TY - JOUR
AB - 1. Genetic and clinical heterogeneity in the DNA repair disorder xeroderma pigmentosum Alan Lehmann 1 , Hiva Fassihi 2 , Mieran Sethi 3 , Susie Morley 2 , Shehla Mohammed 4 , Paola Giunti 2 , Adesoji Albiona 2 and Robert Sarkany 2 1 University of Sussex, Famer, Brighton, UK; 2 National XP Service, Guy’s and St Thomas’s Foundation Trust, London, UK; 3 King’s College London, London, UK; 4 Guy’s Hospital, London, UK Xeroderma pigmentosum (XP) is a rare DNA repair disorder characterized by increased susceptibility to UV-induced skin pigmentation, skin cancers, ocular surface disease, and, in some patients, sunburn and neurological degeneration. Genetically, it is assigned to eight complementation groups (XP-A to -G and variant). XP-A to G are defective in nucleotide excision repair, whereas XP variants are defective in translesion synthesis past DNA damage. For the last six years, the UK national multidisciplinary XP service has provided follow-up for 89 XP patients, representing most of the XP patients in the United Kingdom. Causative mutations, DNA repair levels, and more than 60 clinical variables relating to dermatology, ophthalmology, and neurology have been measured, using scoring systems to categorize disease severity. This deep phenotyping has revealed unanticipated heterogeneity of clinical features, between and within complementation groups. Skin cancer is most common in XP-C, XP-E and XP-V patients, previously considered to be the milder groups based on cellular analyses. These patients have normal sunburn reactions and are therefore diagnosed later and are less likely to adhere to UV protection. XP-C patients are specifically hypersensitive to ocular damage, and XP-F and XP-G patients appear to be much less susceptible to skin cancer than other XP groups. Within XP groups, different mutations confer susceptibility or resistance to neurological damage. Our findings on this large cohort of XP patients under long-term follow-up reveal that XP is more heterogeneous than has previously been appreciated. Our data now enable provision of personalized prognostic information and management advice for each XP patient, as well as providing new insights into the functions of the XP proteins. 2. Signatures of mutational processes in human cancer Mike Stratton Wellcome Trust Sanger Institute, Hinxton, UK All cancers are caused by somatic mutations. However, the processes underlying the genesis of somatic mutations in human cancer are remarkably poorly understood. Recent large-scale cancer genome sequencing initiatives have provided us with new insights into these mutational processes through the mutational signatures they leave on the cancer genome and into the underlying mutational processes that have been operative. 3. Interrogating the architecture of cancer genomes Peter Campbell Wellcome Trust Sanger Institute, Cancer Genome Project, Hinxton, UK Cancer is driven by mutation. Using massively parallel sequencing technology, we can now sequence the entire genome of cancer samples, allowing the generation of comprehensive catalogues of somatic mutations of all classes. Bespoke algorithms have been developed to identify somatically acquired point mutations, copy number changes and genomic rearrangements, which require extensive validation by confirmatory testing. The findings from our first handful of genomes illustrate the potential for next-generation sequencing to provide unprecedented insights into mutational processes, cellular repair pathways and gene networks associated with cancer development. 4. Cellular responses to DNA double-strand breaks Stephen Jackson University of Cambridge, Gurdon Institute, Cambridge, UK Work in my laboratory aims to decipher the mechanisms by which cells respond to DNA damage, particularly DNA double-strand breaks (DSBs). Much of our work addresses the functions of proteins that mediate such processes, and how the activities of these proteins are controlled by post-translational modifications (PTMs). We are also investigating how defects in various DNA repair and various other cellular processes cause mutagenesis. Some of our recent work shows how PTMs control DSB repair, for example by promoting molecular transitions in DNA-damage response (DDR)-protein complex assembly and disassembly. We and our collaborators are using existing yeast gene knockout cell collections as well as mammalian cells engineered by CRISPR-Cas9 approaches, to assess how various DDR/DNA repair defects give rise to distinct mutational signatures. 5. Customized exome-sequencing screen of first and late-onset second cancers in Belgian aristolochic acid nephropathy patients Xavier Castells 1 , Maude Ardin 1 , Sandrine Rorive 2 , Nilufer Broeders 3 , Adriana Heguy 4 , Pierre-Paul Bringuier 5 , Thierry Quackels 6 , Thierry Roumeguere 6 , Joëlle Nortier 3 and Jiri Zavadil 1 1 International Agency for Research on Cancer (WHO), Molecular Mechanisms and Biomarkers, Lyon, France; 2 ULB-Erasme University Hospital, Department of Pathology, Brussels, Belgium; 3 ULB-Erasme University Hospital, Department of Nephrology, Brussels, Belgium; 4 New York University Langone Medical Center, Genome Technology Center, New York, USA; 5 Hôpital Edouard Herriot, Laboratoire de Biologie des Tumeurs, Lyon, France; 6 ULB-Erasme University Hospital, Department of Urology, Brussels, Belgium Exposure to IARC Group 1 carcinogen aristolochic acid (AA) leads to aristolochic acid nephropathy (AAN) and a sequential development of multiple urothelial carcinomas. We used exome sequencing analysis of same-patient urinary tract tumours of women from a unique, prototypical AAN population in Belgium, to characterize the mutual relationship of the first and second neoplasms, as well as the link of the late-onset tumours to the exposure to AA. We analysed first cancers (upper tract urothelial carcinomas, UTUC) and second delayed cancers (carcinomas of bladder or ureteral meatus) in AAN patients who developed cancer within eight years following the exposure to AA. All patients had received a kidney transplant before developing the second cancers and had a functional renal graft prior to prophylactic nephroureterectomy. Genomic DNAs were isolated from FFPE sections of the renal cortex and the upper and lower tract tumours using macrodissection and laser-capture microdissection. Low-coverage exome sequencing was performed using Illumina HiSeq2500. Customized somatic variant calling and non-negative matrix factorisation were used to extract mutational signatures. The mutational signature of AA (COSMIC signature 22) was identified in all the first UTUC as well as second, lower-tract neoplasms. Furthermore, the first and second cancers harboured considerable overlaps in exposure-specific somatic mutations, suggesting that the delayed onset of bladder carcinomas is due to distal seeding of cancer cells of the primary UTUC origin. Our results highlight the relevance of genomic and bioinformatic approaches for cancer aetiology research, prevention and treatment, and underline the importance of long-term bladder follow-up in high-risk populations with established or suspected environmental or iatrogenic exposure to AA. 6. Massive C. elegans whole-genome sequencing for profiling mutational signatures of carcinogens and DNA repair deficiency Bettina Meier* 1 , Moritz Gerstung* 2,3 , Susanna L. Cooke* 3 , Nadezda Volkova* 2 , Ye Hong 1 , Joerg Weiss 1 , Victor Gonzalez-Huici 1 , Simone Bertolini 1 , Aymeric P. Bailly 1 , Ludmil B. Alexandrov 3 , John Marshall 3 , Keiran Raine 3 , Mark Maddison 3 , Elizabeth Anderson 3 , Michael R. Stratton 3 , Peter J. Campbell* 3,4,5 and Anton Gartner* 1 1 University of Dundee, Dundee UK; 2 European Bioinformatics Institute (EMBL-EBI), Hinxton, UK; 3 Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, UK; 4 Department of Haematology, University of Cambridge, Cambridge, UK; 5 Department of Haematology, Addenbrooke’s Hospital, Cambridge, UK; * these authors contributed equally Mutation is associated with developmental and hereditary disorders, ageing and cancer. While some mutational processes operative in human disease are well understood, many remain enigmatic. We developed C. elegans whole genome sequencing to investigate genetic and environmental contributions to mutational signatures. In an initial study, we analysed 183 worm populations across 17 DNA repair-deficient backgrounds, propagated for 20 generations or exposed to carcinogens [1]. The baseline mutation rate in C. elegans was ~1/genome/generation, stable across wild-type and 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, a process akin to chromotripsis. Similar genome rearrangements have been reported in lymphoblastic leukemia. Aflatoxin B1 induced guanine substitutions in a GpC context consistent with mutations 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 that XPF 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. To date we have sequenced and are currently analysing the mutation profiles of over 2000 genomes, propagated over generations or following exposure to a variety of DNA damaging agents, including ionizing radiation, methylating and ethylating agents and UV. Our results provide a mechanistic understanding how various DNA lesions are repaired in-vivo and how DNA repair mechanism both prevent, but occasionally also contribute to mutagenesis. Reference 1. Meier, B., Cooke, S.L., Weiss, J., Bailly, A.P., Alexandrov, L.B., Marshall, J., Raine, K., Maddison, M., Anderson, E., Stratton, M.R., Gartner, A. and Campbell, P.J. (2014), C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency, Genome Res., 24, 1624–1636. 7. Advances in the understanding of mutational signatures in human cells Serena Nik-Zainal Wellcome Trust Sanger Institute, Hinxton, UK Mutational signatures are the imprints of the biological processes that have gone awry in human cells. We previously outlined the methods for identifying and quantifying base substitution mutational signatures present in primary human cancers ( http://cancer.sanger.ac.uk/cosmic/signatures ). Here, using a highly-curated cohort of 560 whole genome sequenced breast cancers, we extend the understanding of mutational signatures to include six novel rearrangement signatures. We further demonstrate the variation in genomic distribution of mutational signatures of breast tissue, relative to replication time and strands, transcriptional strands and chromatin organisation. These mutational signatures distinguish clinical cohorts in breast cancer and have intriguing genomic properties with potential for clinical application as a biomarker. 8. Use of the Muta™Mouse transgenic rodent assay to investigate the mutagenic and carcinogenic hazards of polycyclic aromatic hydrocarbon mixtures Paul White, Alexandra Long and Christine Lemieux Health Canada, Mechanistic Studies Division, Ottawa, ON, Canada Soils and other complex environmental matrices are frequently contaminated with polycyclic aromatic hydrocarbons (PAHs). Effective Human Health Risk Assessment (HHRA) of PAH-contaminated materials necessitates hazard assessment; however, hazard assessment of PAH mixtures is challenging. The standard, chemically-specific paradigm focuses on a small number of prioritized PAHs, and calculates total hazard as the incremental sum of the contributions from each targeted PAH. However, there is a paucity of evidence to support the validity of this additive paradigm. This work employed the Muta™Mouse system to assess the mutagenic activity of targeted PAHs, complex mixtures of PAHs, and simplified mixtures of PAHs. Initial in-vitro results indicate that the mutagenic activity of soil-derived PAH mixtures are largely within 2-fold of predictions based on the sum of targeted PAH contributions. Follow-up analyses, which assessed adducts, mutations and micronuclei in Muta™Mouse specimens orally exposed to PAHs, complex PAH mixtures, and synthetic PAH mixtures, indicate that the effect on site of contact and related tissues are generally greater than additive predictions (i.e., up to 4.9-fold in small intestine and 12.3-fold in liver), with the reverse pattern observed for bone marrow (i.e., 0.2 to 0.8-fold). The results were also employed to determine bioassay-derived concentrations of benzo[a]pyrene (BaP) equivalents for estimation of excess lifetime cancer risk (ELCR). The in-vitro results indicate that a Potency Equivalence Factor (PEF)-based additive approach yields ELCR values that are generally greater than those based on bioassay-derived BaP equivalent values. Differences are generally less than 5-fold, and the results suggest that the additive paradigm may yield conservative risk estimates. The in-vivo results indicate that for site of contact tissues, PEF-based concentrations of BaP equivalents are generally greater than bioassay-derived values, with the reverse being true for remote tissues such as bone marrow. However, with a single exception, differences were within 5-fold. 9. Evaluation of a transgenic mouse derived in-vitro model to assess mutagenic hazard Mirjam Luijten 1 , Edwin Zwart 1 , Martijn Dollé 1 , Maaike de Pooter 1 , Julie Cox 2 , Paul White 2 and Jan van Benthem 1 1 National Institute for Public Health and the Environment (RIVM), Centre for Health Protection, Bilthoven, The Netherlands; 2 Health Canada, Environmental Health Sciences and Research Bureau, Ottawa, ON, Canada Assessment of genotoxic potential is an important step in the safety evaluation of chemical substances. Under most regulatory jurisdictions, the first tier of testing comprises a standard battery of in-vitro genotoxicity tests in bacterial and mammalian cells. However, the mammalian cell tests commonly used exhibit a relatively high rate of misleading positive results, which may lead to unnecessary in-vivo testing. We previously established a proof-of-concept for the LacZ reporter assay in proliferating primary hepatocytes as a promising alternative genotoxicity test. Here, cryopreserved instead of freshly isolated hepatocytes were used and the assay was evaluated in more detail. We examined the effect of cryopreservation on phenotype and metabolic capacity of the LacZ hepatocytes, and assessed the predictive performance of the assay by testing a set of substances comprising true positive, true negative and misleading positive substances. Additionally, a historical negative control database was created and the type of mutations induced was analysed for two of the substances tested, i.e. benzo[a]pyrene and bleomycin. Our findings indicate that proliferating cryopreserved primary hepatocytes derived from LacZ plasmid mice retain their hepatocyte-specific characteristics and metabolic competence. Furthermore, we demonstrate that both gene mutations and chromosomal aberrations can be detected with the LacZ reporter assay. The assay also seems to have a lower rate of misleading positive test results compared to the assays currently used. Together, our findings strongly support the use of the LacZ reporter assay in cryopreserved primary hepatocytes as follow-up to the standard in-vitro test battery for genotoxicity testing. 10. The impact of p53 on DNA damage and metabolic activation of environmental carcinogens: effects in Trp53(+/+ ), Trp53(+/ -) and Trp53(-/ -) mice Volker M. Arlt King’s College London, Analytical and Environmental Sciences Division, London, UK Many environmental carcinogens require metabolic activation in order to exert their genotoxic properties. We used Trp53(+/+ ), Trp53(+/ -) and Trp53(-/ -) mice to investigate the metabolism of benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and found that the genotoxicity (i.e. DNA adduct formation) of BaP and PhIP clustered according to Trp53 genotype in several tissues whereas 3-NBA genotoxicity was not dependent on p53 status. For example, BaP-DNA adduct levels were significantly higher in the livers of Trp53(-/ -) mice than Trp53(+/+ ) mice and higher adduct levels in the livers of Trp53(-/ -) mice correlated with increased Cyp1a enzyme activity in these animals. Complementarily, higher amounts of BaP metabolites were formed ex vivo in hepatic microsomes from BaP-pretreated Trp53(-/ -) mice. Treating the mice with BaP-7,8-dihydrodiol-9,10-epoxide (BPDE) resulted in similar adduct levels in the livers in all mouse lines confirming that the influence of p53 is on the Cyp1a-mediated biotransformation of BaP. In contrast, PhIP-DNA adduct levels were significantly lower in livers of Trp53(-/ -) mice than Trp53(+/+ ) mice and lower PhIP-DNA adduct levels in the livers of Trp53(-/ -) mice correlated with a decrease in Cyp1a enzyme activity in these animals. We found that in addition to Cyp1a enzymes, sulfotransferases (Sults) can contribute to PhIP-DNA adduct formation. Interestingly, PhIP-DNA adduct levels were significantly higher in kidney and bladder of Trp53(-/ -) mice than Trp53(+/+ ) mice which was accompanied by increased sulfotransferase (Sult) 1a1 enzyme activity in the kidney of these animals. Our results show that the cellular p53 genotype can impact on carcinogen metabolism in-vivo and indicate that p53 status might be a critical determinant contributing to organotropism. Further, our results indicate that the influence of p53 on carcinogen biotransformation depends on the agent studied and that besides phase I enzymes (i.e. Cyp) also phase II enzymes (i.e. Sult) can be regulated by p53. 11. Recent developments in the analysis of genetic toxicity dose-response data: what can we learn from them when developing a risk assessment paradigm for genetic effects? Wout Slob National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands Currently, genotoxicity tests have been used in a strictly qualitative way by providing the information whether a given compound should be considered as genotoxic or not. However, it has recently been proposed that genotoxicity dose-response data may just as well be used as a basis for quantitative risk assessment. When genotoxic effects are considered as adverse health outcomes, BMDLs can also be calculated for genetic endpoints like micronucleus counts or mutation rates, which may then be used as PODs for risk assessment. The key question to be answered is: What value of the Benchmark response (BMR) is appropriate for the various genetic endpoints? This question can be addressed by the following two approaches. One is the use of compounds that are “data-rich”, by providing good dose-response information on various genetic endpoints. This information may be used to validate a new theory on how BMRs should be scaled based on information regarding the “maximum” response of that endpoint. If dose-responses of different genetic endpoints result in similar BMD estimates when the BMR is scaled according to that theory this would be a clear indication of how to define BMRs in a relative sense, such that they are comparable among different genetic endpoints. The other approach for substantiating the value of the BMR is based on correlating genetic BMDs to cancer BMDs. These correlations have been shown to exist, and, when further completed, can be used to relate a BMR for a specific genetic endpoint to a BMR for cancer endpoints. In this way, the genetic BMR can be anchored to a given cancer risk. Both approaches were applied to a number of analyses of existing data, showing that they may substantiate the question of how to deal with BMR values in genetic toxicology. 12. Using standard in-vivo genotoxicity studies and benchmark dose modelling to determine acceptable daily intakes for mutagenic impurities George Johnson 1 , Angela White 2 , John Wills 3 , Paul White 3 , Wout Slob 4 , Julia Kenny 2 and Jim Harvey 2 1 Swansea University, Institute of Life Science, Swansea, UK; 2 GlaxoSmithKline, Ware, UK; 3 Health Canada, Mechanistic Studies Division, Ottawa, ON, Canada; 4 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands The ICH M7 guideline describes a safety and quality risk management framework that permits the control of mutagenic impurities in pharmaceuticals to levels that would be expected to pose negligible carcinogenic risk to patients. In certain cases the control of a mutagenic impurity to acceptable levels (i.e. concentrations below the associated ‘Threshold of Toxicological Concern’) can be problematic due to the possible presence of drug degradants. In such cases ICH M7 states that the results from an appropriate in-vivo genotoxicity assay may be used to support the determination of “compound specific impurity limits”. However, the procedure for derivation of compound- specific impurity limits is not defined in ICH M7. The No-Observed-Genotoxic-Effect-Level (NOGEL) has been proposed as a point of departure (PoD) metric for determining limits; however, this metric suffers from the same weaknesses as the NOAEL used in non-cancer risk assessment. International organisations (e.g., USEPA, EFSA, JECFA) are promoting the benchmark dose (BMD) methodology as a superior approach for quantifying the PoD, and this approach was applied in the present study. In-vivo micronucleus dose response data for 32 DNA reactive genotoxicants from 3 common classes of impurities (i.e., aromatic nitro compounds, aromatic amines and alkylating agents) were compiled from publicly available databases (e.g. NTP, ECHA). BMD-covariate analysis was used to simultaneously calculate BMD metrics for the genotoxicants examined. The approach provided improved BMD precision, and where there was no clear increase in MN frequency across the tested doses, the lower bound of the BMD confidence interval (BMDL) was determined. Alongside the in-vivo MN BMDL metrics, which could be used to directly determine the PDE (permissible daily exposure), the data also provided an opportunity to validate published correlations between micronucleus induction BMDs and carcinogenicity BMDs. 13. BMD-covariate analyses for multiple genotoxicity endpoints permit robust, cross-tissue comparisons of Muta™Mouse responses to orally-delivered benzo[a]pyrene Alexandra S. Long 1,2 , John Wills 1 , Volker M. Arlt 3 , Stephen D. Dertinger 4 and Paul A. White 1,2 1 Health Canada, Mechanistic Studies Division, Ottawa, ON, Canada; 2 University of Ottawa, Department of Biology, Ottawa, ON, Canada; 3 King’s College London, Analytical and Environmental Sciences Division, London, UK; 4 Litron Laboratories, Rochester, NY, USA Genetic damage is recognized as a key event in tumorigenesis and chemically-induced genotoxic effects are a human health concern. Historically genetic toxicity test results have been qualitative, using a “screen and bin” approach to identify genotoxic substances that are potential carcinogens. However, there is increasing interest in quantitative analysis of genetic toxicity data, and more specifically of applying the benchmark dose (BMD)-covariate approach to empirically rank genotoxic potency across covariates such as compound, sex, and tissue. Using benzo[a]pyrene (BaP) as a model genotoxic carcinogen, we examined the dose-response relationships for four genetic damage endpoints over an extended dose range, and employed BMD-covariate analysis to conduct potency ranking. Muta™Mouse specimens were exposed sub-chronically to 10 doses of BaP via oral gavage (i.e., 0.1–50mg/kg BW/day and vehicle control). The frequency of stable DNA adducts and LacZ mutants were measured in bone marrow (BM), glandular stomach (GS), small intestine (SI), liver (Lv), lung (Lg), spleen (Sp), and kidney (Kd), and the frequency of Pig-a mutations and micronuclei were assessed in reticulocytes and red blood cells (RBCs). BMD100 estimation was carried out independently across all tissues and endpoints using PROAST v.50.9. The cross-tissue BMD pattern for LacZ mutations (i.e., SI < SP / BM < GS / Lg < Lv / Kd) is consistent with the route of exposure and the published trend in tumour BMD. When the tissue-specific BMDs for DNA adducts are plotted against those for LacZ mutations, a significant linear trend is observed (r2=0.79, F=19.0, p=0.007). Pig-a and micronucleus upper and lower BMD bounds overlapped for reticulocyte- and RBC-based data, suggesting comparable utility for study designs that involve protracted exposures. The BMD approach permitted robust comparisons of responses across tissues and endpoints; the latter comparisons generate hypotheses regarding tissue-specific handling of DNA damage and mutation formation. 