TY - JOUR
AU - Semple, Kirk, T.
AB - Abstract Speakers: John Ashby (Syngenta CTL, UK), Peter A.Behnisch (Eurofins GfA, Germany), Paul L.Carmichael (Unilever Colworth, UK), Curtis C.Harris (National Cancer Institute, USA), Kevin C.Jones (Lancaster University, UK), Andreas Kortenkamp (School of Pharmacy, London, UK), Caroline J.Langdon (Reading University, UK), Anthony M.Lynch (GlaxoSmithKline, UK), Francis L.Martin (Lancaster University, UK), Trevor J.McMillan (Lancaster University, UK), David H.Phillips (Institute of Cancer Research, UK), Huw J.Ricketts (University of Cardiff, UK), Michael N.Routledge (University of Leeds, UK), J.Thomas Sanderson (Utrecht University, The Netherlands) and Kirk T.Semple (Lancaster University, UK) The effects of many environmental exposures to either single contaminants or to mixtures still remain to be properly assessed in ecotoxicological and human toxicological settings. Such assessments need to be carried out using relevant biological assays. On a mechanistic basis, future studies need to be able to extrapolate exposure to disease risk. It is envisaged that such an approach would lead to the development of appropriate strategies to either reduce exposures or to initiate preventative measures in susceptible individuals or populations. To mark the opening of a new Institute, the Lancaster Environmental Centre, an environmental health workshop was held over 2 days (9–10 September 2003) at Lancaster University, UK. The fate, behaviour and movement of chemicals in the environment, together with environmental exposures and human health, biomarkers of such exposures, hormone-like compounds and appropriate genetic toxicology methodologies, were discussed. Marking the opening of a new Institute, the Lancaster Environmental Centre (LEC), an environmental health workshop was held over two days (9–10 September 2003) at Lancaster University, UK. This was a forum in which to discuss environmentally relevant exposure effects and how these may be assessed. Living organisms are continuously exposed to natural and man-made chemicals and to endogenous substances. Traditional (geno)toxicological testing of individual chemicals, often at high concentrations, may not take into account (i) the effects of chemical mixtures or (ii) whether high doses may be truly extrapolated to low dose effects. The first day's session was entitled ‘Environmental toxicology: mechanisms, measurement and meaning’. Research on the fate, behaviour and movement of chemicals in the environment, biotic–chemical interactions and mechanisms of impact and resistance were presented. The second day's session was entitled ‘Genetic toxicology: DNA damage, prevention and biomarkers’. Environmental exposures and human health, biomarkers of such exposures, hormone-like compounds and appropriate genetic toxicology methodologies were discussed. Toxicological exposures were assessed in a global environmental setting and then discussed in the context of predicting their effects on human health and understanding the mechanisms underlying the aetiology of particular disease phenotypes, e.g. cancer. The first day's session, chaired by Kirk T.Semple (Lancaster University), started with a source to exposure overview of persistent organic pollutants (POPs) in a talk on the ‘Movement of chemicals through the environment’ delivered by Kevin C.Jones (Lancaster University). More than 200 POPs occur at low concentrations in the environment, often as mixtures, and are characterized by their persistence, ability to accumulate in food chains, ability to move long distances globally via complex transport and exposure pathways and the fact that low concentrations of these contaminants appear to trigger biochemical processes (Kalantzi et al., 2004a). They include the older organochlorine (OC)-type compounds and newer brominated compounds (Kalantzi et al., 2004b), and production may exceed 10 000 tonnes/year (Harrad et al., 1994). Determining in which ecosystem compartment a chemical will be found is important for risk assessment. As a function of chemical properties and discharge from source, residues may accumulate, there may be dynamic exchange through the air into ocean or terrestrial systems and less persistent contaminants are degraded. Regulators distinguish between controllable sources and non-controllable sources, e.g. pesticides leaching out of the soil into the atmosphere. From the late 1960s onwards OC levels in human tissues have in general decreased, whereas polybrominated diphenyl ether (PBDE) concentrations have continued to increase (Norén and Meironyté, 2000; Kalantzi et al., 2004b). A global inventory requires knowledge of formulations, usages and emissions (Alcock et al., 2000; Sweetman et al., 2000; Prevedouros et al., 2004). A pulse of contaminants may concentrate in soils or an air system exchange might drive a global movement. Contaminants may condense onto mountain or polar ecosystems (where they are very persistent), concentrate in soils high in organic matter (e.g. the Northern Hemisphere), or pass into oceans where they are coupled into phytoplankton cycles. Soils from background environments at varying latitudes may thus be a marker of contaminant profile deposition (Meijer et al., 2003). Heavier contaminants (less volatile) are strongly associated with source area, whereas lighter hexachlorocyclohexanes (HCHs) volatize out of the soil and may move more freely (Prevedouros et al., 2004). Passive air samplers distributed throughout Europe showed that the highest concentrations of POPs tend to occur in urban centres and, 30 years after being banned, there are still substantial emissions from primary source regions (Jaward et al., 2004). Despite localized emissions, HCH air residue concentrations are similar throughout Europe. POPs are fat-soluble contaminants that are found in meat, dairy products and fish; drinking water or inhalation are minor