Studies using specific biomarkers for human exposure assessment to exogenous and endogenous chemical agents.

The extent of formation of carcinogen adducts with DNA and protein may be used to assess the biologically effective dose of these carcinogens in the tissue under study. In normal human tissues, such carcinogen adducts arise in part from exposures to exogenous genotoxic compounds, although it has been shown that endogenously formed carcinogens also make a significant contribution to the observed DNA and protein damage. Although, highly sensitive analytical methods, such as immunoassay, 32P-postlabelling and mass spectrometry have been developed and successfully applied to measure carcinogen adducts, further methodological advances are making these methods more amenable to molecular epidemiological studies. Thus, the use of immunoslot blot assays allows a higher sample throughput for adduct quantification. Liquid chromatographic separations of adducts, either for their radiochemical detection following 32P-postlabelling or for their determination by mass spectrometry, improves the specificity and applicability of these techniques. In this review, the sensitivities and specificities of the analytical methods used for adduct detection are compared and the limitations of these methods described.     

DNA adducts as markers of exposure and risk

Many carcinogens exert their biological effects through the formation of DNA adducts by metabolically activated intermediates. Detecting the presence of DNA adducts in human tissues is, therefore, a tool for molecular epidemiological studies of cancer. A large body of evidence demonstrates 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 target tissues. Monitoring accessible surrogate tissues, such as white blood cells, also provides a means of investigating occupational or environmental exposure in healthy individuals. Such exposure to carcinogens, e.g. to polycyclic aromatic hydrocarbons, has been demonstrated in several industries and in defined populations, respectively, by the detection of higher levels of adducts. Adducts detected in many tissues of smokers are at levels significantly higher than in non-smokers, although the magnitude of the elevation does not predict the magnitude of the risk. While such associations do not demonstrate causality, they do, importantly, lend plausibility to observed associations between smoking and cancer. However, there is still 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 years of being considered a confounding factor; yet smoking-related adducts have been known to be present in cervical epithelium for some time. In the few prospective studies thus far, elevated adduct levels have been found in individuals who subsequently developed cancer compared with individuals who did not. The potential for biomarker measurements, such as DNA adducts, to provide answers to the origin of many cases of human cancer for which an environmental cause is suspected, needs to be exploited more fully in future epidemiological studies.     

Application of new techniques for the detection of carcinogen adducts to human population monitoring

Several techniques have recently been developed for the detection and quantitation of carcinogen-DNA or -protein adducts without the requirement for radioactive carcinogens. These assays can be used to detect adducts in animals or cultured cells exposed to test compounds or in humans exposed to environmental carcinogens. Immunologic, 32P-postlabeling and fluorescence techniques, used on human samples for DNA adduct measurement, are reviewed here. Methods for the detection of carcinogen-protein adducts on human samples are also summarized.     

Use of DNA adducts to identify human health risk from exposure to hazardous environmental pollutants: the increasing role of mass spectrometry in assessing biologically effective doses of genotoxic carcinogens

The carcinogens to which humans are exposed are normally in the form of complex mixtures, and much effort has gone into determining the nature of the most significant carcinogenic components in these mixtures and their mechanisms of action. Essential to achieving this aim in exposed populations is the use of biomarkers, which can characterize the chemical nature of the carcinogens involved and identify key biological effects that result from the exposure. DNA adducts are particularly appropriate as biomarkers in the case of genotoxic carcinogens as they indicate the biologically effective dose of the genotoxin in the target tissue under study. This review considers in particular the use of mass spectrometry (MS), which is having an increasing role in the determination of DNA adducts. Compared to other existing DNA damage detection methods, such as 32P-postlabeling, HPLC-fluorescence or electrochemical detection, immunoassay-based techniques and modified Comet assays, MS provides improved structural characterization of adducts. Greater selectivity in the analyses is achieved by the use of tandem MS with selected reaction monitoring or constant neutral loss of ions. Use of capillary/nano liquid chromatography and micro/nano electrospray ionization improves the analytical sensitivity and higher throughput may be obtained by the use of online-column switching. The application of microfluidics technology offers exciting new possibilities for interfacing sample preparation to the mass spectrometer. Despite these improvements in the use of MS for adduct detection, the main current requirement is to validate these methods both analytically and in molecular epidemiology studies. More knowledge of the stability of stored samples is required. Development of sensitive mass spectrometric DNA adductomic screening systems, and of long-term biomarkers (e.g., phosphotriester adducts that are not repaired efficiently) seems important areas for the future assessment of the effects of human exposure to environmental genotoxins, together with studies of dose-response relationships at low doses.     