14. The influence of dose-spacing and choice of critical effect size (CES) for benchmark-dose modelling Andreas Zeller, Gonzalo Duran-Pacheco and Melanie Guérard F. Hoffmann-La Roche Ltd., pRED Innovation Center Basel, Pharmaceutical Sciences, Basel, Switzerland Analysing dose-response relationships with benchmark dose (BMD) models is becoming more popular with easy-to-use statistical tools like “PROAST”. However, published guidance in the field of genotoxicity is sparse and often remains vague. Critical effect sizes (CES, comparable to benchmark-response BMR) currently recommended by IWGT are 10% and 1SD [1]. We suggest that this approach may be overly simplistic and unjustifiably conservative (10%) or dependent on the quality of study conduct (1SD). Typical datasets obtained in genetic toxicology have a low, but often highly variable background and compound induced effects may be rather substantial. That is in contrast to other toxicological endpoints (e.g. organ weights, tumor incidences) from which the BMD concept originates. We therefore propose to use endpoint-specific CES values taking into account their unique characteristics. In our experience, the in-vivo micronucleus (MN) assay has a relatively low background noise while its maximum response rarely exceeds 10% micronucleated cells. Pig-a mutant phenotype frequencies on the other hand have a greater background variability but can increase up to more than several hundredfold over controls. Comet assay tail intensity background depends on the tissue type but the response can never exceed 100% (see also [2]). Here, we applied PROAST methodology to several datasets obtained with reference carcinogens like methyl methanesulfonate, temozolomide and dibenzo[a,l]pyrene. The resulting BMDs and BMDLs are compared to other PoD metrics. Moreover, we compared the impact of experimental dose spacing on no-genotoxic-effect-level (NOGEL) and BMD. While the NOGEL obviously is a function of study design, BMD modelling takes into account the whole dose range and is therefore suggested to be more robust in this context. We found that exclusion of dose levels can considerably impact the PoDs obtained with both methods. References 1. MacGregor, J.T., Frötschl, R., White, P.A., Crump, K.S., Eastmond, D.A., Fukushima, S., Guérard, M., Hayashi, M., Soeteman-Hernández, L.G., Kasamatsu, T., Levy, D.D., Morita, T., Müller, L., Schoeny, R., Schuler, M.J., Thybaud, V. and Johnson, G.E. (2015) IWGT report on quantitative approaches to genotoxicity risk assessment I. Methods and metrics for defining exposure-response relationships and points of departure (PoDs). Mutation Res.,783 , 55–65. 2. Zeller, A., Tang, L., Dertinger, S.D., Funk, J., Duran-Pacheco, G. and Guérard, M. (2015) A proposal for a novel rationale for critical effect size in dose–response analysis based on a multi-endpoint in-vivo study with methyl methanesulfonate, Mutagenesis,31 , 239–253. 15. Mutation signatures of carcinogen exposure in the genome Jill E. Kucab 1 , Eszter Nagy 1 , Madeleine Joel 1 , Serena Nik-Zainal 2 , Michael R. Stratton 2 , Volker M. Arlt 1 and David H. Phillips 1 1 King’s College London, Analytical and Environmental Sciences Division, London, UK; 2 Wellcome Trust Sanger Institute, Hinxton, UK Thousands of human cancers have now been sequenced at the whole genome level, revealing dozens of distinct mutational signatures. Some of these signatures are correlated with known environmental exposures, endogenous mutagenic processes or defects in DNA repair, but the causative origins of many signatures remain unknown. In an effort to understand how exposure to mutagenic agents in the environment may contribute to the signatures observed in cancers, we are assessing whole genome mutation spectra in mouse and human cells treated with known and suspected carcinogens. As proof-of-principle, we examined the whole genome mutation profiles of mouse embryo fibroblasts immortalised following exposure to benzo[a]pyrene (BaP), ultraviolet irradiation (UV) and aristolochic acid I (AAI). Thousands of mutations were detected in treated cells, revealing a characteristic mutation pattern for each agent: predominantly G>T mutations for BaP, C>T and CC>TT for UV and A>T for AAI. Furthermore, mutation signatures extracted from the data were highly similar to signatures previously extracted from, respectively, smoking-associated lung cancers, skin cancers associated with UV exposure or urothelial cancers caused by ingestion of AA. Currently, we are expanding this work to examine genome-wide mutagenesis in human induced pluripotent stem (hiPS) cells, treated with approximately 100 environmental mutagens and DNA-damaging chemotherapeutics. Initially, we are determining the effect of each agent on cellular viability and induction of the DNA damage response to identify appropriate treatment conditions for mutagenesis studies. Single-cell clones from hiPS cell cultures treated with each agent are being isolated for whole genome sequence analysis, which is currently ongoing. This work will generate detailed mutational signatures connected with specific mutagenic exposures. These signatures of known cause can then be compared with signatures found in human tumours, providing new insight into the aetiology of cancer. 16. A new era of genetic toxicity testing Rhiannon David and Ann Doherty AstraZeneca, Cambridge, UK The standard genotoxicity test battery (bacterial mutagenesis, in-vitro cytogenetics, and in-vivo genotoxicity) is well established. While these assays reliably predict genotoxicity, there are positive genotoxicity results observed only in the in-vivo rodent micronucleus (MN) assay with no obvious explanation; one reason may be the complex nature of the bone marrow (BM). Moreover, new therapeutics such as oligonucleotides and modified RNA present safety challenges that will require alternative assays. Ongoing work at AstraZeneca aims to develop techniques that could complement and enhance current safety testing. Treatment with oligonucleotides or modified RNA could perturb the nucleotide pool, resulting in a non-DNA reactive positive. To demonstrate this mechanism, pyrimidine/purine ratio measurement was ‘added-on’ to an in-vitro MN assay. Cells were treated with o-phenylenediamine (4.3–16.2 µg/mL) or solvent control (0.1% v/v) for 24h followed by 24h recovery. Cells were analysed for MN or pyrimidine/purine ratios by LC-MS/MS. Results showed a dose-dependent increase in MN (14-fold induction; 13 µg/mL) with a concordant decrease in the pyrimidine/purine ratio (1.7-fold, C-G; 1.9-fold, C-A; 13 µg/mL) compared to solvent control. Three-dimensional cell cultures better represent in-vivo tissue structure, and microfluidic organ-on-a-chip systems recapitulate organ-level functions. Therefore, development of a BM-on-a-chip may improve in-vivo genotoxicity prediction. Our results showed populations of human mesenchymal stem cells (MSCs) and haematopoietic stem cells (HSCs) can be maintained within a scaffold under-flow for 21 days, with a 10% increase in the percentage of HSCs of the total population over 7 days. These results indicate the ability to ‘add-on’ analyses to established, regulatory-approved assays to provide mechanistic information, and the development of a BM-on-a-chip may help reduce unpredicted in-vivo positives. Future genetic toxicity testing strategies may be more dynamic, with add-on assays selected according to the test compound, and organ-on-a-chip systems providing better translational models for safety assessment. 17. Impact of TP53 status on the metabolic activation of the air pollutant 3-nitrobenzanthrone in human cells Laura E. Wohak 1 , Ann-Christin Baranski 1 , Annette M. Krais 1 , Osman Sozeri 1 , Heinz H. Schmeiser 2 , David H. Phillips 1 and Volker M. Arlt 1 1 King’s College London, MRC Centre for Environment and Health, London, UK; 2 German Cancer Research Center (DKFZ), Division of Radiopharmaceutical Chemistry, Germany P53 is known as the ‘guardian of the genome’ due to its prominent role in cancer prevention. We previously demonstrated a novel role for p53 in the CYP1A1-mediated bioactivation of polycyclic aromatic hydrocarbons (PAHs) [1]. Here we investigate p53’s impact on the metabolic activation of the nitro-PAH 3-nitrobenzanthrone (3-NBA), its human metabolite 3-aminobenzanthrone (3-ABA) and their reactive intermediate N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA). Since 3-NBA is a potent mutagen and suspected human carcinogen that is widely distributed throughout the environment understanding its mode of action is of critical importance. To investigate the role of TP53 in the bioactivation of 3-NBA a panel of isogenic colorectal HCT116 cells was used, differing only in their TP53 status. Cells having TP53(+/+ ), TP53(+/ -) or TP53(-/ -) were treated with 1 μM 3-NBA, 10 μM 3-ABA or 1 μM N-OH-3-ABA for up to 48hr. DNA adduct formation, determined by 32 P-postlabelling, revealed similar adduct levels (~800 adducts/10 8 nucleotides) after 3-NBA treatment. This correlated with a similar induction (measured by qRT-PCR and Western blotting) of NQO1, which is considered the most efficient nitroreductase activating 3-NBA. Additionally, we found similar CYP1A1 mRNA induction for all cell lines. In contrast, adducts induced by 3-ABA and N-OH-3-ABA were significantly decreased (~15-fold) in TP53(+/ -) and TP53(-/ -) cells compared with TP53(+/+ ) cells indicating that metabolism of these compounds depends on wild-type p53 function. This corresponded with up to 5 times higher CYP1A1 mRNA induction and higher basal SULT1A1 protein expression in TP53(+/+ ) cells linking wild-type p53 to CYP1A1/SULT1A1-mediated adduct formation of 3-ABA and N-OH-3-ABA. Collectively, these results demonstrate how cellular TP53 status impacts the bioactivation of 3-NBA through their metabolising enzymes thereby revealing a novel role for p53 in xenobiotic metabolism. Reference 1. Wohak, L.E., Krais, A.M., Kucab, J.E., Stertmann, J., Øvrebø, S., Seidel, A., Phillips, D.H. and Arlt, V.M. (2016) Carcinogenic polycyclic aromatic hydrocarbons induce CYP1A1 in human cells via a p53-dependent mechanism. Arch. Toxicol.,90 , 291–304. 18. In-vitro alternatives: evaluation and recommendations for the reconstructed 3D skin micronucleus assay (RSMN assay): 72 hour protocol Sarah Phillips, Darren Kidd, Teresa Chirom, Heather Lyon, James Whitwell, Robert Smith, Debbie Wood and Julie Clements Covance Laboratories Ltd, Harrogate, UK There is literature in the public domain that fully describes the 48h exposure protocol [1] for the 3D Reconstructed Skin Micronucleus assay (3D RSMN) and as a result of our in-house validation, we speculated that increasing the exposure period to 72h may increase the assay’s sensitivity. Concerning detection of metabolically activated chemicals in the 3D RSMN, the available literature is very limited [2] so our additional work was designed to investigate this aspect further. We have generated new data for 6 chemicals using the 72hr exposure protocol: mitomycin C (MMC), vinblastine (VIN), N- ethylnitrosourea (ENU), benzo[a]pyrene (BaP), cyclophosphamide (CPA) and 4-nitroquinoline-N-oxide (4-NQO). We had previously seen mixed results when testing the above chemicals in the 48h protocol but the results from the 72h protocol demonstrated an increased cell turnover (measured by Replication Index (RI)) and increased micronucleated binucleate (MNBN) cell frequencies compared to the 48h protocol. These improvements have shown that the 48h protocol is insufficient to allow optimal cell turnover to allow suitable detection of micronuclei following exposure. This is supported by clear positive results for the metabolically activated chemicals (CPA and BaP). These results extend our previous work and support an argument that a 72h protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. References 1. Dahl, E.L., Curren, R.D., Barnett, B.C., Khambatta, Z., Reisinger, K., Ouedraogo-Arras, G., Faquet, B., Ginestet, A.C., Mun, G.C., Hewitt, N.J., Carr, G., Pfuhler, S. and Aardema, M.J. (2011) The reconstructed skin micronucleus assay (RSMN) in EpiDerm™: Detailed protocol and harmonised scoring atlas. Mutation Res.,720 , 42–52. 2. Aardema, M.J., Barnett, B.B., Mun, G.C., Dahl, E.L., Curren, R.D., Hewitt, N.J. and Pfuhler, S. (2013) Evaluation of chemicals requiring metabolic activation in the EpiDerm™ 3D reconstructed human skin micronucleus (RSMN) assay. Mutation Res.,750 , 40–49. 19. HDACi-induced DNA damage: identifying potential endpoints for safety assessment Wenbin Wang 1 , Anthony Lynch 2 and Simon Reed 1 1 Cardiff University, Institute of Cancer and Genetics, Cardiff, UK; 2 GlaxoSmithKline R&D, Safety Assessment, Ware, UK Classes of inhibitors have been designed to alter the actions of epigenetic modifiers with the aim of ‘reprogramming’ the epigenome of diseased tissues back to their normal disease-free state. However, factors that make epigenetic targets fruitful avenues for disease intervention may also require a different approach to preclinical studies supporting human safety. Alterations in epigenetic control of gene expression may result in effects that result in overt toxicity, but also, may change physiological processes over time in a way that leads to a disease prone phenotype (e.g., changes in insulin sensitivity). Addressing these epigenetic changes in Safety Assessment requires knowledge of the underlying biology. Histone deacetylase inhibitors (HDACi’s) are a class of epigenome-targeting drugs shown to trigger profound changes in transcription and replication as well as causing DNA damage through unknown mechanisms leading to genome instability. Consequently, conflicts between transcription and replication can result in the formation of stable DNA:RNA structures (R-loop), which if unresolved can form DNA lesions. To better understand how these epigenome-targeting drugs can elicit damage at the genomic level, we used DNA microarray studies to map changes in markers of transcription (RNA pol II), and replication (ORC1) on chromosome 17 in response to the pan-HDAC inhibitor Trichostatin A (TSA). We show that TSA causes the redistribution of RNA pol II and ORC1, with previously un-enriched coding and non-coding genes (~500–1000) exhibiting protein binding, while the overall enrichment is significantly lower following treatment (p<0.01). We further identified regions exhibiting both high levels of RNA pol II and ORC1 binding following TSA treatment. Using existing software (Qm-RLFS finder) we show that a number of these regions contain predicted R-loop forming sequences, implicating a potential for TSA induced formation of DNA:RNA hybrid structures. 20. Genotoxicity, DNA repair and innate immune responses in sea urchins Helena C. Reinardy and Andrea G. Bodnar Bermuda Institute of Ocean Sciences, Molecular Discovery Laboratory, Bermuda The ability to protect the genome from genotoxic insult is critical for maintaining genome integrity, and differences in this ability might underlie the wide discrepancy in cancer susceptibility across animal groups. Sea urchins are noted for the absence of neoplastic disease despite a wealth of fisheries and life history data and therefore represent a novel model to investigate cellular and systemic cancer protection mechanisms. Our previous studies have shown that the circulating immune cells (coelomocytes) are relatively resistance to DNA damage and have efficient DNA repair capabilities. The immune system plays an important role in detection and ablation of potentially cancerous cells and sea urchins have a complex and expanded innate immune gene repertoire; however, the role of innate immunity in the DNA damage response of sea urchins is not known. In this study the response of the innate immune system was investigated following in-vivo genotoxicant-induced DNA damage in adult sea urchins. Intracoelomic injection of the DNA alkylating agent, methyl methanesulfonate (0, 100, or 300mg MMS/kg body weight) resulted in a significant concentration-dependent increase in DNA damage (analysed by the fast micromethod) in coelomocytes over a 24-h exposure period. After 24h exposure, similar levels of DNA damage were detected in all tissues examined (coelomocytes, muscle, oesophagus, ampullae, and gonad). Additionally, coelomocytes were analysed for expression of a panel of innate immune genes ( traf, ikk2, ikb, rel, nfkb, rae, tbk1, irf4, 185/333 , and gadd45 ); many genes were significantly upregulated at the highest treatment, including genes involved in NF-κB signalling. These results suggest a role of innate immunity in mounting an effective DNA damage response, and we speculate that genotoxicity resistance, DNA repair, and innate immune responses might underlie the naturally-occurring resistance to neoplastic disease in sea urchins. 21. Occupational exposures to polycyclic aromatic hydrocarbons and other environmental mutagens in Ottawa firefighters Jennifer L. Keir 1 , Umme S. Akhtar 1 , Dave Matschke 2 , Paul A. White 1,3 and Jules M. Blais 1 1 University of Ottawa, Department of Biology, Ottawa, ON, Canada; 2 Ottawa Fire Service, Ottawa, ON, Canada; 3 Health Canada, Environmental Health Science and Research Bureau, Ottawa, ON, Canada Firefighters experience above average risks of injury and chronic disease, including kidney, ureter and pancreatic cancer, respiratory diseases and heritable genetic effects. Exposures to carcinogens during firefighting, including combustion emissions and by-products, vary depending on the nature of the fire and the individual’s role during fire suppression. Polycyclic aromatic hydrocarbons (PAHs) are a group of compounds commonly associated with combustion emissions and include several well known mutagenic carcinogens. Few studies have examined firefighters’ exposures to combustion emissions, and those that have been conducted studied exposures during training exercises. This work assessed exposures associated with “on-shift” fire suppression in OFS (Ottawa Fire Service) firefighters using paired urine samples (i.e., pre- and post-fire event) and examining urinary excretion of PAH metabolites and other organic mutagens. PAH exposures were quantified by GC/MS-MS analysis of urinary PAH metabolites. Urinary mutagenicity, which assesses total exposure to organic mutagens, was measured using the Salmonella mutagenicity assay (Ames Test). The results show that post-event levels of 1-hydroxypyrene, a urinary metabolite of pyrene, increase by an average of 3.7-fold (i.e., compared to pre-event, p<0.0001). Similarly, post-event levels of phenanthrene, fluorene and naphthalene metabolites showed significant average increases of 5.3-, 3.9- and 2.9-fold, respectively (p<0.0001). Post-event levels of urinary mutagenicity showed a significant average increase of 4.3-fold (p<0.001). Duration of fire suppression activities was found to be empirically related to post-event levels of urinary PAH metabolites (r2=0.17 to 0.28, p<0.05). Overall, the results indicate that on-shift firefighting is associated with significant increases in exposures to PAHs and other organic mutagens. The project is also examining levels of PAHs and selected metals in personal air collected during the events, and surface wipes of PPE (personal protective equipment) and skin collected both before and after the events. 22. The functional role of STEAP2 in prostate cancer progression Stephanie Burnell 1 , Claire Morgan 1 , Huw Summers 2 , Helen Whiteland 1 , Howard Kynaston 3 and Shareen Doak 1 1 Swansea University Medical School, Institute of Life Science, Swansea, UK; 2 Swansea University, College of Engineering, Swansea, UK; 3 Cardiff University School of Medicine, Cardiff, UK Prostate cancer is one of the most common male malignancies in the world and is associated with poor prognosis if aggressive disease is developed. Unfortunately, it is difficult to distinguish between these patients and those with benign disease. Recently, the six-transmembrane epithelial antigen of the prostate 2 (STEAP2) has been suggested as a diagnostic tool for prostate cancer, as its expression is shown to be increased in prostate cancer when compared to normal prostate cells. Regrettably, the role of STEAP2 remains unknown; therefore, the aim of this project was to determine the role STEAP2 plays in prostate cancer progression. STEAP2 was knocked down in PC3 (bone metastatic prostate cancer) cells by siRNA treatment. The cells then underwent a battery of tests to analyse the functional changes in response to the reduction of STEAP2 expression. This included evaluation of proliferation, migration, invasion, bioenergetics and gene expression analysis utilising a tumour metastasis gene array (Qiagen). Proliferation and invasion of the PC3 prostate cancer cell line was significantly reduced when treated with STEAP2 siRNA by 53% and 36%, respectively. Migration was also reduced in STEAP2 knockdown cells as they failed to close a 500 µm gap within 24h. In addition to this, glycolysis, oxidative phosphorylation and ATP production were all significantly reduced in STEAP2 knockdown PC3 cells by 44%, 25% and 25%, respectively. Furthermore, gene array analysis indicated that the expressions of 9 genes involved in tumour metastasis were altered in response to reduced STEAP2 expression. This study demonstrates that STEAP2 is involved in driving the proliferation, migration and invasion of PC3 cells. Moreover, STEAP2 expression has an impact on mitochondrial function. Thus, STEAP2 expression is important in driving the aggressive behaviour of prostate cancer cells and could serve as a novel therapeutic target for treatment. 23. Sequencing in-vivo Pig-a mutations Vasily Dobrovolsky National Center for Toxicological Research/FDA, Jefferson, AR, USA The in-vivo Pig-a gene mutation assay is an emerging tool for regulatory safety assessment. Pig-a is an endogenous X-linked gene involved in the synthesis of GPI anchors. Mutation in the Pig-a gene results in disruption of GPI synthesis and a deficiency in GPI-anchored markers at the surface of mutant cells. It is the marker deficiency that is detected by flow cytometry in the Pig-a assay. Theoretical considerations and human data support a close relationship between PIG-A mutation and anchor/marker deficiency. Yet there is a concern that deficiency of GPI-anchored markers may be caused by events other than mutation in the Pig-a gene (e.g., mutations in other genes in the GPI synthesis pathway, or epigenetic events). In order to gain regulatory acceptance, it is necessary to demonstrate that an assay detects what it claims to detect – in case of the Pig-a assay, mutation in the Pig-a gene. Various approaches employing flow cytometry, sorting and sequencing analysis have been employed for linking the mutant surface marker-deficient phenotype to mutation in the Pig-a gene. The identification of mutagen-specific Pig-a mutations in individual mutant-phenotype cells, and mutation spectra in bulk-sorted mutant-phenotype cells, suggest that deficiency of GPI-anchored markers is mainly caused by inactivating mutations in the endogenous Pig-a gene. Thus, the available evidence is consistent with the idea that that the various flow cytometry-based Pig-a assays, including the Pig-a assay using peripheral red blood cells, detect gene mutation. 24. OECD requirements for historical control ranges and control charts Carol Beevers Covance Laboratories Ltd, Harrogate, UK The 2014 OECD test guideline revisions included additional requirements for the presentation and use of historical control ranges and control charts for the core genotoxicity assays; the intention of these requirements is to enable a laboratory to demonstrate adequate proficiency in the tests being undertaken thereby increasing confidence in the reliability of test data. In 2015 OECD released the draft Guidance Document on Revisions to OECD Genetic Toxicology Test Guidelines, which provides further guidance and expectations for the generation of historical control ranges and control charts. These recommendations present laboratories with a number of challenges for robust HCR and control chart generation, as well as their maintenance and use in routine assays. 