exposure routes (Kalantzi et al., 2001). Air deposition on vegetation tends to be the start of the food chain transfer process, with livestock such as dairy cattle acting as a conveyor belt carrying POPs into the human food chain (Thomas et al., 1999). Absorbed passively through the gastrointestinal tract, POPs may circulate unmetabolized until they are absorbed into the body fat. As the fat store changes capacity, contaminants may re-mobilize and re-enter the circulation, resulting in delayed effects (Juan et al., 2002). Humans have extremely variable tissue concentrations, dependent, for example, on cumulative exposure, absorption efficiency, age, gender, fat composition and metabolic efficiency (Alcock et al., 2000). Additional factors, including occupational exposures or habitats close to landfill sites, may also be confounding factors. Caroline J.Langdon (Reading University, UK) discussed ‘Processing of arsenic by tolerant and non-tolerant earthworm species’. Several earthworm species inhabiting sites with arsenic (As)-rich metalliferous soils in the UK exhibit resistance to As toxicity (Langdon et al., 1999, 2001a). However, the mechanisms of uptake, accumulation and resistance are not clearly understood (Langdon et al., 1999, 2001a,b, 2002, 2003a,b; Piearce et al., 2002). Early studies showed that the LC50 concentration of As for Lumbricus rubellus from Devon Great Consols (an abandoned arsenic and copper mine) in the UK (1510 mg As/kg) was significantly higher than that in earthworms originating from an uncontaminated site (96 mg As/kg). There is some evidence that earthworms can accumulate As from contaminated soils. Measuring As loss following transfer of populations of L.rubellus originating from contaminated sites (Devon Great Consols and Carrock Fell in the UK) to uncontaminated soil, Langdon et al. (2003a) calculated the half-life in the earthworm body to be 10.4 days. By investigating the rate and extent of As uptake and depuration by resistant and non-resistant earthworm populations, earthworms native to contaminated sites were found to have a residual As burden even after transfer for up to 6 months to uncontaminated soil (Langdon et al., 2003a). Furthermore, Langdon et al. (2002) described the presence of arsenobetaine in L.rubellus from contaminated soil being predominantly bound to sulfur in the form of As (V)-O, in a glutathione-type environment, suggesting metallothionein complexation. Through changes in the As speciation, earthworms may be able to partially regulate endogenous concentrations, possibly through increased elimination rates at higher levels. The mode of As detoxification in earthworm tissues is presently unclear. Biochemical analyses indicate that As3+–thiol complexes are probably involved (Langdon et al., 2002, 2003a) with the cysteine-rich metalloprotein metallothionein, the most probable thiol donor (Langdon et al., 2003c). However, the mechanisms of resistance remain unclear and may be adaptive, i.e. genetic, or represent physiological acclimation. Langdon et al. (2003b) observed that As resistance was evident in F1 generations of resistant populations whilst resistance to copper toxicity was not, suggesting different mechanisms of resistance within the same organism. Huw J.Ricketts (Cardiff University, UK) described ‘Reproductive molecular genetic biomarkers in earthworms: from single organisms to ecology’. Because acute toxicity measurements are insufficient in isolation, the use of molecular biomarkers to assess the risk that toxic chemicals may pose within the environment is increasing. Sublethal end-points, such as growth or reproduction, are sensitive measurements of toxicity (Ricketts et al., 2004). Although these may not readily allow ecotoxicologists to assess ecologically relevant exposures, such problems may be overcome by using molecular biomarkers that are either linked in a specific manner to demography or a higher functional level, i.e. growth or reproduction (Ricketts et al., 2004). Biomarker data may then be used in tandem with chemical analysis to develop cause/effect relationships. One such biomarker in the earthworm, a sentinel soil invertebrate, is the sex hormone annetocin. Measurement of annetocin gene expression in earthworms exposed to contaminated soils has been compared with traditional growth and reproductive indices, including cocoon production (Ricketts et al., 2004). A reduction in cocoon production was accompanied by a significant reduction in annetocin expression, suggesting that it may have ecotoxicological predictive potential (Ricketts et al., 2004). Andreas Kortenkamp (School of Pharmacy, University of London, UK) dealt with ‘Mixture effects of oestrogenic chemicals at levels below “no effect concentrations” (NOECs)’. Chemical risk assessment has traditionally focused on individual agent effects, as mixture effects are often dismissed on the grounds that adverse reactions are unlikely when components are present at levels around ‘no observable adverse effect levels’ (NOAELs) (Silva et al., 2002). Silva et al. (2002) tested whether multi-component mixtures of xeno-oestrogens would produce significant effects when each component was combined at concentrations below its individual NOEC. The oestrogenic effects of a mixture of eight environmentally relevant chemicals, including hydroxylated PCBs, benzophenones, parabenes, bisphenol A and genistein, were recorded in a recombinant yeast estrogen screen (YES). To exclude the possibility of one chemical contributing disproportionately to the overall combination effect, a mixture was prepared at a mixture ratio proportional to the potency of each individual component. The combined effects of the chemicals were well predicted by the concept of concentration addition. Substantial mixture effects were observed in the YES even though each chemical was present at levels well below its NOEC. This suggests that