20+/+ cells (p<0.01). Preliminary data indicate that the levels of DNA damage (γ-H2AX and 53BP1) induced by coffee and caffeic acid in PD20-/- cells were higher than in parental PD20+/+ cells. Conclusion: Fanconi anemia cells deficient in FANCD2 are hypersensitive to the cytotoxic effects of coffee and caffeic acid. Fanconi anemia patients may be more susceptible than healthy people to the possible toxic effects of coffee. Symposium 7: Poster 041 Modified Comet assay in human skin models for a mechanistic approach Markus Schulz, Anna Tobias, Marc Bartel, Robert Landsiedel BASF SE, Exp. Toxicology & Ecotoxicology, Ludwigshafen, Germany For most cosmetic products the human skin is first site of contact. Yet, all standard (OECD test guideline) genotoxicity tests in vitro are performed in submersed cell cultures (or bacteria). Skin models may, however, be a more relevant model to study the potential genotoxicity of cosmetic ingredients. 3D human skin models have already been used to study the formation of chromosomal aberrations (micronucleus assay, E.L.Dahl et al., Mut. Res. 720, 2011). More recently, the method of single cell gel electrophoresis (Comet assay) has been applied to skin models. In a project funded by the German government (BMBF) in co-operation with Cosmetics Europe a prevalidation trial is currently performed. Beyond the prevalidation of the standard method, we were interested in identifying specific DNA modifications like 8-oxo-guanine or alkylated bases caused by reactive oxygen species and small electrophiles, respectively. For this mechanistic approach we used bacterial endonuclease formamidopyrimidine DNA glycosylase (Fpg) which is part of the base excision repair complex (S.S.Wallace, Environ. Mol. Mutagen. 54, 2013). This enzyme recognizes the specific DNA base modifications and cuts the DNA strand in order to repair. Hence more strand breaks are detected by the Comet assay, and the relative tail intensity increases. The Fpg Comet assay was adapted to 3D human skin models using EMS and MMS. A significant increase of the relative tail intensity in 3D human skin models was already observed after incubation with the two direct acting mutagens using the standard method. However, using Fpg in the Comet assay resulted in significantly higher tail intensities. In conclusion, including Fpg in Comet assays in 3D human skin models may offer additional information on the mechanism of DNA damage (such as alkylation or oxidative damage). Sponsored by the Federal Ministry of Education and Research, Germany Symposium 7: Poster 042 PARP1 as mediator of the enzyme activities processing apurinic/apyrimidinic sites Olga Lavrik Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia The apurinic/apyrimidinic (AP) sites are considered to be mutagenic and cytotoxic DNA lesions. The specific capacity of poly(ADP-ribose) polymerase 1 (PARP1) to interact with the AP sites has been demonstrated by us. The identity of PARP1 cross-linked to AP sites in DNA in cell extracts was determined by mass spectrometry. PARP1 was weakly activated to conduct poly(ADP-ribose) synthesis upon binding to the AP site containing DNA. PARP1 interacting with AP sites catalyzes DNA strand incision by its AP lyase activity and shows 5’dRP lyase activity. The interaction of PARP1 with AP sites modulates activity of apurinic/apyrimidinic endonuclease 1 (APE1) in the case of DNA substrate containing single or clustered AP sites. The capacity of the other enzyme, tyrosyl-DNA phosphodiesterase (Tdp1), to the AP site cleavage was determined. Tdp1 catalyzes the cleavage of AP site and its synthetic analog, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5’-adjacent phosphate. The product of the Tdp1 cleavage in the case of AP site is unstable and is hydrolyzed with the formation of 3’- and 5’-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase β and DNA ligase. In the case of THF, Tdp1 generates break with the 5’-THF and the 3’-phosphate termini. PARP1 stimulates activity of Tdp1 in the AP site cleavage. The protein–protein interactions of PARP1 with APE1 and Tdp1 detected by various techniques were suggested as a key of the modulation of APE1 and Tdp1 activities by PARP1 in the AP site processing. Project funded by RFBR (13-04-93107, 13-04-40197-comfi and 14-04-00268), MCB Program of RAS, grant RF for scientific schools (420.2014.4) Symposium 7: Poster 043 Polyphenolic antioxidants that produce positive results in the GADD45a-GFP assay reduce levels of oxidised DNA tested using 8OxodG antibody Christopher Addinsell1,2, Richard Walmsley1,2 1University of Manchester, UK;2Gentronix Ltd, UK Polyphenolic antioxidants (PPAs) can produce positive results in in vitro mammalian genotoxicity assays, but test negative in vivo genotoxicity tests. Epidemiological evidence shows little or no risk of carcinogenicity from the PPAs consumed regularly in our diets. 11 of 12 PPAs produced positive results for genotoxicity in the GADD45a-GFP assay, and exposure to 10 of those 11 lead to an increase in intracellular reactive oxygen species (ROS). Cells were then analysed using flow cytometry to detect 8-Oxo-dG (FITC-conjugated antibody). Positive controls, tert-butyl hydroperoxide and hydrogen peroxide as well as 2 of the 11 compounds increased the level of antibody binding. 8 of the 11 compounds however decreased the level of antibody binding. The observed generation of ROS suggests that PPAs do indeed act as pro-oxidants. However the 8-oxo-dG study suggests that either the ROS generated do not cause oxidative damage to nuclear DNA or that GADD45a is up-regulated in response to another mechanism of genetic damage, or that cells with an induced oxidative damage response achieves a lower base level of 8-Oxo-dG residues. In this case GADD45a is induced by the initially high levels of 8-oxo-dG. The repair of 8-Oxo-dG however can be mutagenic: 8-Oxo-dG itself can mis-pair with Adenine as well as pairing with Cytosine. The human health significance of these findings is put into perspective, by considering difference between dietary exposure and test concentrations this study, as well as the likely significance of exposure in comparison to other antioxidants in the blood, such vitamins A, C and E and uric acid. Further study of the relationship between the positive genotoxicity results, intracellular ROS generation and oxidative DNA damage would help in the understanding of the plausibly misleading positive results generated for in vitro genotoxicity by PPAs. Symposium 7: Poster 044 Structural and dynamic basis for substrate specificity and non-substrate discrimination by 8-oxoguanine DNA glycosylases Dmitry Zharkov Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia 8-oxoguanine, a pre-mutagenic oxidative DNA lesion, is often encountered in a genome due to leaks of aerobic respiration intermediates and various environmental assaults. Normally, 8-oxoguanine is excised from DNA by dedicated DNA glycosylases, Fpg in bacteria and OGG1 in eukaryotes. While recognizing the common substrate, these proteins share no homology and belong to different structural families. In addition, a bacterial DNA glycosylase MutY removes adenine misincorporated by DNA polymerases opposite 8-oxoguanine, recognizing it through yet another fold. In order to clarify the source of specificity for the lesion vs non-damaged guanine, we performed molecular dynamic simulations of normal and damaged DNA bound by Fpg from Lactococcus lactis and Geobacillus stearothermophilus, human OGG1, and MutY from Geobacillus stearothermophilus, and supplemented the computation with steady-state and stopped-flow kinetic studies of the corresponding wild-type proteins or mutants in which we modified critical residues at the protein–DNA interface. In both Fpg and OGG1, the ability of the enzyme to evert the damaged nucleotide into a catalytically competent conformation, rather than the events within the active site, seem to be the deciding factor in their substrate specificity; mutant forms of these glycosylases that are unable to evert 8-oxoguanine retain their activity on abasic substrates that are intrinsically partially extrahelical. Destabilization of the everted pre-catalytic complex of Fpg or OGG1 by amino acid substitutions or introducing adenine opposite 8-oxoguanine may be tolerated due to DNA backbone adjustment. In MutY, we have identified a set of interactions both within the p13 domain responsible for 8-oxoguanine specificity and between p13 and other domains that distinguish 8-oxoguanine from guanine in the complex. Overall, early events in the path to the catalytically competent complex seem to play a predominant role in 8-oxoguanine recognition by DNA glycosylases. Wednesday, Symposium 8: Talk 1/5 RNA-mediated heredity of paramutation and acquired phenotype in the mouse Minoo Rassoulzadegan Inserm1091/CNRS7277, Nice, France Our laboratory reported three instances of RNA induced hereditary transmission of variations in the mouse (Nature 441:469, 2006; Dev Cell 14:962, 2008; Development 136:3647, 2009). Increased transcriptional activities of the Cdk9 and Sox9 genes result in heart hypertrophy and gigantism, respectively, and a decreased expression of Kit, in fur colour variation and diabetes. Designated ‘paramutations’ by analogy with the known hereditary epigenetic variants in plants, they are induced by small RNAs homologous in sequence to the transcript, either the cognate microRNAs or oligoribonucleotides, injected in fertilized eggs. Sperm RNA, also active in the microinjection assay, appears as the likely transgenerational signal responsible for paternal transmission. The genuine interest for the phenomenon of paramutation led often to ask questions about the molecular mechanisms of the change in gene expression. We could not so far cover all aspects of this complex issue in in vivo mouse models by classical genetic tools, we concentrate on the initial period of establishment in the very early embryo, leading to the entirely new concept of a control by methylation of the stability of a small noncoding RNA and thus of their signaling ability. Questions relative for instance to the state of the chromatin in the resulting state of expression in the paramutant, important as they are, were deliberately not considered. Finally, we are asking whether one could consider the same mode of transgenerational determination for complex disease with transgenerational epigenetic variations as well characterized in ‘diet-induced” mammals models. As in our previous studies, a main analytical tool was microinjection of RNA molecules into one-cell embryos, an efficient and well-tolerated procedure. We now report the transgenerational transfer of metabolic pathologies- ie obesity and type II diabetes- by injection in naive embryos of testis RNA prepared from obese and diabetic males raised on high fat diet and demonstrate that sperm RNA act as a vector of paternal inheritance. Wednesday, Symposium 8: Talk 2/5 Epigenetic Regulation of Genetic Integrity in Germ Cells and Stem Cells John McCarry University of Texas, San Antonio, USA We have used a mutation-reporter (lacI) transgene system to show that germ cells and pluripotent cells maintain genetic integrity more stringently than do somatic cells or differentiated cells. Thus, male and female germ cells in the mouse show a frequency of spontaneous point mutations that is 5–10 fold lower than that detected in somatic cells from the same animals. Pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) show a spontaneous mutation frequency that is similar to that seen in germ cells, whereas cells derived from these pluripotent cells by either directed or spontaneous differentiation display a mutation frequency similar to that detected in somatic cells. The enhanced genetic integrity observed in germ cells and pluripotent cells has been attributed to elevated levels of DNA repair and/or cell death activities in these cell types. To further investigate this, we mined existing transcriptome data bases comparing gene expression patterns in ESCs, iPSCs and differentiated somatic cells (fibroblasts) from humans and mice. We found a general up-regulation