25. Achieving 3Rs benefits from detailed OECD guidance: the in-vitro micronucleus test as a case study Hedwig Braakhuis 1 , Paul Fowler 2 , David Kirkland 3 , Jan van Benthem 1 and Mirjam Luijten 1 1 National Institute for Public Health and the Environment (RIVM), Centre for Health Protection, Bilthoven, The Netherlands; 2 Unilever, Leatherhead, UK; 3 Kirkland Consulting, Tadcaster, UK In several regulatory frameworks, the in-vitro micronucleus test (MNvit) is the recommended test for detecting chromosome aberrations (clastogenicity and aneugenicity). Most regulations refer to OECD Test Guideline (TG) 487 for study protocol and interpretation of test results. In many regulatory frameworks, a positive response in an in-vitro genotoxicity test triggers subsequent in-vivo testing. A misleading positive result in-vitro may therefore lead to unnecessary animal testing. Recent studies suggested that p53-competent human cells are to be preferred over p53-deficient rodent cells for conducting the MNvit. Here, we re-evaluated the accuracy of the MNvit - using an updated list of chemical substances that are known to induce misleading positive results in-vitro - with the aim of identifying the best protocol to reduce the number of misleading positive results. Based on this evaluation, we conclude that the MNvit is a robust mammalian cell genotoxicity test that is unlikely to produce misleading positive results, provided that relevant cell types (preferably p53-competent human cells) are used in combination with a cytotoxicity measure that allows selection of test concentrations below the range in which apoptosis occurs (preferably relative population doubling [RPD] or relative increase in cell count [RICC]). OECD TG 487 provides recommendations on the selection of measures of cytotoxicity but not on the choice of cell type. Based on this case study, we recommend that OECD includes specific requirements in its Test Guidelines to further reduce the number of misleading positive results and thus prevent unnecessary animal testing. 26. Is non-disjunction a more sensitive marker than chromosome loss for aneugens? Ann Doherty AstraZeneca, Cambridge, UK Currently, the most common method used for the detection of aneugens is the in-vitro micronucleus assay, in combination with either centromere or kinetochore detection. However, this method does not take into account non-disjunction; a mechanism in which studies using primary human lymphocytes has suggested is the predominant mechanism of spindle poison induced aneugenicity. If true, this implies that the micronucleus assay may potentially be relatively insensitive in detecting aneuploidy. To investigate whether sensitivity of detection of aneugenicity could be improved, an assay which incorporates the analysis of chromosome distribution, specifically non-disjunction, using chromosome-specific centromeric probes was used to test the effects colchicine, vinblastine, Taxol, chloral hydrate and diethylstilbestrol, and Noscapine with differing modes of action on human lymphoblastoid TK6 cells. The results show that in TK6 cells, chromosome loss rather than non-disjunction appears to be the predominant process leading to aneuploidy, differing from results from studies in human lymphocytes which showed that non-disjunction is the major process induced by spindle poisons leading to aneuploidy. Only colchicine showed an increase in non-disjunction which was higher than chromosome loss at the equivalent concentration. In summary, the results from this investigation show that although compound specific effects on non-disjunction were detected by the reference aneugens tested, there was no increase in sensitivity of this assay over the in-vitro micronucleus assay, as chromosome loss and non-disjunction were detected at the same or lower concentrations, even when non-disjunction levels where comparatively high. Therefore, the methods in this investigation do not provide evidence that the use of chromosome-specific centromeric FISH probes provides a more sensitive assay for the detection of aneugenicity, when compared to the use of a micronucleus based approach. 27. Can weak carcinogens be detected using an integrated in-vitro carcinogenicity test? Katherine E. Chapman 1 , Eleanor Wilde 1 , Leanne Stannard 1 , Anna L. Seager 1 , Katja Bruesehafer 1 , James Tonkin 2 , M. Rowan Brown 2 , George E. Johnson 1 , Shareen H. Doak 1 and Gareth J. S. Jenkins 1 1 Swansea University, Institute of Life Science, Swansea, UK; 2 Swansea University, College of Engineering, Swansea, UK There is no clear dividing line between weak carcinogens and biologically inactive substances. This renders weak carcinogens inherently difficult to detect using standard genotoxicity and carcinogenicity tests. Identification of weak carcinogens is crucial for evaluating the risk of human exposure to high concentrations, such as those potentially encountered during occupational exposure. Furthermore, in-vitro test strategies for the detection of carcinogenesis provide advantages over in-vivo tests, particularly with respect to the implementation of the 3Rs Principle. The present study’s objective was to use a novel, multiple-endpoint in-vitro approach to observe whether weak carcinogens could be identified. Three weak carcinogens were studied: acetaldehyde, a metabolite of ethyl alcohol; hydrogen peroxide (H 2 O 2 ), a pro-oxidant used in bleach and as an antiseptic; quinacrine dihydrochloride, an anti-malarial drug. The aforementioned integrated in-vitro approach incorporated six techniques to measure alterations in chromosome damage, cell cycle kinetics, cell signalling, cell morphology and mitochondrial activity following exposure to the test chemicals. H 2 O 2 and acetaldehyde induced significant increases in micronucleus frequency in human lymphoblastoid TK6 cells at 4h and 23h, respectively. Only acetaldehyde altered cell cycle kinetics, inducing a G2 block at 500 µM after 23h. H 2 O 2 induced p53 and phospho-p53 expression at 4h, and also increased p21 mRNA expression at 12 µM. Cell morphology and mitochondrial activity were not altered relative to untreated controls. These data suggest some heterogeneity in terms of weak carcinogens’ responses for the studied endpoints, yet unlike potent genotoxic carcinogens, they tend to only produce notable changes for a limited number of endpoints. This therefore demonstrates that assessment of a single endpoint, such as micronucleus formation, may be insufficient for detecting weak carcinogens and could lead to “misleading negative” results. Use of multiple endpoints could aid the improvement of in-vitro tests, reducing the number of animal-based tests required. 28. Development of a multi-end point genotoxicity assessment screen (mega-screen) Joanne Elloway, Stephanie Ling, Rhiannon David and Ann Doherty AstraZeneca, Cambridge, UK Screening for genotoxicity using standard regulatory guidelines [1] is time consuming and expensive, and is currently unfeasible for the early stages of drug development where compounds are only available in small amounts. Therefore, a high throughput screen which assesses compound genotoxicity would be beneficial. The regulatory-approved [2] in-vitro micronucleus assay (IVM) has already shown potential for higher throughput [3]. This work describes a complimentary method of analysis using an IVM high-content biological screen, the Multi-End Point Genotoxicity Assessment Screen (MEGA-Screen) which enables the identification of multiple genotoxicity phenotypes in addition it also aims to provide mechanistic understanding of positive responses. The MEGA-Screen is a high content biology assay developed in 384-well format, thus far with two cell lines, A549 (p53-wt) and HGC27 (p53-mutant) assessed in 24h treatment regime compared to 24 hour treatment followed by 24h recovery regime using a validation compound set comprising 7 aneugens, 3 clastogens, 2 nucleoside-analogues and 4 negative controls. Compounds were acoustically dosed, therefore compound requirement is approximately 2mg. Confocal image acquisition was completed using a CellVoyager™ C7000 (Yokogawa, Japan) and images obtained were analysed via Columbus™ software (PerkinElmer, UK).The MEGA-Screen includes the detection of micronuclei, cytotoxicity, cell-cycle dynamics, kinetochore labelling for aneugenicity, H2AX for clastogenicity and apoptosis and validation results have shown changes in these in-line with the known mechanism of action of the compound. In conclusion, preliminary results from the MEGA-Screen have demonstrated its potential to provide early genotoxic mechanistic information to aid the selection of compounds with the best safety profile for pharmaceutical development. References 1. ICH S2 (R1) (2011) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. International Conferences on Harmonization of the Toxicological Requirements for Registration of Pharmaceuticals for Human Use. (ed ICH). 2. OECD (2014) Test No. 474: Mammalian Erythrocyte Micronucleus Test. (OECD Publishing). 3. Bryce, S.M., Bernacki, D.T., Bemis, J.C. and Dertinger, S.D. (2016) Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach. Environ. Mol. Mutagenesis,57 , 171–189. 29. Urban air pollution: here we go again Frank J. Kelly King’s College London, NIHR HPRU Heath Impact of Environmental Hazards, London, UK There was a time when the UK led the world in cleaning up its air - passing the Clean Air Act in 1956 to reduce smoke and sulphur dioxide. In recent years air quality improvements have stalled. We have been breeching European Union (EU) limit values every year since 2010 for the modern day pollutants – namely nitrogen dioxide (NO 2 ) and (WHO guidelines for) particulate matter (PM) – and currently there is no prospect of achieving compliance for NO 2 in some areas until 2025. What is more worrying, over the same period of time, evidence to support the detrimental short and long-term effects on health has increased substantially: data for 2008 estimate that the air pollution burden is equivalent to 29,000 premature deaths in the UK – in 2012 the International Agency for Research on Cancer classified particulates in diesel fumes as a known carcinogen – in 2013 a WHO report concluded that health effects of PM and NO 2 can occur at concentrations lower than the health-based Guideline values which of note for PM, are lower than the EU limits we fail to adhere to. In addition, other than the well-documented risks to cardiopulmonary health, increasing evidence exists that air pollution exerts a wider threat, negatively influencing reproductive outcomes and neurological health. Solving the air pollution problem is a huge challenge. It is highly unlikely that our major cities will ever be able to boast ‘pure air’ especially if strategies focus on small areas of an overall road network. With bold, realistic and moral leadership however, enormous potential exists to reduce air pollution so that it no longer poses a damaging and costly toll on public health. 30. Asthma, genetic susceptibility and environmental pollutant exposures Martin Leonard Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, Didcot, UK Asthma is a life threatening disease affecting 5.4 million people in the UK. A number of factors both genetic and environmental have been associated with modifying the risk for developing asthma. These include exposures such as farm living, environmental tobacco smoke, air pollution and respiratory viral infection. The identification of specific hazards relevant for asthma is an important part of risk assessment and risk management strategies for the condition. Recent developments in experimental modelling, systems biology and mechanistic toxicology as well as advances in genetic epidemiological approaches are important new tools with the potential to provide novel insights into how hazard potential is examined in complex disease states. These developments will also include the ability to identify specific genetic-environmental interactions important for the development of disease in susceptible populations. In addition, it is important to consider population genetics beyond polymorphism based linkage to include epigenetic information as this is likely to provide detailed information not only for biomarkers of specific environmental exposures but also as potential modifiers for asthma risk development. 31. Assessment of the toxic response to PM and diesel exhaust in human airway epithelial cultures Ian W.H. Jarvis 1,2 , Zachary Enlo-Scott 1 , Eszter Nagy 1 , Volker M. Arlt 1,2 and David H. Phillips 1,2 1 King’s College London, Division of Analytical and Environmental Sciences, London, UK; 2 NIHR HPRU in Health Impact of Environmental Hazards at King’s College London in Partnership with PHE, London, UK Combustion-derived chemicals such as polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are released into the environment from industrial activities and traffic emissions. Outdoor air pollution and diesel engine exhaust have been classified as carcinogenic to humans (Group 1) by the International Agency for Research on Cancer. The primary route of human exposure is through inhalation of such chemicals after they become adsorbed onto particulate matter (PM), and exposure has been linked to the development of lung cancer and other respiratory diseases, though the mechanisms involved in disease pathogenesis remain unclear. The aim of this study was to assess the effects of coarse (2.5–10 µm) and fine (<2.5 µm) PM on DNA damage and inflammatory responses in comparison to the carcinogenic nitro-PAH 3-nitrobenzanthrone (3-NBA) present in diesel emissions. Genotoxic and inflammatory effects were assessed in human alveolar type 1 like cells (TT1) and a mucociliary bronchial tissue model (EpiAirway, MatTek Corp.) cultured at an air/liquid interface. TT1 cells showed a concentration-dependent increase in DNA damage signalling activation (e.g. phosphorylation of Chk1, Chk2 and H2AX evaluated by Western blotting) after 3-NBA treatment that corresponded with increased DNA damage measured by the comet assay. Exposure to coarse PM induced comparable effects to 3-NBA; fine PM did not induce any detectable effects. In agreement, only coarse PM induced secretion of IL-6 and IL-8 assessed by ELISA. In contrast, no discernible effects on DNA damage and signalling were observed in EpiAirway cultures exposed to either 3-NBA or PM mixtures. Furthermore, no increase in IL-6 or IL-8 secretion was detected. Our results highlight the genotoxic potential of the air pollutant 3-NBA and show that coarse PM exhibits higher genotoxicity than fine PM in lung cells. Work is ongoing to understand the discordance in the data relating to EpiAirway cultures. 32. Pulmonary toxicity of silver nanoparticles in a human relevant in-vitro air liquid interface exposed epithelium model Chang Guo, Martin Leonard and Rachel Smith Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, Didcot, UK Silver nanoparticles (AgNPs) are increasingly being used as anti-microbial agents in consumer products, particularly personal hygiene products, wound dressings and disinfectant sprays. Use of these nanoparticles has raised concerns about the potential for harmful effects on exposure in humans. The majority of experimental modelling to identify specific inhalation hazards of AgNPs have used rodent models, which do not fully capture human relevant toxicities, but also have the potential to cause animal distress and suffering. Advances in in-vitro modelling have seen systems developed which combine ‘inhalation-like’ nanoparticulate aerosol deposition with relevant human cell and tissue cultures. We have previously identified the small airway epithelium as an important target for AgNPs deposition in-vivo . In this work we have established and optimised an air-liquid-interface aerosol-exposure (ALI-AE) system to facilitate hazard identification of AgNPs in human relevant culture models. Organotypic reconstituted 3D human primary small airway epithelial (HPSAE) cell cultures were exposed to AgNP aerosols and characterised for adverse effects (general toxicity and gene expression on selected mRNA and miRNA). Key pathways reflective of specific adverse effects relevant for human hazard identification were identified that provided further information on the potential health effects of AgNPs exposure. 33. Pulmonary bioreactivity of engineered nanoparticles Terry Tetley Imperial College London, National Heart and Lung Institute, London, UK The development and production of engineered nanoparticles is increasing rapidly and with it, the range of applications. It is predicted that the workforce will increase 3-fold between 2015 and 2020, and the global market will increase to 3 trillion dollars by 2020. Engineered nanomaterials are unlike their bulk particles and exhibit a wide range of characteristics that confer alternative biophysical properties, the impact of which is largely unknown. They are being included in many more consumer goods, in health care, cosmetics, sports, clothing, engineering, construction and in many other areas of everyday life. Currently, there is little information on occupational exposure and even less on exposure during use and disposal. There is some concern that some of these materials could have adverse health effects; if airborne, upon inhalation it is predicted that a high number of nanosized particles of relatively little mass would access the deep lung, causing local irritation, and possibly cross the gas blood barrier to access other lung compartments and the vasculature, where they may have systemic effects. This presentation will describe work on the interaction of carbon based nanomaterials, metal oxides and nanomaterials in consumer products (e.g. silver and zinc) with in-vitro models of the human alveolus, using primary human lung cells. Studies of particle uptake and translocation across the gas-blood barrier, modifying effects of lung secretions and lung surfactant and effects on the pulmonary vasculature will be presented. Although ideally, it would be useful to be able to predict the bioreactivity of engineered nanoparticles based on generic features of the particles, our work, and that of others, indicates that the situation is complex and there are no simple solutions to predicting toxicity. 34. Genotoxic and inflammatory effects of silver nanoparticles in the lung Andrew Thornley Imperial College London, National Heart & Lung Institute, London, UK The development of silver nanoparticles for therapeutic applications, and their incorporation into commercial products as an effective antimicrobial agent, has rapidly increased over the last decade such that it is estimated that approximately 320 tons are produced every year, making them the one of the most commonly used engineered nanoparticles. Their use in medical care is already apparent in wound dressings, face masks, medical catheters, surgical instruments and, of potential concern, unregulated disinfectant sprays, nasal sprays and solutions for home nebulization. Approximately 50% of inhaled nanoparticles preferentially deposit in the alveolar respiratory units. Thus, inhalation of airborne silver nanoparticles might induce deleterious effects on the peripheral human lung. We investigated the bioreactivity of silver nanoparticles on human alveolar epithelial cells, and how pulmonary surfactant might modulate the cellular response. Our studies have demonstrated that whilst not acutely cytotoxic, silver nanoparticles induce inflammation, autophagy and DNA damage which is driven by generation of reactive oxygen species, and subsequent activation of p38 MAP kinase signalling. However, importantly, we have also demonstrated that pulmonary surfactant is able to significantly inhibit the pro-inflammatory effects of silver nanoparticles via suppression of oxidative stress and p38 phosphorylation and may therefore act as a protective screen to inhaled silver nanoparticles in-vivo . Finally, we have also investigated how silver nanoparticles interact with the pulmonary innate immune response to bacterial and viral ligands and have elucidated pathways by which the presence of viral infection may increase the deleterious effects of silver nanoparticles on the lung. 35. Transcriptomics responses among environmentally exposed study populations Jos Kleinjans Maastricht University, Department of Toxicogenomics, Maastricht, The Netherlands The advent of the so-called ‘omics technologies has inspired the human biomonitoring research field, in particular in Europe, to explore in an increasing number of projects this new generation of biomarkers for identifying environmental health risks. To date, data sets have been largely derived from whole genome gene expression analysis applying microarray technology. Currently we witness more ‘omics platforms being applied, e.g. epigenomics, global microRNA analysis, metabolomics, and (targeted) proteomics, presenting complex challenges for comprehensive data analysis. A decade of performing transcriptome analysis in a range of multi-partner investigations among environmentally exposed populations across Europe (FLESH, NewGeneris, EnviroGenoMarkers, Exposomics) has yielded large ‘omics data sets which now can be exploited for cross-study, cross-platform investigations. Relatively new insights refer to gender-related susceptibilities showing relatively large differences between the sexes in ‘omics responses while levels of environmental exposure are similar, and age differences which for instance demonstrate that children are more susceptible towards air pollution than their parents. While results so far still are rather associative, future challenges relate to the possibility of inferring causality from these data. 36. The exposome in the context of causal reasoning and risk assessment Paolo Vineis Imperial College London, London, UK To address the new challenges of epidemiology, the concept of the “exposome” has been proposed, initially by Wild [1], with more recent detailed development in relation to its application to population-based studies [2]. The original concept was expanded by others, particularly Rappaport and Smith [3] who functionalized the exposome in terms of chemical signals detectable in biospecimens. The canonical exposome concept refers to the totality of exposures from a variety of sources including chemical agents, biological agents, radiation, and psychosocial components from conception onward, over a complete lifetime [2,3]. The large amount of data we have collected through the Exposomics project allow us to build a unifying framework to incorporate omic data into causal models, referring to a position called “evidential pluralism”, according to which causal reasoning is based on both “difference-making” and the underlying biological mechanisms (Russo and Williamson). Examples from our recent projects in the field include: new omic approaches such as adductomics; new long-term methylation biomarkers (in relation to smoking and air pollution); markers related to early life exposure and the role of socio-economic differentials. In addition, exposome research contributes to risk assessment, e.g. in analysing the health impacts of air pollution. Exposome research can contribute to elucidate key aspects of risk assessment, such as dose-response relationships, and provide mechanistic insights. References 1. Wild, C.P. (2005) Complementing the genome with an “exposome”: the outstanding challenge of environmental exposure measurement in molecular epidemiology. Cancer Epidemiol. Biomarkers Prev.,14 , 1847–1850. 2. Wild, C.P. (2012) The exposome: from concept to utility. Int. J. Epidemiol.,41 , 24–32. 3. Rappaport, S.M. and Smith, M.T. (2010) Epidemiology. Environment and disease risks . Science,330 , 460–461. 