oestrogenic agents are able to act together to produce significant effects when combined at concentrations below their individual NOECs (Silva et al., 2002). Although it remains to be seen whether these observations also apply to apical end-points, including cell proliferation, the results highlight the limitations of the traditional focus on single agent effects. Hazard assessments that ignore the possibility of additive effects of oestrogenic chemicals are likely to significantly underestimate risk. J.Thomas Sanderson (Institute for Risk Assessment Sciences, Utrecht University, The Netherlands) continued and discussed the ‘Effects of xenobiotics and natural compounds on steroid hormone synthesis and metabolism: implications for genotoxicity and the development of hormone-dependent cancers’. Environmental contaminants that interact with sex hormone receptors may be partly responsible for the rise in endocrine-related adverse health effects. Oestrogen exposures are associated with increased risk of breast cancer (Henderson and Feigelson, 2000), enhance cell proliferation and oestrogen metabolites may be genotoxic (Yared et al., 2002). Other than receptor interactions, effects on endocrine functions may include inhibition or induction of cytochrome P450 (CYP) enzymes that are involved in steroid hormone synthesis and metabolism. Sanderson et al. (2002) and van Duursen et al. (2003) investigated the expression levels and catalytic activities of aromatase (CYP19), CYP1A1, CYP1B1 and catechol O-methyltransferase (COMT) following exposure of mammalian cells to POPs. Herbicides and fungicides variously induced CYP19 in human adrenocortical carcinoma (H295R) cells in vitro (Sanderson et al., 2000, 2002). Dioxin-like polychlorinated aromatic hydrocarbons preferentially increased the formation (CYP1A1-catalysed) of less genotoxic 2- over more genotoxic (CYP1B1-catalysed) 4-hydroxyoestrogens in human breast cancer MCF-7 and MCF-10A cell lines (van Duursen et al., 2003). Natural flavonoids, potential chemopreventative agents, were generally weak inhibitors of CYP19 activity in H295R cells; quercetin was an inducer whilst being an effective inhibitor of COMT activity (Sanderson et al., 2002). Such observations suggest that complex mixtures may modulate oestrogen metabolism and consequently may affect breast cancer risk. Michael N.Routledge (University of Leeds, UK) discussed the ‘Genotoxic effects of a binary mixture of mutagens’. Humans are exposed to mixtures of mutagens. Routledge et al. (2001) investigated the genotoxicity of a binary mixture of benzo[a]pyrene (BP) and UV irradiation. UV induction of strand breaks in plasmid DNA was enhanced by prior adduction with a BP diol epoxide (BPDE). In the supF gene forward mutation assay the mutation frequency for BPDE followed by UVC was 197 in 104 transformants; this compared with 12 in 104 for single treatments of BPDE and 26 in 104 for single treatments of UVC. Signature mutations predominant in individual BPDE (GC→TA) and UVC (GC→AT) spectra were present following this binary exposure; there was a relatively high increase in GC→TA mutations, although the BPDE adduct levels remained unchanged (Routledge et al., 2001). When the order of exposure was reversed, the enhanced mutation frequency induced by UVC followed by BPDE treatment compared with the individual treatments was less profound (McLuckie et al., 2004). Thus, UV-irradiation appears to enhance mutation frequency via a direct effect on BPDE adducts. In cultured A549 cells, a metabolically active lung epithelial cell line, BP followed by UVC treatment resulted in an increase in DNA single-strand breaks, as measured using the alkaline comet assay, that was greater than the additive effects of BP and UVC treatments (Misl'anova et al., 2003). However, 8-oxo-deoxyguanosine levels (measured by HPLC–ECD) were additively elevated. For the day's final presentation, Peter A.Behnisch (Eurofins GfA, Germany) discussed ‘Bioanalytical screening methods for dioxins and dioxin-like compounds’. Since the Stockholm Convention on POPs (2001), many governmental bodies are re-evaluating the real threats from old and new environmental contaminants. Because of environmental pollution incidents and increasing worldwide awareness of environmental health, it is now necessary to combine both chemical and biological testing strategies in order to better understand impact and mechanism (Behnisch et al., 2001a). New screening tools, based on the binding of dioxin-like compounds to the arylhydrocarbon or oestrogen receptors, have been developed and are generating new interpretations that are additional to the existing chemical analysis database. These include gene reporter assays (CALUX) and ELISA (Ah Immunoassay) methods. They allow the evaluation of POPs such as dioxins and/or PCBs as well as other contaminants such as PBDEs and they have already been applied to feed/food crises in Europe and to biomonitoring projects (Behnisch et al., 2001b). These cost-effective, quick, sensitive and accurate methods are based on well-known mechanisms utilizing well-defined end-points. The first session on the second day was chaired by Trevor J.McMillan (Lancaster University) and started with the keynote lecture entitled ‘Molecular carcinogenesis and molecular epidemiology of human cancer’ delivered by Curtis C.Harris (National Cancer Institute, USA). Hypotheses generated from observations in epidemiological studies may be tested experimentally. Environment may be classified as macro-environment (e.g. exogenous) or micro-environment (e.g. endogenous). The ultimate goal of molecular epidemiology is to identify high cancer risk individuals based on gene–environment–lifestyle interactions. Although lung cancer risk is reduced following a cessation of smoking, it never returns to levels observed for never smokers (Peto et al., 2000). Genetic susceptibility probably plays a more