The uses of carcinogen-DNA adduct measurement in establishing mechanisms of mutagenesis and in chemoprevention

DNA adducts generated by carcinogenic chemicals reflects human exposure and DNA adducts are related to tumor formation. Most chemical carcinogens require activation to reactive intermediates that bind to nucleophilic centers in proteins and nucleic acids thereby forming covalent adducts. Also, many of the chemicals considered carcinogenic for humans form covalent DNA adducts. Therefore, such DNA damage is generally considered to be causative and linked to tumor formation. In this article we have summarized the work done for many years on the role of DNA adduct formation as an indicator of their carcinogenicity. We have also addressed the important role for measurement of DNA adducts in studies with potential chemopreventive agents for which it is central to have a marker that can be measured more rapidly than changes in cancer incidence.     

DNA adduct profiles: chemical approaches to addressing the biological impact of DNA damage from small molecules

Diverse small molecules alkylate DNA and form covalently linked adducts that can influence crucial biological processes, contributing to toxicity and mutation. Understanding the chemical reactivity dictating DNA alkylation and interactions of adducts with biological pathways can impact disease prevention and treatment. The ambident reactivity of DNA-alkylating small molecules, and of DNA itself, often results in formation of multiple adducts. Determining which structures impart biological responses is important for understanding the underlying relationships between small-molecule structure and biology. With application of sensitive and structure-specific experimental and analytical methodology, such as heteronuclear NMR spectroscopy and mass spectrometry, there are increasing numbers of studies that evaluate DNA alkylation from the perspective of resulting adduct profiles. DNA adduct profiles have been examined for both exogenous and endogenous reactive small molecules. Examples of recent findings are in the areas of tobacco-specific carcinogens, lipid peroxidation products, environmental and dietary chlorophenols, and natural-product-derived antitumor therapies. As more profile data are obtained, correlations with biological impact are being observed that would not be identified by a simplified single agent/single adduct approach.     

High-performance liquid chromatographic analysis of 32P-postlabeled DNA-aromatic carcinogen adducts

The technique of 32P postlabeling of DNA-carcinogen adducts is a useful and extremely sensitive method of detecting and quantitating DNA damage by carcinogens. We have adapted the 32P method to analysis by high-pressure liquid chromatography, making the procedure more rapid and convenient than when thin-layer chromatography is used. Following DNA isolation and hydrolysis, nucleotide-carcinogen adducts are enhanced relative to normal nucleotides by solvent extraction and then labeled with high-specific-activity [gamma-32P]ATP. The resulting 32P-postlabeled nucleotides are resolved by reverse-phase ion-pair HPLC. After as little as 3 h of exposure to carcinogens, DNA adducts can be demonstrated from 1 microgram or less of mouse hepatic DNA. Acetylated and nonacetylated adducts can be resolved from hepatic DNA of mice treated with 2-aminofluorene. Differences in DNA damage as measured by adduct formation were demonstrated between "rapid" and "slow" acetylator mouse strains. Rapid-acetylator C57BL/6J mice had three times the amount of hepatic DNA adducts as slow-acetylator A/J mice 3 h after a 60 mg/kg dose of 2-aminofluorene. 4-Aminobiphenyl and 2-naphthylamine each showed an adduct peak with retention time similar to that of the nonacetylated 2-aminofluorene adduct, while benzidine gave a major adduct that eluted somewhat earlier as would be expected for an acetylated adduct. The alkenylbenzenes, safrole and methyleugenol, also formed DNA adducts detectable by this method. DNA prepared from skin of mice painted with benzo[a]pyrene also contained carcinogen-DNA adducts detectable and resolvable by HPLC analysis following 32P postlabeling. The combination of HPLC with 32P postlabeling appears to be a useful technique for the rapid detection and quantitation of DNA damage caused by several classes of aromatic carcinogens.     