of expression of many DNA repair genes in plurpipotent cells relative to that in differentiated cells, and a particular up-regulation of genes involved in intrinsically regulated proapoptotic activity in these same cells. This suggests a mechanistic link between the gene networks responsible for establishing and/or maintaining pluripotency and maintaining enhanced genetic integrity, respectively. To test this hypothesis, we mined available cistrome databases describing genome-wide binding patterns of key pluripotency factors in ESCs and iPSCs from humans and mice. Our data provide evidence that enhanced maintenance of genetic integrity is fundamentally linked to the epigenetic state of pluripotency at the genomic level. In addition, our findings demonstrate how a small number of key regulatory factors can regulate large numbers of downstream genes involved in genetic integrity pathways. Wednesday, Symposium 8: Talk 3/5 Selfish mutations in spermatogenesis and paternal age effects Andrew Wilkie1, Geoffrey Maher1, Eleni Giannoulatou1, Gil McVean2, Anne Goriely1 1Weatherall Institute of Molecular Medicine, University of Oxford, UK;2Wellcome Trust Centre for Human Genetics, University of Oxford, UK Parental age is well known as a factor affecting mutation frequency. Whereas maternal age is the key contributor to rates of meiotic nondisjunction, paternal age was recently shown to be the major variable affecting the frequency of point mutations. This talk will focus on a specific mechanism that gives rise to strong paternal age effects, which we term selfish spermatogonial selection. The work is founded on the observation that a handful of human congenital disorders are caused by highly localised, heterozygous de novo mutations that are virtually always of paternal origin and occur at apparent rates up to 1000-fold above the background. These phenomena are currently well documented for five genes: FGFR2 (causing Apert syndrome and related craniosynostoses), FGFR3 (achondroplasia and other short-limbed bone dysplasias), RET (multiple endocrine neoplasia II), PTPN11 (Noonan syndrome) and HRAS (Costello syndrome). These observations have led to efforts to measure the levels of these mutations directly in human sperm and testes. The major conclusion from this work is that these mutations do not arise from true hotspots, but become enriched in spermatogonial stem cells or long-lived progenitors (ie mitotic cells) because of a selective proliferative or survival advantage. Significantly, the encoded mutant proteins all have gain-of-function properties, and the mutations correspond to documented hotspots in known oncogenes. The challenge now is to ask whether this phenomenon of ‘selfish’ mutations is limited to a handful of genes influencing a single pathway in spermatogenesis (involving signalling by selected receptor tyrosine kinase proteins through the RAS-MAPK pathway), or could be much more pervasive. I will review two complementary approaches that we are currently taking to addressing this issue. Wednesday, Symposium 8: Talk 4/5 Alternative in vitro approaches to the in vivo assessment of genetic, reproductive toxicity Martin Brinkworth, Khaled Habas, Chongye Guo, Yonghong Peng, Diana Anderson University of Bradford There is a pressing need for alternative approaches to the identification of reproductive genotoxins since the field is currently heavily dependent on in vivo studies. Many factors are driving this, including a commitment to the principles embodied by the 3R’s, the time-consuming nature and high cost of animal experiments, and new opportunities presented by modern molecular analytical methods. In addition, there is currently no suitable model of full mammalian spermatogenesis in vitro yet under the REACH initiative, any new tests introduced to assess (geno)toxicity should be in vitro. Accordingly, we have been developing a battery of tests used in conjunction with unit-gravity sedimentation (Staput) purification of male germ cells, validated by immunocytochemistry, to investigate reproductive genotoxicity in specific types of spermatogenic cells. Freshly isolated and purified, mouse spermatogonia, spermatocytes and spermatids were cultured overnight then exposed to H2O2 or Doxorubicin followed by assessment using the TUNEL and Comet assays. All were strikingly positive but spermatogonia the most sensitive, followed by spermatocytes, with spermatids the least affected. These experiments were repeated in rat cells with the same results. Rat spermatogenic cells were exposed to 6 different genotoxins known from in vivo studies to be selectively active in specific germ cell types and also tested in the TUNEL and Comet assays. Again, the isolated cells proved very sensitive, indicating that this is a robust system suitable for testing a wide range of genotoxic compounds. In order to explore this sensitivity further, we have investigated whether specific genes can be monitored as indicators of damage. Spermatids progressively lose all transcriptional activity throughout their development and actively transcribed genes are believed to be particularly susceptible to genetic damage. Therefore, we have examined the effect of genotoxin exposure of spermatocytes and spermatids on RNA expression levels of three different transcription factors using RT-qPCR. Levels were significantly reduced in all cases suggesting that they may be useful biomarkers of such damage. The genes were then cloned into a bacterial expression system, tested in the same way and comparable results obtained. In parallel, we have developed a computational approach to the analysis of microarray data using pathway analysis and applied it to data from testes with spermatogenic problems in men and appropriate controls. The analysis identified a particular protein whose over-expression was associated with spermatogenic defects and this was confirmed using immunohistochemistry and RT-qPCR. This approach can be used in conjunction with microarray data from treated isolated germ cells in order to identify key proteins within specific pathways that are affected by genotoxin exposure. This will lead to a deeper understanding of how genetic damage is induced, to the identification of new biomarkers of damage, and to potential routes to protection from such damage. Thus, by building increasingly sophisticated end-points on to a relatively simple culture system, it may be possible to develop valuable in vitro screens for reproductive genotoxicity. Wednesday, Symposium 8: Talk 5/5 Germline transmission of enhanced susceptibility to radiation-induced brain tumors Francesca Pacchierotti ENEA, Rome, Italy The germline transmission of enhanced tumor susceptibility has been debated for a long time. Experimental data in support of this hypothesis have not always been reproducible and the limited statistical power of human epidemiological studies hindered their weight. However, recent findings about transgenerational epigenetic inheritance in laboratory rodents offer a possible mechanistic understanding of the phenomenon that might be instrumental to resolve the debate. We exploited a tumor susceptible mouse model, heterozygous for a Ptch1 gene null mutation, prone to develop spontaneous and radiation-induced medulloblastoma, to test the hypothesis of transgenerational carcinogenesis induced by irradiation of differentiating spermatogonia. Ptch1 wild-type mice were irradiated with 1 Gy X-rays and mated 42 days later with unirradiated Ptch1+/- females. Their Ptch1+/- progeny was tested for the spontaneous incidence of medulloblastoma and for the incidence of tumors induced by irradiation (1 Gy) at postnatal day 2, a well-characterized window of cerebellum radiation sensitivity. We showed a borderline significant increase of tumors induced by neonatal irradiation in the progeny of irradiated fathers, whereas the spontaneous tumor incidence was unaffected by paternal irradiation. By comet test we excluded that cerebellar cells of irradiated fathers’ offspring suffered more radiation-induced DNA damage or were impaired in DNA repair. Further, by LOH analysis of all tumors, we ruled out epigenetic inactivation of the Ptch1 wild-type allele as a driver of the increased tumor incidence in the irradiated fathers’ progeny. Finally, we demonstrated a switch between proliferating/differentiating pathways in the preneoplastic lesions of irradiated fathers’ offspring. This suggested that the paternal irradiation legacy consisted in an epigenetically modulated shift of gene expression promoting proliferation of the external granular layer of neonatal cerebellum, which made it more sensitive to radiation-induced carcinogenicity. Studies are in progress to characterize the molecular messenger of epigenetic inheritance in sperm of irradiated fathers. Symposium 8: Poster 045 Detection of germ cell mutagens in the MutaMouse transgenic mutation assay Carol Beevers Covance Laboratories Ltd, UK OECD test guideline 488 describes optimum study designs for determination of mutagenicity in rodent tissues. For male germ cells sampling times should be carefully selected to ensure the cells are adequately exposed at all stages of germ cell development. The guideline recommends one of two approaches for germ cell mutation assessment: 1) to dose for 28 days plus 3 days expression period and sample both developing germ cells from the seminiferous tubules and mature sperm from the cauda epididymis/vas deferens or 2) to dose for 28 days and sample mature sperm only a minimum of 7 weeks (mice) or 10 weeks (rat) after last treatment. Option 1 has significant advantages in terms of logistics, duration and cost of the study. Even more importantly where somatic tissues are investigated alongside germ cells, Option 1 is more ethically justified as it would use up to half the number of animals as Option 2. However, it has been questioned whether all stages of germ cell development are adequately covered by the 28 plus 3 day study design to ensure accurate detection of germ cell mutagens. We treated male MutaMice with water or the potent mutagen ethylnitrosurea (ENU). Animals were treated orally by gavage for 28 consecutive days at 10mg/kg/day. On Day 31, animals were necropsied and developing germ cells from the seminiferous tubules and mature sperm from the cauda epididymis/vas deferens were isolated. Both cell types were examined for mutation in the neutral lacZ transgene using positive selection methods. Clear increases in mutant frequency were detected in developing germ cells, however, no significant increases were detected in the mature sperm, supporting the idea that a 28 plus 3 day study design may not be optimal for robust detection of germ cell mutagens. Symposium 8: Poster 046 Epigenetics and aneuploidy Kayleigh Bozon, Katie Wood, Ann Doherty AstraZeneca, UK Aneuploidy can arise from a range of different mechanisms. This work attempts to gain more understanding of the potential epigenetic origin of, or contribution to, aneuploidy. Immunodeficiency-centromeric instability-facial anomalies syndrome 1 (ICF1) is caused by mutations in the genes encoding DNA methyltransferase-3B. An increase in micronuclei containing chromosomes 1, 9 and 16 are seen ICF patients. Pericentromeric satellite regions have been shown to be hypomethylated in patients and are associated with the centromeric instability identified. This project aims to see if an increase in the number of centromere positive micronuclei of these chromosomes to see if pericentromeric region hypomethylation has an impact on aneuploidy by checking for an over-involvement of these chromosomes in micronuclei compared to chance alone. 