prominent role in lung cancer risk in cases of lower carcinogen exposure, e.g. passive smokers. Approximately 50% of the population are homozygous carriers of the glutathione S-transferase M1 (GSTM1) null allele. A statistically significant elevation of lung cancer risk following environmental tobacco smoke exposure was observed amongst homozygous null as compared with heterozygous or homozygous carriers of the wild-type GSTM1 allele (Bennett et al., 1999). Future studies will allow a comparison of phenotypes (e.g. cancer) with genotypes using various gene expression and bioinformatics platforms. Commonly mutated in human cancers, the molecular archaeology archived within the mutation spectrum of TP53 might reveal clues about mutation-inducing agents, exogenous or endogenous, that are implicated in clonal expansion from preneoplastic to neoplastic to tumour to metastases. The p53 protein is at the crossroads of the cellular stress response (Hussain et al., 2001). Mutant cells within tumours might hypothetically be selected on the basis that, of all possible mutations that occur, only those that give a survival/growth advantage would be archived. The majority of TP53 mutations are missense, giving rise mostly to loss of tumour suppressor function, although sometimes to a gain of oncogenic function. Molecular epidemiology studies clearly link aflatoxin B1 exposure and codon Ser249 mutations in the aetiology of hepatocellular carcinoma (HCC) (Hussain et al., 2001). Case–control studies demonstrated that an interaction between hepatitis B virus infection and aflatoxin B1 exposure dramatically increased HCC risk (Montesano et al., 1997). Micro-environmental factors such as inflammation can also play an important role in the aetiology of cancer (Ames, 1989). TP53 mutational hotspots at CpG dinucleotides may be the result of endogenous 5-methylcytosine deamination (Hussain et al., 2001). Increased nitric oxide (NO) production by nitric oxide synthase-2 (NOS2) is associated with TP53 C→T transitions and may be a crucial pathway in inflammatory-mediated carcinogenesis (Hussain et al., 2003). Ulcerative colitis (UC) is an oxyradical overload disease that shows an association between increased TP53 mutations and NOS2 activity and an elevated risk of colon cancer (Hussain et al., 2000). NO induces DNA damage via p53 post-translational modifications in the activation of the DNA damage stress response pathway and occurs in chronic inflammatory diseases such as UC (Hofseth et al., 2003). A DNA damage response pathway resulting in the phosphorylation, acetylation and activation of p53 is induced by NO both in vitro (in MCF-7 cells exposed to NO-generating drugs or co-cultured with NO-releasing macrophages) and in UC (Hofseth et al., 2003). Characteristic TP53 mutation spectra from non-tumour tissue may be used as a biomarker of carcinogen effect or as a predictor of cancer risk (Olivier et al., 2004). Francis L.Martin (Lancaster University) discussed ‘Clever carcinogens: actions and interactions’. The aetiology of cancers of the breast and prostate remain obscure (Grover and Martin, 2002). Candidate agents that are proven rodent mammary carcinogens have been identified, but typical exposures question their relevance to human health. Increased cumulative exposure to oestrogen has been linked to increased risk of breast cancer (Henderson and Feigelson, 2000). The cytokinesis block micronucleus assay and the alkaline comet assay were used to examine the oestrogen receptor-positive breast carcinoma MCF-7 cell line for chromosomal damage and DNA single-strand breaks (SSBs), respectively (Yared et al., 2002; Davis et al., 2002). β-Oestradiol, oestrone and oestriol were tested for genotoxicity in the 10−10–10−2 M concentration range. β-Oestradiol, following a 24 h treatment, induced increases (up to 3-fold) in micronuclei (MNi) at a concentration of 10−9 M; at higher concentrations dose-related decreases were observed. Oestrone induced dose-related increases in MNi (up to 5-fold), whereas oestriol appeared to be inactive (Yared et al., 2002). All three oestrogens induced dose-related increases in per cent binucleate cells, suggesting that they enhance mitotic rate. In the comet assay both β-oestradiol and oestrone induced dose-related increases in SSBs (up to 7-fold over control comet tail length) and were significantly comet forming (P < 0.0001) at concentrations as low as 10−9 and 10−8 M, respectively, whereas oestriol was less genotoxic (Yared et al., 2002). Oestrogens as modulators of carcinogen-induced DNA damage may increase breast cancer risk. The effects of BP in MCF-7 cells incubated in the presence or absence of β-oestradiol, oestrone or oestriol were also investigated (Davis et al., 2002). Marked increases in MNi (3-fold) occurred following a 24 h treatment with 10−6 M BP; lower concentrations appeared to be inactive. Co-treatment with either 10−9 M β-oestradiol, 10−8 M oestrone or 10−8 M oestriol induced 2- to 3-fold increases in MNi with 10−8 M BP. Co-treatment with β-oestradiol enhanced levels of BP–DNA adducts whilst reversing BP-induced decreases in cell proliferation (per cent binucleate cells) and plating efficiency (Davis et al., 2002). A characteristic feature of low dose exposure to hormone-like compounds is an elevated percentage of cells staining positive for the anti-apoptotic Bcl-2 protein and a reduced percentage of cells staining positive for the pro-apoptotic Bax protein (Kalantzi et al., 2004a). In combination with BP, low dose mixture effects, detectable by both immunocytochemical methods and quantitative real-time RT–PCR, were apparent. This work suggests that whilst hormone-like compounds appear to enhance carcinogen-induced DNA damage, they may also trigger survival mechanisms. Paul L.Carmichael (Unilever, UK) reported on ‘Mechanisms of “selective oestrogen receptor modulators” (SERM)-induced carcinogenesis’. In 1996 a