Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant O6-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an O6-Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S-methyl, S-ethyl, S-propyl, S-hydroxyethyl, S-carboxymethyl, S-benzyl, and S-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-radioimmunoassay (r2 = 0.74; p = 0.014). O6-CMG, a putative O6-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information about cancer pathogenesis.

Impact of occupational pesticide exposure assessment method on risk estimates for prostate cancer, non-Hodgkin's lymphoma and Parkinson's disease: results of three meta-analyses.

Assessment of occupational pesticide exposure in epidemiological studies of chronic diseases is challenging. Biomonitoring of current pesticide levels might not correlate with past exposure relevant to disease aetiology, and indirect methods often rely on workers' imperfect recall of exposures, or job titles. We investigated how the applied exposure assessment method influenced risk estimates for some chronic diseases. In three meta-analyses the influence of exposure assessment method type on the summary risk ratio (sRR) of prostate cancer (PC) (25 articles), non-Hodgkin's lymphoma (NHL) (29 articles) and Parkinson's disease (PD) (32 articles) was investigated. Exposure assessment method types analysed were: group-level assessments (eg, job titles), self-reported exposures, expert-level assessments (eg, job-exposure matrices) and biomonitoring (eg, blood, urine). Additionally, sRRs were estimated by study design, publication year period and geographic location where the study was conducted. Exposure assessment method types were not associated with statistically significant different sRRs across any of the health outcomes. Heterogeneity in results varied from high in cancer studies to moderate and low in PD studies. Overall, case-control designs showed significantly higher sRR estimates than prospective cohort designs. Later NHL publications showed significantly higher sRR estimates than earlier. For PC, studies from North America showed significantly higher sRR estimates than studies from Europe. We conclude that exposure assessment method applied in studies of occupational exposure to pesticides appears not to have a significant effect on risk estimates for PC, NHL and PD. In systematic reviews of chronic health effects of occupational exposure to pesticides, epidemiological study design, publication year and geographic location, should primarily be considered.

Evaluation of proteins in natural rubber latex gloves and pulmonary function amongst female nurses in two tertiary hospitals in southern, Thailand.

BACKGROUND: Inhaled natural rubber latex (NRL) allergens in a healthcare environment can cause NRL sensitization and reduce pulmonary functions. OBJECTIVES: To determine the amount of proteins and the effects of NRL gloves on the pulmonary functions of female nurses in two hospitals in the southern Thailand. METHODS: The study included 340 female nurses from two hospitals in which self-reported information and a blood sample were collected. NRL sensitization was determined by using a solid-phase enzyme-labeled fluoroenzyme-immunoassay for anti-NRLIgE antibodies. Proteins in NRL gloves were measured by a modified Lowry method. Pulmonary function was measured by a spirometer. RESULTS: The prevalences of respiratory effects self-attributed to NRL glove use and of NRL sensitization were 6.5 and 4.7%, respectively. Four of the 16 sensitized nurses reported respiratory symptoms. NRL sensitized nurses had lower forced expiratory flow (FEF25-75% predicted value) than those who were non-sensitized (Adj. difference = -12.56, 95% CI = -24.41 to -0.70). Furthermore, examination gloves contained protein levels in the range of 111-250 mg/dm2. Difference types of NRL glove contained with different amount of proteins.NRL sensitization was more prevalent in nurses with high concentrations of proteins in NRL gloves (p = 0.04). CONCLUSION: Sensitization to NRL was associated with a decrease in FEF25-75% predicted value, indicating narrowing of the small airways of the lung. Use of gloves with low proteins can reducing NRL allergen exposure in these hospitals, which may reduce the risk of developing respiratory problems and NRL sensitization.

Insufficient respiratory hazard identification in the safety data sheets for cleaning and disinfection products used in healthcare organisations across England and Wales.

BACKGROUND: Exposure to cleaning and disinfection products has been associated with respiratory disorders such as asthma in cleaning and healthcare workers. Safety data sheets (SDSs) provide information on hazardous chemicals that are present in products to help users with risk assessment and implement appropriate control measures. However, they have potential limitations in identifying respiratory hazards due to a lack of regulatory test methods for respiratory sensitisation and irritation of chemicals. METHODS: SDSs were first used to identify chemicals on the database as respiratory sensitisers and irritants. A quantitative structure-activity relationship (QSAR) model and an asthmagen list established by the Association of Occupational and Environmental Clinics (AOEC) were used to identify potential respiratory sensitisers and irritants (by the AOEC list only) in the cleaning and disinfection products. RESULTS: From a total of 459 cleaning and disinfection products used in healthcare organisations across England and Wales, 35 respiratory sensitisers not labelled as such on the SDS were identified by QSAR or AOEC. Only 2% of cleaning and disinfection products contained at least one respiratory sensitiser as identified by their SDSs; this was increased to 37.7% of products when the QSAR or the AOEC list was used. CONCLUSIONS: A significantly higher proportion of cleaning products contain respiratory hazardous chemicals, particularly respiratory sensitisers than would be expected from the information provided by SDSs alone. Cleaners and healthcare workers may, therefore, be insufficiently protected.