5-azacytidine (clastogen) was included due to its effects on the methylation status of the DNA. Metallic compounds, such as cadmium chloride and nickel sulphate have been examined as potential hypomethylating agents. The in vitro micronuclei assay has been performed in TK6 cells using either a 3 hour dosing regimen or 24 hour dosing regimen. Analysis of micronuclei has been performed after both a 24 and a 40 hour recovery for each compound. Cadmium chloride induced a significant increase in micronuclei after a 3 hour dosing regimen with a 40 hour recovery period. FISH was performed on these slides and showed a significant increase in centromere positive micronuclei over the control (67%). A significant increase in micronuclei has been observed after a 40 hour recovery for 5-azacytidine with a 24 hour dosing regimen. Contrastingly, nickel sulphate induced a significant increase in micronuclei observed after 24 hour dosing with 24 hour recovery. No increased involvement of chromosomes 1, 9 and 16 has been seen in micronuclei from cadmium chloride exposure. Symposium 8: Poster 047 Ethanol-induced primary DNA damages in the cells of the placenta and rat embryos and their correction by Afobazole Ekaterina Shreder, Viktoria Zabrodina, Olga Shreder, Andrew Durnev, Sergey Seredenin Moscow, Russia The purpose of this study is the evaluation of primary DNA damage and the protective effect of the Afobazole on placental and embryonic rat cells of rats exposed to alcohol. Ethanol was administered per os (4.3ml/kg) from the 10th to 13th days from the beginning of pregnancy. Afobazole was administered per os in doses of 1 and 10mg/kg. After either 3 or 6 hrs post final ethanol administration the animals were sacrificed. Evaluation of DNA damage was conducted by an alkaline DNA comet assay. Percentage of DNA in the tails of DNA-comets was used as a measure of DNA damage. Animals were ranged as either low or high-sensitivity. For the group of animals showing high-sensitivity, following 3hr of ethanol treatment the DNA-damage levels in placental and embryonic tissue was significantly increased, 4.5 and 2.6 times. Afobazole significantly reduced ethanol-induced DNA damage for embryos. Values reaching control levels were observed for doses of 10mg/kg. After 6 hrs of ethanol administration a significant increase in placental and embryonic DNA damage was also observed. Afobazole significantly reduced the DNA-damaging effect of ethanol in embryos for doses of 10mg/kg. In a group of low-sensitivity animals following 3hr of ethanol administration the level of DNA damage in placenta and embryos increased by 3.7 and 3 times. In embryos, Afobazole significantly decreased the level of DNA-damage induced by ethanol. A significant increase in DNA damage levels was noted in placentas following 6 hrs of ethanol treatment, in comparison with 3hr of ethanol exposure. The genotoxic effect of ethanol was significantly reduced by 2.7 times in placentas of rats treated with Afobazole. In embryos, Afobazole decreased the DNA-damaging effect of ethanol. Symposium 8: Poster 048 Germ-cell DNA damage measured by the Comet assay in rat cells following in vitro genotoxin exposure Khaled Habas, Diana Anderson, Martin Brinkworth University of Bradford, UK Introduction: The single-cell gel electrophoresis (Comet) assay has been widely used to measure DNA damage in male germ cells in a variety of physiological and pathological conditions. We investigated the effects in vitro, of a variety of known genotoxins; on rat spermatogenic cell types separated using Staput unit-gravity velocity sedimentation, evaluating any DNA damage using the Comet assay. The alkylating agents N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU), 6-mercaptopurine and 5-bromodeoxyuridine and methyl methanesulphonate (MMS) and Ethyl methanesulphonate (EMS) are potent male rodent germ cell mutagens and have been widely used historically to characterise the susceptibility of germ cells to the induction of genetic damage. Method: Purified germ cells (spermatogonia, spermatocytes and spermatids) were assayed for DNA damage using alkaline single-cell electrophoresis (Comet) assay. Briefly, 2.5x105 cell suspensions were mixed with RPMI (total volume 1000 µl) and incubated for 1h at 37°C with different concentrations (0.05, 0.5, and 1mM) of one of six mutagens (see above). An aliquot of 100 µl of each cell suspension was mixed with 100 µl of 1% low melting point agarose. The slides were submerged in cell lysis buffer (2.5M NaCl, 100mM EDTA, 10mM Tris HCl pH 10.0 containing 1% Triton X-100 and 40mM dithiothreitol) for 1h at room temperature and protected from light. Following this initial lysis period, proteinase K was added to the lysis solution (final concentration 10µg/ml) and additional lysis was performed at 37°C for 2.5h. Following lysis, slides were placed in the electrophoresis buffer (0.3M NaOH, 1mM EDTA) for 30min and then subjected to electrophoresis at a setting of 20V (approximately 300 mA) for 30min at 4 °C. After electrophoresis slides were neutralised 3 times for 5min using Tris buffer. Cells were stained using EtBr (20 µl/ml) and slides were covered with cover slips and the DNA integrity of 50–100 cells per slide was analyzed using Comet Assay software. Results: All six compounds were significantly genotoxic in cultured germ cells. Treatment of the isolated germ cells with ENU and MNU produced a dose-related increase in DNA damage in spermatogonia; spermatocytes were most sensitive to 6-mercaptopurine and 5-bromodeoxyuridine. MMS and EMS were found to be most damaging in spermatids. Increases were found when measuring olive tail moment and tail length, but the greatest changes were in olive tail moment. Conclusion: The level of DNA damage is dependent on both the mutagen dose and the type of germ cell, and occurs at doses likely to be relevant to in vivo testicular and reproductive toxicity. Furthermore, these results indicate that Staput-isolated rat testicular germ cells are a suitable model to study genetic damage in different phases of spermatogenesis. Symposium 8: Poster 049 Lifestyle and occupational and environmental exposure and DNA damage in human sperm Bashar Altakroni1, Jill Stocks1, Daniel Brison2, Andrew Povey1 1Centre for Occupational and Environmental Health, University of Manchester, UK;2Department of Reproductive Medicine, Central Manchester University Hospitals NHS Foundation Trust, UK Background and aims: Sperm DNA contains DNA damage but the sources of this damage are poorly characterised. The aim of this study was to examine the associations between lifestyle and occupational exposures and two types of DNA damage namely DNA double strand breaks (DSBs) and N7-methyldeoxyguanosine (N7-MedG). Methods: 53 male partners of couples attending for infertility treatment provided semen samples and answered a questionnaire about lifestyle, occupational and dietary exposures. DSB levels were measured by the neutral Comet assay, the %DNA in the Comet tail quantified and the proportion of sperm with few DSBs (FDSBs) or high levels of DSBs (HDSBs) determined if their Comet tails contained <0.005% or >7.5% DNA, respectively. N7-MedG levels were quantified in sperm DNA by an immuno-slot blot assay. Oneway Anova was used to analyze the data. Results: The mean levels of FDSBs, HDSBs and N7-MedG were 11.4±2.0 %, 13.1±1.6 % and 0.59±0.35 fmoles/µg DNA respectively. There was a weak negative but significant association between %FDSBs and N7-MedG levels. Eating nuts in the three months prior to treatment was associated with increased HDSBs (15.4±1.6% vs 11.5±1.6%, P: 0.031) but exercise with a significant decrease (P: 0.047). N7-MedG levels were positively associated with self-reported occupational exposure to detergents (P: 0.003), printing inks or dyestuffs (P: 0.049) and driving (P: 0.040). DNA damage levels were not associated significantly with common lifestyle exposures (previous smoking habit, alcohol drinking, eating meat). Conclusion: The main determinants of DNA damage in human sperm remain to be determined but subtle effects would not have been detected in a study of this size. The strong positive correlation between N7-MedG levels and occupational exposure to detergents warrants further investigation to determine whether this is the result of some specific exposure or as yet unrecognised confounder. Symposium 8: Poster 050 Transplacental mutagenicity of benzo(a)pyrene in the mouse Francesco Marchetti, Jason O’Brien, Beverly Allan, Marianela Rosales, Marc Beal, Lynda Soper, John Gingerich, Carole Yauk Health Canada, Canada Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon, is a by-product of incomplete combustion of organic compounds and a widespread environmental pollutant. BaP is an established mutagen and carcinogen; however, its ability to induce mutations during prenatal development has not been studied as extensively. We investigated lacZ mutant frequencies (MF) in 10-week old MutaTMMouse transgenic rodents that were exposed in utero to 0, 10, 20 or 40mg/kg/d BaP from gestational day 7 through 16, which represents the major period of organogenesis. MF were measured in three tissues originating from a different primary germ cell layer: the liver (endoderm), bone marrow (mesoderm) and brain (ectoderm). In addition, sperm motility and MF were evaluated to assess potential reproductive effects. We found a statistically significant dose-dependent increase in lacZ MF in all three somatic tissues. The mutagenic response was comparable among the three tissues. However, the response was greatly diminished in male germ cells and lacZ MF was only moderately increased at the middle dose alone; no effect was observed at the lowest dose and MF could not be determined at the highest dose because of a severely reduced sperm count. Additional effects of transplacental BaP exposure on spermatogenesis included significant dose-related decreases in testes weight, sperm concentration and motility in F1 males. These results show that maternal exposure to BaP during gestation is mutagenic to the embryo and that differences in metabolism, DNA repair capacity and proliferation rates among embryonic tissues may influence the magnitude of the response. The results also show that male germ cell precursors may undergo elevated rates of apoptosis resulting in sterility at the high dose. Based on the significant increases in MF from prenatal exposure, the results suggest that in utero exposure to BaP has the potential to increase the risk of tumorigenesis in the adult offspring. Wednesday, Workshop 4: Talk 1/6 Consensus recommendations from the 6th IWGT: Introduction Hans-Joerg Martus1, David J Kirkland2 1Novartis Institutes for BioMedical Research, Basel, Switzerland;2Kirkland Consulting, Tadcaster, UK On October 31 – November 2, 2013, the 6th International Workshop on Genotoxicity Testing (IWGT) was held in Foz do Iguacu, Brazil. For anyone unaware of this workshop format, IWGT has held five previous workshops, as satellite meetings of major meetings in environmental mutagenesis, of which the consensus recommendations have been highly influential in shaping the revisions to OECD guidelines and the recommendations in the ICH S2 guidances. Originally focusing on individual test systems, IWGT has evolved into a forum to discuss strategic issues, and topics like e.g. integration of assays have been prominent. The format is such that panels of invited experts, acknowledged in their field and selected for their ability to advance the discussion, are invited to debate on the topics, and consensus recommendations are published subsequently. For the Brazil IWGT, the following groups were formed (with group chair, co-chair, rapporteur): Liver micronucleus test (Yoshifumi Uno, Takeshi Morita, Mirjam Luijten) Germ cell tests (George Douglas, Carole Yauk, Jan van Benthem) Pig-A assay (Bhaskar Gollapudi, Anthony Lynch, Bob Heflich) Comet assay (Günter Speit, Hajime Kojima, Dan Levy) Quantitative analysis of genotoxicity data (Jim MacGregor, Roland Froetschl, Paul White) Further, a panel discussion on Next generation testing strategies (Kerry Dearfield, Bhaskar Gollapudi, Mirjam Luijten) concluded the workshop. Thus, for those groups who were concerned with new assays, such as the pig-A, comet, or liver micronucleus test, recommendations were developed around assay sensitivity, reliability, and validation issues, whereas for the overarching topics such as the need for germ cell assays or quantitative considerations of genotoxicity data the discussions had a more strategic emphasis. The status of the discussions is such that manuscripts have been drafted for most of the topics and