37. Challenges in investigating the asthma exposome Nicole Probst-Hensch Swiss Tropical and Public Health Institute, Basel, Switzerland The aetiology and life course of asthma remain poorly understood. Asthma encompasses multiple phenotypes (e.g. early onset allergic asthma or obesity related asthma), endotypes (e.g. air flow obstruction caused by obesity) and severity stages which can vary over the life course within an individual. Poor understanding of asthma is in part rooted in asthma complexity. Genome-wide association studies point to different pathways in childhood versus adult-onset asthma. DNA methylation signals explaining a considerable variance of total serum IgE are not broadly overlapping with asthma-related signals. Few epidemiological studies have followed-up individuals from the time of conception and during pregnancy to adult age. Large sample size is critical for studying the complexity of asthma and risks, but limits the precision with which asthma phenotypes as well as external environmental and lifestyle factors can be measured over the life course at reasonable cost and low loss to follow-up. The cross-sectional and longitudinal (cross)-omic profiling of asthma and exposures offers opportunities for novel insight into asthma phenotypes, specific modifiable risks, disease course and treatment responsiveness. Blood-based asthma-related omic profiles as easily accessible in epidemiological studies need to be combined across studies in different age groups and must be complimented with omic profiles in asthma-related target tissues and cells. Bioinformatics analysis needs to provide proof-of-principle for the relevance of peripheral blood profiles in identifying specific asthma phenotypes and risk factors as recently evidenced for tobacco smoking. Unfortunately other exposures in the environment (e.g. air pollution) or life style (e.g. nutrition, physical activity) are more challenging to capture due to long latency of effects and considering that for asthma both acute and long-term effects are of interest. Decreasing measurement error in the external exposome with the help of novel sensoring and modelling methods is as essential as molecular profiling in improving disease understanding. 38. Exposure to disinfection by-products in swimming pools and associated early effect biomarkers Cristina Villanueva 1,2,3,4 , Laia Font-Ribera 1,2,3,4 , Esther Marco 5 , Joan O. Grimalt 5 , Susanna Pastor 6 , Ricard Marcos 6 , Lilliane Abramsson-Zetterberg 7 , Marie Pedersen 8 , Tamara Grummt 9 , Esther Barreiro 4 , Dick Heederik 10 , Jack Spithoven 10 , Rossana Critelli 11,12 , Alessio Naccarati 11 , Christina Schmalz 13 , Christian Zwiener 13 , Jiaqi Liu 14 , Xiangru Zhang 14 , William Mitch 15 , Lourdes Arjona 1,2,3,4 , Jeroen de Bont 1,2,3 , Lluïsa Tarès 1 and Manolis Kogevinas 1,2,3,4 1 Center for Research in Environmental Epidemiology (CREAL), Barcelona, Spain; 2 CIBER Epidemiología y Salud Pública, Barcelona, Spain; 3 Universitat Pompeu Fabra, Barcelona, Spain; 4 Hospital del Mar Medical Research Institute, Barcelona, Spain; 5 Institute of Environmental Assessment and Water Research, Barcelona, Spain; 6 Universitat Autònoma de Barcelona, Barcelona, Spain; 7 National Food Administration, Uppsala, Sweden; 8 Kræftens Bekæmpelse, Copenhagen, Denmark; 9 Federal Environmental Agency, Bad Elster, Germany; 10 Institute for Risk Assessment Sciences, Utrecht, The Netherlands; 11 Human Genetics Fundation, Torino, Italy; 12 University of Turin, Department of Medical Science, Turin, Italy; 13 University Tübingen, Tübingen, Germany; 14 Hong Kong University of Science and Technology, Department of Civil and Environmental Engineering, Hong Kong, China; 15 Stanford University, Department of Civil and Environmental Engineering, Palo Alto, CA, USA Swimming is a healthy activity entailing a high exposure to irritant and carcinogenic disinfection by-products (DBPs). We assessed changes in biomarkers of genotoxicity and lung epithelium permeability after swimming, and evaluated the association with individual DBP exposure. 116 non-smoker adults swam 40min in an indoor chlorinated pool. We measured changes in the following biomarkers before and at different times after swimming: trihalomethanes (THMs) in exhaled breath (five min after), trichloroacetic acid (TCAA) in urine (30min), serum club cell protein (CC16) (one hour), urine mutagenicity (two hours) and micronuclei in reticulocytes (four days). Several DBPs in water and trichloramine in air were measured. Physical activity was assessed with distance swum, energy expenditure and heart rate. Median exhaled total THMs, brominated THMs and creatinine adjusted urine TCAA increased by 13.4 µg/m 3 , 2.6 µg/m 3 and 3.1 µmol/mol, respectively. Effect biomarkers did not significantly increase after swimming and DBP exposure was not related to the change in urine mutagenicity. Exhaled THMs were positively related to an increase in reticulocytes micronuclei (β Coefficient 0.82 [95% Confidence Interval = 0.21, 1.43] for an interquartile range increase in brominated THMs). Energy expenditure, but not trichloramine in air or exhaled THMs explained the change in serum CC16 in multivariate models. DBP exposure in swimming pools at the observed levels did not affect urine mutagenicity or lung epithelium permeability biomarkers. A positive association with micronuclei in reticulocytes, a sensitive test, was detected in a subsample. 39. Metabolomics and the exposome in epidemiology Augustin Scalbert International Agency for Research on Cancer, Lyon, France Cancers are largely governed by lifestyle and environmental factors. Major risk factors have been identified but the causes of several cancers still remain largely unknown. Epidemiological studies conducted so far have been largely hypothesis-driven and focused on limited numbers of risk factors. Today highly sensitive mass spectrometry techniques and metabolomics allow to simultaneously measure hundreds or thousands of chemicals and metabolites in blood, urine and other biospecimens. Such measurements should provide a more comprehensive picture of the exposome and precious information on exposures as diverse as diet, air pollution, contaminants or drugs. This data on exposure biomarkers is now curated in the Exposome-Explorer database ( http://exposome-explorer.iarc.fr ). This knowledge can be exploited in exposome-wide association studies for an informed selection of panels of exposure biomarkers to be specifically measured in blood, urine or other biospecimens. It can also be used to identify a large diversity of biomarkers in complex metabolic fingerprints in untargeted metabolomic studies and classify individuals according to exposures. However a number of challenges related in particular to the sensitivity and robustness of the analytical methods and to the stability over time of the biomarkers measured need to be addressed. Recent progress in this rapidly moving field should contribute to the identification of novel risk factors for cancers and to the improvement of our understanding of aetiology of these diseases. 40. Measuring personal exposure to ultra-fine particles in the EXPOsOMICS project John Gulliver 1 , Gerard Hoek 3 , Roel Vermeulen 3 , Erik van Nunen 3 , Kees Meliefste 3 , Jelle Vlaanderen 3 , David Donaire-Gonzalez 2 , Ariadna Curto 2 , Marta Cirach 2 , Laia Font-Ribera 2 , Alex Ineichen 4 , Mark Davey 4 , Nicole Probst-Hensch 4 , Paolo Vineis 1 , Andre F.S. Amaral 1 , David Morley 1 , Alessio Naccarati 6 , Laura Zini 6 , Sabrina Bertinetti 6 , Mark J. Nieuwenhuijsen 2 , Frank Kelly 6 and Ming Tsai 4 1 Imperial College London, MRC-PHE Centre for Environment and Health, London, UK; 2 ISGlobal, Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain; 3 University of Utrecht, Institute for Risk Assessment Sciences, Utrecht, The Netherlands; 4 University of Basel, Swiss Tropical and Public Health Institute, Basel, Switzerland; 5 Molecular and Genetic Epidemiology Unit, HuGeF Foundation, Torino, Italy; 6 King’s College London, Environmental Research Group, London, UK To assess 24-hour personal exposures to ultra-fine particles (UFP) of individuals from five areas in Europe taking part in the EU-funded EXPOsOMICS ( http://www.exposomicsproject.eu ) project, and to provide these data to studies relating UFP exposures to a series of omics (e.g. metabolomics, adductomics) being undertaken on blood samples that were taken immediately after each UFP exposure measurement. Adult and child personal exposure monitoring of UFP using the DiSCmini sensor (Matter Aerosol AG, Switzerland) was undertaken in five areas in Europe (Adults: Basel (Switzerland), Amsterdam and Utrecht (The Netherlands), Turin (Italy); and Norwich (UK); Children, Sabadell (Spain) during 2014 and 2015. 24-h measurements were repeated up to three times on each participant in different seasons. Raw 1-second data from the DiSCmini was cleaned using R scripts developed for the project. Using a completeness threshold of > 75% in the 1-second data, a total of 495 UFP personal exposure measurements from 162 adults and 42 children were retained to calculate 24-h mean and median exposures. Average and median 24-h particle number counts (PNC) (particles cm 3 ) were produced for each individual. For adults, mean UFP PNC were highest in Turin (20434; n = 108) followed by Amsterdam/Utrecht (15429; n = 124), Norwich (14303; n = 54) and Basel (11632; n = 127). For children (Sabadell), mean UFP were 18208 (n = 82). Values of median UFP PNC were lower than means due to exposures overall being positively skewed: Turin (16627), Amsterdam/Utrecht (11308), Norwich (11112), Basel (8981) and children in Sabadell (15823). Eleven of the 24-h mean PNC exceeded 50000 with nine of these from Amsterdam/Utrecht and Turin. Mean UFP exposures varied up to about two fold between study areas. The EXPOsOMICS project has a rich dataset on UFP exposures to determine the contribution of different microenvironments (e.g. home, journeys, work) to average/total exposures and undertake omic analysis. 41. Albumin adductomics: a methodology for probing the internal exposome George W. Preston 1 , Osman Sozeri 1 , Lilian Bastian 1 , Michelle Plusquin 2 , Karin van Veldhoven 2 , Marc Chadeau-Hyam 2 and David H. Phillips 1 1 King’s College London, Analytical and Environmental Sciences Division, London, UK; 2 Imperial College London, London, UK It is widely acknowledged that the aetiologies of many chronic diseases have substantial environmental components, yet the agents responsible and their mechanisms of action often remain elusive. Albumin adductomics is a promising new methodology for discovering biomarkers of exposure and/or biomarkers of effect. The approach is based on the idea that electrophilic chemicals in blood can form stable adducts with long-lived circulating proteins such as human serum albumin. In 2011, Rappaport and co-workers [1] reported a mass-spectrometry-based workflow for untargeted analysis of albumin adducts, focusing on a 21-residue peptide containing a single cysteine that is the presumed site of modification. As part of a wider investigation (EXPOsOMICS), we have implemented this workflow in studies relating to air and water pollution. To enable this, Rappaport and co-workers’ methods were adapted for higher throughput, and a quality control strategy was developed. An extensive evaluation of accuracy, precision and stability was undertaken, and methods were developed to screen the data based on their quality. A number of putative adducts have emerged as candidates for further qualitative (e.g., structural) and quantitative analyses. Our results to date represent a significant step forward in the use of untargeted protein adductomics for epidemiological studies. 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. 42. 3D culture of human bone marrow mesenchymal stem cells protects from genotoxic effects of melphalan exposure Saeed Kabrah 1 , Jenny May 2 , Craig Donaldson 1 and Ruth Morse 1 1 University of the West of England, Centre for Research in Biosciences, Bristol, UK; 2 Plymouth University, Centre for Research in Translational Biomedicine, Plymouth, UK Melphalan is a chemotherapeutic drug belonging to the class of nitrogen mustard alkylating agents used in bone marrow cancer therapy. It causes cross-links between DNA strands leading to genotoxicity events that can be measured by the micronucleus assay. This study aimed to compare HS-5 cultured in 2D with 3D systems (Biomerix, Nanofiber and Alvetex) for genotoxicity after treatment with the clinically relevant dose of melphalan (32.8µM) and was assessed using the micronucleus assay. Cells (1×10 5 ) were statically seeded into each system and maintained for one week to allow cells to populate the scaffolds. Subsequently, cells were cultured with and without the drug for 1hr prior to being cytospun onto clean slides, stained with acridine orange and examined for genotoxic events (2000 cells per test condition). Melphalan induces micronucleated, binucleated, lobed and multinucleated cells in all treated versus untreated environments, however all these events were reduced in 3D compared with 2D treated cultures. Binucleation had a trend towards reduction in all 3D systems, but was only significant in Biomerix (vs 2D; p<0.05) and Alvetex had a non-significant lower binucleation in treated compared with untreated cultures. Micronuclei were significantly reduced in all treated 3D compared to 2D (2D 8.6%, Biomerix 0.9%, Nanofiber 2.6% and Alvetex 5.1%; p<0.05) despite all environments (except Biomerix) having a significantly raised induction of micronuclei with treatment (p<0.05). Similarly, both lobed and multinucleated cells were significantly raised in all treated versus untreated cultures, but significantly reduced in 3D treated cultures compared to 2D, with more than 50% reduction of multinucleation in Biomerix and Alvetex. The results indicated that 3D cultures protect the HS-5 cells and reduce genotoxic damage. We conclude that the use of 2D in-vitro culture environments should be used with caution in estimating risk of genotoxicity. 43. 3D model of the bone marrow protected HS-5 stromal cell line from melphalan damage Saeed Kabrah 1 , Jenny May 1 , Craig Donaldson 2 and Ruth Morse 1 1 University of the West of England, Centre for Research in Biosciences, Bristol, UK; 2 Plymouth University, Centre for Research in Translational Biomedicine, Plymouth, UK Genotoxic chemotherapeutic agents are central to the treatment of cancer cells however, they induce damage to the normal proliferative cell, including bone marrow mesenchymal stem cells (BM-MSCs). BM-MSCs are primary cells that form bone marrow, which is a highly proliferative organ, continuously forming the haematopoietic and immune systems, and thus are environments for the investigation of drug cytotoxicity and genotoxicity. In-vitro assessment is usually conducted on flat monolayer culture systems. In-vivo genotoxicity tests used to verify in-vitro outcomes regularly show different results. Due to the lack of BM models that mimic the in-vivo situation, this study aimed to investigate the impact of melphalan on HS-5 (stromal cell line) in 2D and 3D (Biomerix, Nanofiber and Alvetex) cultures. Cytotoxic damage was evaluated by comparing cellular morphology, proliferation and viability. Moreover, multilineage differentiation capacities were assessed by flow cytometry. Melphalan induces morphological changes in 2D culture where cells had more granulation and lost their fibroblast morphology, but cells in 3D culture maintained their structure and increased cell size by approximately 2mm. Also, cell proliferation reduced in 2D (6%), Biomerix (3%) and Nanofiber (13.6) but increased in Alvetex by 37% compared to the normal cultures. Cell viability was reduced after exposure except for cells in Biomerix with high viability (>82%). HS-5 colony formation (CFU-F) were reduced after melphalan exposure by more than 5% in all cultures, however, total CFU-F was higher in Biomerix (64%) compared to other environments (2D 41%, Nanofiber 57% and Alvetex 55%). The results demonstrated that 3D cultures were more protective than the tradition 2D culture system especially the multi-porous scaffolds more than the polymers fibres. 44. Cluster of differentiation (CD) phenotype in the immortalized stromal cell line HS-5 in 2D and 3D environments and post-melphalan exposure Saeed Kabrah 1 , Jenny May 2 , Craig Donaldson 2 and Ruth Morse 1 1 University of the West of England, Centre for Research in Biosciences, Bristol, UK; 2 Plymouth University, Centre for Research in Translational Biomedicine, Plymouth, UK Mesenchymal stem cells (MSCs) are cells involved in the formation of the bone marrow (BM) microenvironment, and are characterized by being CD105, CD166, CD44, CD73, CD90 and CD29 positive, but CD45 negative [1]. Current research conducted on traditional monolayer (2D) cultures indicate that the functionality of these cells are damaged by alkylating chemotherapeutic agents such as melphalan. This project aimed to evaluate the CD marker phenotype of the immortalized stromal cell line HS-5 in three different three dimensional (3D) systems (Biomerix, Alvetex and Nanofiber) compared with the 2D system. In addition, it aimed to compare the CD expression after exposing to the clinically relevant dose of melphalan (32.8 µM). The results showed different expression profiles of the positive CD markers (CD44, CD90, CD105 and CD73) for the untreated MSCs in the 3D cultures compared to the traditional cultures. There was a reduction in expression of almost 1% in CD44 and more than 16% in CD105 in all 3D systems, while a 5% reduction of CD90 was observed in Biomerix and Alvetex cultures only. Moreover, melphalan changed the expression level of these markers relative to the untreated for each environment where CD44 was elevated in the 2D cultures by almost 0.2% but significantly decreased in Alvetex by 16.5%. A significant reduction in CD105 was observed in 2D and 3D cultures after treatment (2D 58.5%, Biomerix 41.95%, Nanofiber 34.9% and Alvetex 39.8%). A major decrease in CD90 was observed in 2D environments by 27.4%, Nanofiber 18.1% and Alvetex 24.3%. Finally, CD73 expression reduced by 17.8% in Alvetex. These data demonstrate that guidelines on CD marker expression as a tool to characterize MSC must be reviewed in light of the observed changes in 2D and 3D cultures, taking into consideration that 3D culture are more likely to reflect the in-vivo situation. Reference 1. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D. and Horwitz, E. (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy,8 , 315–317. 45. A comparison of nano and bulk NSAIDs (aspirin and ibuprofen) in breast cancer patients and healthy individuals Osama Dandah 1 , Mojgan Najafzadeh 1 , Muhammad Isreb 1 , Adolf Baumgartner 1 , Richard Linforth 2 and Diana Anderson 1 1 University of Bradford, Bradford, UK; 2 Bradford Hospitals NHS Trust, Bradford, UK Various recent studies have suggested that regular intake of some non-steroidal anti-inflammatory drugs (NSAIDs) have a preventative effect against several types of tumours including breast cancer. This work studied the effect of both ibuprofen and aspirin on DNA damage using lymphocytes obtained from breast cancer patients and comparing the result with healthy females as a control. Nanoparticles (NPs) and bulk sizes were used in this study, in the comet and micronucleus assays. Two hundred and fifty ng/mL of ibuprofen (NPs and bulk) and 500ng/mL of aspirin were used to treat the lymphocytes. It was observed that, both aspirin and ibuprofen caused a reduction in DNA damage and micronucleus formation. Aspirin, both bulk and NP sizes, showed a significant reduction in DNA damage in the comet and micronucleus assays. However, the effect of aspirin NPs (P≤0.01) was more significant in DNA damage reduction compared to aspirin bulk (P≤0.05). Ibuprofen, on the other hand, showed a significant reduction in micronucleus frequency with the micronucleus assay (ibuprofen NPs (P≤0.001) versus, ibuprofen bulk (P≤0.01)), whilst its preventative effect with the comet was weak or insignificant. This observation suggests that NPs have better penetration through the nuclear membrane due to their smaller sizes compared to their bulk size. Finally, in both assays, aspirin was more effective than ibuprofen in the reduction of DNA damage and MNi formation in the comet and micronucleus assays respectively. NPs of both agents were more effective than bulk sizes. The results of this work are consistent with the view that NSAIDs, particularly aspirin and ibuprofen, could have a promising role in cancer treatment, including breast cancer. 46. A novel in-vitro approach to study effects of complex carcinogens ochratoxin A and urethane Jatin Verma, Eleanor Wilde, Kate Chapman, Shareen Doak, Gareth Jenkins and George Johnson Swansea University, Institute of Life Science, Swansea, UK Ochratoxin A (OTA) and urethane are known to induce tumours in rodents with genotoxicity as a primary mechanism. Furthermore altered cell-cycle, p53 down-regulation and apoptosis have been recorded in response to OTA and urethane. In contrast to in-vivo studies, negative or contradicting results have been reported in various in-vitro studies for these chemicals. In this study, genotoxicity testing was combined with cell signalling analysis in p53 competent cells in an attempt to improve in-vitro testing and elucidate cellular targets modulated by complex carcinogens. Lymphoblastoid cells TK6 and metabolically active MCL-5 were treated with OTA or Urethane for 4 or 23h and allowed to recover for 23 or 26h. The Cytokinesis-Block Micronucleus (CBMN) assay was used to assess genotoxicity. Western blotting was utilised to detect p53 and phospho-p53 protein abundance and gene expression changes were monitored using real-time PCR. Finally the MicroFlow™ approach was implemented to assess cell-cycle arrest. In the case of OTA, a significant increase in micronucleus (MN) was observed in TK6 cells at 40 and 45mM. Furthermore, a substantial increase in p53 and phospho-p53 protein abundance was seen in response to 35, 40 and 45mM OTA. Gene expression analysis showed a significant down-regulation in the expression of p21, SGK1 and TNFS9 at similar concentrations. However, no cell cycle arrest was seen in response to OTA. In contrast to OTA, Urethane did not cause significant increase in MN induction or alter cellular parameters such as p53 and phospho-p53 protein, cell cycle and changes in gene expression in MCL-5 cells. These results clearly highlight the issues in detecting the precise mechanism for complex carcinogen such as urethane. However this high-throughput multiplexed approach may be used to construct a reference library of in-vitro results, from chemicals with complicated mechanisms such as OTA. 47. An investigation into the potential disruption of mitosis by thalidomide and lenalidomide in human lymphoblast cell lines Jennifer Razik and Ruth Morse University of the West of England, Centre for Research in Biomedical Science, Bristol, UK Thalidomide (THAL) and lenalidomide (LEN) are used to treat multiple myeloma, however, prolonged use causes peripheral neuropathy and therapy-related malignancies [1]. THAL is thought to target microtubule structures present in nerve axons [2]. This study hypothesizes that THAL and LEN disrupt microtubule assembly, and subsequent spindle fibre formation during mitosis, giving rise to multinucleated cells and altered chromosome segregation, causing the therapy-related side effects. To investigate potential mitotic disruption, TK6 cells were exposed to THAL (20-500ng/ml) and LEN (1–8μM). Principles of the in-vitro micronucleus assay were employed to quantify multinucleation, apoptosis and necrosis with dose and time (3-96hours). A thymidine-block protocol was utilised to synchronise cells in M-phase, and Giemsa stained metaphase spreads were prepared to detect aneugenic potential of drugs, correlated with multi-nucleation presence. THAL and LEN exposed TK6 cells demonstrated multinucleation up to eight nuclei, with increasing frequency of multinucleated cells in a time and dose dependent manner (p<0.001). These cells survived up to a week post treatment. Control samples saw an average of 1 multinucleated cell at 72 hours, compared to 16 and 15 for THAL and LEN, respectively. In synchronised cells treated with THAL (200ng/ml) and LEN (4μM), several cells with anaphase bridges and three-way mitotic disruption were observed. Chromosome analysis found fluctuations in all samples, and demonstrated a peak of spreads with a chromosome number of 473. Average chromosome counts were 35 for controls (n=32), 52 for THAL (n=18) and 44 for LEN (n=16). Results suggest that THAL and LEN have a potential aneugenic effect. Observed survival of multinucleated cells, proposes that viable aneuploidy could explain the peripheral neuropathy and carcinogenesis seen in multiple myeloma patients. References 1. Koeppen, S. (2014) Treatment of multiple myeloma: thalidomide-, bortezomib-, and lenalidomide-induced peripheral neuropathy. Oncol. Res. Treat.,37 , 506–513. 2. Delforge, M., Bladé, J., Dimopoulos, M.A., Facon, T., Kropff, M., Ludwig, H., Palumbo, A., Van Damme, P., San-Miguel, J.F. and Sonneveld, P. (2010) Treatment-related peripheral neuropathy in multiple myeloma: the challenge continues. Lancet Oncol.,11 , 1086–1095. 