Geochemical compositional controls on DNA strand breaks induced in in vitro cell-free assays by crushed rock powders from the Panasqueira mine area, Portugal.

DNA strand breaks are a common form of DNA damage that can contribute to chromosomal instability or gene mutations. Such strand breaks may be caused by exposure to heavy metals. The aim of this study was to assess the level of DNA strand breaks caused by µm-scale solid particles of known chemical composition with elevated heavy metals/metalloids, notably arsenic, using an in vitro cell-free DNA plasmid scission assay. These samples were incubated with and without H2O2 to see whether damage occurs directly or indirectly through the Fenton reaction. Levels of DNA damage in the absence of H2O2 were < 10%, but in the presence of H2O2, all samples showed higher levels of damage ranging from 10 to 100% suggesting that damage was being incurred through the Fenton reaction. Using bivariate correlation analysis and multiple linear regression, manganese oxide (MnO), sulphur (S), copper (Cu), and zinc (Zn) concentrations in the particulates were found to be the most significant predictors of DNA damage. The mechanism of this DNA damage formation has yet to be thoroughly investigated but is hypothesised to be due to reactive oxygen species formation. Further work is required to assess the extent of contribution of reactive oxygen species to this DNA damage, but this study highlights the potential role of chemistry and/or mineralogy to the extent and/or nature of DNA damage caused by particulates.

Systematic review of methods used to assess exposure to pesticides in occupational epidemiology studies, 1993-2017.

OBJECTIVE: Numerous exposure assessment methods (EAM) exist for investigating health effects of occupational exposure to pesticides. Direct (eg, biomonitoring) and indirect methods (eg, self-reported exposures) are however associated with degrees of exposure misclassification. We systematically reviewed EAM in studies of occupational pesticide exposure. METHODS: We searched for articles reporting observational epidemiological studies in MEDLINE and Embase published 1993 to 2017. The relative frequency of EAM was analysed according to EAM type (direct and indirect methods), health outcome, study design, study location (country) and specificity of assessment. Temporal trends in EAM were analysed. RESULTS: In 1298 included articles 1521 EAM occurrences were documented. Indirect EAM (78.3%), primarily self-reported exposures (39.3%) and job titles assessments (9.5%), were mainly applied in case-control studies (95.0%), in high-income countries (85.0%) and in studies of doctor-diagnosed health outcomes (>85%). Direct EAM (20.8%), primarily biomonitoring of blood (15.6%) or urine (4.7%), were predominantly applied in cross-sectional studies (29.8%), in lower middle-income countries (40.9%) and in studies of neurological (50.0%) outcomes. Between 1993 to 2017 no distinct time trends regarding the ratio indirect to direct methods was seen. Within the category of indirect methods use of self-reported exposures and job exposure matrices increased while assessments by job titles and registers decreased. The use of algorithms showed no trend. The specificity of pesticide assessment increased since studies assessing exposure by using job title as a proxy declined. Assessments of type of pesticide increased. CONCLUSION: Over the last 25 years, the ratio (5:1) of indirect to direct EAM applied in articles on occupational pesticide epidemiology stayed relatively constant; changes were mainly attributable to increasing use of self-reported exposures and job exposure matrices. This review, combined with studies assessing EAM validity, will inform on magnitudes of exposure misclassification and help improve the quality of studies on occupational pesticides exposure.

Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines.

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER.

Cytotoxicity and genotoxicity of urban particulate matter in mammalian cells.

Ambient air particulate matter (PM)-associated reactive oxygen species (ROS) have been linked to a variety of altered cellular outcomes. In this study, three different PM samples from diesel exhaust particles (DEPs), urban dust standard reference material SRM1649a and air collected in Manchester have been tested for their ability to oxidise DNA in a cell-free assay, to increase intracellular ROS levels and to induce CYP1A1 gene expression in mammalian cells. In addition, the cytotoxicity and genotoxicity of PM were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and alkaline comet assay, respectively. All PM samples catalysed the Fenton reaction in a cell-free assay, but only DEP resulted in the generation of ROS as measured by dichlorodihydrofluorescein diacetate oxidation in mammalian cells. However, there was no evidence that increased ROS was a consequence of polycyclic aromatic hydrocarbon metabolism via CYP1A1 induction as urban dust, the Manchester dust samples but not DEP-induced CYP1A1 expression. Urban dust was more cytotoxic in murine embryonic fibroblasts (MEFs) than the other PM samples and also induced expression of GADD45a in the GreenScreen Human Cell assay without S9 activation suggesting the presence of a direct-acting genotoxicant. Urban dust and DEP produced comparable levels of DNA damage, as assessed by the alkaline comet assay, in MEFs at higher levels than those induced by Manchester PM. In conclusion, results from the cytotoxic and genotoxic assays are not consistent with ROS production being the sole determinant of PM-induced toxicity. This suggests that the organic component can contribute significantly to this toxicity and that further work is required to better characterise the extent to which ROS and organic components contribute to PM-induced toxicity.