publication of the recommendations is foreseen for later this year. Wednesday, Workshop 4: Talk 2/6 IWGT Workgroup Report on the In Vivo Pig-a Mutant Phenotype Assay Bhaskar Gollapudi Exponent, Inc., Center for Toxicology and Mechanistic Biology, MI, USA The in vivo Pig-a assay measures phenotypic variants for antibody binding to cell surface glycosylphosphatidylinositol (GPI)-anchored proteins. A Workgroup formed by the International Workshop on Genotoxicity Testing evaluated the assay development in the context of its utility in safety assessment studies. The Workgroup reached consensus on the state of scientific knowledge regarding the Pig-a assay and various aspects pertaining to a protocol for the assay conduct. There is adequate knowledge confirming that the absence of antibody binding to cell surface is the result of a mutation in the X-linked Pig-a gene. Currently the assay is optimized only for measuring Pig-a mutant phenotype in peripheral blood erythrocytes (reticulocytes and red blood cells) of rats. The recommended study design in the rat consists of 5–6 males/dose, treatment duration of 28 days, sampling of blood within 3 days of the end of treatment (with optional earlier or later samples), and flow cytometric analysis of at least 1–5 million erythrocytes (or preferably larger sample, if feasible). The Workgroup concluded that there is no mandatory requirement for the inclusion of concurrently treated positive control animals. With adequate demonstration of exposure of the bone marrow (or plasma) to the test material and/or its metabolites, the assay can be considered as a follow-up to positive findings (with or without metabolic activation) in the Ames or in vitro mammalian cell gene mutation assays. The assay, however, is not recommended as a follow-up to in vitro clastogenicity or aneugenicity findings. Finally, the Workgroup made several recommendations to further enhance the assay’s utility for hazard identification and risk assessment studies. Wednesday, Workshop 4: Talk 3/6 6th IWGT Meeting: Report from the Comet Assay Working Group, ‘Critical Issues with the in vivo Comet Assay’ Brian Burlinson HLS, UK In this presentation is a report on the outcome of the comet assay workgroup who reviewed critical issues with the in vivo comet assay. The members of the workgroup were: Günter Speit (Chair), Hajime Kojima (Co-chair) and Dan Levy (Rapporteur). A number of speakers were invited to the workshop and these were: Brian Burlinson, Andrew Collins, Peter Kasper, Ulla Plappert, Yoshifumi Uno and Marie Vasquez along with a number of invited participants, namely: Carol Beevers, Marlies de Boeck, Patricia Escobar, Sachiko Kitamoto, Kamala Pant, Stefan Pfuhler and Jin Tanaka. In what was a very interesting meeting some of the perceived issues which could possibly impair the progression of an OECD guideline for the comet assay were discussed and in many cases resolved. A broad range of issues were covered, from the application of positive/ negative controls, reproducibility of the assay, through to regulatory acceptance. In this presentation I will review the issues and the subsequent resolutions and conclusions reached by the working part. Wednesday, Workshop 4: Talk 4/6 A report of the liver micronucleus test working group in the 6th IWGT Yoshifumi Uno1, Takeshi Morita2, Mirjam Luijten3, Carol Beevers4, Shuichi Hamada5, Satoru Itoh6, Wakako Ohyama7, Hironao Takasawa5. 1Mitsubishi Tanabe Pharma Co, Japan;2National Institute of Health Sciences;3National Institute for Public Health and the Environment (RIVM);4Covance Laboratories Ltd.;5LSI Medience;6Daiichi Sankyo Co, Ltd.;7Yakult Honsha Co, Ltd At the 6th IWGT, the liver micronucleus test working group discussed practical aspects of the in vivo rodent liver micronucleus test (LMNT). The group members focused on the three methodologies currently used, i.e., a partial hepatectomy (PH) method, a juvenile/young rat (JR) method, and a repeated-dose (RD) method in adult rodents. LMNT is expected to detect clastogens, especially those that form unstable reactive metabolites, and aneugens. Based on current data the three methods seem to have a high sensitivity and specificity, but more data, especially on non-genotoxicants and/or non-carcinogens including toxic substances, would be needed to fully evaluate the test performance. The three methods can be combined with micronucleus (MN) tests using bone marrow (BM) and/or peripheral blood (PB). The ability of the PH method to detect both clastogens and aneugens has already been established, but the methodology is technically challenging. The JR method is relatively straightforward, but animal metabolism might not be fully comparable to adult animals and data on aneugens are limited. These two methods also have the advantage that the testing period is short. The RD method is also straightforward and can be integrated into repeated-dose (e.g. 2 or 4 weeks) toxicity studies, but again data on aneugens are limited. The LMNT is assumed to be used as a second in vivo test when a relevant positive result (chromosome aberration) in in vitro mammalian cell genotoxicity tests is noted, especially under the condition of metabolic activation, and a negative result is observed in the in vivo BM/PB-MN test. The group members discussed LMNT protocols and reached consensus about many aspects of test procedures. However, data gaps were also pointed out through the discussion and further data would be needed to fully establish the LMNT protocol. Wednesday, Workshop 4: Talk 5/6 A toolbox to estimate point-of-departure metrics in genetic toxicology Paul White Health Canada, Canada Interpretations of genetic toxicity test results have traditionally involved dichotomous hazard identification (i.e., positive versus negative) rather than quantitative dose-response analyses and point-of-departure (PoD) determination. Recent work conducted under the auspices of the ILSI/HESI Genetic Toxicology Technical Committee (GTTC) has established a foundation for routine dose-response analyses and PoD determination. GTTC members collected and analyzed dose-response data for four potent alkylating agents across a wide range of in vitro and in vivo endpoints and evaluated various techniques for PoD determination. Routine PoD determination necessitated the development of a standardized analytical methodology. The initial work (Gollapudi et al., 2013), which examined dose-response data for MMS (methyl methane sulfonate) and EMS (ethyl methanesulfonate), employed published methods to determine PoDs such as the no-observed-genotoxic-effect-level (NOGEL), the breakpoint-dose (BPD; previously Td), and the benchmark dose (BMD). Subsequent analyses of data for ENU (1-ethyl-1-nitrosourea) and MNU (1-methyl-1-nitrosourea) (i.e., Johnson et al., 2014) expanded the range of analytical tools via inclusion of two R-based procedures for bilinear analysis (i.e., segmented) and non-linear smoothing (i.e., mgcv). An R-based tool called drsmooth, which is now available through CRAN (Comprehensive R Archive Network), and a companion guidance document, were created to facilitate routine analyses of dose-response data and derivation of numerous PoDs. The Guidance Document contains complete instructions regarding initial data screening and processing (e.g., requirements for transformations), installation of required R-based tools, and determination of PoDs via drsmooth (i.e., NOGEL, BPD, BMD and STD). The drsmooth package includes analytical tools to examine variable distributions and variance homogeneity, statistical tools for NOGEL determination, and analytical tools for dose-response ‘shape testing” and subsequent modelling to determine BPD, STD and BMD. BPD determinations employ two modelling techniques, the Lutz and Lutz ‘hockey-stick’ approach and the R-based segmented procedure. Similarly, BMD determinations employ two non-linear modelling tools, BMDS (USEPA) and PROAST (RIVM). Wednesday, Workshop 4: Talk 6/6 Approaches for Identifying Germ Cell Mutagens: Report of the 2013 IWGT Workshop on Germ Cell Assays George Douglas1, Carole L Yauk1, Jan Van Benthem2, Marilyn J Aardema3, Jack B Bishop4, Kerry L Dearfield5, David M DeMarini6, Yuri Dubrova7, Masamitsu Honma8, James R Lupski9, Francesco Marchetti1, Marvin Meistrich10, Francesca Pacchierotti11, Jane Stewart12, Michael D Waters13 1Health Canada, Canada;2RIVM, The Netherlands;3BioReliance, USA;4NIEHS, USA;5USDA, USA;6University of North Carolina, USA;7University of Leicester, UK;8NIHS, Japan;9Baylor College of Medicine, USA;10University of Texas M.D. Anderson Cancer Center, USA;11ENEA, Italy;12AstraZenica, UK;13Integrated Laboratory Systems Inc., USA This workshop reviewed the current science to recommend best evidence-based approaches on the use of germ cell genotoxicity tests. The workshop questions and key outcomes are: 1) Do genotoxicity and mutagenicity assays in somatic cells predict germ cell effects? Limited data suggest that somatic cell tests detect most germ cell mutagens, but there are strong concerns that dictate caution in drawing conclusions. 2) Should germ cell tests be done, and when? If there is evidence that a chemical, or its metabolite(s), will not reach target germ cells or gonadal tissue, it may not be necessary to conduct germ cell tests notwithstanding somatic outcomes. However, it was recommended that negative somatic cell mutagens with clear evidence for gonadal exposure and evidence of toxicity in germ cells should be considered for germ cell mutagenicity testing. For positive somatic mutagens that are known to reach the gonadal compartments and expose germ cells, the chemical could be assumed to be a germ cell mutagen without further testing. Nevertheless, germ cell mutagenicity testing would be needed for quantitative germ cell risk assessment. 3) What new assays should be implemented and how? There is an immediate need for research on the application of whole genome sequencing in heritable mutation analysis for genome wide assays of de novo mutational events in humans and animals, and to integrate germ cell assays with somatic cell genotoxicity tests. We must focus on environmental exposures that can cause de novo mutations, especially newly recognized types of genomic changes. Mutational events, which may occur by exposure of germ cells during embryologic development before they are protected within the germ cell milieu, should also be investigated. And finally, the outcomes of reproductive toxicology tests that are indicative of germ cell toxicity should be leveraged to inform of possible germ cell genotoxicity. Thursday, Symposium 9: Talk 1/6 Mitochondrial DNA mutations in health and disease Patrick Chinnery Newcastle University, UK Following the identification of the first pathogenic mutation in mitochondrial DNA in human disease in 1990, mitochondrial DNA diseases have emerged as a common cause of inherited neurological and multi-system disorders. Remarkably, pathogenic mutations are found in approximately 1:200 of general population providing a reservoir of mutations for maternally inherited disease affecting future generations. Unlike nuclear DNA, there are many thousands of copies of mitochondrial DNA in most nucleated mammalian cells. In any one given cell the proportion of mutated mitochondrial DNA can vary (heteroplasmy). Many asymptomatic carriers can harbour low levels of mutated mitochondrial DNA, and these only cause disease when the level reaches a critical threshold. The mechanisms responsible for the change in level of heteroplasmy within the human population are only just being defined. In parallel, somatic mutations of mitochondrial DNA have been found in many late-onset, chronic common human diseases, such as Alzheimer’s disease, Parkinson’s disease and type II diabetes. The role of these mutations in the pathogenesis has yet to be defined, but it is conceivable they contribute to the disease mechanism through bioenergetic compromise. Ultra-deep sequencing methods have recently shown that very low levels of mitochondrial DNA mutations are common in the human population, potentially contributing to these common late-onset human diseases. This may be