48. Application of flow cytometry: scoring an in-vivo bone marrow micronucleus study in under 1 hour! Luke Foster, Darren Kidd, Chris Rothwell and Carol Beevers Covance Laboratories Ltd, Harrogate, GB In the 2014 revision to OECD Test Guideline 474 ( in-vivo micronucleus assay), the requirements for the number of immature (polychromatic, PCE) erythrocytes to be assessed for micronuclei (MN) was increased from 2000 to 4000 PCE. The impact of this change was an increase in both cost and time for MN analysis, which prompted us to consider flow cytometric methods for MN assessment in bone marrow. Flow cytometry categorises cell populations by their fluorescence profile; by using antibodies with fluorescent conjugates and/or dyes that fluoresce when activated, different fluorescent profiles can be assigned to each of numerous cell types in the same sample. By analysing more than 20,000 cells per second for multiple fluorescence profiles, a flow cytometer becomes a powerful analysis tool. Using four known genotoxicants with different mechanisms of action (cyclophosphamide [CPA], vinblastine [VIN], methyl methanesulfonate [MMS] and 5-fluorouracil [5-FU]), we assessed our proficiency in bone marrow sample preparation and analysis using flow cytometry by comparing the results with traditional MN analysis of bone marrow smears prepared from the same animals. For each sample, 20,000 PCEs were scored by flow cytometry and 4,000 PCEs scored by microscope analysis. As expected, all chemicals were positive, with both scoring methods demonstrating significant and dose-related linear increases in micronucleated PCEs. The results showed good concordance between the two methods (e.g. Vehicle [%MN-PCE = 0.35 (flow): 0.15 (microscope)] CPA [%MN-PCE = 2.91 (flow); 3.12 (microscope)]) and it is anticipated flow cytometry assessment could decrease assay turn-around time, strengthen data interpretation and increase flexibility in certain study designs. 49. Assessment of nanomaterial induced secondary genotoxic mechanisms using in-vitro airway models Stephen Evans 1 , Neenu Singh 1 , John Wills 1 , Nicole Hondow 2 , Tom Wilkinson 1 , Gareth Jenkins 1 and Shareen Doak 1 1 Swansea University, Institute of Life Science, Swansea, UK; 2 University of Leeds, Institute for Material Research, Leeds, UK Inhalation of nanomaterials is a substantial occupational exposure risk. Of vital consideration is deducing the chronic exposure risk to nanomaterials, namely their ability to promote the onset of genotoxicity. The majority of in-vitro studies assessing this risk have been based on mono-cellular cultures applied to evaluate primary genotoxic mechanisms. This eliminates the ability to explore secondary mechanisms of genotoxicity, such as those initiated immune cells. The purpose of this study therefore, was to elucidate the ability of both Fe 2 O 3 and Fe 3 O 4 iron oxide nanoparticles (SPION) (2–100 µg/mL) to promote secondary mechanisms of genotoxicity in lung-based 3D in-vitro models. Initially, 16HBE14o- bronchial monocultures were used as a baseline for a 3D co-culture model consisting of 16HBE14o- and THP-1 macrophages. The ability for the different nanomaterials to cause genotoxicity was measured via the promotion of chromosomal damage, quantified by the micronucleus assay. Cellular uptake was investigated by transmission electron microscopy. Monoculture treatments showed only Fe 2 O 3 was capable of promoting increased micronucleus frequency, the lowest observed effect being at 4 µg/mL, and increasing in a dose-dependant manner. In comparison co-cultures showed increased micronucleus frequency following exposure to both nanomaterials, the lowest observed effect at 10 µg/mL with a dose-dependent increase also seen. Macrophages within the co-culture system demonstrated uptake of both SPIONs, however only Fe 2 O 3 were observed to be present within the 16HBE14o- cells. Macrophage SPION internalisation consequently promoted oxidative radical production leading to oxidative DNA damage within 16HBE14o-. The use of alternative models in this study has clearly demonstrated the potential of SPION to promote DNA damage via secondary mechanisms through immune cell activation. The influence of immune cells therefore needs to be a vital consideration when assessing the genotoxic potential of a nanomaterial in-vitro . 50. Auranofin-induced genotoxicity and cytotoxicity positively correlates with membrane fluidity: a comparative study in ovarian cancer cell lines Deepu Oommen 1 , Nicholas Dodd 1 , Dennis Yiannakis 2 and Awadhesh Jha 1 1 Plymouth University, School of Biomedical and Biological Sciences, Plymouth, UK; 2 Derriford Hospital, Plymouth Oncology Centre, Plymouth, UK Auranofin, an organogold compound classified as an anti-rheumatic agent is under phase 2 clinical trials for re-purposing to treat recurrent epithelial ovarian cancer. We have reported earlier that Breast cancer, early onset (BRCA1) mutant ovarian cancer cells exhibit increased sensitivity to auranofin. BRCA1 is a DNA repair protein whose functional status is critical in the prognosis of ovarian cancer. Apart from DNA repair capability of cancer cells, membrane fluidity is also implicated in modulating resistance to chemotherapeutics. We report here that membrane fluidity influences the sensitivity of ovarian cancer cell lines (OVCAR5, OVCAR4, and IGROV1) to auranofin. Electron spin resonance (ESR) analysis revealed a more fluidized membrane in IGROV1 compared to OVCARs. Interestingly, IGROV1 cells were more sensitive to auranofin induced cytotoxicity than OVCARs. In comparison to OVCARs, IGROV1 cells also exhibited increased number of DNA double strand breaks (DSBs) upon auranofin treatment as assessed by 53BP1 immunostaining. Adding further, Pearson correlation analysis demonstrated a positive correlation (r = 0.855) between membrane fluidity and auranofin sensitivity in these cell lines. Anti-oxidant N-acetyl cysteine (NAC) inhibited the cytotoxic and genotoxic effects of auranofin in ovarian cancer cells suggesting reactive oxygen species (ROS) mediates the anti-cancer properties of auranofin. Taken together, our data suggest that ROS mediated auranofin cytotoxicity and genotoxicity in ovarian cancer cells positively correlates with membrane fluidity. 51. Bioaccumulation of polycyclic aromatic hydrocarbons and genotoxic effects in fish ( Mugil platanus ) from Cananéia Island, Southern Brazil Daniel Bussolaro, Gislaine de Fátima Filla and Flavia Duarte Ferraz Sampaio Instituto Federal do Paraná, Curitiba/PR, Brazil Estuaries are routinely exposed to pollutants from human activities originating from discharges of industrial, agricultural and urban wastes. Aquatic organisms, such as fish, accumulate pollutants directly from contaminated water and indirectly by feeding on contaminated aquatic organisms. Mugil platanus is an estuarine-dependent fish which can be found from shallow estuaries to deep offshore water. This species has been selected in this study because these fish spend some of their life stages near the shore and feed mainly on detritus and are largely consumed by human population as part of their diet. In order to investigate genotoxic effects and polycyclic aromatic hydrocarbons (PAHs) accumulation in these fish, ten adult specimens of Mugil platanus were caught in an intermediate-polluted area in the Cananéia Island -São Paulo State–Southern Brazil. Peripheral blood and bile samples were taken in order to perform micronucleus test and PAHs accumulation, respectively. Other nuclear abnormalities were also registered. PAHs in the bile were quantified via fixed-wavelength fluorescence in the spectrofluorometer (Sunrise-Tecan). PAHs (2-ringed, 4-ringed, and 5-ringed) were found in the bile of all fish studied and they are probably related to the nautical activities in the estuary. The presence of micronucleus was observed only in four individuals, however, other nuclear abnormalities (blebbed, lobed, vacuolated and notched) were found in all blood samples. The frequency of micronucleus (±2%) and nuclear abnormalities (±8%) was considered low in this study when compared to other studies using the same fish species. However, as the micronucleus test has the potential to detect clastogenic and aneugenic effects of genotoxic substances in aqueous media, these results could indicate the presence of genotoxic components besides PAHs in the estuary and that these organisms are reacting to the genotoxicants. 52. Butyrate alters bioactivation and genotoxicity of benzo[a]pyrene in colon cancer cell models Ondrej Zapletal 1 , Zuzana Tylichova 1 , Jiri Neca 2 , Jiri Kohoutek 2 , Miroslav Machala 2 , Alena Milcova 3 , Jan Topinka 3 , Jirina Hofmanova 1 , Alois Kozubik 1 and Jan Vondracek 1 1 Institute of Biophysics AS CR, Department of Cytokinetics, Brno, Czech Republic; 2 Veterinary Research Institute, Department of Chemistry and Toxicology, Brno, Czech Republic; 3 Institute of Experimental Medicine AS CR, Department of Genetic Ecotoxicology, Prague, Czech Republic The development of colorectal carcinoma (CRC), one of the most prevalent forms of cancer in the Western world, is affected not only by individual genetic background, but also by environmental factors. Butyrate, a short-chain fatty acid produced by fermentation of dietary fibre, is an important regulator of colonic epithelium homeostasis. Butyrate can act as an inhibitor of histone deacetylases (HDACs), thus altering both chromatin structure and gene expression. Benzo[a]pyrene (BaP), classified by the IARC as a group 1 human carcinogen, is an abundant PAH found in processed food. Like other PAHs, it must be bioactivated by cytochrome P450 family 1 (CYP1) enzymes, in order to yield genotoxic metabolites forming covalent DNA adducts. We found that sodium butyrate strongly potentiated the ligand-induced expression and activity of CYP1A1 in human colon cancer HCT116, AA/C1 and HT-29 cell lines. Importantly, this corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts by its ultimate genotoxic metabolite, anti-BPDE. The effects of butyrate on CYP1A1 expression and BaP metabolism were related to its impact on histone acetylation, since additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. Butyrate induced acetylation of histones H3 (at Lys14) and H4 (at Lys16), two histone modifications known to be modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This study suggests that butyrate, through modulation of histone acetylation, could significantly up-regulate CYP1A1, which may in turn modulate bioactivation and genotoxicity of carcinogenic compounds within colon epithelium. 53. Changes in gene expression in HHL-16 cells induced by aflatoxin B1 Ya Xu 1 , Yun Yun Gong 2 and Michael Routledge 1 1 University of Leeds, LICAMM, Leeds, UK; 2 Institute for Global Food Security, Queen’s University Belfast, Belfast, UK Aflatoxins, a group of naturally occurring contaminants, are produced by Aspergillus flavus and A. parasiticus , which are frequent contaminants of groundnuts, maize, rice and other grains, especially in Africa. Aflatoxin B1 (AFB1) has been confirmed as the most toxic type of aflatoxin and classified by the International Agency for Research on Cancer as a Group 1 human carcinogen, causing liver cancer. Aflatoxins have also been associated with growth impairment in children and immunosuppression in animal models. HHL-16 cells are a human hepatocyte line obtained from healthy human liver and retain the same phenotype as the primary hepatocytes. In the current study, the impact of aflatoxin exposure on gene expression in HHL-16 cells was investigated. Cells were grown in 75cm 2 flask to approximately 80% confluence and treated with 5, 20 and 30 µg/mL of AFB1 for 24 and 48h. Non-treated cells and cells treated with 0.1% DMSO were used as controls. RT-PCR was used to determine the expression of four genes ( CCL20, NF- κB, IL-6 and STAT3 ). After 24h treatment, significant dose-dependent increases of mRNA expression were found in all four genes, at all doses of AFB1, with increases in expression of over fivefold for CCL20 , up to fourfold for IL-6 and almost twofold for NF- κB and STAT3 . However, after 48h treatment, there was little difference in gene expression between treated and control cells, except for NF- κB . The changes in gene expression seen in the liver cells show that aflatoxins may transiently increase expression of genes involved in pathways related to inflammatory response ( CCL20, IL6 ) and cancer ( NF- κB, STAT3 ). 54. Characterisation of DNA damage induced by N-nitrosated spermine Liam T. Hanson 1 , David M. Williams 2 , Daniel R. Brison 3 and Andrew C. Povey 1 1 University of Manchester, Institute of Population Health, Manchester, UK; 2 University of Sheffield, Department of Chemistry, Sheffield, UK; 3 Univerisity of Manchester, Institute of Human Development, Manchester, UK Higher levels of spermatozoa DNA alkylation, in the form of N7-methylguanine, have previously been associated with male infertility. The increased levels suggest that DNA alkylation could be a significant contributor towards cases of male infertility, although the source of the alkylation is not identified [1]. Polyamines, such as spermine, in the seminal plasma can be N-nitrosated to give rise to a number of mutagenic species and the aim of this study was to examine the nature of DNA damage induced by N-nitrosated spermine. N-nitrosated spermine caused DNA strand breaks as detected using a supercoiled plasmid nicking assay. Strand breaks increased in a dose-dependent fashion from 5% at 0mM to 51% at 30mM of N-nitrosated spermine, spermine alone did not induce strand breaks above background. To characterise DNA base damage induce by N-nitrosated spermine, oligodeoxyribonucleotides were synthesised to contain a single O 6 -(3-aminopropyl)guanine adduct, a putative DNA adduct formed upon reaction with N-nitrosated spermine. This adduct is shown to bind to alkyl transferase-like protein 1 (Atl1) using an electrophoretic mobility shift assay (EMSA). In addition, this adduct is repaired by the DNA repair protein O 6 -methylguanine DNA methyltransferase (MGMT) using a tritium based assay measuring functional MGMT activity. The IC50 value of 21±6.4nM was higher than that of O 6 -methylguanine in an oligonucleotide of the same sequence. N-nitrosated spermine causes DNA damage and has the potential to cause O 6 -alkylguanine lesions that require further characterisation, but which may increase the load of O 6 -alkylguanines in sperm. Reference 1. Stocks, S.J., Agius, R.M., Cooley, N., Harrison, K.L., Brison, D.R., Horne, G., Gibbs, A. and Povey, A.C. (2010) Alkylation of sperm DNA is associated with male factor infertility and a reduction in the reduction in the proportion of oocytes fertilised during assisted reproduction. Mutation Res.,698 , 18–23. 55. Chemotherapy treatments induce oxidative DNA damage and reactive oxygen species in lymphoblast cell lines Louise Shilton, Kelechi Okeke and Ruth Morse University of the West of England, Centre for Research in Biosciences, Bristol, UK Reactive oxygen species (ROS) can oxidise DNA leaving lasting damage in the cell [1] and reduce chemotherapy effectiveness [2]. The lymphoblast cell lines AHH1, TK6 and TK6 with S9 were incubated with subclinical doses of chemotherapeutics, then assessed for ROS and 8-oxo-deoxyguanosine (8-oxo-dG). Cells were analysed by flow cytometry for 8-oxo-dG and by dichlorofluorescein-diacetate assay for ROS using potassium bromate (PB) and hydrogen peroxide (H 2 O 2 ) as positive controls respectively. AHH1 and TK6-S9 failed to produce a ROS standard curve through H 2 O 2 , whereas a standard curve was possible with TK6. However, comparison of fluorescence for drug exposed cells demonstrated higher ROS release with AHH1. Interestingly ROS for TK6-S9 was only slightly higher than for TK6, suggesting ROS release is not due to metabolic competence alone. Chlorambucil with AHH1 released the highest ROS over 20h. Drug mode of action correlated with 8-oxo-dG induction in AHH1, with crosslinkers producing the highest levels; interestingly the nitrosourea carmustine produced similar levels to PB, but cyclophosphamide, mechlorethamine and cisplatin induced about 20% higher fluorescence. The antimetabolites (cytosine arabinoside, hydroxyurea, 5-fluorouracil and methotrexate) and topoisomerase inhibitors (etoposide, mitoxantrone) produced similar levels of 8-oxo-dG in AHH1 to PB. All drugs induced 8-oxo-dG levels equivalent to or lower than PB in TK6 cells, with 5-fluorouracil and methotrexate producing the lowest level. Of note, baseline 8-oxo-dG in AHH1 cells was over 550% higher than baseline levels in TK6, but this was still doubled with drug and positive control treatment. It is clear that all chemotherapeutics have the capacity to induce oxidative DNA damage, even at subclinical doses, however further evaluation is required of the role of metabolic competence in order to fully assess the in-vivo risk. References 1. Cooke, M.S., Evans, M.D., Dizdaroglu, M. and Lunec, J. (2003) Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J.,17 ,1195-1214. 2. Conklin, K.A. (2004) Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integr. Cancer Ther.,3 , 294–300. 56. Coarse particulate matter induces a genotoxic and inflammatory response in human alveolar type 1 like TT1 cells Ian W.H. Jarvis 1,2 , Zachary Enlo-Scott 1 , Eszter Nagy 1 , Volker M. Arlt 1,2 and David H. Phillips 1,2 1 King’s College London, Division of Analytical and Environmental Sciences, London, UK; 2 NIHR HPRU in Health Impact of Environmental Hazards at King’s College London in Partnership with PHE, London, UK Many health effects associated with human exposure to airborne particulate matter (PM) are driven by genotoxic and inflammatory responses. Many PM-associated organic pollutants such as polycyclic aromatic hydrocarbons (PAHs) or nitro-PAHs are known or suspected human carcinogens. Nitro-PAHs are closely associated with diesel exhaust emissions, although they are not routinely monitored in environmental samples. The aim of this study was to assess the effects of coarse (2.5–10 µm) and fine (<2.5 µm) PM on DNA damage and inflammatory responses in comparison with benzo[a]pyrene (BaP) and 3-nitrobenzanthrone (3-NBA), a model PAH and nitro-PAH, respectively. Human alveolar type 1 like cells (TT1) were exposed to BaP (0–10 µg/ml [0–39.6 µM]), 3-NBA (0–1 µg/ml [0–3.6 µM]) or PM mixtures (10 µg/ml) for 24 hrs. Activation of DNA damage signalling was assessed by Western blotting and genotoxicity by the comet assay. Secretion of interleukins 6 (IL-6) and 8 (IL-8) was assessed by ELISA. TT1 cells were exposed to non-cytotoxic concentrations of BaP, 3-NBA and PM mixtures (confirmed by AlamarBlue assay). Exposure of TT1 cells to 3-NBA (>0.1 µg/ml [>0.4 µM]) caused a significant concentration-dependent increase in phosphorylation of Chk1 and H2AX. In agreement, 3-NBA caused a significant increase in genotoxicity; no DNA damage or effect on signalling was observed with BaP. Exposure to coarse PM caused genotoxicity and a significant increase in phosphorylation of Chk1, Chk2 and H2AX, coupled with a large increase in IL-8 secretion. In contrast, fine PM caused a lower but significant increase in IL-6 secretion, but no detectable effect regarding DNA damage. Taken together our data indicate that coarse PM exhibits higher genotoxicity than fine PM in lung cells and that the contribution of nitro-PAHs to mixture toxicity is an important consideration for future health risk assessment. 57. Combining micronucleus induction and nuclear morphology endpoints enable differentiation of carcinogens from non-carcinogens Eleanor Wilde, Kate Chapman, Anna Seager, George Johnson, Rowan Brown, James Tonkin, Shareen Doak and Gareth Jenkins Swansea University, Institute of Life Science, Swansea, UK Chromosomal defects or gene mutations are the most commonly used endpoint during in-vitro genotoxicity testing. However currently some misleading-positive results remain problematic during pharmaceutical drug development. This difficulty could be overcome by combining multiple endpoints to determine carcinogenic mechanisms. This project aims to improve in-vitro safety assessment, testing effects of known genotoxic carcinogens and non-carcinogens on DNA-damage, cell-cycle and cell phenotype.Human-lymphoblastoid TK6 cells were treated for 4 or 23h with the non-carcinogens 2,4-dichlorophenol (2,4-DP) and oestradiol and the genotoxic carcinogens methyl-methanesulfonate (MMS) and N-nitroso-N-methylurea (MNU). The Cytokinesis-Block Micronucleus (CBMN) Assay was then used to assess genotoxicity and changes in cell morphology were detected using high-throughput automated image analysis (InCell-Analyzer 2000). Cell-cycle alterations were assessed using a flow cytometric-based platform and p53/ phospho-p53 protein expression were analysed utilising Western Blotting. The non-carcinogens each showed a positive (but non-significant) trend in the CBMN Assay, despite being inconsistent between replicates. However, a significant MN induction was initiated by both MMS and MNU at 0.7 and 0.3 μg/mL (Dunnett’s t-test, p < 0.05). These genotoxic carcinogens also caused a G2/M phase cell-cycle block and an increased p53/ phospho-p53 protein abundance. Finally a significant decrease in nuclear solidity was seen in response to genotoxic carcinogens. In contrast to this the non-carcinogens caused an increasing trend in nuclear solidity. The contrasting effect on nuclear solidity and micronucleus induction for the genotoxic and the non-carcinogens provides a potential differentiating feature for these groups. The increase in genotoxicity shown by MMS and MNU then chimes with their increased p53 protein abundance and cell-cycle block, since these chemicals are known to cause G2/M cell-cycle arrest to initiate DNA repair/ apoptosis. Linking these data-sets could help to indicate how effects of specific compounds relate to their mechanisms of action, potentially improving genotoxic identification in-vitro . 58. Comparing the use of isolated peripheral lymphocytes and human whole blood in the comet assay before and after UVA+B exposure Mojgan Najafzadeh and Diana Anderson University of Bradford, School of Life Sciences, Division of Medical Sciences, Genetic and Reproductive Toxicology Group, Bradford, UK The comet assay is a sensitive method used to detect DNA damage in eukaryotic cells and can also measure DNA breaks and alkali labile lesions. For the last five decades or so, human peripheral lymphocytes have been used in many genotoxicological studies. In this study, the use of human lymphocytes was compared with whole blood in the alkaline gel electrophoresis method. Two hundred and seventy blood samples from individuals: 120 healthy individuals, 65 suspected or pre-cancerous individuals and 85 cancer patients. Each blood sample was divided into two identical volumes in different falcon tubes. The lymphocytes were isolated from one tube and frozen at-80°C, and whole blood was prepared and stored by adding the same amount of RPMI and 1% DMSO. The cells were exposed to UVA+B with the intensity ~ 1.20 mW/cm 2 for 15min and the results were compared to results without treatment in the comet assay. After applying the Student’s t-Test, the data showed a p value = 0.59 for Olive tail moment (OTM) and a p value= 0.16 for % tail DNA, with no statistically significant differences between the two methods. Also, in both methods after inducing DNA damage by exposing the cells to UVA+B, there was a significant difference by comparison with unexposed cells (p value = 0.01), which showed the response to UVA+B in WBC from whole blood is the same as in the isolated lymphocytes from the different groups of the study. In conclusion, using whole blood instead of isolated lymphocytes saves time, is still very sensitive and requires less than 20 µL of blood from each individual. 