P-Postlabelling and mass spectrometric methods for analysis of bulky, polyaromatic carcinogen—DNA adducts in humans

There has been significant recent progress toward the development of human carcinogen—DNA adduct biomonitoring methods. ³²P-Postlabelling is a technique which has found wide application in human studies. ³²P-Postlabelling involves enzymatic preparation and labelling of DNA samples, followed by chromatographic separation of carcinogen—nucleotide adducts from unadducted nucleotides. Thin-layer ion-exchange and high-performance liquid chromatography (HPLC) have been utilized. This paper critically reviews ³²P-postlabelling methods for analysis of bulky, polyaromatic carcinogen—DNA adducts and details a strategy to optimize this technique for monitoring human samples. Development of a human carcinogen biomonitoring method requires that the biomarker meet certain criteria: that the biomarker be responsive to exposures known to increase human cancer risk, to reductions in those exposures, and to the influence of metabolic differences. In addition, reliable samples must be available by non-invasive means. The ability of ³²P-postlabelling to meet these criteria is traced in the literature and discussed. Identification of specific carcinogen—DNA adducts is a difficult task due to the low (femtomole) levels in human target tissues. Because co-chromatography in thin-layer chromatography (TLC) is generally not considered to be proof of chemical identity, both synchronous fluorescence and HPLC in conjunction with ³²P-postlabelling and TLC are used to confirm the identity of specific carcinogen-DNA adducts in human samples. Mass spectrometry is a highly specific method, the sensitivity of which has been improved to the point which may allow its use to confirm the identity of carcinogen—DNA adducts isolated by ³²P-postlabelling and other methods. The literature relating to the use of mass spectral techniques in carcinogen—DNA adduct analysis is reviewed.     

Repair of exocyclic DNA adducts: rings of complexity

Exocyclic DNA adducts are mutagenic lesions that can be formed by both exogenous and endogenous mutagens/carcinogens. These adducts are structurally analogs but can differ in certain features such as ring size, conjugation, planarity and substitution. Although the information on the biological role of the repair activities for these adducts is largely unknown, considerable progress has been made on their reaction mechanisms, substrate specificities and kinetic properties that are affected by adduct structures. At least four different mechanisms appear to have evolved for the removal of specific exocyclic adducts. These include base excision repair, nucleotide excision repair, mismatch repair, and AP endonuclease-mediated repair. This overview highlights the recent progress in such areas with emphasis on structure-activity relationships. It is also apparent that more information is needed for a better understanding of the biological and structural implications of exocyclic adducts and their repair.     

DNA adducts, DNA repair genotype/phenotype and cancer risk

It is well established that the initiating event in chemical carcinogenesis is the binding of reactive carcinogens to DNA. Thus, a number of analytic methods have been developed for determining levels of carcinogen-DNA adducts in humans as a marker of individual exposure and, potentially, of risk for cancer development. We have developed monoclonal and polyclonal antibodies to carcinogen-DNA adducts and highly sensitive ELISA and immunohistochemical assays for determining levels of adducts in human tissues. These methods have been combined with genotyping and phenotyping methods for DNA repair to study gene-environment interactions in cancer risk. Recent studies on breast cancer have utilized two large biorepositories. The first is blood and tumor tissues collected as part of the Long Island Breast Cancer Study Project, a population-based case-control study of environmental risk factors. The second is the Metropolitan New York Registry of Breast Cancer Families, one of six sites funded by the National Cancer Institute as a part of the Breast Cooperative Family Registry (CFR). Analysis of samples from these two studies have demonstrated the utility of measurement of DNA adducts as biomarkers of exposure and that DNA repair capacity, measured by genotyping or phenotyping, can influence risk.     

Polycyclic aromatic hydrocarbon-DNA adducts in humans: relevance as biomarkers for exposure and cancer risk