SERM-type drug, tamoxifen, one of the most successful and widely prescribed antineoplastic agents worldwide, was classified as a group one human carcinogen (International Agency for Research on Cancer, 1996). Studies of the mechanistic basis for this carcinogenicity have largely focused on the potential, as seen in experimental animals, for the drug to be metabolized to genotoxic species that bind covalently to DNA and cause mutations (White et al., 1992). The presence of DNA adducts in human tissues and most importantly in the target tissue, the endometrium, has been the source of considerable debate (Poirier and Schild, 2003). Initial analyses, by 32P-post-labelling, of DNA isolated from tissues of patients treated with the drug failed to find any evidence of adducts (Carmichael et al., 1996). These findings were supported by HPLC analysis of 32P-post-labelled DNA from endometrial explants from a further cohort of patients treated in culture with tamoxifen (Carmichael et al., 1999). However, later studies have suggested that adducts may be detectable in the endometrium of some women on tamoxifen therapy (Hemminki et al., 1996; Shibutani et al., 1999). Regardless of the presence or absence of DNA adducts, tamoxifen is often responsible for rapidly induced benign changes in endometrial pathology, such as hyperplasia and polyp formation (Carmichael et al., 2000). This has been attributed to oestrogen agonist activity in the tissue, but rapid tamoxifen-specific induction of adenomyosis in mice and endometrial tumours in neonatal rats, in the absence of hormone-induced hyperplasia, suggests other epigenetic or non-genotoxic mechanisms (Waddell et al., 2004). Tamoxifen induces a mitogenic environment in the human endometrium based on dysregulation of TGFβ and cognate receptors (Carmichael et al., 2000). In studies exploring gene expression profiles in cultures of human endometria treated with tamoxifen, raloxifene (a non-carcinogenic SERM) or 17β-estradiol, tamoxifen was found to induce the largest number of gene expression changes that were common to those characteristic of endometrial neoplasia. To label tamoxifen as an adduct-forming genotoxin is an oversimplification of the carcinogenic profile of this drug. Anthony M.Lynch (GlaxoSmithKline, UK) presented ‘A brief overview of regulatory genetic toxicology testing’. The accumulation of mutations in somatic cells is implicated in cancer and, to some extent, in other multifactorial diseases, e.g. heart disease. Novel agents introduced into the environment by humans represent a potential source of damage to DNA and thus appropriate testing strategies are required in order to minimize the risk of genotoxicity (Smith et al., 2003). The minimum package of assays recommended by regulatory agencies for the assessment of genotoxicity usually consists of: (i) an in vitro bacterial assay for gene mutation; (ii) an in vitro mammalian cell assay for gene mutation and/or cytogenetic damage; (iii) for drugs, an in vivo mammalian assay for cytogenetic damage (Cahill et al., 2004). A negative result in all of these is usually sufficient evidence for a lack of genotoxic potential, although additional testing may be required for substances with structural chemical alerts or when structurally similar compounds are carcinogenic (Snyder et al., 2004). More testing is required in the event of positive findings. Taking into account the significance and limitations of each assay, a risk assessment is made based on all the test results and indications as to whether genetic damage occurs directly or indirectly. In the final session, chaired by Francis L.Martin, John Ashby (Syngenta CTL, UK) discussed ‘Mixtures, low dose effects, mechanisms and the regulation of endocrine active chemicals’. Concerns over endocrine disrupters cannot be dismissed, but need refining. In order to validate test systems that accurately measure the effects of endocrine disrupters, low dose effects, mixture effects and assumptions regarding mechanisms need to be considered. Although many low dose effects data exist for bisphenol A (BPA), it remains impossible to locate a single study that is capable of independent confirmation (Ashby et al., 2004). Hence, either BPA possesses subtle low dose hormone toxicities that are only evident under as yet undefined experimental conditions or these observations reflect a failure to define and understand control variability, thus generating artefactual positive results. This question requires urgent resolution so as to enable reliable low dose risk assessments of endocrine disrupters to be made. Additive or synergistic mixture effects are equally of primary environmental importance (Tinwell and Ashby, 2004). However, pharmacokinetic or pharmacodynamic considerations indicate that it will be critical to distinguish in vitro from in vivo mixture effects. Mixtures of BPA and genistein in the immature rat uterotrophic assay indicate substantially less than additive uterotrophic effects. The nature of the activities of a range of androgens in the immature rat uterotrophic assay suggests a complex aetiology for uterotrophic (gravimetric) activity. Weihua et al. (2002) recently reported that the ability of 17β-oestradiol to increase luminal epithelial hyperplasia in the immature rat uterus could be blocked by the anti-androgen flutamide. However, flutamide failed to modulate 17β-oestradiol-induced uterotrophic activity in the immature rat. These observations indicate that the uterotrophic assay is not a simple and specific test for oestrogens. In the final presentation, David H.Phillips (Institute of Cancer Research, UK) considered ‘Biomarkers of exposure with particular emphasis on DNA adducts’. Many carcinogens exert their biological effects through covalent binding of their metabolically activated intermediates to DNA. DNA adduct analysis in human tissues is, therefore, a potential tool for molecular