59. Oxaliplatin and vinflunine inhibit survivin expression while inducing cytogenetic damage in-vitro in lymphocytes from colon cancer patients and healthy individuals Mojgan Najafzadeh 1 , Amal A.A. Alotaibi 1 , Justin Davies 2 , Adolf Baumgartner 1 and Diana Anderson 1 1 University of Bradford, Bradford, UK; 2 Bradford Hospitals NHS Trust, Bradford, UK Chemotherapy drugs usually inflict a lethal dose to tumour cells with the consequence that these cells are being killed by cell death. However each round of chemotherapy also inflicts damage to normal somatic cells. The DNA cross-linking agent oxaliplatin which causes DNA double-strand breaks and vinflunine which disrupts the mitotic spindle are two of these chemotherapy drugs which were evaluated in-vitro using peripheral lymphocytes from colorectal cancer patients and healthy individuals to determine any differential response. Endpoints examined included micronucleus (MN) induction using the cytokinesis-blocked micronucleus (CBMN) assay and pancentromeric fluorescence in situ hybridisation. Also survivin expression was monitored since it regulates the mitotic spindle checkpoint and inhibits apoptosis. MN induction was greater in lymphocytes from cancer patients than those from healthy individuals. Oxaliplatin produced micronuclei in binucleated cells via its clastogenic and aneugenic action whilst vinflunine produced micronuclei in mononucleated cells because of incomplete karyokinesis. Survivin expression was found to be reduced / down-regulated in a concentration-dependent manner not only by oxaliplatin but also by vinflunine. This was observed in the large numbers of multinucleated cells found with the CBMN assay. As survivin is upregulated in cancers, eliminating apoptosis inhibition might provide a more targeted chemotherapy approach; particularly, when considering vinflunine, which only affects cycling cells by inhibiting their mitotic spindle, possibly alongside with other pro-apoptotic compounds. With this newly found property of vinflunine also inhibiting survivin expression, it might not inflict the amount of DNA damage to normal somatic cells that a more generally acting compound does. 60. Correlating the cytotoxic and genotoxic potential of few-layer graphene flakes with their physico-chemical features Michael J. Burgum 1 , Stephen J. Evans 1 , Alex Lord 3 , Sandra Bustamante-Lopez 3 , Afshin Tarat 2 , Kenith E. Meissner 3 , Martin J.D. Clift 1 and Shareen H. Doak 1 1 Swansea University, Institute of Life Sciences, Swansea, UK; 2 Swansea University, Centre for NanoHealth, Swansea, UK; 3 Swansea University, College of Engineering, Swansea, UK Few-layer graphene (FLG), defined as possessing three or more atomic layers of graphene, is a revolutionary material stronger than diamond and more conductive than copper. Applications include touch-screen technology, enhanced properties composites reinforced with FLG, resistive heaters and biosensors. Yet, despite the high demand, resulting in an increasing occupational exposure, there is a limited understanding as to the potential human health risks associated with inhalatory exposure to FLG. Thus, the present study sought to assess the toxicological impact of 700m 2 /g FLG flakes engineered with (i) no specific functionality, (ii) amine groups or (iii) carboxyl groups. Each FLG was then exposed to a representative human airway epithelial monoculture of 16HBE140- cells. Size and morphology of the flakes were characterised with scanning electron microscopy (SEM) and atomic force microscopy (AFM), lateral dimensions of the flakes measured 3–5 µm and possessed a depth of <450nm. Cell viability and chromosomal damage were assessed by relative population doubling (RPD) and the cytokinesis block micronucleus (CBMN) assay respectively. A one-way analysis of variance (ANOVA) with post hoc Dunnett’s test (P<0.05) was applied to evaluate statistical significance. FLG induced no significant cytotoxicity at any of the tested concentrations (0–100 µg/ml). In contrast, at sub-lethal concentrations, significant genotoxicity was induced by both non- and amine-functionalised FLG from 20 µg/ml. The non-functionalised FLG showing a two-fold increase over the control whilst the carboxyl-functionalised FLG induced a significant response at 100 µg/ml. Genotoxicity at sub-lethal concentrations suggests FLG deposition in the airway model may induce adverse biological responses such as reactive oxygen species (ROS) production that could result in DNA damage, the level of which is dependent upon the surface chemistry. 61. Cytochrome b 5 impacts on cytochrome P450-mediated metabolism and DNA adduct formation by benzo[a]pyrene: studies in Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice Lindsay Reed 1 , Iveta Mrizova 2 , Frantisek Barta 2 , Radek Indra 2 , Michaela Moserova 2 , Colin Henderson 3 , Marie Stiborova 2 , David H. Phillips 1 and Volker M. Arlt 1 1 King’s College London, Analytical and Environmental Sciences Division, London, UK; 2 Charles University, Department of Biochemistry, Prague, Czech Republic; 3 University of Dundee, Division of Cancer Research, Dundee, UK Benzo[a]pyrene (BaP) is an environmental carcinogen that exerts its effects after metabolic activation by cytochrome P450 (CYP) enzymes, particularly CYP1A. Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice were used to study the role of P450-mediated bioactivation of BaP. In HRN mice cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 (Cyb 5 ), which can also act as an electron donor from cytochrome b 5 reductase to P450 enzymes, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated i.p. with 125mg/kg body weight of BaP for 24h. BaP-DNA adduct levels were measured using 32 P-postlabelling. Hepatic microsomal fractions were used to study BaP metabolism and BaP-DNA adduct formation in-vitro using NADPH and NADH as cofactors. BaP metabolites formed in-vitro were determined by HPLC analysis. Cyp1a enzyme activity in hepatic microsomes was measured using EROD and Sudan I oxidation. BaP-DNA adduct levels were ~7-fold higher in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. In contrast, BaP-DNA adduct formation in-vitro was significantly lower with microsomal fractions from HRN (~2-fold) or HBRN mice (~7-fold) than with those of WT mice, which correlated with total BaP metabolite formation and Cyp1a activity when NADPH was used as cofactor. Our results in WT and HBRN mice demonstrate that deletion of Cyb 5 on a POR null background does not alter P450-mediated BaP bioactivation in the livers of HBRN mice in-vivo . Results using hepatic microsomal fractions from HRN and HBRN mice confirmed the importance of Cyb 5 in catalysing P450-mediated activation of BaP in-vitro . 62. Detection and single-cell sequencing of rat bone marrow-derived Pig-a mutant erythroid cells Javier Revollo 1 , Daniel Roberts 2,3 , Laura Custer 2 and Vasily Dobrovolsky 1 1 National Center for Toxicological Research/FDA, Jefferson, AR, USA; 2 Bristol Myers Squibb, New York, NY, USA; 3 Rutgers University, Joint Graduate Program of Toxicology, New Brunswick, NJ, USA The flow cytometry-based Pig-a assay detects cells deficient in GPI-anchored surface markers (e.g., CD59) and provides a rapid and cost-efficient enumeration of cells that are presumed to contain mutations in the endogenous X-linked Pig-a gene. The red blood cell (RBC)-based Pig-a assay is a promising tool for regulatory safety assessments because of its remarkable sensitivity for detecting potential mutagens. While the link between the measured mutant phenotype and actual Pig-a mutant genotype has been established in lymphocytes, this link is lacking in nucleic acid-free RBCs. To determine whether CD59-deficiency in RBCs is caused by Pig-a gene mutation, we established a Pig-a assay for bone marrow erythroid cells, the direct DNA-containing precursors of RBCs. Sequencing genomic Pig a of single erythroid cells derived from ENU-treated rats suggests that CD59(-) erythroid cells do carry mutations in the Pig-a gene. Importantly, these mutations are consistent with types of sequence changes induced by ENU. No mutations were found in the Pig-a gene of sorted CD59(+) cells. Overall, our data support the hypothesis that GPI-anchored marker-deficient peripheral RBCs are derived from precursor cells having Pig-a gene mutation, which further supports the hypothesis that the phenotypic RBC-based Pig-a assay detects bona fide Pig-a gene mutation. 63. Determining the mechanism of chromatin remodelling in GC-NER demonstrates that DNA repair is organised in the yeast genome Shuvro Prokash Nandi, Patrick van Eijk and Simon H. Reed Cardiff University, Cardiff, UK Whole genome sequencing of cancer genomes is revealing the non-random nature of somatic mutations acquired during tumorigenesis. We are investigating how nucleotide excision repair (NER) is organised in the genome to establish how genomic DNA repair rates contribute to this. We measured the distribution of NER rates for UV-induced lesions throughout the yeast genome in the context of gene structure and Abf1 binding sites. We reveal that in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organised within the genome. We have shown that GG-NER is organised and initiated from Abf1 binding sites within the yeast genome. To get a nucleotide resolution map of Abf1 binding sites throughout the genome we performed Abf1 ChIP-Seq of wild-type cells. We found around 4,000 Abf1 binding sites that indicate Abf1 is widely distributed throughout the yeast genome, in line with previously published data. To understand DNA repair associated chromatin remodelling in response to UV irradiation, we also measured genome-wide nucleosome positioning using MNase-Seq in wild-type and RAD16 deleted cells and noted no role for Rad16 in nucleosome positioning, either before or after UV irradiation. We conclude that the GG-NER complex does not promote wholesale nucleosome sliding during chromatin remodelling. However, these observations do not rule out the possibility that the GG-NER complex might determine the UV-induced recruitment of other chromatin remodellers such as SWI/SNF and INO80, whose function could include UV-induced nucleosome sliding at specific regions of the genome. These observations may have important implications for the effect of histone and chromatin modifiers on the distribution of mutations acquired throughout the genome. 64. Development of a method for the in-vitro Pig-a assay in L5178Y cells Bethany Allen, Rhiannon David and Ann Doherty AstraZeneca, Discovery Safety, Cambridge, UK Assessing candidate drugs for genotoxicity is a key part of pharmaceutical safety testing. The Pig-a protein is a catalytic subunit of N-acetylglucosaminyltransferase, an enzyme essential for GPI-anchor synthesis. Mutations in the hemizygous Pig-a gene result in GPI-anchor loss, measured by the absence of GPI-linked cell surface proteins. An in- vivo Pig-a assay has been developed, and an in-vitro assay may complement this and represent a useful tool in genetic toxicity testing. Therefore, the aim of the current project was to develop an in-vitro Pig-a assay in L5178Y mouse lymphoma cells. To develop the assay, cells were treated with a dose range (1.5–4.5mM) of ethyl methanesulfonate (EMS) or media (negative control) for 24h, and the loss of GPI anchor determined by measuring the presence of the GPI-anchored protein CD90.2 and the stably expressed protein CD45 (to prevent measurement of non-specific cell-surface alterations) using flow cytometry. The following parameters were tested and optimised: antibody concentration (0.05–0.5 µg/mL CD90.2; 0.25–1 µg/mL CD45) and incubation period (2x15min vs 1x30min), phenotypic expression period (2–14 days), inclusion of a viability marker (7-AAD vs Zombie Violet), and analysis of fresh compared to fixed samples. The results showed that 2x15min incubations gave increased staining over 1x30min with 0.25 µg/mL of both CD90.2 and CD45 optimum. For optimal phenotypic expression, it was found that cells should be maintained for 8 days. Results from fixed cells (1% formaldehyde) were not different from freshly stained samples. The viability marker Zombie Violet was used (at 0.5% for 30min) rather than 7-AAD to reduce spectral overlap. Analysis of EMS-treated cells using this method showed a dose-dependent increase in the percentage of GPI(-) mutant cells of the total viable population from 0.034% (untreated) to 0.184% (4.5mM). In conclusion, an in-vitro Pig-a assay has been developed, which has the potential to complement established genotoxicity assays. 65. Epigenetic effects of polycyclic aromatic hydrocarbons Francesca Galea 1 , Paolo Vineis 1 , James Flanagan 2 and Michelle Plusquin 1 1 Imperial College London, Department of Epidemiology and Biostatistics, London, UK; 2 Imperial College London, Department of Surgery and Cancer, London, UK Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants formed from the incomplete combustion of organic substances. Human exposure to these compounds results in downstream mutagenic and carcinogenic consequences with the primary sources of exposure being the diet in non-smokers, tobacco smoke in smokers, and lastly inhalation of air pollution. The hypothesis for this study is that PAHs may induce epigenetic changes which may play a role in PAH-induced carcinogenesis. To achieve this, an epigenome-wide association study (EWAS) was carried out in a cohort of 159 women from the Dutch European Prospective Investigation into Cancer and Nutrition (EPIC) study. Estimates for the concentrations of a sum of 8 PAHs at the home address of each subject were calculated using land-use regression models built in the European Study of Cohorts for Air Pollution Effects (ESCAPE) project (exposure range: 0.56 – 2.26ng/m 3 ΣPAHs). Using a beta regression model, 64 CpG sites were found to be differentially methylated as a consequence of PAH exposure at a Bonferroni 5% cut-off level (p < 1.44 x 10 –7 ). Of these, 38 were found to be hypomethylated and the remaining 26 hypermethylated. Transcription-related roles were found to be the most common function of the genes associated with these CpG sites, as well as apoptosis, cell cycle and p53 regulation, angiogenesis, and tumorigenesis. Seven of these differentially methylated CpG sites are associated with gene promoters, while expression of TMEM33 (hypomethylation) and TRIM4 (hypermethylation) may be altered by PAHs. The functions for these genes are not well-established but the latter is thought to be involved in cellular protein localisation and assembly of the nuclear pore complex. Future work will seek to replicate these findings in a second cohort and dietary PAH exposure estimates. 66. Evaluation of DNA damage response of chemotherapeutic drugs and environmental mutagens in human induced pluripotent stem cells for whole genome sequencing Madeleine Joel, Jill E. Kucab, Volker M. Arlt and David H. Phillips King’s College London, Analytical and Environmental Sciences Division, London, UK DNA damage can cause permanent changes to the genome (e.g. mutations) if not repaired. The DNA damage response (DDR) is an integrated signalling network to ensure genome stability in response to DNA damaging agents. Many proteins are phosphorylated during DDR (e.g. CHK1, CHK2, p53, H2AX), coordinating an outcome of cell cycle arrest, DNA repair or apoptosis in the case of severe genomic damage. We examined cytotoxicity and DNA damage induction in human induced pluripotent stem (hiPS) cells treated with DNA damaging chemotherapeutics (i.e. cisplatin [CIPL], cyclophosphamide [CP], melphalan [MPH], mitomycin C [MMC], temozolomide [TMZ]) and environmental carcinogens (i.e. benzo[a]pyrene [BaP] and its activated metabolite BaP-7,8-diol-9,10-epoxide [BPDE]). Whole genome mutation analysis of treated hiPS cells will subsequently be performed and mutational signatures will be compared to those found in human tumours to study cancer aetiology. All compounds except CP and BaP induced concentration-dependent cytotoxicity. As BPDE was highly cytotoxic, this indicated that hiPS cells lack the metabolic capacity (e.g. cytochrome P450 enzymes) to activate agents like BaP to reactive intermediates. By co-treating the hiPS cells with an optimised exogenous rat liver S9 system, the metabolic activation of CP and BaP was achieved and cytotoxicity was observed. Induction of DDR or apoptosis after treatment was assessed by Western blotting for the protein expression of p-H2AX (S139), p-CHK1 (S345), p-CHK2 (T68), p53, p-p53 (S15), p21 and cleaved PARP. All DDR proteins were expressed or induced for CIPL, MPH, MMC, TMZ and BPDE in the absence of S9 and for CP and BaP in the presence of S9. After identifying a concentration of each compound that resulted in 40–60% viability and induction of DDR, pools of hiPS cells were treated with the test agents for whole genome mutation analysis, and are currently being processed. 67. Exposing iron oxide nanoparticles to an in-vitro liver 3D model to assess nano(geno)toxicity Jefferson de Oliveira Mallia 1,2 , Neenu Singh 1 , Sylvain Bohic 2 , Ume-Kulsoom Shah 1 , Gareth Jenkins 1 and Shareen H. Doak 1 1 Swansea University, In-vitro Toxicology Group, Swansea, UK; 2 European Synchrotron Radiation Facility INSERM U-836 Equipe 6, Grenoble, France Novel nanomaterials are being developed and used across various fields causing further need to investigate and assess possible toxicity pathways. Sites of nanomaterial accumulation, such as the liver, are areas of higher risk and greater concern. The hepatic 3D in-vitro model consists of HepG2 spheroids grown using the hanging drop method. Initially surface area, cell viability and metabolite secretion (albumin, urea and aspartate transaminase) were assessed to evaluate their growth and metabolic characteristics throughout a 7 day period. Urea and aspartate transaminase secretion increased gradually as the days progressed and a 100-fold higher than the expression in 2D HepG2 cells by day 7. A cytokinesis block micronucleus assay was integrated with this model, to assess magnetite (Fe 3 O 4 ) and maghemite (γ-Fe 2 O 3 ) nanoparticle genotoxicity after 24h of exposure. Spheroids were exposed to these nanomaterials after 4 days of growth due to the optimal growth and metabolic characteristics observed. Mitochondrial respiration was assessed for this period using a Seahorse XF Cell Mito Stress test. Micronucleus formation increased by 1.68 fold for the 1 µg/mL maghemite dose with respect to control and in the absence of cytotoxicity. Scanning electron microscopy and synchrotron X-ray fluorescence were used to assess the distribution of these nanoparticles. It was observed that the maghemite nanoparticles accumulated mostly at the periphery of the spheroids tissues. As spheroid microtissues more closely resemble human tissues than 2D cell cultures, the nanoparticle genotoxic responses observed are likely to be more representative. This 3D model therefore provides an improved and more realistic test environment to conduct in-vitro nano(geno)toxicity studies. 68. Extended treatment with recovery is required for detection of certain chemicals in the human peripheral blood lymphocyte micronucleus assay Teresa Chirom, James Whitwell, Robert Smith, Debbie Wood, Heather Lyon, Sarah Phillips and Julie Clements Covance Laboratories Ltd., Department of Genetic Toxicology, Harrogate, UK The in-vitro micronucleus (IVMN) test was endorsed for regulatory genotoxicity testing with the adoption of OECD TG 487 in 2010. This guideline included two equally acceptable options for extended treatment in the absence of metabolic activation: a treatment for 1.5–2.0 cell cycles with harvest at the end of treatment (Option A) or treatment for 1.5–2.0 cell cycles followed by a recovery for 1.5–2.0 cell cycles prior to harvest (Option B). Although no preferences were discussed, TG 487 warned that Option B may not be appropriate for human lymphocytes (HuLy) where exponential growth may be declining at 96h post PHA stimulation. With the revision of the TG 487 in 2014, a strong emphasis has been placed on performing extended treatments without recovery (Option A) with Option B only used where justified. However, we have shown that an extended treatment without recovery is not optimal for sensitive detection of certain classes of chemical. By measuring replication index (with cytochalasin B) and average generation time (via BrdU incorporation) we show that HuLy are actively dividing beyond 96h PHA stimulation and therefore appropriate for the extended treatments with recovery (Option B). We have added to our previous work using both Option A and Option B treatment schedules and further demonstrate that some aneugens and nucleoside analogues are only detected in HPBL following extended treatment with a recovery period. In addition, the inclusion of a recovery period generally results in a reduced toxicity gradient, making selection of suitable concentrations (within acceptable toxicity limits) for MN analysis more practical. 69. Fluence of low level laser therapy affects proliferation and DNA damage in THP-1 cells Ruwaidah A. Mussttaf 1 , Awadhesh Jha 2 and David Jenkins 1 1 Plymouth University, School of Computing, Electronics and Mathematics, Plymouth, UK; 2 Plymouth University, School of Biological Sciences, Plymouth, UK Low-level laser therapy (LLLT) is a form of phototherapy used to promote wound healing, inflammation and pain reduction in different clinical conditions. LLLT at the correct wavelength, intensity, and dose can accelerate tissue repair. However, there is still conflicting information about the effect of multiple irradiations on the cellular response of wounded cells. This study investigates the potential role of LLLT in cellular proliferation of human monocytic leukaemia cells (THP-1) under in-vitro conditions. Cells were irradiated with an 850nm diode laser using different powers (0 -11 mW) and different exposure times (0.3 ̶ 15min) to produce energy densities (fluence or dose) of 0–31 J/cm 2 . After irradiation, cells were incubated for 12h and 24h to allow time for proliferation. Comet assay was conducted to evaluate genotoxicity of the irradiated cells. Trypan blue was used to estimate cytotoxicity, which peaked at the highest dose as expected. Preliminary results suggest that cell counts increase at intermediate doses, whereas a decrease in cell number at high exposure time was noted compared to controls. Comet assay showed no significant difference between irradiated and non-irradiated cells at low doses. In contrast, DNA damage increased at doses ≥ 10.37 J/cm 2 and was comparable to the 100 μM H 2 O 2 positive control at the highest fluence. It could be concluded that LLLT has the ability to stimulate the THP-1 cell line to proliferate if supplied with the correct energy and dose. It is interesting to note that below the threshold of 0.1 J/cm 2 the cells are not stimulated to proliferate, therefore further investigation into optimal conditions for cell stimulation is needed. 70. Generation of a novel bioluminescent biosensor for therapeutic drug monitoring of acquired resistance to Zidovudine (AZT) in HIV treatment Wijnand Ng 1 , Finn Padmore 1 , Jennifer May 1 , Gareth Robinson 1 , Cherith Reid 2 and Elizabeth Anderson 1 1 University of the West of England, Centre for Research in Biosciences, Bristol, UK; 2 Randox Laboratories, Crumlin, County Antrim, UK Zidovudine (AZT), when administered as Combined Antiretroviral Therapy (cART) faces eventual resistance, and 50% of HIV patients will move from first-line cART within two years. UNAIDS-Lancet Commission pose insufficiency in patient monitoring an undermining factor in the United Nations’ aim to eradicate HIV as a threat to public health by 2030 [1]. AZT passes into the cytosolic compartment in a prodrug form, either by passive diffusion or active uptake by sodium-coupled-nucleoside-transporters and is sequentially phosphorylated by cellular kinases to active AZT-triphosphate (AZT-TP). Non-uptake, non-metabolism, mis-metabolism or efflux of the drug by multidrug resistance pumps at any stage renders the patient resistant, and no anti-viral effect will be observed. This study evaluated two bioluminescent bacterial biosensors ( E.coli HA-1 and KAB-1) for monitoring of AZT-TP generation and prediction of drug resistance in a lymphoblastic cell line, TK6. Biosensor E. coli HA-1 was designed for assessment of drug efficacy of the pyrimidine analogue cytarabine (Ara-C) in treatment of acute myeloid leukaemia. E.coli KAB-1 was derived from HA-1 to increase specificity & sensitivity to thymidine-based drugs such as AZT. E.coli HA-1 showed a bactericidal trend under exposure to AZT, while E.coli KAB-1 showed bacteriostatic trends. Both biosensors were able to provide a minimum 337-fold increase in concomitant bioluminescence, indicating sensitivity to AZT at clinically-relevant doses. Exposure of supernatants of lysed TK6 cells prior treated with AZT for 1h to both biosensors in the absence and presence of exogenously-added alkaline phosphatase produced a differential increase in bioluminescence that was indicative of AZT-TP generation within these cells. With further refinement, results show potential for the provision of a bacterial biosensor able to provide clinical results within a working day, reducing patient risk of remaining on a highly cytotoxic treatment regimen lacking efficacy for 15 million patients worldwide [2]. References 1. Piot, P., Karim, S., Hecht, R., Legido-Quigley, H., Buse, K., Stover, J., Resch, S., Ryckman, T., Møgedal, S., Dybul, M., Goosby, E., Watts, C., Kilonzo, N., McManus, J. and Sidibé, M. on behalf of the UNAIDS-Lancet Commission. (2015) Defeating AIDS advancing global health. Lancet,386 , 171–218. 