The methodology applied for DNA adducts in humans has become more reliable in recent years, allowing to detect even background carcinogenic adduct levels in environmentally exposed persons. Particularly, combinations of the various methods now allow the elucidation of specific adduct structures with detection limits of 1 adduct in 10⁸ unmodified nucleotides or even lower. The quantification of polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in human tissues and cells has been achieved with a number of highly sensitive techniques: immunoassays and immunocytochemistry using polyclonal or monoclonal antisera specific for DNA adducts or modified DNA, the ³²P-postlabelling assay, and adduct identification using physicochemical instrumentation. The results summarized in this review show that PAH-DNA adducts have been detected in a variety of human tissues, including target organs of PAH- and tobacco-associated cancers. Although dosimetry has not always been precise, a large number of data now clearly show that lowering exposure to carcinogenic PAH results in decreasing PAH-DNA adduct levels. In most studies, however, bulk DNA of a certain tissue or cell type has been examined, and there were relatively few studies in which mutations as a consequence of DNA damage at specific genes have been investigated. Promising as these biomarker studies seem for epidemiology and health surveillance, future biomonitoring and molecular epidemiological studies should be directed to combine several endpoint measurements: i.e., adduct formation (preferably at specific sites), mutational spectra in cancer-relevant genes, and genetic markers of (cancer) susceptibility in a number of cancer-predisposing genes.     

What is the significance of increases in background levels of carcinogen-derived protein and DNA adducts? Some considerations for incremental risk assessment

Improvements in analytical methodology have led to the detection and quantification of 'background' levels of a number of DNA and protein adducts. Many of these adducts are derived from 'low molecular weight' reactive species which may be generated during normal physiological processes, metabolic pathways or inflammatory processes. The adducts have been detected using gas chromatography-mass spectrometry, HPLC in combination with various detection systems, 32P-postlabelling and immunoassay methods. The reliability and accuracy of many widely used methods for adduct measurements are discussed with reference to several examples where human data is available, namely 4-aminobiphenyl, malondialdehyde, methylating agents, ethylene oxide and hydroxyl radical damage. The accurate and specific quantitation of 'background' levels of damage is essential if reliable estimates of increases in risk associated with incremental increases in exposure to exogenous agents are to be calculated. In experimental studies using low dose exposures to carcinogens, such as N-nitrosodimethylamine, adduct levels in liver correlate closely with tumour incidence. In all likelihood, such relationships need to be established for each exposure and, in order to be relevant to human risk assessment, need to take into account factors such as DNA repair and mutagenic efficiency. Finally, in order to estimate the increase in cancer attributable to a given level of external exposure, it is clearly important to establish background levels of corresponding DNA damage so that the scale of the incremental increase can be calculated.     

Carcinogen DNA adducts and the risk of colon cancer: case-control study

Colorectal cancer represents 8.5% of all tumours at the King Faisal Specialist Hospital & Research Centre. Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) have long been suspected to play a prominent role in colon cancer aetiology. We designed a case-control study to test the hypothesis of whether or not the presence of DNA adducts can play a role in the aetiology of colon cancer. DNA adducts were measured in 24 cancerous and 20 non-cancerous tissue samples of newly diagnosed colon cancer patients by ³²P-post-labelling technique. Normal tissue from 19 hospital patients served as controls. The mean levels of adducts per 10¹⁰ nucleotides in cancerous and non-cancerous tissue were 151.75±217.27 and 114.81±186.10, respectively; however, only adducts in cancerous tissue were significantly higher than controls (32.78±57.51 per 10¹⁰ nucleotides) with p-values of 0.017. No BPDE-DNA adducts were found. No relationship was found between urinary cotinine as a marker of tobacco smoke and 1-hydroxypyrene as an indicator of an individual's internal dose of PAHs and DNA adducts. In a logistic regression model, only adducts in cancerous tissue were associated with the subsequent risk of colon cancer, with an odds ratio of 3.587 (95% confidence interval 0.833-15.448) after adjustment for age and the duration of living in the current region, but of a borderline significance (p=0.086). Although it is difficult to arrive at a definite conclusion from a small dataset, our preliminary results suggest the potential role of DNA adducts in the colon carcinogenesis process. Additional studies with larger sample sizes are needed to confirm our preliminary finding. It is also important to identify the structural characterization of these unknown DNA adducts in order to have a better understanding of whether or not environmental carcinogens play a role in the aetiology of colon cancer.     