exacerbated by different drugs which promote de novo mutation events or lead to the preferential amplification of mutated species. The interaction between somatic mutations of mitochondrial DNA and mutations in the underlying nuclear genome is a totally unexplored area which may also be relevant for age-related human disorders, and the ageing process itself. Thursday, Symposium 9: Talk 2/6 The accumulation of mutant mitochondrial DNA in disease and ageing – Beyond oxidative stress Ian Holt, Daniel Ives, Catherine Nezich, Jiuya He, Chloe Moss, Gokhan Akman, Mark Bowmaker MRC, UK Mitochondria produce energy for our cells and tissues via electron transfer, creating free radicals as a by-product. Hence, the mitochondrion is often perceived as a white-hot crucible of free radical production. This perspective gave rise to the free radical theory of ageing, which places at its centre the mitochondrion. An obvious target of free radicals is the DNA inside mitochondria. However, recent deep sequencing studies have failed to find any evidence of an age-related accumulation of oxidative damage to mitochondrial DNA. Nevertheless, there is ample evidence of a decline in mitochondrial function with age that remains to be explained. Clonal expansion of mitochondrial DNA mutants has been documented in a variety of post-mitotic cell types including muscle, colonic crypts and dopaminergic neurons, and disease-causing mutants are specific to individual patients. We use reductionist and unbiased approaches to elucidate the pathways and processes that link mitochondrial DNA to organelle function. These studies aim to elucidate how cells prevent the accumulation of deleterious mitochondrial DNA, which is an ever-present threat. One conceptually important advance was our discovery of a physical coupling between the mitochondrial protein synthesis machinery and mitochondrial DNA, as this provides a direct link between mitochondrial genes and their products. Consequently uncoupling of DNA and protein synthesis in the mitochondria could release the negative selective pressure on deleterious mutants. Hence, our characterization of the mitochondrial nucleoid and associated proteins has identified candidates for regulating mtDNA segregation, which we are actively pursuing. In other work we are using bioinformatics to distinguish human cells that select wild-type mitochondrial DNA from those that select mutant molecules. These studies have identified endoplasmic reticulum stress as a prominent feature of cells that select wild-type mtDNA. Currently we are testing compounds that induce ER-stress to determine if they have the capacity to alter the mutant mitochondrial DNA load of cells. Finally we are developing new protocols to quantify abnormalities of mitochondrial DNA in order to determine the impact of a range of parameters from gene ablation to drug treatments and ageing on this vital piece of DNA. Thursday, Symposium 9: Talk 3/6 The role of mitochondrial dysfunction in human syndromes defective in DNA repair Eugenia Dogliotti Istituto Superiore di Sanità, Rome, Italy A growing body of evidence indicate that cells derived from patients affected by DNA repair/DNA damage response defective diseases such as Xeroderma pigmentosum group A and C, Cockayne syndrome, Ataxia telangiectasia, and Fanconi’s anemia, present changes in the redox balance, altered metabolism and dysfunctional mitochondria. How these newly identified mechanisms participate to the development/progression of disease is still an open question. The response to oxidative DNA damage of Cockayne syndrome cells will be presented as a prototype of a disease with mutations in well-characterized proteins, CSA and CSB, involved in the transcription-coupled nucleotide excision repair and with an unanticipated role in the repair of mitochondrial DNA damage and in the electron transport chain. The observation that CS proteins are physical components of mitochondria puts Cockayne syndrome into context with many other human neurodegenerative diseases that are caused by mutations in ‘mitochondria-shaping’ proteins. This complexity needs to be addressed by mechanistic studies but also provides a number of new therapeutic targets that may be of promise for future clinical studies. Thursday, Symposium 9: Talk 4/6 Mitochondrial DNA damage in smoking related pulmonary disease William Rumsey GlaxoSmithKline, USA Exposure of somatic cells to DNA damaging agents may lead to genomic instability and degenerative disease. Restoration of base pair sequence avoids mutation of nuclear or mitochondrial (mt) DNA. Chronic Obstructive Pulmonary Disease (COPD) is a growing cause of global disability. Although most smokers will suffer from smoking-related pathology, the incidence and course of development of COPD is highly variable, indicative of a heritable component. Oxygen consumption by mt oxidative phosphorylation supplies energy for cellular processes. Gene expression analysis of key components of the electron transport chain in sputum samples collected from a subset of subjects from ECLIPSE (Gold stage II-IV COPD) showed decreased levels of these mRNA biomarkers with increasing severity. For example, lower expression levels of COX5B were found in patients with GOLD stage III-IV as compared to those with stage II. These decrements were associated with the loss of lung function over 3 years. Variations in ECLIPSE mtDNA were explored using semi-conductor sequencing. Preliminary data indicated that the lineage H-HV-V may be an important risk factor for COPD. Somatic mtDNA mutations were greater in the COPD subjects (vs age-matched controls). In separate studies, higher concentrations of mt 8-oxoguanine (8-oxoG) were found in COPD (vs control) alveolar macrophages. Exposure of human pulmonary cells to cigarette smoke or mt toxins increased the levels of reactive oxygen species and 8-oxoG while impacting cytochrome c translocation, and levels of Ogg1. Pulmonary instillation of paraquat to rodents resulted in mtDNA damage, cell death and loss of airway function. Notably, Ogg1 expression responded to the insult. MtDNA variations and dysfunction may contribute to the heritable component of COPD clinical disease. Our non-clinical data may improve understanding of its progression. Support: ECLIPSE (Clinicaltrials.gov <http://clinicaltrials.gov/> identifier NCT00292552; GSK Code SCO104960) was funded by GlaxoSmithKline. Thursday, Symposium 9: Talk 5/6 Towards a mitochondrial fragilome Chloe Moss, Ian Holt National Institute for Medical Research, UK I am developing new protocols to measure fragile sites of mammalian mitochondrial DNA in order to determine its susceptibility to gene ablation and drug treatments. DNA damage often occurs at fragile sites, such as the single-stranded DNA that typically forms at the replication fork. Initial analyses of mammalian mitochondrial DNA fragments, generated by single-stranded nuclease treatment, yielded a non-random distribution of fragile sites, but with some inconsistencies. Therefore, we are taking advantage of a gene knockout that produces a substantial increase in fragment ends at a defined site in the mitochondrial genome to refine the protocol. High throughput sequencing of fragment ends from the model cell line produced a prominent peak in the expected region, although the ends were more disparate than was predicted by other analytical methods. In this way I have shown that the current nuclease treatment is excessive and so it will be reduced to improve the signal to noise ratio, thereby enabling us to identify less dramatic changes in the mtDNA and produce ‘fragility’ maps of the mitochondrial genome in normal and disease states. Thursday, Symposium 10: Talk 1/5 Impact Of Moving From Qualitative To Quantitative Approach On Genotoxicity Risk Assessment Veronique Thybaud Sanofi, Vitry-sur-Seine, France For most toxicological endpoints, it is assumed that there is a dose below which no biologically significant effects are induced, and appropriate uncertainty and safety factors are applied to determine acceptable exposures. For genotoxic end-points, the prevailing paradigm is linear extrapolation of effects to low doses and the assumption that these substances could be mutagens and induce deleterious effects via non-threshold mechanisms. Non-linear dose-responses are generally accepted for non-DNA reactive agents, but criteria for determining acceptable exposures have not been clearly defined. Recent data suggest that nonlinear dose-responses also occur with some DNA-reactive agents (e.g., alkylating agents) and they exhibit doses (point of departures) below which exposure does not add appreciably to existing background rates of DNA damage. It has been acknowledged that certain biological process (e.g., DNA repair, apoptosis) may contribute to low-dose non-linearity. Moving from qualitative to quantitative approaches across test systems and species would require (1) the use of all available data for the selection of reliable models and experimental designs to obtain robust dose-response data, (2) a consensus on the appropriate mathematical models and statistical analysis for characterizing dose-response relationships and identifying the relevant points of departure parameters to be used for risk assessment, 3) an understanding of possible mechanisms/modes of action underlying non-linear dose-response relationships for moving away from default assumptions and determining the uncertainty factors, and 4) the development of reliable dose-metrics to facilitate comparison of data across test systems to derive acceptable safety factors, acceptable exposures and risk levels depending on human exposure scenarios. This quantitative approach to the relevance of positive results in genotoxicity assays would move genetic toxicology from a purely qualitative science to a risk-based paradigm, and allow for a better use of genotoxicity data as an apical endpoint in the prevention of genetic diseases (e.g., cancer) and risk management. Thursday, Symposium 10: Talk 2/5 Dose-Response Assessment in Genetic Toxicology: Thresholds or Points of Departure? Bhaskar Gollapudi Exponent, Inc., Center for Toxicology and Mechanistic Biology, MI, USA Genetic toxicology studies have historically been designed/used for the purpose of qualitative hazard identification and binary (positive or negative) decision making. In general, very little emphasis was placed in defining a full dose response across a range of doses/concentrations. The default paradigm has been that there are no thresholds for genotoxic effects, especially those induced by DNA reactive chemicals. The acceptance of the ‘one-hit” model of radiation mutagenesis by a large section of genetic toxicologists resulted in a lack of interest in quantitative assessment of genetic toxicology data. In recent years, however, the validity of this assumption has come under intense scrutiny with a number of investigators reporting detailed dose-response studies using both DNA-reactive and DNA non-reactive substances. In addition, expert groups under the auspices of International Life Sciences Institute -Health and Environmental Sciences Institute (ILSI-HESI) and the International Workshop on Genotoxicity Testing (IWGT) have been examining the issue of quantitative dose response assessment in genetic toxicology. From a pragmatic risk assessment standpoint, the issue is no longer to identify absolute threshold doses for genotoxic effects, but rather to evaluate various approaches for the identification point-of-departure (PoD) metrics that can be used to inform the beginning of extrapolation to lower, environmentally relevant doses. A number of direct acting alkylating agents examined to date (MMS, MNU, EMS, and ENU) showed non-linear dose-responses for both gene mutations and chromosomal aberrations. Among the PoD metrics evaluated, the lower confidence limit of a benchmark response rate of 10% (BMDL10) is determined to be an acceptable metric for application to genetic toxicology data. It is conceivable that the PoD estimate for genotoxicity can be used, similar to other toxicology endpoints, to identify margin-of-exposure to prioritize