2. Granich, R., Williams, B. and Montaner, J. (2013) Fifteen million people on antiretroviral treatment by 2015: treatment as prevention. Curr. Opinion HIV AIDS,8 , 41–49. 71. Genotoxic evaluation of nano- and bulk forms of aspirin and ibuprofen in lymphocytes from prostate cancer patients and healthy individuals Azeza Guma, Mojgan Najafzadeh, Muhammad Isreb, Adi Baumgartner and Diana Anderson University of Bradford, Medical Sciences, Bradford, UK Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly used over-the-counter medications worldwide. All NSAIDs act through inhibiting COX enzyme activity. In recent years, inflammation has been found to play a major role in cancer development. Therefore, the anti-inflammatory properties of these drugs might play an indirect role in the suppression of cancer by inhibiting inflammatory processes. The past decade saw a huge development in nano-biotechnology that uses nanoparticles of 10 -1000nm in size that have different physico-chemical properties compared to the large particles of the respective bulk compound. Hence, this makes it a potential alternative for cancer treatment. This study investigated the effects of nanoparticle and bulk forms of both aspirin and ibuprofen, in peripheral blood lymphocytes of prostate cancer patients and healthy volunteers using the comet and micronucleus assay. Nano-forms of aspirin and ibuprofen did not induce any damage but even showed a trend towards a decrease in DNA damage. Micronuclei (MNi) decreased after aspirin nano (ASP N) and aspirin bulk ASP B in the healthy individual and prostate cancer patients. These findings suggests that the nanoform of aspirin does not cause DNA damage and can possibly facilitate repair. However, other events are possible, hence the need for testing more in the future. Treating cancer patients with the nanoform of aspirin instead of the conventional aspirin might be more beneficial. 72. Genotoxic potential of chemotherapeutic agents in an in-vitro bystander model Kelechi Okeke, Myra Conway and Ruth Morse University of the West of England, Bristol, UK A long-term complication post stem cell transplant is malignancy in donated cells. This suggests that the microenvironment can induce genotoxicity through a bystander effect [1–3]. To investigate this possibility, the stromal cell line HS-5 was exposed for 1hr to clinically relevant doses of 22 chemotherapeutic agents with different modes of action then co-cultured with lymphoblast cells (TK6 or AHH1) for 24h. Bystander cells were assessed for viability and genotoxicity (micronucleus [MN] and comet assay). Cell viability was comparable to controls for AHH1, with proliferation increased for alkylating agents and antimetabolites, but reduced for antibiotics and plant alkaloids. Viability and proliferation was non-significantly reduced in TK6. All drugs increased average MN per 2000 cells in both AHH1 and TK6, with average MN higher in AHH1 than TK6; of note baseline MN in AHH1 were approximately 50% higher than TK6. Only Ara-C, daunorubicin and irinotecan did not increase MN between TK6 and AHH1. Alkylating agents and antimetabolites were consistent in MN induction within groups, whereas antibiotics, topoisomerase inhibitors and plant alkaloids were variable in response. Within the comet assay, 5 drugs for TK6 (hydroxyurea, bleomycin, mitoxantrone, teniposide, vincristine) increased percentage DNA in the tail (p<0.0001), whereas 12 drugs increased the tail with AHH1 (p<0.001). For TK6, notable positives were the topoisomerase inhibitors, whereas for AHH1 these were the antibiotics. Data from the MN assay strongly support a genotoxic effect for all chemotherapeutics, but different outcomes for the comet assay suggest differing modes of action to induce bystander. It is currently unknown how this phenomenon occurs, however the data from these assays will help interpret the exposure risk to patients. References 1. Merle, P., Morvan, D., Caillaud, D. and Demidem, A. (2008) Chemotherapy-induced bystander effect in response to several chloroethylnitrosoureas: an origin independent of DNA damage? Anticancer Res.,28 , 21–28. 2. Asur, R.S., Thomas, R.A. and Tucker, J.D. (2009) Mutation Res.676 , 11–16. 3. Alexandre, J., Hu, Y., Lu, W., Pelicano, H. and Huang, P. (2007) Novel action of paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species. Cancer Res.,67 , 3512–3517. 73. Genotoxicity assessment of environmental pollutants in the invertebrate model Caenorhabditis elegans Mustafa Abbass, Volker M. Arlt and Stephen Stürzenbaum King’s College London, Analytical and Environmental Sciences Division, London, UK Chemicals are an essential component of most aspects of modern life, but chemical exposures can also pose a risk to health. Induction of genetic damage is widely recognised as a key component of cancer initiation. Invertebrate models such as the nematode Caenorhabditis elegans have been shown to be a useful model for studying toxicological responses to environmental pollutants at the molecular level as well as at the level of the organism. In this study, C. elegans was used to assess the genotoxicity of the environmental pollutant benzo[a]pyrene (BaP). Wild-type C. elegans worms were exposed to BaP, added via their food source E. coli bacteria (OP50), at concentrations of 0.1, 1, 5, 10, and 20 µM. Nematodes were stained with Nile Red (which stains intracellular lipid droplets) and LysoTracker ® -Green (which stains acidic compartments, predominantly lysosomes) vital dyes and fluorescent microscopy was utilised to assess changes in lysosomal morphology. The single-cell gel electrophoresis (comet) assay was used to assess BaP-induced DNA damage. Our results suggest that exposure to BaP diminishes the lysosomal lipid levels and dose-dependently increases the number of lysosomes. A method for cell isolation from the nematodes was established and optimised for use in the comet assay. A 4-fold induction of DNA damage (i.e. increase in % tail DNA) was observed in cells isolated from nematodes exposed to BaP (compared to untreated controls). This demonstrates that BaP induces genotoxicity in C. elegans . However, the precise mechanism(s) and dose-responsiveness by which BaP damages DNA in C. elegans remain to be determined. Our study also revealed that BaP exposure impacts on the lysosomes manifesting as a novel marker of BaP toxicity in C. elegans . These studies will be combined with toxicogenomic approaches (e.g. gene expression analysis) in order to define the relationship between toxicological and phenotypic end-points and identifying mechanism of toxicity. 74. In-vitro cytotoxic assessment of single and binary combinations of mycotoxins and persistent organochloride pesticides in MA-10 murine Leydig cell Line Ukpai Eze 1,2 , Lisa Connolly 1 , Sheena Lewis 2 and Yun Yun Gong 1 1 Queen’s University Belfast, Institute for Global Food Security, Belfast, UK; 2 Queen’s University Belfast, Institute of Pathology, Belfast, UK There is increasing evidence for the deterioration of male sperm quality worldwide. Of emerging concern is the contribution of environmental and food toxins, especially mycotoxins and persistent organochlorides, in causing developmental and reproductive disorders in males. Currently, toxicity studies evaluating mixtures of contaminants are often restricted to their respective contaminant group thereby limiting the study of complex mixtures of environmental and food contaminants from different groups. However, humans and animals are simultaneously exposed to mixtures of several groups of environmental and food toxicants and it is necessary to consider the impact of combined effects. Here, the MA-10 murine Leydig cell line was used as a model to evaluate the cytotoxic effects of mycotoxins and persistent organochloride pesticides on Leydig cells. The cytotoxic effects of single and binary combinations of aflatoxin B1 (AFB1), ochratoxin A (OTA), deoxynivalenol (DON) and zearalenone (ZEN), dichlorodiphenyltrichloroethane (DDT) and dichlorodiphenyldichloroethylene (DDE) on MA-10 Leydig cell line were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-dipenyltetrazolium bromide (MTT) assay after 48h of exposure. Individual toxicity showed that ZEN was the most cytotoxic, significantly decreasing cell viability in a dose-dependent manner while DDT and DDE significantly increased cell viability in a dose-dependent manner. In the binary mixtures, AFB1/DDT and DON/DDE significantly increased cell viability in a dose-dependent fashion. A combination of ZEN and DDT led to significant reduction in cell viability in a dose-dependent manner while lower concentrations of ZEN/DDE had no significant effect but were cytotoxic at the highest combination tested (64 µM). The interactive effects of the combined mycotoxins and pesticides were mainly synergistic and additive in nature. The results of this study indicate that exposure to single or mixtures of mycotoxins and pesticides may cause adverse effects on the Leydig cells thereby impairing male reproductive function. 75. In-vitro determination of the effect of chemotherapeutic agents on leukaemic T-cell transmigration through the vascular endothelial barrier Sultan Almalki, Saeed Kabrah, Jinsheng Xu, Craig Donaldson and Ruth Morse University of the West of England, Bristol, UK T-lymphoblastic leukaemia is a blood-borne cancer where the bone marrow produces excess lymphocytes that can leak into the circulatory system and infiltrate other organs, such as the liver and spleen. Such leukaemia is usually treated with a combination of chemotherapy and radiation. However, there is high rate of relapse where the disease spreads to difficult locations such as the central nervous system; such complications can arise unfortunately from the use of chemotherapy. The current study aimed to develop an in-vitro model to investigate the changes in leukaemic T-cell transmigration rates through the endothelial cell layer and to highlight the effect of cyclophosphamide on cell extravasation. The ECV304 cell line was used to establish a confluent endothelium monolayer on a hanging transwell insert. Furthermore, T-lymphoblastic leukaemic (MOLT-4) cells were co-cultured with the confluent monolayer to evaluate cell infiltration. Cyclophosphamide (0.0001, 0.5 and 10mM) effects on cell extravasation were evaluated with/without HepG2 spheroids. This study demonstrated that the ECV304 monolayer formed a barrier in the 5 m and 8 m pore insert but not 10 m. Additionally, MOLT-4 cells established a firm contact with the monolayer and infiltrated through the membrane. High dose cyclophosphamide (10mM) non-significantly reduced monolayer permeability by almost 1.1 fold in the presence of HepG2. In addition, results indicated that cyclophosphamide induced cell infiltration in a dose dependent manner exclusively in the presence of HepG2 (2.9, 2.2 and 5.9 fold increase in 0.0001, 0.5 and 10mM respectively), thus suggesting changes in the adhesion molecule expression associated with drug exposure and subsequently altering the cellular interactions. In conclusion, the current findings suggested that ECV304 and 8 m pore insert can be used as in-vitro model to evaluate T-cell interaction with the monolayer as well as cell infiltration. Moreover, it showed that cyclophosphamide with HepG2 has more impact on cell migration than those without HepG2. 76. Industrialising the in-vitro micronucleus assay for applied screening in human relevant cells Danielle Beeson, Julia Kenny, Anthony Lynch and Natalia Sumption GlaxoSmithKline, Genetic Toxicology and Photosafety, Ware, UK Designing and developing a new in-vitro assay for use in an industrial setting is subject to intense focus on quality, consistency and accuracy. These aspects are vital to enable full deployment of an assay suitable for good laboratory practice (GLP) testing and predictive screening with new assays often employing new and emerging technologies to diversify testing strategies and meet ever evolving business needs. Flow cytometry has progressed significantly over the past decade and as such has been the focus of recent Genetic Toxicology capability studies at GlaxoSmithKline. Here, we have used the development of a flow cytometry-based in-vitro micronucleus test in primary human lymphocytes as a case study for the processes involved in preparing and validating a new assay for use in pharmaceutical testing. These included culture optimisation where exponential growth of cultures was assured by adjusting cell densities (optimised at 7x10 5 /ml), length of phytohaemagglutinin (PHA) stimulation (optimised as 5 µl/ml of 1mg/ml HA16 for 48h following culture initiation) & harvest time points (optimal 24h continuous treatment with harvest at 72h). In addition, complex flow cytometry gating strategies were implemented, in order to remove the likelihood of false positive results being observed. This was achieved through sequential staining of cells to exclude necrotic cells and debris as well as nuclear staining to correctly identify micronuclei. Lastly, preliminary statistical analysis found that there were no systematic plate effects (enabling the whole plate to be utilised) and the within plate variability was comparable to the between plate variability. Understanding and implementing these control measures is essential to the definition of a robust, high quality assay which can be deployed to make Go/NoGo decisions in early Drug Discovery. 77. Inhalational exposure of deuterium-labelled bis(2-ethylhexyl)phthalate [DEHP] and subsequent biomonitoring of DEHP metabolites in human urine Annette Krais 1,3 , Christina Andersen 2 , Axel Eriksson 2 , Christian Lindh 1 , Bo A.G. Jönsson 1 , Joakim H. Pagels 2 , Aneta Wierzbicka 2 and Anders Gudmundsson 2 1 Division of Occupational and Environmental Medicine, Lund University, Lund, Sweden; 2 Ergonomics and Aerosol Technology, Lund University, Lund, Sweden; 3 Swedish Toxicology Sciences Research Center (Swetox), Södertälje, Sweden Phthalates are ubiquitous contaminants in indoor environments. They are found in a wide range of applications, such as food packaging, building materials, cleaning products and children’s toys. Phthalates can easily leach into the environment and are thus constantly emitted to the air. There are large concerns that phthalates might have impact on human health due to disruption of the endocrine system. The aim of this study was to assess the uptake of the phthalate ester bis(2-ethylhexyl) phthalate [DEHP] into the human body after inhalational exposure. To avoid interference by background exposure the deuterium labelled DEHP analogue was used. In a first test experiment two participants were exposed to elevated air concentrations of D4-DEHP. The two male participants spent three hours in an exposure chamber filled with an air concentration of 15 µg/m 3 D4-DEHP, as quantified using online high resolution time of flight aerosol mass spectrometer (HR-ToF-AMS). Liquid chromatrography tandem mass spectormetry (LC-MS/MS) was used to analyze the major metabolites of D4-DEHP, namely deuterium labelled mono-ethyl phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), mono-(2-ethyl-5-oxo-hexyl) phthalate (5-oxo-MEHP), mono-(2-ethyl-5-carboxypentyl) phthalate (5-cx-MEPP) and mono-[2-(carboxymethyl) hexyl] phthalate (2-cx-MMHP). Due to its long half-life and high abundance D4-5-cx-MEPP served as excellent biomarker for this study. The absorption and distribution phase was in between 9h and 20h; half-life times of excretion ranged from 8h to 10h. In this human biomonitoring study 5-cx-MEPP was assessed as biomarker for inhalational exposure of the phthalate ester DEHP. Knowledge on metabolism, uptake and biodistribution of phthalates is crucial for estimating our daily exposure as well as for correct risk assessment in humans. 78. Investigating the possibility of redefining the Derek rule for benzimidazole containing structures Amy Lane 1 , Sharon Robinson 1 , Julia Kenny 1 , Mark Burman 1 , Anthony Lynch 1 , Geraldine Whelan 2 , Angela White 2 , Richard Williams 3 and Jim Harvey 2 1 GlaxoSmithKline, Genetic Toxicology and Photosafety, Ware, UK; 2 GlaxoSmithKline, Computational Toxicology, Ware, UK; 3 Lhasa Ltd, Leeds, UK Benzimidazole structures are heterocyclic aromatic organic compounds consisting of the fusion of benzene and imidazole. They constitute the core structure of marketed antihelminthic treatments including albendazole, mebendazole and thiabendazole, which act by binding to spindle microtubules and blocking nuclear division, leading to cell death. They also have the potential to induce cell division aberrations including chromosomal aneuploidy. Based on this, a generic chromosome damage alert was implemented in the Derek knowledge base to identify benzimidazole structures of concern. However, this structure-activity relationship was found to be oversensitive as many compounds with this alert do not induce genotoxicity in mammalian tests in-vivo or in-vitro ; therefore coverage of benzimidazoles was restricted to 2-thiobenzimidazoles and derivatives of 2-aminobenzimidazoles. At GlaxoSmithKline, we have recently tested a new chemical series which contain the benzimidazole substructure. These do not fire any relevant SAR alerts for aneuploidy in Derek Nexus v4.1.0 and yet produce positive in-vitro and in-vivo micronucleus results via an aneugenic mode of action (i.e. increase in the ratio of kinetochore positive micronuclei). These data will be shared with Lhasa Ltd. to investigate the possibility of redefining the Derek rule for benzimidazole containing structures. We would also like to request other Lhasa members to share relevant genotoxicity data on compounds with benzimidazole structures that may help to refine the existing SAR rule on this chemical class. 79. Is nickel chloride really a non-genotoxic carcinogen? Leanne Stannard 1 , Kate Chapman 1 , Eleanor Wilde 1 , Dmitrij Sachno 2 , Shareen Doak 1 and Ann Doherty 3 1 Swansea University Medical School, Institute of Life Science, Swansea, UK; 2 Charité, Universitätsmedizin Berlin, Berlin, Germany; 3 AstraZeneca, Cambridge, UK Nickel chloride (NiCl 2 ) is a toxic and carcinogenic widespread environmental and occupational pollutant; human exposure to which occurs through air, water, food and tobacco smoke. A wider study aimed at improving current in-vitro genotoxicity tests, which do not detect non-genotoxic carcinogens, has been carried out. This study focussed on combining genotoxicity data to other mechanistic aspects of cell biology to obtain more predictive data for assessing carcinogenicity. A multi endpoint approach was important in this study, and key elements of this included studying the DNA damaging potential as well as alterations to the cell cycle, cell morphology, bioenergetics and cell signalling. NiCl 2 has previously been classified as a non-genotoxic carcinogen (NGTC); however, after studying the effect of NiCl 2 on these many mechanistic endpoints, it has become clear that NiCl 2 behaves more like a genotoxic carcinogen. The genotoxic carcinogens cadmium chloride (CdCl 2 ) and methyl methanesulfonate (MMS) showed very similar results to NiCl 2 in these mechanistic studies. Although NiCl 2 induced no increase in micronuclei frequency, as CdCl 2 and MMS did, these chemicals all induced an increase in the number of cells in G2 phase of the cell cycle, an increase in both p53 and phospho-p53, an increase in p21 activity, an increase in cell size and a decrease in mitochondrial respiration rate. In comparison, the NGTC methyl carbamate had opposing results such as a decrease in cell area and p21 activity. The induction of reactive oxygen species (ROS) after treatment with NiCl 2 , along with positive micronuclei results from a preliminary 5-day chronic dose micronucleus study further support that NiCl 2 has been misclassified as a NGTC. It is possible that NiCl 2 causes indirect DNA damage by the production of ROS and requires a longer, chronic exposure, which is more similar to that of human exposure. 80. Mass spectroscopic analysis of MGMT tryptic peptides allows detection of O 6 -alkylguanine adducts in oligodeoxyribonucleotides and calf thymus DNA treated with temozolomide Rasha Abdelhady 1 , Perdita Barran 2 , David Williams 3 and Andy Povey 1 1 University Of Manchester, Centre of Occupational and Environmental Health, Manchester, UK; 2 Manchester Institute of Biotechnology, The Michael Barber Centre for Collaborative Mass Spectrometry, Manchester, UK; 3 University of Sheffield, Department of Chemistry, Sheffield, UK Red and processed meat consumption increases human colorectal cancer risk, potentially by haem catalysed formation of carcinogenic N-nitrosocompounds (NNOC) that form mutagenic O 6 -alkylguanine DNA adducts. NNOC formation cannot be accurately estimated from meat consumption alone due to in situ processing required for their formation. We are thus developing a novel approach to assessing NNOC exposure by quantifying O 6 -alkylguanine DNA adducts that result from their exposure, by using the known action of the DNA repair protein, O 6 -alkylguanine O 6- alkyltransferase (MGMT), to transfer the O 6 -alkyl to an active site cysteine residue in MGMT. This is a proof-of principle study. Oligodeoxyribonucleotides (ODNs) containing O 6 -methylguanine (O6-MeG), O 6 -carboxymethylguanine (O6-CMG) adducts as well as calf thymus (CT) DNA treated with temozolomide (TMZ) (containing 5.7 fmoles of O6-MeG per µg CT DNA) were used as standards to examine whether O 6 -alkyl groups can be detected via MS analysis of tryptic peptides of MGMT. Maltose binding protein-MGMT (MBP-MGMT) fusion protein was expressed, purified, and MGMT functional activity determined by tritium transfer using calf thymus DNA methylated with 3 H-N-Nitrosomethylurea. Double stranded ODNs containing O6-MeG and O6-CMG, an unmodified ODN and alkylated CT DNA were incubated with MBP-MGMT and the resulting alkylated MGMT digested with trypsin and analysed using MALDI-TOF. MBP-MGMT tryptic digests contained multiple peptides including the active site peptide fragment (GNPVPILIPCHR) with m/z 1315.7 and in case of MBP-MGMT incubated with ODNs containing O6-MeG and O6-CMG the active site peptide containing S-methylcysteine and S-carboxymethylcysteine, with m/z 1329.7 and 1373.7, respectively. In addition, mass spectroscopic analysis of tryptic digests of MBP-MGMT incubated with CT DNA treated with TMZ showed the active site peptide containing S-methylcysteine with m/z 1329.7. These results demonstrate proof of principle and suggest that this approach might be used to characterise O 6 -alkylguanine adducts in human DNA. 81. Optimisation and validation of an in-vitro passive dosing system using hydrophobic carcinogens Fiona Chapman 1 , Andrew Scott 2 , David Sanders 2 , Roger van Egmond 2 , Shareen Doak 1 and Gareth Jenkins 1 1 Swansea University, Swansea, UK; 2 Unilever, Colworth, UK Global regulatory guidelines for in-vitro genotoxicity tests, such as the micronucleus assay, use a large chemical dose, effectively saturating cellular uptake. This is unrepresentative of most human exposures, which are often repeated or prolonged, and at low doses. If low doses of hydrophobic chemicals, such as benzo[a]pyrene (B[a]P), are added to aqueous cell cultures the system is subject to large chemical losses. These include aggregation to serum proteins, sorption into surrounding plastic and volatilisation. Therefore, current in-vitro dosing requires refinement to counter these losses, and biologically available exposure concentrations need better definition. Passive dosing (PD), the constant, defined chemical release from an in-test silicone source, can act to replenish chemical losses. This study used model pro-carcinogens, B[a]P and 1-nitropyrene (1-NP) and examines some of the chemical depletion suffered within cell medium. Medium samples were analysed by fluorescence HPLC. Silicone discs were loaded with B[a]P or 1-NP and MCL-5 cells were exposed to the discs for 48h. Subsequent genotoxicity was assessed using the in-vitro Micronucleus Assay. Horse serum within culture medium maintains a near constant B[a]P concentration after 24h if 10 μM B[a]P is spiked in. However, B[a]P is undetectable from a 2 μM spiked dose after 24h. Silicone discs can be loaded with an 80% saturated B[a]P or 1-NP solution, and subsequent dilutions, to release corresponding chemical concentrations into aqueous cell medium. For example, 14mm 3 discs loaded with an approximately 20% saturated solution can maintain concentrations below 2 μM B[a]P for at least 48h. Exposure of cells to B[a]P released from discs for 48h results in genotoxicity. Medium and the plastic container in which cells are cultured deplete the chemical available to cells, therefore an acute chemical exposure is not at concentrations expected. PD can replace chemical losses to maintain concentrations less than 2 μM, which better represents human exposure concentrations. 