epidemiological studies of cancer (Perera et al., 2002). A large body of evidence now suggests that DNA adducts are useful markers of carcinogen exposure, providing an integrated measurement of carcinogen intake, metabolic activation and delivery to the target macromolecule in the target tissue (Phillips, 2002). Monitoring accessible surrogate tissues, such as white blood cells, also provides a means of monitoring healthy individuals (Perera et al., 2002). Environmental exposure to carcinogens, for example to polycyclic aromatic hydrocarbons, in several defined populations has been demonstrated by the detection of higher levels of DNA adducts (Butkiewicz et al., 1999). Smoking-related adducts are detectable in many human tissues at levels that are significantly higher than in non-smokers, although the magnitude of the elevation does not always predict the magnitude of the risk (Perera et al., 2002; Benhamou et al., 2003; Phillips et al., 2004). While such associations do not demonstrate causality, they do, importantly, lend plausibility to observations of associations between smoking and cancer. However, there is still some resistance to the notion that such monitoring can inform, rather than merely confirm, epidemiological investigations of cancer causation. Interestingly, smoking was recently causally linked to cervical cancer after many years of being considered a confounding factor (Plummer et al., 2003); yet smoking-related DNA adducts have been known to be present in cervical epithelium for some years (Phillips, 2002). In the limited number of prospective studies performed thus far, elevated levels of DNA adducts have been found in exposed individuals who subsequently developed cancer compared with exposed individuals who did not (Perera et al., 2002). The precise reasons for the large differences in DNA adduct levels among individuals apparently exposed to similar levels of carcinogens (e.g. smokers smoking similar numbers of cigarettes/day) are currently unclear. To some extent, polymorphisms in genes for xenobiotic metabolism or DNA repair may be influential, but so also may many non-genetic factors, such as nutritional status and antioxidant levels (Palli et al., 2003). The Workshop discussed the movement of chemicals in the environment, the biomonitoring of these in different systems and current thoughts on underlying pathophysiological mechanisms. Key questions dealt with included the relative importance of measuring total contaminant or the bioavailable concentrations in environmental matrices. With regard to organism exposures, it was emphasized that dose alone might not be critical but that other factors, such as timing or mixture effects, may well be important. Thus a mixture of agents (e.g. endocrine disrupters) may affect the same pathway or, alternatively, two different mechanisms may be involved, thus predicting outcomes is difficult. Traditionally, the view has often been taken that if human exposures are reduced then, as a by-product, the environment will also be protected. Clearly, the environmental ecological impact might be quite different from the human scenario. Susceptibility to biologically effective doses of agents may be a function of endogenous activation or detoxification mechanisms. Whether disease risk following exposures, either on a population or on a more individually predictive basis, extrapolates to disease needs to be determined. Environmental agents, singly or in mixtures, still require proper assessment at environmentally relevant levels using relevant biological assays. Future studies need to distinguish heterogeneous intra- as well as inter-individual risk variations and dose versus susceptibility. Understanding disease mechanisms will allow the development of strategies to reduce exposures or, through retrospective salvaging treatments, to neutralize such processes. The meeting was supported by the Institute of Environmental and Natural Sciences (Lancaster University), the LEC and UKEMS. We are most grateful to Sue Taylor (Environmental Science) for administrative organization and Rebecca Hewitt and Kirstie J.Ford for assistance. The authors also wish to acknowledge the contribution made by participants to the preparation of this article through the provision of abstracts prior to the workshop. References Alcock,R.E., Sweetman,A.J., Juan,C.Y. and Jones,K.C. ( 2000 ) A generic model of human lifetime exposure to persistent organic contaminants: development and application to PCB-101. Environ. Pollut. , 110 , 253 –265. Ames,B.N. ( 1989 ) Mutagenesis and carcinogenesis: endogenous and exogenous factors. Environ. Mol. Mutagen. , 14 (suppl. 16), 66 –77. Ashby,J., Tinwell,H., Odum,J. and Lefevre,P. ( 2004 ) Natural variability and the influence of concurrent control values on the detection and interpretation of low-dose or weak endocrine toxicities. Environ. Health Perspect. , 112 , 847 –853. Behnisch,P.A., Hosoe,K. and Sakai,S. ( 2001 ) Bioanalytical screening methods for dioxins and dioxin-like compounds a review of bioassay/biomarker technology. Environ. Int. , 27 , 413 –439. Behnisch,P.A., Hosoe,K. and Sakai,S. ( 2001 ) Combinatorial bio/chemical analysis of dioxin and dioxin-like compounds in waste recycling, feed/food, humans/wildlife and the environment. Environ. Int. , 27 , 495 –519. Benhamou,S., Laplanche,A., Guillonneau,B., Mejean,A., Desgrandchamps,F., Schrameck,C., Degieux,V. and Perin,F. ( 2003 ) DNA adducts in normal bladder tissue and bladder cancer risk. Mutagenesis , 18 , 445 –448. Bennett,W.P., Alavanja,M.C., Blomeke,B. et al. ( 1999 ) Environmental tobacco smoke, genetic susceptibility and risk of lung cancer in never-smoking women. J. Natl Cancer Inst. , 23 , 2009 –2014. Butkiewicz,D., Cole,K.J., Phillips,D.H., Harris,C.C. and Chorazy,M. ( 1999 ) GSTM1, GSTP1, CYP1A1 and CYP2D6 polymorphisms in lung cancer patients from an environmentally polluted region of Poland: correlation with lung DNA adduct levels. Eur. J. Cancer Prev. , 8 , 315 –323. Cahill,P.A., Knight,A.W., Billinton,N., Barker,M.G., Walsh,L., Keenan,P.O., Williams,C.V., Tweats,D.J. and Walmsley,RM. ( 2004 ) The GreenScreen genotoxicity assay: a screening validation programme. Mutagenesis , 19 , 105 –119. Carmichael,P.L., Ugwumadu,A.H., Neven,P., Hewer,A.J., Poon,G.K. and Phillips,D.H. ( 1996 ) Lack of genotoxicity of tamoxifen in human endometrium. Cancer Res. , 56 , 1475 –1479. Carmichael,P.L., Sardar,S., Crooks,N. et al. ( 1999 ) Lack of evidence from HPLC 32P-post-labelling for tamoxifen-DNA adducts in the human endometrium. Carcinogenesis , 20 , 339 –342. Carmichael,P.L., Pole,J.C. and Neven,P. ( 2000 ) Modulation of endometrial transforming growth factor beta (TGFbeta) by tamoxifen. Eur. J. Cancer , 36 (suppl. 