chemicals for risk management decisions as well as an integral component in setting permitted daily exposure standards. Thursday, Symposium 10: Talk 3/5 Roles of DNA repair in points of departure for genotoxicity George E Johnson1, Shareen H Doak1, Bernd Kaina2, Owen G Bodger1, Karl-Heinz Tomaszowski2, Lya G Soeteman-Hernández3, Paul D Lewis1, Ben J Rees1, Adam T Thomas2, Cathy A Thornton1, Jatin R Verma1, Paul A White4, Zoulikha M Zair1, Gareth J Jenkins1 1Swansea University, UK;2University of Mainz;3National Institute for Public Health and the Environment (RIVM);4Health Canada, Canada An expert group at the 2013 International Workshops on Genetic Toxicology (IWGT) in Brazil was made up by industrial, academic and regulatory scientists who re-addressed the default assumption of linearity for direct acting genotoxic agents. Our work in Swansea University helped to initiate this potential paradigm shift using the model alkylating agents, methyl- and ethyl-methane sulphonate (EMS and MMS), as well as methyl- and ethyl- nitrosourea (MNU and ENU). Consequently, there is now a wealth of data for these chemicals for both gene mutation and chromosome damage endpoints in vitro and in vivo. These high power data sets can be used to calculate point of departure (PoD) metrics using statistical modelling packages, hence showing non-linearity. These extensive statistical analyses have been carried out by the ILSI-HESI Genetic Toxicology Technical Committee (GTTC) Quantitative Subgroup, and clear PoD have been defined for all agents both in vitro and in vivo. However, to accept a range of low doses as biologically irrelevant by defining a PoD, a plausible mechanism of action must be shown experimentally. We are addressing this data gap, and have been investigating the roles of DNA repair in relation to their genotoxic PoD. Specific DNA repair enzymes have been shown to be up-regulated by low doses of alkylating agents, and knocking down specific DNA repair enzymes in vitro alters the shape of the dose response e.g. to EMS and MNU. In conclusion, DNA repair has been shown to be a mode of action for the PoD exhibited by mono-functional alkylating agents. Thursday, Symposium 10: Talk 4/5 Analyses of genotoxicity dose-responses: pros and cons of the different approaches and biological relevance Paul White Health Canada, Canada Interpretations of genetic toxicity test results have traditionally involved dichotomous hazard identification (i.e., positive versus negative) rather than quantitative dose-response analyses and point-of-departure (PoD) determination. Recent work conducted under the auspices of the ILSI/HESI Genetic Toxicology Technical Committee (GTTC) has established a foundation for routine dose-response analyses and PoD determination. GTTC members collected and analyzed dose-response data for four potent alkylating agents across a wide range of in vitro and in vivo endpoints and evaluated various techniques for PoD determination. The techniques employed permitted determination of several PoDs including the no-observed-genotoxic-effect-level (NOGEL), the breakpoint-dose (BPD; previously Td), the benchmark dose (BMD), and the non-linear slope-transition-dose (STD). The initial work (Gollapudi et al., 2013), which examined dose-response functions for MMS (methyl methane sulfonate) and EMS (ethyl methanesulfonate), indicated that PoD metrics determined using several approaches are within the same order of magnitude, with somewhat more variability for in vivo data. The follow-up work (Johnson et al., 2014), which examined dose-response data for ENU (1-ethyl-1-nitrosourea) and MNU (1-methyl-1-nitrosourea), confirmed that, although complementary, the different modelling approaches each have noteworthy advantages and disadvantages. For example, the NOGEL and BMD metrics can readily be calculated for most gene mutation and chromosomal damage endpoints, but BPDs and STDs cannot always be determined due to data limitations and methodological constraints. The two studies concluded that the BMD approach, which is more flexible and more somewhat more familiar, is preferred for routine analyses of genetic toxicity dose-response data. The latter study further noted that the lower confidence limit of BMD10 values were often lower than the other PoDs. The latter work described a framework for the use of genetic toxicity PoD metrics (i.e., the BMDL10) to calculate regulatory limits (e.g., TDI, PDE, RfD) that can be used for human health risk assessment and/or risk management. Thursday, Symposium 10: Talk 5/5 Threshold of Genotoxic Carcinogens: Consideration from No-Effect Doses for Cancer-Related Markers in Chemical Carcinogenesis Shoji Fukushima1, Min Gi2, Anna Kakehashi2, Hideki Wanibuchi2 1Japan Bioassay Research Center, Japan Industrial Safety & Health Association, Japan;2Department of Molecular Pathology, Osaka City University Graduate School of Medicine The presence or absence of a carcinogenic threshold will determine the reliability of risk assessment of chemical carcinogens, particularly genotoxic carcinogens when extrapolated from high dose rodent testing. Therefore, it is essential to verify scientifically whether the non-threshold concept is valid for genotoxic carcinogens. Herein, we present low-dose carcinogenicity data based on medium-term rat liver bioassays for genotoxic hepatocarcinogens: 1) heterocyclic amines, 2-amino-3, 8 dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2) N-nitrosocompounds, N-nitrosodiethylamine (DEN) and N-nitrosodimethylamine (DMN). Very low doses of MeIQx induced formation of DNA-MeIQx adducts; somewhat higher doses caused elevation of oxidative DNA damage, gene mutations and initiation activity; and the more higher doses induced formation of glutathione S-transferase placental form (GST-P) positive foci in the liver, a well-known preneoplastic lesion marker in rat hepatocarcinogenesis. Similarly, only the higher doses of IQ caused an increase in the number of GST-P positive foci in the liver, the lower doses had no effect. Furthermore, the finding that p21Cip1/WAF1 was significantly induced in the liver at doses well below those required for IQ-mediated carcinogenic effects, suggests that induction of p21Cip1/WAF1 is one of the mechanisms responsible for no-effect doses in IQ carcinogenicity. We also demonstrated that low doses of DEN and DMN did not induce either GST-P positive foci formation or gene mutations in the liver. Thus, based on the findings of existence of no-effect doses for markers that cells typically acquire as they move through the initiation and promotion stages of carcinogenesis, we argue strongly for the existence of a threshold, at least a practical threshold, for the carcinogenic effects of these genotoxic carcinogens in the rats. Symposium 10: Poster 051 Demonstration of non-threshold of 8-oxoG inducing genotoxicity by targeted mutagenesis Masamitsu Honma1, Manabu Yasui1, Yuki Kanemaru1, Nagisa Kamoshita1, Tetsuya Suzuki1, Toshiya Arakawa2 1National Institute of Health Sciences;2Health Sciences University of Hokkaido We developed a system for tracing DNA adducts in targeted mutagenesis (TATAM) and investigated the consequent mutations (Yasui et al., DNA Repair,15,11-20, 2014). The TATAM system site-specifically replaces endogenous DNA bases with bases carrying synthetic adducts using targeting vectors. It was enabled by introduction of site-specific DNA double strand breaks (DSB), which strongly enhanced targeting efficiency through homologous recombination (HR). Human TSCER122 cells are compound heterozygous for the thymidine kinase gene (TK−/−), and have a homing endonuclease I-SceI site in intron 4 of the TK gene. Using the TATAM system, we targeted the TK− allele with the I-SceI site by a synthetic TK+ allele containing an 8-oxo-7,8-dihydroguanine(8-oxoG) adduct, a typical product of oxidative DNA damage. The targeted clones (TK+/−) were then isolated by drug selection. Site specific HR for DSB induced by I-SceI improved targeted integration of the synthetic allele by five orders of magnitude (from 10−7 to 10−2). Subsequent analyses of approximately 800 target clones revealed that 8-oxoG was restored to G in 86% clones, probably reflecting base excision repair or translesion synthesis without mutation. Lesions of the remaining clones (14%) were associated with mutations. The mutation spectrum corresponded closely with that of oxidative DNA damage inducers reported, in which G:C to T:A transversion (5.9%) were predominant. Over-expression of MutY homologs in cells, which prevents G:C to T:A transversion by removing 8-oxoG:A mispairing, decreased the frequency of mutations to 2.6%, but not zero. These results indicate that only single 8-oxoG adduct in the human genome produces a gene mutation at high probability, and the DNA repair mechanism does not contribute to establish the threshold of 8-oxoG inducing genotoxicity. Thursday, Symposium 11: Talk 1/5 Translation into a clinical setting: the studies that would be needed Pierre Martin-Hirsch MAC Early diagnosis, effective treatment and monitoring of disease status are the cornerstones for effective management of cancer. In cervical screening, the morphological changes on a Papanikolaou cervical smear first reported in 1928 have led to a dramatic fall in the incidence and mortality from cervical cancer where organized screening programmes exist. Assessment of morphological changes of cervical cells on a smear is subjective and can lead to wide variation in interpretation. In countries where quality assurance programmes do not exist the sensitivity of cytology can be below 50% compared to the 85% achieved in regulated laboratories. New objective screening biomarkers have been developed to not only improve diagnostic accuracy but also reduce costs. Cervical cancer is the second most common cancer in low resource countries and low cost effective screening programmes need to be developed to reduce incidence and mortality from this disease. In my presentation, I will present the steps of validating new biomarkers developed through translational research. The presentation will focus on assessing the diagnostic accuracy of biomarkers and developing the evidence base for implementing in clinical practice. Thursday, Symposium 11: Talk 2/5 Biospectroscopy as a novel approach to diagnose underlying disease: from biofluids to cytology Francis L Martin Centre for Biophotonics, LEC, Lancaster University, UK Biospectroscopy sensor approaches offer the possibility of lending novel insights into complex biological questions in a fashion that could be complimentary to conventional techniques, e.g., genomics, proteomics. Such approaches often allow one to derive fingerprint spectra of biological material based on constituent chemical bond vibrations. Of significance is the fact that sample preparation is minimal and mostly reagent-free, generally requiring application to a substrate that can be glass. Depending on the question being addressed, fingerprint spectra can be derived in a spatial resolution of 1 µm × 1 µm to 250 µm × 250 µm (this can be expanded) to allow for sub-cellular imaging of chemical constituents to deriving an integrated measure, respectively. From application of a physical sciences sensor to a biological question, this inter-disciplinary approach is further extended by the necessity to design sophisticated computational frameworks to handle large and complex datasets that are generally generated. Individual fingerprint spectra consist of wavenumber-absorbance intensities giving rise to hundreds of points/spectrum; increasingly, a typical experiment may consist of 105 to >106 spectra. Typical experiments involve classification of normal versus disease variants (e.g., pre-cancer to cancer) or determination of effects induced by a particular exposure compared to control. Given the heterogeneity of biological systems, objective classification according to health status or induced effect is challenging; even more so is identifying underlying biomarker alterations that prove to be robust. Consequently multivariate tools are often applied. Readily-obtainable cytology can be from the cervix whereas