82. Placental DNA repair activity and air pollution Nurulshyha Md Yatim 1 , Geoffrey Margison 1 , Edward Johnstone 2 and Andrew Povey 1 1 University of Manchester, Institute of Population Health, Manchester, UK; 2 St. Mary’s Hospital, Maternal and Fetal Health Research Centre, Manchester, UK The genotoxic effects of DNA damaging present in air pollution are likely to be ameliorated, in part, by DNA repair pathways. One such pathway uses O 6 -methylguanine DNA methyltransferase (MGMT) to remove toxic and mutagenic O 6 -alkylguanine adducts from DNA formed following exposure to alkylating agents as potentially generated by the reaction of nitrogen oxides (NO x ) with amines. The aims of this pilot study are to quantify MGMT functional repair activity in placenta and examine its associations with air pollutants. Tissue samples from peripheral, middle and cord areas of placenta were collected from 20 mothers and stored at ‒80°C until processed. MGMT repair activity was quantified in cell-free sonicates of 100–120mg tissue using a standard radioistotopic methyl transfer assay and protein levels were quantified by Bradford assay. Initial results on ten samples of middle area of placenta indicated almost a 5-fold variation in MGMT activity with levels ranging from 0.04 to 0.19 fmoles/µg extract protein. MGMT standard curves indicate a theoretical lower limit of quantitation of 10 fmoles MGMT. Postcodes of the mothers were geocoded by ArcGIS and air pollution data obtained from the Department for Environment, Food and Rural Affairs. The levels of exposure during pregnancy for particulate matter diameter less than 10 µm (PM 10 ), particulate matter diameter less than 2.5 µm (PM 2.5 ), nitrogen dioxide (NO 2 ), NO x and sulphur dioxide (SO 2 ) ranged from 10–20 µg/m 3 , 5–15 µg/m 3 , 11–30 µg/m 3 , 20–50 µg/m 3 and 3–6 µg/m 3 respectively. In this small study, there was no significant association (p>0.05) observed between level of MGMT repair activity with level of all air pollutants for the whole pregnancy period. An association (p<0.05) was found between NO 2 and NO x but not with other pollutants. This initial study indicates that the placenta contains functional MGMT activity that may affect susceptibility to alkylating agent exposure. 83. Preliminary genomic analysis of L5178Y tk+/- cells and mutagen-induced tk-/- mutant colonies using next generation sequencing Daniel J. Smart, James N. Battey, Jérôme Thomas, Nicolas Sierro, Damian McHugh, Patrick Vanscheeuwijck and Nikolai V. Ivanov Philip Morris International Research & Development, Neuchâtel, Switzerland The genetic basis that underpins the mouse lymphoma assay (MLA) is ostensibly well understood; inactivation of the functional thymidine kinase (tk+) allele in L5178Y cells via mutation or deletion induces trifluorothymidine (TFT) resistance and tk-/- mutants can be selected for against a background of tk+/- wild-type cells via TFT-mediated enrichment. However, despite its widespread use over the past 20 years, few studies have sought to characterise in greater detail the colonies of cells which propagate as a result of mutagenesis in the assay. The recent advent of whole genome sequencing has the potential to enhance mutagenicity testing since genetic changes can now be investigated with high fidelity on a global genome level rather than at just one reporter gene. To this end, preliminary investigations using next generation sequencing (NGS) technology were carried out to sequence the genome of a L5178Y cell line frequently used in hazard identification studies (clone 3.7.2C; IVGT). This approach revealed numerous pre-existing small polymorphisms within the cell line’s genome, many of which are shared with various mouse strains. These polymorphic loci were then used to map loss of heterozygosity in tk-/- mutant clones induced by the prototypical mutagens methyl methanesulfonate and 7,12-dimethylbenz[a]anthracene. Several breakpoints in chromosome 11 were identified. Furthermore, putative deleterious mutations in genes relating to DNA repair were also uncovered. We believe NGS-mediated genomic analysis of tk-/- mutant cells may shed further light on the genetic basis of the MLA, while also potentially providing additional evidence for the risk assessment of mutagenic compounds. 84. The impact of p53 status on the metabolic activation of benzo[a]pyrene in the mouse Hepa1c1c7 cell line Mateja Sborchia 1 , Hector Keun 2 and Volker M. Arlt 1 1 King’s College London, Analytical & Environmental Sciences Division, London, UK; 2 Imperial College London, Department of Surgery & Cancer, London, UK The transcription factor p53 responds to a variety of stress signals by activating tumour suppressive mechanisms. p53 is frequently inactivated in human cancers and such deregulation can be linked to environmental exposures. Studies involving benzo[a]pyrene (BaP), an environmental carcinogen metabolised by cytochrome P450 (CYP) 1A1, demonstrate that its genotoxic effects cluster according to p53 genotype [1, 2]. The main aim of this study is to investigate the impact of p53 status on the metabolic activation of BaP in the mouse Hepa1c1c7 cell line. Hepatoma cells (Trp53+/+ ) were exposed to a range of BaP concentrations (0.01–1 µM) over 48h and cell viability was assessed by Trypan Blue. Such cells were used to investigate the expression of p53 and Cyp1a1 by Western blotting and the formation of DNA adducts by 32 P-postlabelling. We also optimised p53 siRNA transfections in these cells to conduct preliminary BaP exposure experiments in a Trp53(-/ -) scenario. Cell viability decreased following BaP exposure in Hepa1c1c7 cells; whereas, p53 and Cyp1a1 expression increased at higher BaP concentrations. BaP-DNA adduct formation increased in a concentration-dependent manner and correlated with Cyp1a1 protein levels over 48h. Given a cell viability of ≥60%, the induction of Cyp1a1 and formation of DNA adducts (particularly at 24h), BaP concentrations of 0.25 µM and 0.5 µM were selected for knock down experiments in these cells. We characterised the effects of BaP exposure in the Hepa1c1c7 cell line, which will serve to assess the impact of p53 status on BaP bioactivation. References 1. Krais, A.M., Speksnijder, E.N., Melis, J.P., Indra, R., Moserova, M., Godschalk, R.W., van Schooten, F.J., Seidel, A., Kopka, K., Schmeiser, H.H., Stiborova, M., Phillips, D.H., Luijten, M. and Arlt, V.M. (2016). The impact of p53 on DNA damage and metabolic activation of the environmental carcinogen benzo[a]pyrene: effects in Trp53(+/+ ), Trp53(+/ -) and Trp53(-/ -) mice. Arch. Toxicol.,90 , 839–851. 2. Wohak, L.E., Krais, A.M., Kucab, J.E., Stertmann, J., Øvrebø, S., Seidel, A., Phillips, D.H. and Arlt, V.M. (2016). Carcinogenic polycyclic aromatic hydrocarbons induce CYP1A1 in human cells via a p53-dependent mechanism. Arch. Toxicol.,90 , 291–304. 85. The influence of the cellular p53 status on the chemotherapeutic drug-induced expression of CYP1A1 in human cells and its impact on the metabolism of benzo[a]pyrene Alexandra Willis 1 , Laura Wohak 1 , Radek Indra 2 , Iveta Mrizova 2 , Kerstin Feser 1 , Marie Stiborova 2 , David H. Phillips 1 and Volker M. Arlt 1 1 King’s College London, Analytical and Environmental Sciences Division, London, UK; 2 Charles University, Department of Biochemistry, Prague, Czech Republic The effect of the p53-inducing chemotherapeutic drugs etoposide (ETOP), cisplatin (CIPL) and ellipticine (ELLI) on CYP1A1 expression, and the resultant effect on the metabolism of the environmental pollutant (benzo[a]pyrene) BaP, was investigated in isogenic human colorectal HCT116 cell lines that differed only by their p53 status. HCT116 cells that were TP53(+/+ ), TP53(+/− ) and TP53(−/− ) were initially incubated with 0–100 µM ETOP, 0–100 µM CIPL and 0–10 µM ELLI over 48h to measure cell viability using crystal violet staining and p53 expression using Western blotting. For further experiments 50 μM ETOP, 60 μM CIPL and 5 μM ELLI were chosen as they all produced 60–80% cell viability and high p53 induction. ETOP and ELLI induced CYP1A1 expression in TP53(+/+ ) cells after 48h, whereas CIPL did not. Next TP53(+/+ ) and TP53(−/− ) cells were exposed to the drugs in co-incubation experiments with 2.5 μM BaP. In these co-incubations, ETOP was found to increase CYP1A1 expression, CIPL did not alter CYP1A1 expression and ELLI decreased CYP1A1 expression compared to TP53(+/+ ) cells treated with BaP alone. The effect of ETOP on BaP-mediated CYP1A1 induction was stronger pronounced in TP53(+/+ ) cells compared to TP53(−/− ) cells; no CYP1A1 induction was observed in co-incubations of BaP and CIPL/ELLI in TP53(−/− ) cells. An HPLC method to detect the CYP1A1-mediated metabolite BaP-7,8-dihydrodiol was established which confirmed previous results showing that the formation of BaP-7,8-dihydrodiol is considerably lower in TP53(−/− ) cells than in TP53(+/+ ) cells; however, the potential effects of drugs on BaP metabolism remain to determined. Our results show that chemotherapeutic drugs influence CYP1A1 expression in a p53-dependent manner that could potentially affect BaP bioactivation and subsequently alter BaP genotoxicity (i.e. DNA adduct formation). This could especially be critical in smokers during chemotherapy who are exposed to increased BaP levels via cigarette smoke. 86. The ToxTracker reporter system is a mechanistic genotoxicity platform that discriminates clastogenic from aneugenic compounds Giel Hendriks 1 , Remco Derr 1 , Branislav Misovic 2 , Bruno Morolli 2 , Fabienne Calléja 2 and Harry Vrieling 2 1 Toxys B.V., Robert Boyleweg 4, 2333 CG, Leiden, The Netherlands; 2 Leiden University Medical Center, Department of Human Genetics, Leiden, the Netherlands With the increasing production of new chemicals for a wide range of applications in health care, food and as cosmetics, the demand for rapid and reliable toxicity assessment is growing. Novel innovative in-vitro systems should ideally not only identify genotoxic properties of chemicals, but also provide insight into the type of cellular damage inflicted in order to more reliably predict human health hazard of novel compounds. The ToxTracker assay is a mammalian stem cell-based assay that detects activation of specific cellular signalling pathways as the result of biological reactivity of the tested compounds. ToxTracker discriminates between induction of DNA damage, oxidative stress and protein damage by detection of DNA replication stress, NF-kB associated DNA damage signalling, various cellular anti-oxidant responses and activation of the unfolded protein response. In addition, ToxTracker could discriminate clastogenic genotoxicants from eugenic compounds based on the differential induction and kinetics to the different genotoxicity reporters. For data analysis, we have developed the software tool ToxPlot. Automated data analysis and graphical representation of the test results allow clear and rapid assessment of the reactive properties of compounds. The integrative approach of the ToxTracker assay provides a powerful tool for in-vitro carcinogenic hazard identification of chemicals by unveiling activation of specific cellular signalling pathways upon exposure and deliver insight into the underlying mechanism of toxicity. 87. Tissue-specific differences in transcriptomic and genotoxic responses of marine mussels exposed to benzo[a]pyrene Lorna J. Dallas 1 , Volker M. Arlt 2 , Susanna Sforzini 3 , Mohamed Banni 3 , Yann Animot 4 , Jim W. Readman 4 , Aldo Viarengo 3 and Awadhesh N. Jha 1 1 Plymouth University, School of Biological Sciences, Plymouth, UK; 2 Kings College London, Analytical and Environmental Sciences Division, London, UK; 3 University of Eastern Piedmont, Department of Environmental and Life Sciences, Alessandria, Italy; 4 Plymouth University, School of Geography, Earth and Environmental Sciences, Plymouth, UK It is well established that marine mussels exposed to the ubiquitous polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) show genotoxic responses. However, the underlying mechanistic causes of this (geno)toxicity require further investigation. This study combined measurements of DNA damage with the use of a new targeted low density microarray with an aim to elucidate the pathways that may be behind this effect. Mussels ( Mytilus galloprovincialis , shell length 50.76±2.84mm) were exposed to waterborne BaP concentrations of 0, 5, 50, and 100 µg/L (in 0.02% DMSO). After 3 days, gill and digestive gland samples were digested with dispase, DNA damage was assessed using the alkaline comet assay and DNA adducts were quantified using 32 P-postlabelling. An additional concentration (1000 µg/L BaP) was also used for DNA adduct analysis only. In addition, mussels from the same exposure were analysed by hybridisation to a 476 probe microarray (described elsewhere) and data for those genes related to DNA metabolism and repair were examined. The results of this study confirm that BaP is genotoxic to mussels, with significant increases in % tail DNA for the comet assay in both tissues. It was, however, clear that this is a tissue-specific response, with gill cells showing less damage than those of the digestive gland (2- and 4-fold increases respectively, compared to the control). DNA adducts also showed considerable differences between tissues, and suggest a possible sex-specific response, with female mussels having higher numbers of dG- N2 -BPDE adducts. The expression of DNA metabolism/repair genes was significantly altered after exposure to 50 and 100 µg/L BaP when compared to the control treatment, but with tissue and treatment-specific differences. For example, in the gills, members of the ras family of oncogenes were upregulated only at the higher concentration, in contrast to the digestive gland where this occurred in both treatments. A variety of caspase genes were also upregulated except for after the 100 µg/L BaP exposure in gills, whereas downregulated genes were more variable between the two concentrations and tissues. 88. Uncovering mutation rates and signatures caused by DNA damaging agents in wild type and DNA repair mutant strains by large scale C. elegans next generation sequencing (NGS) Victor Gonzalez Huici 1 , Bettina Meier 1 , Moritz Gerstung 2-3 , Nadezda Volkova 2 , Peter Campbell 3 and Anton Gartner 1 1 University of Dundee, School of Life Sciences, Dundee, UK; 2 EMBL-EBI, Wellcome Genome Campus, Hinxton, UK; 3 Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, UK DNA damaging agents cause reduced cell survival, which is further compromised when DNA repair is defective. However, very little is known about its correlation with the rates, types and signatures of mutations caused by various mutagens in wild-type and DNA repair mutant defective lines. We use C. elegans as a model to correlate reduced survival and the number and nature of mutations as determined by massive NGS. To date we have already sequenced more than 1000 worm genomes, and are currently sequencing and analysing an even higher number. We studied the effect of alkylating agents (MMS, DMS and EMS), finding extreme sensitivity in pol-η and pol-ζ translesion polymerases (TLS) mutants, moderate for pol-θ and mild for rev-1 and pol-κ mutants. The latter, however, shows an extremely high mutation accumulation rate, 1 to 2 orders of magnitude above the other TLS mutants. Hypersensitivity is also observed in mus-81 and slx-1 recombination mutants, and moderately in mutants defective in DNA damage checkpoint and apoptosis induction. While NHEJ, BER and NER pathways mutants exhibit null or mild alkylator sensitivity, NGS reveals a significant accumulation of point mutations, predominantly T>A transversions, in NER mutants. Bulky adduct generators aflatoxin and aristolochic acid significantly decrease cell viability both in GG-NER and TLS mutants; however, the sensitivity profile differs: Pol-ζ has a major role in the response to aflatoxin, while Pol-θ and Rev-1 to aristolochic acid. We have also studied the effect of UV light, observing dramatic hypersensitivity in pol-η and mus-81, but not slx-1, mutants. Mutants in GG-NER and the Bloom helicase mutant him-6 are also impaired. In addition we are studying further damaging agents: chemotherapeutic drugs as streptozotocin, mitomycin, cyclophosphamide, bleomycin, etoposide and camptothecin, atmospheric contaminants such as nitrobenzanthrone and dinitropyrene, and food contaminants such as glycidamide and potassium bromate. 89. Use of the PigA mutation assay to study genomic instability in humans Rachel Lawrence 1 , Hasan Haboubi 1,2 , Lisa Williams 2 , Owen Bodger 1 , Cathy Thornton 1 , Shareen Doak 1 and Gareth Jenkins 1 1 Swansea University, Institute of Life Science, Swansea, UK; 2 ABM University NHS Trust, Department of Endoscopy, Swansea, UK The X-linked PIG-A gene has been widely employed as a reporter gene for in-vivo somatic cell mutagenicity within the pharmaceutical industry. PIG-A mutant erythrocytes lack GPI anchors and are easily recognised by flow cytometry. Recent research at Swansea University has shown that PIG-A mutation levels significantly increase from healthy individuals to patients with oesophageal adenocarcinoma. The aim of the current work is to further explore using this blood test in early oesophageal cancer detection and to identify background levels of PIG-A mutation in a healthy population to understand why these levels may fluctuate between individuals. Oesophageal cancer patients (and pre-malignant patients) as well as colorectal cancer patients are currently being collected for analysis. In the past 6 months, blood was collected from consenting healthy volunteers, stained with fluorescently conjugated antibodies to CD235a, CD55 and CD59 cell surface markers and analysed on a flow cytometer. Mutants were defined as cells that were positive for the erythrocyte specific marker CD235a but negative for both CD55 and CD59. After collection, blood was stored for increasing time periods to see how storage may affect the PIG-A mutant frequency. Confounding variables such as BMI, sex and age were compared in order to identify any factors that may need to be considered when measuring an individual’s mutant frequency. Storing the blood showed that the sample can be kept at room temperature for up to 5 days before there is a significant increase in mutant frequency. There was a significant positive correlation between PIG-A mutant frequency and BMI. This supports the hypothesis that obesity may contribute to genomic instability and perhaps even cancer. Furthermore, a significant positive correlation was observed between age and mutant frequency. This is in line with what we may expect: genetic instability and mutation frequency increases with age. 90. Using gamma-H2AX to identify tissue-specific DNA damage Ulla Plappert-Helbig, Silvana Libertini, Wilfried Frieauff and Hans-Joerg Martus Novartis Institutes for BioMedical Research, Basel, Switzerland DNA double strand breaks trigger the phosphorylation of the histone protein H2AX in Serine 139, generating gamma-H2AX. Therefore, gamma-H2AX is used widely as a DNA damage marker in-vitro . In our study, we evaluated the usefulness of this marker for detecting tissue-specific effects in-vivo . Since gamma-H2AX can be detected on histopathological sections, a genotoxicity read out of multiple organs/tissues can be integrated into general toxicity studies. In contrast to the comet assay or micronucleus test, no specific preparation of the organ is required; thus, paraffin blocks for histopathology can be used, saving animals (3Rs) and resources. To evaluate gamma-H2AX staining as valid genotoxicity endpoint, rats were treated orally with EMS, doxorubicin or ENU. Organs were sampled 3h and 24h after the last treatment and either prepared for the comet assay (in accordance with OECD guideline) or formalin-fixed/paraffin embedded for immunofluorescent staining with gamma-H2AX. An image analysis system was developed to count the number of gamma-H2AX positive cells over total number of cells and assess the relative intensity of the signal in positive cells. EMS-induced DNA damage was detected in the jejunum and the liver with both approaches, but gamma-H2AX staining displayed a lower sensitivity than the comet assay. Conversely, doxorubicin showed a weak positive result in the comet assay in liver only, while gamma-H2AX staining indicated DNA damage induction in all organs analysed. Interestingly, for doxorubicin gamma-H2AX positivity was not limited to the crypts but also observed in the muscolaris externa of the jejunum. For ENU, DNA damage was more strongly detected with the comet assay. Thus, different compounds are detected with different sensitivity. In conclusion, our experiments showed that gamma-H2AX staining could be a useful tool to detect DNA damage on histopathological slides, however further studies on more chemicals are required to validate its use. 91. Comet assay in rat nasal tissue Kamala Pant 1 , Shannon Bruce 1 , Sandra Springer 1 , Michelle Klug Laforce 1 , Louette J. Rausch 2 and Rohan Kulkarni 1 1 BioReliance by SAFC, Rockville, MD, USA; 2 Akzo Nobel, Inc. Chicago, IL, USA The in-vivo alkaline comet assay is used for the detection of DNA strand breaks in cells or nuclei isolated from multiple tissues of animals, usually rodents that have been exposed to potentially genotoxic materials. One of the main advantages of the comet assay is that it allows use of any tissue of interest provided single cell population is prepared from that tissue. For inhalation studies, nasal tissue is important as it is the first site of contact. Due to the size of the nasal tissue, collection and preparation of single cell population requires skilled technicians and validated technique, and hence the comet assay in nasal tissues is less routinely performed. To satisfy the validation requirements as per the OECD comet assay guideline 489 and in order to build historical database in nasal tissue, male Sprague Dawley rats were dosed with vehicle (saline) and the positive control ethyl methanesulfonate (EMS) at 200mg/kg using oral gavage. Five independent experiments with two groups of five animals in each were conducted. Nasal tissues were collected and processed to prepare single cell suspensions as per the Test Guideline. The comet assay results from these five experiments demonstrated that oral dosing of EMS resulted in significant DNA damage in the nasal tissue (% tail DNA range from 9.63 to 20.55) and the response is significantly higher than the vehicle control (% tail DNA range from 0.18 to 0.39).The work is a proof of concept experiment to validate nasal tissue extraction, cell suspension preparation in detail and to demonstrate that the comet assay can detect the DNA strand breaks in nasal tissues. In summary, this technique can be used to prepare single cell population to perform the comet assay on nasal tissues for inhalation studies. © The Author 2016. 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. © The Author 2016. 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 39th Annual Meeting of the United Kingdom Environmental Mutagen Society, 26th - 29th June 2016 at King’s College London, UK
JF - Mutagenesis
DO - 10.1093/mutage/gew044
DA - 2016-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/abstracts-of-the-39th-annual-meeting-of-the-united-kingdom-Loch3E46tX
SP - 703
EP - 734
VL - 31
IS - 6
DP - DeepDyve
ER -