Studies on biomarkers in cancer etiology and prevention: a summary and challenge of 20 years of interdisciplinary research

Sensitive, specific methods have been developed that allow quantitative measurements of the metabolites of carcinogen metabolites and of DNA and protein adducts in humans exposed occupationally, environmentally and endogenously to genotoxic agents. The interrelationship between exposure to carcinogens, host risk factors and the responses of biomarkers has been examined in cross-sectional, ecological and case-control studies which provided new insights into the causes of cancer and the mechanisms of carcinogenesis. The identification of hitherto unknown DNA-reactive chemicals formed in the human body from dietary precursors and of carcinogenic components of complex mixtures has increased the possibility of establishing causal relationships in etiology. The identification of individuals and subgroups heavily exposed to carcinogens has led to the development of measures for avoiding or decreasing exposure to carcinogenic risk factors. New, ultrasensitive methods for measuring DNA adducts allow the quantification and structural elucidation of specific DNA damage in humans arising from oxidative stress and lipid peroxidation (LPO), which have been found to be the driving forces in several human malignancies. Background DNA damage in "unexposed" individuals has been shown unequivocally to be due to LPO products, and a significant interindividual variation in adduct levels has been shown in individuals with comparable exposure to carcinogens. Thus, pharmacogenetic variants with higher susceptibility to carcinogenic insults, due to genetic polymorphism in xenobiotic-metabolizing enzymes, have been characterized by a combination of genotyping and measurements of macromolecular adducts. Dosimetry has been used in human studies to evaluate the efficacy of interventions with chemopreventive agents like ascorbic acid, dietary phenols and green tea. Advances in the application of selected biomarkers in human studies are reviewed and illustrated by examples from the author's research conducted during the past two decades.     

32P-Postlabeling analysis of lipophilic DNA adducts resulting from interaction with (+/-)-3-hydroxy-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene.

Bay-region diol epoxides are considered the putative ultimate carcinogens of polynuclear aromatic hydrocarbons. However, the results of studies on tumorigenesis and DNA binding of benzo[a]pyrene (BP) and its bay-region diol epoxide, (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyren e [(+)-anti-BPDE] suggest that, in addition to anti-BPDE, other reactive metabolite(s) of BP may also be involved in BP-induced carcinogenesis. Recent studies have demonstrated that 3-hydroxy-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a ]pyrene (anti-BPTE) is another highly reactive metabolite of BP. In order to identify syn- and anti-BPTE-derived DNA adducts and their base selectivity, we synthesized both compounds by two different methods and reacted in vitro with calf thymus DNA and individual nucleotides. The resultant adducts were analyzed by nuclease P1-enhanced 32P-postlabeling. Anti-BPTE produced three major and several minor adducts with DNA; dAp and dGp were the preferred substrates, while dCp and dTp were the least reactive. In contrast, syn-BPTE produced two major adducts each with DNA and dGp; dAp generated only one adduct. Co-chromatography of anti-BPTE-derived DNA adducts with those of mononucleotide adducts revealed that the major adducts in DNA were guanine derived. Further, co-chromatographic results revealed that the anti-BPTE-DNA adducts were distinctly different from that of anti-BPDE-DNA adducts. These observations indicate that both syn- and anti-BPTE can react with DNA bases and these DNA adducts may also contribute to BP-induced carcinogenesis.     

Carcinogen DNA adducts and the risk of colon cancer: case–control study

Colorectal cancer represents 8.5% of all tumours at the King Faisal Specialist Hospital & Research Centre. Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) have long been suspected to play a prominent role in colon cancer aetiology. We designed a case–control study to test the hypothesis of whether or not the presence of DNA adducts can play a role in the aetiology of colon cancer. DNA adducts were measured in 24 cancerous and 20 non-cancerous tissue samples of newly diagnosed colon cancer patients by ³²P-post-labelling technique. Normal tissue from 19 hospital patients served as controls. The mean levels of adducts per 10¹⁰ nucleotides in cancerous and non-cancerous tissue were 151.75±217.27 and 114.81±186.10, respectively; however, only adducts in cancerous tissue were significantly higher than controls (32.78±57.51 per 10¹⁰ nucleotides) with p-values of 0.017. No BPDE-DNA adducts were found. No relationship was found between urinary cotinine as a marker of tobacco smoke and 1-hydroxypyrene as an indicator of an individual's internal dose of PAHs and DNA adducts. In a logistic regression model, only adducts in cancerous tissue were associated with the subsequent risk of colon cancer, with an odds ratio of 3.587 (95% confidence interval 0.833–15.448) after adjustment for age and the duration of living in the current region, but of a borderline significance (p=0.086). Although it is difficult to arrive at a definite conclusion from a small dataset, our preliminary results suggest the potential role of DNA adducts in the colon carcinogenesis process. Additional studies with larger sample sizes are needed to confirm our preliminary finding. It is also important to identify the structural characterization of these unknown DNA adducts in order to have a better understanding of whether or not environmental carcinogens play a role in the aetiology of colon cancer.     