biofluids may include blood plasma/serum. We demonstrate that biospectroscopy identifies disease that conventional methods (i.e., visual screening) fail to detect. Objective classification in excess of 95% was achieved for low-grade alterations in cervical cytology and plasma/serum for ovarian disease. Given the insidious nature of disease progression, biospectroscopy approaches offer the potential of a high throughput but inexpensive screening method. Thursday, Symposium 11: Talk 3/5 Systematic toponome decoding of disease mechanisms in human tissue Walter Schubert1-3 1Molecular pattern recognition research group, OvG-University Magdeburg; Germany;2International faculty, CAS-MPG partner institute for computational biology, Shanghai, China;3Human toponome project, TNL, Munich, Germany The recent development of parameter-unlimited functional super-resolution microscopy (Imaging Cycler Microscopy (ICM)) provides direct access to protein networks at nanometer 2D and 3D resolution in a single tissue section. ICM is a device that overcomes the spectral resolution limit of conventional light microscopy without having to change hardware. It is the first ready-to-use technology for dimension- and parameter-unlimited histological diagnostics and systematic decoding of the toponome at super-resolution (toponome: defined as the spatial protein network code in morphologically intact cells and tissues). Using ICM-data it will be shown that large arrays of supermolecules, detected here for the first time, together forming a network of several million multi protein clusters inside one single tissue section, can be directly imaged by ICM leading to direct subcellular and transcellular disease mechanisms, which were hitherto undetectable. ICM is a highly flexible machine that can adapt to the needs of the researcher: a 4-in-one microscope including (1) routine transmitted light functions, (2) conventional fluorescence functionalities, (3) parameter-unlimited visualization of protein networks composed of millions of multi protein clusters in real time, and (4) super-resolution of subcellular structures and multi protein clusters in tissue sections (subnanometer to 40nm resolution of neighboring large protein profiles). The human toponome project based on ICM has at its goal to unravel the complete toponome in all cell types and tissues in health and disease (www.huto.toposnomos.com). The technology is scalable as large cooperative parallel screening devices (ICM clusters) extracting the most relevant disease targets from protein network hierarchies in situ: a novel efficient way to find selective drugs, by escaping the present low content trap in drug target and diagnostic marker discovery (Ref: e.g. Nat Biotechnol 2006, Nat Protoc 2007, N. Biotechnol 2012; Toponomics. Encyclopedia of Systems Biology, 2014). Genome sequencing and functional disease mechanism decoding are now done in parallel. Thursday, Symposium 11: Talk 4/5 Nanomaterials in complex biological systems: insights from surface enhanced Raman scattering (SERS) Janina Kneipp1,2, Daniela Drescher1,2, Tina Büchner1,2, Ingrid Zeise1 1Humboldt-Universität zu Berlin, Germany;2BAM Federal Institute for Materials Research and Testing Based on their plasmonic properties, silver and gold nanoparticles can serve as new optical probes of chemistry and sub / microscopic structure inside living cells, in particular by supporting surface enhanced Raman scattering (SERS). In SERS, inelastic (Raman) scattering of light from molecules, characteristic of specific molecular vibrations, is enhanced by many orders of magnitudes, since the local optical fields of noble metal nanostructures lead to an enhancement of both the excitation and the scattered light. Unprecedented, with SERS one can observe the formation of a specific biomolecule corona and monitor its processing inside a live cell. We have applied this approach to several different problems related to the process of endocytosis, with two main aims: (i) In the context of nanotoxicity and nanomedicine, the uptake of nanomaterials, their localization, and processing must be related to the molecular composition of the nanoparticle corona and (ii) The efficiency of gold nanoaggregates to act as SERS nanoprobes has to be assessed in the cellular context. This has been achieved combining information about the SERS characteristics with spatially resolved nanoaggregate quantification and ultrastructural characterization. Specifically, the endosomal maturation has been evidenced by the different kinds of SERS experimental results. Most important, there is evidence of a biochemical composition, and molecular processing characteristic of endosomes and lysosomes based on the SERS spectral fingerprints. In the case of silver nanoparticles, we found that a specific, very stable biomolecule corona is formed from components of the cell culture medium. By combination of SERS with nanoscale cryo soft X-ray tomography we found that the composition at the silver nanoparticle surface can be associated with the ring-shaped organization of silver nanoparticles in the endosomes. Also in the case of silica nanoparticles, the interactions at the nano-bio interface can be characterized employing silica nanoparticles with plasmonic cores, termed BrightSilica. Financial support from ERC Grant No. 259432 (D.D., T.B., J.K.) and Einstein Stiftung Berlin (I.Z., J.K.) is gratefully acknowledged. Thursday, Symposium 11: Talk 5/5 Metabolomics study in a Population-Based Health Risk Assessment of Chemical-Related Male Infertility: Epidemiology Study of Arsenic Heqing Shen, Jie Zhang, Liangpo Liu Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China In environmental health research, different strategies are applied for risk assessment and disease factor identification. When exposure assessment and dose-response assessment are conducted, the exposure data may come from top-down environmental monitoring or bottom-up human bio-monitoring; the value of LOAEL or NOAEL derived from dose-response curves usually made on different tiers of in vitro test systems or in vivo animal studies may be employed to this end. In epidemiological studies, bio-monitoring data has been used to identify the role of chemical exposures in disease risk, in which the relationship between the exposure data and health outcome is established solely on statistically relevant associations. To refine such risk assessment and to improve the understanding of the toxicology behind a statistical correlation of exposure data and disease incidence, biomarkers from human metabolomic data may be useful. As an example, arsenic (As) was investigated in the present work. In risk analysis, As-related biomarkers mining were conducted by using an OPLS-DA model. Exposure-related correlations were conducted between As and candidate biomarkers, such as testosterone, hippurate, serine, guanine and acetyl-N-formyl-5-methoxykynurenamine, were established. This was achieved by using As concentrations and the biomarker occurrence odds ratios (ORs). In disease risk identification, the case incidence OR was designed to identify As risk, and the male infertility related biomarker mining was run by an OPLS-DA model too. Elevated As was associated with infertility. Levels of urinary biomarkers, such as acylcarnitines, aspartic acid and hydroxyestrone, correlated with both male infertility and As exposure. In summary, As-related biomarkers may be used directly in population-based risk assessment combined with biomonitoring data. Disease-related biomarkers may indicate the mechanism of toxicity underlying observed statistically relevant epidemiological associations. Symposium 11: Poster 052 Alterations in Infrared Spectral Signature of Heterobrachus bidorsalis Reflects Polyaromatic Hydrocarbon Concentrations in Ovia River, Nigeria Blessing E Obinaju, Francis L Martin Centre for Biophotonics, LEC, Lancaster University, UK Environmental and chemical stress interferes with physiological and biochemical functions in biological organisms and could lead to alterations in metabolic rate and/or biochemical constituents. Most polyaromatic hydrocarbon (PAHs) compounds e.g. benzo[a]pyrene are known chemical stressors. Highly sensitive techniques, e.g., infrared (IR) spectroscopy have been employed to observe biological responses to chemical stressors at low exposure levels [1]. Using IR spectroscopy this study evaluated the effect of PAHs in the liver, brain, heart, gonads and gills of the sentinel Heterobrachus bidorsalis sampled along the Ovia River in the Niger Delta of Nigeria at points representing polluted and relatively clean sites [2]. Water samples were analysed for PAHs using gas chromatography coupled to a mass spectrometer (GC-MS). Tissues were interrogated using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. The area under distinguishing peaks was determined as a measure of biomolecule concentration within the samples. Results show significant (P < 0.0001, P = 0.0001 and P ≥ 0.0001 < 0.005) increase and decrease in biomolecule concentrations for Amide I & II, Lipids, Nucleic acids and Glycogen. Spectra and alterations were reflective of the total PAH concentrations (ΣPAH) at the sampling sites. This study provides evidence suggesting IR spectroscopy as a suitable tool for real time monitoring of toxicity in real-world situations. References 1. Llabjani, V., Trevisan, J., Jones, K. C., Shore, R. F., & Martin, F. L. (2011). Derivation by Infrared Spectroscopy with Multivariate Analysis of Bimodal Contaminant-Induced Dose-Response Effects in MCF-7 Cells. Environmental Science & Technology, 45(14), 6129–6135. 2. Obinaju, B. E., Alaoma, A., & Martin, F. L. Novel sensor technologies towards environmental health monitoring in urban environments: A case study in the Niger Delta (Nigeria). Environmental Pollution. doi: http://dx.doi.org/10.1016/j.envpol.2014.02.004 Symposium 11: Poster 053 Attenuated total reflection Fourier-transform infrared spectroscopy coupled with multivariate analysis discriminates subtypes of human ovarian cancer Georgios Theophilou1, Helen F Stringfellow2, Pierre L Martin-Hirsch2, Francis L Martin1 1Centre for Biophotonics, LEC, Lancaster University, Lancaster, UK;2Lancashire Teaching Hospitals NHS Foundation Trust, UK Surgical management of ovarian masses largely depends on their histo-pathological diagnosis. Currently, screening for ovarian malignancy with tumour markers in conjunction with radiological investigations has a low specificity for discriminating benign from malignant tumours. Also, pre-operative biopsy of ovarian masses increases the risk of intra-peritoneal dissemination of malignancy. Intra-operative frozen section, although sufficiently accurate in differentiating tumours according to their histological type, increases operation times. This results in increased surgery-related risks to the patient and additional burden to resource allocation. We set out to determine whether attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, combined with multivariate analysis can be applied to discriminate between different ovarian tumour types according to the unique spectral signatures of their molecular composition. Formalin fixed, paraffin-embedded tissue blocks of different ovarian tumour types were de-waxed, mounted on Low-E slides and desiccated before being analysed using ATR-FTIR spectroscopy. Multivariate analysis in the form of principal component analysis (PCA) followed by linear discriminant analysis (LDA) of the obtained spectra revealed clear segregation between benign versus malignant tumours as well as segregation between different histological tumour subtypes. TR-FTIR spectroscopy coupled with multivariate analysis has the potential to provide a novel diagnostic approach in the accurate intra-operative diagnosis of ovarian tumours assisting surgical decision making to avoid under-treatment or over-treatment, with minimal impact to the patient. Symposium 11: Poster 054 Comparative analysis of DNA damage and repair in human T lymphocytes and peripheral blood mononuclear cells (PBMC) Julia Bausinger, Günter Speit Universität Ulm, Germany The comet assay with whole blood or isolated peripheral blood mononuclear cells (PBMC) is increasingly used in human biomonitoring to examine DNA damage after exposure to genotoxic agents in vivo. Cytogenetic assays are also frequently performed in human biomonitoring and phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are usually investigated. The cytogenetic endpoints (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, immunomagnetic cell sorting was used for the first time to comparatively investigate DNA damage and its repair in PBMC and T cell cultures. Cells were exposed to mutagens with different modes of genotoxic action (methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromide). DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. Additionally, the modified comet assay with formamido pyrimidine glycosylase (FPG) protein was performed with MMS and potassium bromide. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and repair of induced DNA lesions occurs to a comparable extent. The damage distribution patterns across cells in the comet assay with PBMC did not reveal subpopulations with different sensitivity towards the mutagens tested. We conclude that there are no fundamental differences between different types of white blood cells with regard to the induction and repair of DNA damage and the comet assay with PBMC is suited to predict DNA damage and its repair in T cells. Symposium 11: Poster 055 Development of EURL-ECVAM Genotoxicity and Carcinogenicity Database of positive Ames results: new resource for predictive toxicology and hazard assessment Federica Madia1, Raffaella Corvi1, David Kirkland2, Errol Zeiger3, Andrew Worth1, Maurice Whelan1 1EURL ECVAM, ST Unit, European Commission, DG JRC Italy;2Kirkland Consulting, UK;3Errol Zeiger Consulting 800 Indian Springs Road Chapel Hill, USA A consolidated and structured database of available genotoxicity and carcinogenicity data of Ames positives compounds is presented as a resource for evaluating the predictivity of the Ames test for in vivo genotoxicity and carcinogenicity when considered alone or in association with a mammalian cell assay (chromosome damage, gene mutations) and for a better characterization of those cases where the Ames test seems to lead to irrelevant results. Indeed, the Ames test conducted in bacteria is the most commonly used genotoxicity test within the current accepted in vitro battery as it is considered able to reveal DNA reactivity and DNA reactive compounds. It is used to assess almost all types of substances. Therefore, knowing whether in vitro positive results are accurate indicators of in vivo mutagenic potential or carcinogenicity is extremely important in determining whether follow-up in vivo tests are needed. Data were collected from disseminated databases: chemicals, pharmaceuticals and cosmetic industry; EU and international regulatory agencies; academia, etc. Only compounds with a known chemical identity (structure, purity, molecular weight, CAS number) were included, while substances such as complex hydrocarbons, gasoline fractions, paraffins, were not. Since many chemicals appeared to be in more than one database, it was necessary to define an ‘overall call’ for each endpoint in vitro and in vivo, following defined criteria as reliability and quality of data. Where information was missing, scientific literature was also consulted. By using a harmonized format to capture the information, the resulting database of approximately 770 compounds is a powerful resource for data analysis and can be used to guide a thorough evaluation of genotoxicity and carcinogenicity. The database development implements part of recently published EURL ECVAM strategy aimed at avoiding and reducing animal use in genotoxicity testing. The database will be made freely accessible via the EURL ECVAM website. Symposium 11: Poster 056 Impact of p53 functionality for in vitro mammalian cell toxicity testing Robert Smith, James Whitwell, Julie Clements Covance Laboratories Ltd, UK There are a high number of reported in vitro mammalian cell positives that do not correlate with in vivo genotoxicity and carcinogenicity testing1. p53 competent human cells may provide more predictive data for assessment of human hazard and risk and less misleading in vitro positives compared to traditionally used rodent cell lines lacking wild-type p53 function2. However, it remains unclear whether cell species origin or p53 status is most relevant. Compared to p53 functional cells, cells lacking wild-type p53 may underestimate cytotoxicity resulting in analysis of inappropriately high (irrelevant) concentrations for genotoxicity. Three closely related human cell lines were tested; TK6 express wild-type p53, NH32 are p53 null, WTK1 over-express mutant p53. Ethyl methanesulfonate (EMS), etoposide and paclitaxel (taxol) were tested and cytotoxicity determined using relative populating doubling. Apoptosis was also determined with caspase-3/7 activity. Expected patterns of cytotoxicity were observed with etopoiside and taxol; TK6 was cytotoxic at lower concentrations with elevated caspase-3/7 activity (typical wild-type p53 response) than both NH32 and WTK1. With etoposide, WTK1 (mutant) toxicity was, however, more similar to TK6 than NH32. In contrast, p53 null NH32 were cytotoxic at lower concentrations of EMS than both TK6 and WTK1. With EMS, NH32 also demonstrated elevated caspase-3/7 activity similar to TK6, but was similar to the low levels observed in WTK1 with etoposide and taxol. In summary, the results showed that p53 compromised cell lines do not underestimate cytotoxicity with all chemicals and cytotoxicity using cells with different p53 functionality is drug specific. Other factors associated with loss of p53 function or species origin may be more relevant to the reported high number of in vitro positives. 1. Kirkland, et al (2005) Mut Res, 584, 1–256 2. Fowler, et al (2012) Mut Res, 742, 11–25 Symposium 11: Poster 057 Investigations into the γ-H2AX Assay as a Potential Screening Tool for the Assessment of Genotoxicity of Potential New Medicines Jessica Bateson, Julia Kenny, Anthony Lynch Genetic Toxicology, GlaxoSmithKline, UK Histone proteins are highly conserved in eukaryotic cells due to their involvement in DNA packaging and specialised biological functions. Detection of serine 139-phosphorylated H2AX, a H2A histone variant (γ-H2AX), is emerging as a useful biomarker of DNA double-strand breaks (DSBs) and has the potential to be applied as an early genotoxicity screen to detect clastogenic chemicals. Due to the high-content nature of the technology, quantification of the γ-H2AX foci by flow cytometry (where one γ-H2AX focus is equivalent to one DSB), provides a measure of DNA damage, but also additional information on cell cycle kinetics and cytotoxicity, relative to concurrent controls. Investigations into assay performance have continued from previous work1 to provide data for over 40 compounds (selected from the ECVAM recommended list of genotoxic and non-genotoxic compounds, together with additional compounds with less characterised genotoxicity). Specificity and sensitivity figures were obtained by the comparison of positive and negative results with two well established genotoxicity pre-screens, the Ames test and the Mouse Lymphoma assay (MLA) which are routinely used to screen potential drug candidates at GSK. Utilisation of L5178Y cells for the γ-H2AX assay enabled direct comparison to the MLA data generated in the same cell line. γ-H2AX immunostaining showed good predictivity of the Ames (100% sensitivity; 75 % specificity; 87% concordance) and MLA (85% sensitivity; 91% specificity; 86% concordance). Recent additional compound data slightly improved performance values compared to values previously reported1. γ-H2AX has shown to be a reproducible, high-throughput pre-screen, reducing time and resource compared to current screening assays. Assay limitations identified were incompatibility with kinase-inhibitory compounds due to the γ-H2AX mechanism and limitation to the detection of chemicals with a predominant clastogenic mode of action. 1. Smart et al Mutat Res. 715:25–31, 2011 Symposium 11: Poster 058 Mapping the 3-D location of the stem cells in the human prostate Antonis Antoniou, Nigel J Fullwood, Francis L Martin Lancaster University, UK The prostate gland is a walnut-shaped gland in the human male, which surrounds the urethra and the neck of the urinary bladder. Prostate gland diseases and cancer constitute a huge problem in older men and is a major cause of mortality. Understanding the cell lineage in the prostate may lend insights into the causes of cancer. Prostate stem cells have conventionally been studied using methodologies such as immunochemistry employing antibody-direct epitope markers such as CD133. In this study, we employ a novel imaging approach incorporating Raman spectroscopy with scanning electron microscopy followed by 3-D image re-construction to better characterise the in situ location of stem cells. Our observations point to a ring of undifferentiated cells uniquely located at a particular region within glandular elements; these cells appear to be migrating away from this region towards a landscape of differentiated cells. This suggests a progenitor-cell population, which gives rise to a hierarchy of cells responsible for replenishing and maintaining the differentiated secretory cells of the gland. This is in contrast to the current model that suggests that stem cells are located into a subpopulation of the basal myoepithelial cells lining the basement membrane. Future investigations employing Raman spectroscopy will determine how the spectral fingerprint of these undifferentiated cells differ from differentiated secretory cells. Given the cancer stem cell hypothesis, these observations could lend novel insights into the patho-physiology of prostate cancer. Symposium 11: Poster 059 Validation of the flow cytometry micronucleus assay in TK6 cells with assessment of metabolic activation in 96-well format Annemette V Thougaard, Joan Christiansen, Tomas Mow, Jorrit J Hornberg H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark In order to support prioritization of compound series and avoid genotoxicity in our drug discovery programs, we employ a battery of three in vitro genotoxicity screening assays: A miniaturized version of Ames test, the GreenScreen assay, and the flow cytometry micronucleus test. Protocol optimizations and inter-laboratory reproducibility have been described for the flow cytometry micronucleus assay. Here, we report further validation of the assay including metabolic activation conditions in a 96-well plate format. The assay conditions were adapted to allow for addition of S9 in the 96-well plate format, and then a set of 48 compounds, consisting of known in vivo genotoxins/carcinogens, non-genotoxins, and marketed drugs, was tested. When screening during the early drug discovery phases it is important to have a low false positive rate to avoid discarding potentially promising compounds. For that reason, we included many compounds which have previously been reported to produce irrelevant positive results in in vitro genotoxicity assays. With the results for the 48 validation compounds, we established the relevant genotoxicity and cytotoxicity cut-off values that would on the one hand identify the in vivo genotoxins (sensitivity 88%) but on the other hand avoid false positives (specificity 87%). The assay was then used to test a set of 16 drugs to further probe assay performance in drug-like chemistry. In conclusion our validation study, the largest to our knowledge for this assay, shows that the assay is well suited for genotoxicity screening during the drug discovery phase. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
TI - Abstracts of the 43rd Annual Meeting of the European Environmental Mutagen Society Lancaster University UK 6–10 July 2014
JF - Mutagenesis
DO - 10.1093/mutage/geu053
DA - 2014-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/abstracts-of-the-43rd-annual-meeting-of-the-european-environmental-mzed05js44
SP - 497
EP - 559
VL - 29
IS - 6
DP - DeepDyve
ER -