4), S42 –S43. Davis,C., Bhana,S., Shorrocks,A.J. and Martin,F.L. ( 2002 ) Oestrogens induce G1 arrest in benzo[a]pyrene-treated MCF-7 breast cells whilst enhancing genotoxicity and clonogenic survival. Mutagenesis , 17 , 431 –438. Grover,P.L. and Martin,F.L. ( 2002 ) The initiation of breast and prostate cancer. Carcinogenesis , 23 , 1095 –1102. Harrad,S.J., Sewart,A.P., Alcock,R. et al. ( 1994 ) Polychlorinated biphenyls (PCBs) in the British environment: sinks, sources and temporal trends. Environ. Pollut. , 85 , 131 –146. Henderson,B.E. and Feigelson,H.S. ( 2000 ) Hormonal carcinogenesis. Carcinogenesis , 21 , 427 –433. Hemminki,K., Rajaniemi,H., Lindahl,B. and Moberger,B. ( 1996 ) Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res. , 56 , 4374 –4377. Hofseth,L.J., Saito,S., Hussain,S.P. et al. ( 2003 ) Nitric oxide-induced cellular stress and p53 activation in chronic inflammation. Proc. Natl Acad. Sci. USA , 100 , 143 –148. Hussain,S.P., Amstad,P., Raja,K. et al. ( 2000 ) Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease. Cancer Res. , 60 , 3333 –3337. Hussain,S.P., Hofseth,L.J. and Harris,C.C. ( 2001 ) Tumor suppressor genes: at the crossroads of molecular carcinogenesis, molecular epidemiology and human risk assessment. Lung Cancer , 34 , S7 –S15. Hussain,S.P., Hofseth,L.J. and Harris,C.C. ( 2003 ) Radical causes of cancer. Nature Rev. Cancer , 3 , 276 –285. International Agency for Research on Cancer ( 1996 ) IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans. Vol. 66. Some Pharmaceutical Drugs. IARC, Lyon, France. Jaward,F.M., Farrar,N.J., Harner,T., Sweetman,A.J. and Jones,K.C. ( 2004 ) Passive air sampling of PCBs, PBDEs and organochlorine pesticides across Europe. Environ. Sci. Technol. , 38 , 34 –41. Juan,C.Y., Thomas,G.O., Sweetman,A.J. and Jones,K.C. ( 2002 ) An input–output balance study for PCBs in humans. Environ. Int. , 28 , 203 –214. Kalantzi,O.I., Alcock,R.E., Johnston,P.A., Santillo,D., Stringer,R.L., Thomas,G.O. and Jones,K.C. ( 2001 ) The global distribution of PCBs and organochlorine pesticides in butter. Environ. Sci. Technol. , 35 , 1013 –1018. Kalantzi,O.I., Hewitt,R., Ford,K.J. et al. ( 2004 ) Low-dose induction of micronuclei by lindane. Carcinogenesis , 25 , 613 –622. doi:10.1093/carcin/bgh048 (accessed 19 December 2003). Kalantzi,O.I., Martin,F.L., Thomas,G.O., Alcock,R.E., Tang,H.R., Drury,S.C., Carmichael,P.L., Nicholson,J.K. and Jones,K.C. ( 2004 ) Different levels of polybrominated diphenyl ethers (PBDEs) and chlorinated compounds in breast milk from two UK regions. Environ. Health Perspect. , 112 , 1085 –1091. doi:10.1289/ehp.6991 (accessed 21 April 2004). Langdon,C.J., Piearce,T.G., Black,S. and Semple,K.T. ( 1999 ) Resistance to arsenic-toxicity in a population of the earthworm Lumbricus rubellus. Soil Biol. Biochem. , 31 , 1963 –1967. Langdon,C.J., Piearce,T.G., Meharg,A.A. and Semple,K.T. ( 2001 ) Survival and behaviour of the earthworms Lumbricus rubellus and Dendrodrilus rubidus from arsenate-contaminated and non-contaminated sites. Soil Biol. Biochem. , 33 , 1239 –1244. Langdon,C.J., Piearce,T.G., Meharg,A.A. and Semple,K.T. ( 2001 ) Resistance of copper toxicity in populations of Lumbricus rubellus and Dendrodrilus rubidus from contaminated mine wastes. Environ. Toxicol. Chem. , 20 , 2336 –2341. Langdon,C.J., Meharg,A.A., Feldmann,J., Charnock,J., Farquhar,M., Piearce,T.G., Semple,K.T. and Cotter-Howells,J. ( 2002 ) Arsenic speciation in arsenate-resistant and non-resistant populations of the earthworm, Lumbricus rubellus. J. Environ. Monitor , 4 , 603 –608. Langdon,C.J., Piearce,T.G., Feldmann,J., Semple,K.T. and Meharg,A.A. ( 2003 ) Arsenic metabolism in the earthworms Lumbricus rubellus and Dendrodrilus rubidus. Environ. Toxicol. Chem. , 22 , 1302 –1308. Langdon,C.J., Piearce,T.G., Meharg,A.A. and Semple,K.T. ( 2003 ) Inherited resistance to arsenate- but not copper-toxicity in a population of Lumbricus rubellus. Environ. Toxicol. Chem. , 22 , 2344 –2348. Langdon,C.J., Piearce,T.G., Meharg,A.A. and Semple,K.T. ( 2003 ) Interactions between earthworms and arsenic in the soil environment: a review. Environ. Pollut. , 124 , 361 –373. McLuckie,K.I., Gaskell,M., Farmer,P.B., Martin,E.A., Jones,G.D. and Routledge,M.N. ( 2004 ) Effects of the order of exposure to a binary mixture of mutagens on the induced mutation spectra in the supF gene. Mutagenesis , 19 , 137 –141. Meijer,S.N., Ockenden,W.A., Sweetman,A., Breivik,K., Grimalt,J.O. and Jones,K.C. ( 2003 ) Global distribution and budget of PCBs and HCB in background surface soils: implications for sources and environmental processes. Environ. Sci. Technol. , 37 , 667 –672. Misl'anova,C., Healey,K., White,K.L., Smith,E.C., Hardie,L.J., Dusinska,M., Wild,C.P. and Routledge,M.N. ( 2003 ) Binary exposure of A549 cells to benzo[a]pyrene and UVC radiation yields enhanced DNA damage in the comet assay but no enhancement of 8-oxo-deoxyguanosine. Environ. Mol. Mutagen. , 42 , 228 –230. Montesano,R., Hainaut,P. and Wild,C.P. ( 1997 ) Hepatocellular carcinoma: from gene to public health. J. Natl Cancer Inst. , 89 , 1844 –1851. Norén,K. and Meironyté,D. ( 2000 ) Certain organochlorine and organobromine contaminants in Swedish human milk in perspective of past 20–30 years. Chemosphere , 40 , 1111 –1123. Olivier,M., Hussain,S.P., Caron de Fromentel,C., Hainaut,P. and Harris,C.C. ( 2004 ) TP53 mutation spectra and load: a tool for generating hypotheses on the etiology of cancer. IARC Sci. Publ. , 157 , 247 –270. Palli,D., Masala,G., Vineis,P. et al. ( 2003 ) Biomarkers of dietary intake of micronutrients modulate DNA adduct levels in healthy adults. Carcinogenesis , 24 , 739 –746. Perera,F.P., Mooney,L.A., Stampfer,M. et al. ( 2002 ) Associations between carcinogen-DNA damage, glutathione S-transferase genotypes and risk of lung cancer in the prospective Physicians' Health Cohort Study. Carcinogenesis , 23 , 1641 –1646. Peto,R., Darby,S., Deo,H., Silcocks,P., Whitley,E. and Doll,R. ( 2000 ) Smoking, smoking cessation and lung cancer in the UK since 1950: combination of national statistics with two case-control studies. Br. Med. J. , 321 , 323 –329. Phillips,D.H. ( 2002 ) Smoking-related DNA and protein adducts in human tissues. Carcinogenesis , 23 , 1979 –2004. Phillips,D.H., Hewer,A., Scholefield,J.H. and Skinner,P. ( 2004 ) Smoking-related DNA adducts in anal epithelium. Mutat. Res. , 560 , 167 –172. Piearce,T.G., Langdon,C.J., Meharg,A.A. and Semple,K.T. ( 2002 ) Yellow earthworms: pigmentation as an indicator of metal resistance in contaminated mine spoil. Soil Biol. Biochem. , 34 , 1833 –1838. Plummer,M., Herrero,R., Franceschi,S., Meijer,C.J., Snijders,P., Bosch,F.X., de Sanjose,S. and Munoz,N. ( 2003 ) Smoking and cervical cancer: pooled analysis of the IARC multi-centric case–control study. Cancer Causes Control , 14 , 805 –814. Poirier,M.C. and Schild,L.J. ( 2003 ) The genotoxicity of tamoxifen: extent and consequences, Kona, Hawaii, January 23, 2003. Mutagenesis , 18 , 395 –399. Prevedouros,K., MacLeod,M., Jones,K.C. and Sweetman,A.J. ( 2004 ) Modelling the fate of persistent organic pollutants in Europe: parameterisation of a gridded distribution model. Environ. Pollut. , 128 , 251 –261. Ricketts,H.J., Morgan,A.J., Spurgeon,D.J. and Kille,P. ( 2004 ) Measurement of annetocin gene expression: a new reproductive biomarker in earthworm ecotoxicology. Ecotoxicol. Environ. Saf. , 57 , 4 –10. Routledge,M.N., McLuckie,K.I., Jones,G.D., Farmer,P.B. and Martin,E.A. ( 2001 ) Presence of benzo[a]pyrene diol epoxide adducts in target DNA leads to an increase in UV-induced DNA single strand breaks and supF gene mutations. Carcinogenesis , 22 , 1231 –1238. Sanderson,J.T., Seinen,W., Giesy,J.P. and van den Berg,M. ( 2000 ) 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity? Toxicol. Sci. , 54 , 121 –127. Sanderson,J.T., Boerma,J., Lansbergen,G.W. and van den Berg,M. ( 2002 ) Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol. Appl. Pharmacol. , 182 , 44 –54. Shibutani,S., Suzuki,N., Terashima,I., Sugarman,S.M., Grollman,A.P. and Pearl,M.L. ( 1999 ) Tamoxifen-DNA adducts detected in the endometrium of women treated with tamoxifen. Chem. Res. Toxicol. , 12 , 646 –653. Silva,E., Rajapakse,N. and Kortenkamp,A. ( 2002 ) Something from “nothing”—eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. Environ. Sci. Technol. , 36 , 1751 –1756. Smith,C.C., Lynch,A.M. and Gooderham,N.J. ( 2003 ) Evaluating the genetic toxicology of DNA-based products using existing genetic toxicology assays. Mutagenesis , 18 , 259 –264. Snyder,R.D., Pearl,G.S., Mandakas,G., Choy,W.N., Goodsaid,F. and Rosenblum,I.Y. ( 2004 ) Assessment of the sensitivity of the computational programs DEREK, TOPKAT and MCASE in the prediction of the genotoxicity of pharmaceutical molecules. Environ. Mol. Mutagen. , 43 , 143 –158. Sweetman,A.J., Alcock,R.E., Wittsiepe,J. and Jones,K.C. ( 2000 ) Human exposure to PCDD/Fs in the UK: the development of a modelling approach to give historical and future perspectives. Environ. Int. , 26 , 37 –47. Thomas,G.O., Sweetman,A.J. and Jones,K.C. ( 1999 ) Metabolism and body-burden of PCBs in lactating dairy cows. Chemosphere , 39 , 1533 –1544. Tinwell,H. and Ashby,J. ( 2004 ) Sensitivity of the immature rat uterotrophic assay to mixtures of estrogens. Environ. Health Perspect. , 112 , 575 –582. van Duursen,M.B., Sanderson,J.T., van der Bruggen,M., van der Linden,J. and van den Berg,M. ( 2003 ) Effects of several dioxin-like compounds on estrogen metabolism in the malignant MCF-7 and nontumorigenic MCF-10A human mammary epithelial cell lines. Toxicol. Appl. Pharmacol. , 190 , 241 –250. Waddell,W.J., Crooks,N.H. and Carmichael,P.L. ( 2004 ) Correlation of tumors with DNA adducts from methyl eugenol and tamoxifen in rats. Toxicol. Sci. , 79 , 38 –40. Weihua,Z., Ekman,J., Almkvist,A., Saji,S., Wang,L., Warner,M. and Gustafsson,J.A. ( 2002 ) Involvement of androgen receptor in 17β-estradiol-induced cell proliferation in rat uterus. Biol. Reprod. , 67 , 616 –623. White,I.N., de Matteis,F., Davies,A., Smith,L.L., Crofton-Sleigh,C., Venitt,S., Hewer,A. and Phillips,D.H. ( 1992 ) Genotoxic potential of tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5 cells. Carcinogenesis , 13 , 2197 –2203. Yared,E., McMillan,T.J. and Martin,F.L. ( 2002 ) Genotoxic effects of oestrogens in breast cells as detected by the micronucleus assay and the Comet assay. Mutagenesis , 17 , 345 –352. Author notes 1Department of Biological Sciences and 2Department of Environmental Science, IENS, Lancaster University, Lancaster LA1 4YQ, UK Mutagenesis vol. 19 no. 5 © UK Environmental Mutagen Society 2004; all rights reserved.
TI - Environmental health impacts: occurrence, exposure and significance, Lancaster University, UK, 9–10 September 2003
JF - Mutagenesis
DO - 10.1093/mutage/geh046
DA - 2004-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/environmental-health-impacts-occurrence-exposure-and-significance-a8BfFfN50U
SP - 423
EP - 429
VL - 19
IS - 5
DP - DeepDyve
ER -