Differences in Metabolism of Chemical Carcinogens in Cultured Human Epithelial Tissues and Cells

The metabolism of chemical carcinogens has been studied in cultured human bronchus, colon, duodenum, pancreatic duct, and esophagus. Metabolite patterns and carcinogen-DNA adducts are generally qualitatively similar among animal species, individuals within a species, and tissues within an individual. However, wide quantitative differences are observed between individuals in out-bred animal species, including humans. These interindividual differences in amounts of carcinogen-DNA adducts and in activities of enzymes that are important in the metabolism of chemical carcinogens are similar in magnitude (10-to 150-fold) to those observed in pharmacogenetic studies of drug metabolism. The role of these differences as risk factors in human cancer is being investigated.     

32P-adduct assay: Short- and long-term persistence of 2-acetylaminofluorene-DNA adducts and other applications of the assay

³²P-Postlabeling techniques have been developed to detect and measure adducts formed by covalent binding of carcinogens of Known or unknown origin with DNA (and RNA). The assay is applicable to various classes of chemical carcinogens and permits detection of many adducts at attomole (10^–18 mol) level using microgram amounts of DNA. Here, we demonstrate the application of the assay for the analysis of short- and long-term persistence of 2-acetylaminofluorene-DNA adducts in rat liver in vivo and also outline examples illustrating the applicability of the procedure to different experimental problems.Abbreviations AAF 2-acetylaminofluorene - N-OH-AAF N-hydroxy-2-acetylaminofluorene     

Bulky DNA adducts in human sperm: relationship with fertility,semen quality,smoking, and environmental factors

The integrity of DNA of spermatogenic cells can be affected by endogenous and exogenous genotoxic factors. Resulting DNA damage in spermatozoa may significantly contribute to impaired fertility. Here, the 32P-postlabeling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 males, either healthy donors or patients with an impaired fertility. When all donors were analyzed, the levels of bulky DNA adducts was 1.2-fold higher in smokers than in non-smokers, but the difference was not statistically significant (P=0.054). However, a statistically significant difference existed between current smokers and never smokers among the healthy individuals (1.7-fold increase, P=0.008). No correlation between alcohol or coffee consumption and sperm DNA adducts was found. The levels of DNA adducts in sperm seemed to be unaffected by environmental and occupational factors. On the other hand, groups of healthy persons and patients with male-factor infertility differed significantly with respect to the level of bulky DNA adducts (P=0.012). A significant negative correlation between DNA adducts and sperm concentration or sperm motility existed among patients with an impaired fertility (n=93; P<0.029, r(S)=-0.225). These results suggest that DNA adducts in sperm cells can be applied as potential biomarkers in studies of human infertility.     

Lung cancer risk and variation in MGMT activity and sequence

O(6)-Alkylguanine-DNA alkyltransferase (MGMT) repairs DNA adducts that result from alkylation at the O(6) position of guanine. These lesions are mutagenic and toxic and can be produced by a variety of agents including the tobacco-specific nitrosamines, carcinogens present in cigarette smoke. Here, we review some of our work in the context of inter-individual differences in MGMT expression and their potential influence on lung cancer risk. In humans there are marked inter-individual differences in not only levels of DNA damage in the lung (N7-methylguanine) that can arise from exposure to methylating agents but also in MGMT activity in lung tissues. In the presence of such exposure, this variability in MGMT activity may alter cancer susceptibility, particularly as animal models have demonstrated that the complete absence of MGMT activity predisposes to alkylating-agent induced cancer while overexpression is protective. Recent studies have uncovered a series of polymorphisms that affect protein activity or are associated with differences in expression levels. The associations between these (and other) polymorphisms and cancer risk are inconsistent, possibly because of small sample sizes and inter-study differences in lung cancer histology. We have recently analysed a consecutive series of case-control studies and found evidence that lung cancer risk was lower in subjects with the R178 allele.