Pesticide residue transfer in Thai farmer families: using structural equation modeling to determine exposure pathways.

Use of pesticides in agriculture may lead to downstream exposure of farmers' families to pesticide residues inadvertently taken home. Identification of the independent contribution of different exposure pathways from the farmer to their children can provide clear targets to reduce exposure of farmers' children. Individual contributions of different pesticide transfer exposure pathways were investigated using structural equation modeling methods, and the benefits of these methods compared to standard multiple regression are described. A total of 72 Thai families, consisting of a farmer, a spouse, and a child, participated in this study. Family members completed a questionnaire and self-collected three spot morning urine samples in the spraying season. Urine samples were analyzed for diethyl phosphate, diethyl thiophosphate, diethyl dithiophosphate, dimethyl phosphate, dimethyl thiophosphate, and dimethyl dithiophosphate. A path model was developed based on an a priori hypothesized framework to examine the individual contributions of different exposure pathways that may directly or indirectly affect transfer of pesticide residues from farmers to their children. Transfer from the farmer to the child occurs indirectly, primarily through transfer to the spouse in the first instance, but also through contamination of the home environment. Clear targets for interventions are directly the reduction of farmers' take-home exposures and indirectly frequent cleaning of the home to avoid buildup of pesticide residues.

The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase.

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O(6)-carboxymethylguanine (O(6)-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O(6)-CMG is not a substrate for the human version of the DNA damage reversal protein O(6)-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O(6)-alkylguanine lesions by removing alkyl groups from the O(6)-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O(6)-methylguanine (O(6)-MeG) or O(6)-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O(6)-alkyl group and its transfer to the active-site cysteine residue of MGMT. O(6)-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O(6)-CMG is a potential causative agent.

Pesticide exposure and screen-positive neuropsychiatric disease in British sheep farmers.

BACKGROUND: Epidemiological evidence linking low dose pesticide exposure and chronic ill-health in UK sheep farmers is limited. Our aim was to examine whether neuropsychiatric disorders were associated with low dose chronic and/or more acute pesticide exposure in sheep farmers. METHODS: A cohort of British farmers working in the 1970s was sent a screening questionnaire which asked about their health and work history. The prevalence of screen-positive depression, dementia, Parkinsonism and neuropathy was determined using a priori algorithms. Self-reported pesticide exposure was assessed by whether the participant had ever handled the pesticide concentrate (for low dose chronic exposure) or sought advice for pesticide poisoning (acute exposure) and participants categorised into those with only acute or chronic exposure, those with both acute and chronic exposure and those with neither acute nor chronic exposure. Associations between acute and chronic pesticide exposure, and screen-positive ill-health were determined after adjustment for demographic, lifestyle, occupation and somatic severity scores and other variables. RESULTS: In those participants, who had never sought advice for pesticide poisoning, handling the pesticide concentrate for treating sheep was associated with elevated ORs for screen-positive neuropathy (ORadi 1.57 95%CI 0.97-2.54) and Parkinsonism (ORadj 1.56 95%CI 0.95-2.56) but not depression or dementia. In those participants who had handled the pesticide concentrate, seeking advice for pesticide poisoning was associated with screen-positive depression (Odds ratio, ORadj=9.97 95%CI 4.76-20.8 ), dementia (OR=6.94 95%CI 3.44-14.0), Parkinsonism (ORadj=4.77 95% 2.39-9.52), and neuropathy (ORadj=4.77 95%CI 2.39-9.52). Adjustment for somatic severity score modified little the associations with pesticide handling in those not acutely exposed but reduced the ORs for seeking advice for pesticide poisoning in those exposed chronically. Furthermore, stratification of results based upon somatic severity score indicated that the highest ORs for handling the pesticide concentrate associated with neuropathy and Parkinsonism were found in those participants whose somatic score was minimal. CONCLUSIONS: Results are consistent with low-dose exposure to pesticides being associated with screen-positive neuropathy and Parkinsonism but the stronger associations between seeking advice for pesticide poisoning and screen-positive ill-health suggest that acute pesticide exposure remains an important determinant of ill-health. Further work is required to better delineate to what extent low dose exposures may contribute to ill-health in populations without acute exposures. Somatising tendency does not appear to play an important role in this population.

Personal exposure to inhalable dust and the specific latex aero-allergen, Hev b6.02, in latex glove manufacturing in Thailand.

OBJECTIVES: Latex product manufacturing is an important industry in south-east Asia but has the potential for considerable occupational exposure of workers to latex allergens. Although exposure to latex allergens can result in adverse health reactions, few studies to characterize this exposure have been conducted to date. This study therefore aimed to characterize current airborne inhalable dust and the specific allergen, Hev b 6.02, exposures in this industry in Thailand. METHODS: Workers were recruited from three factories in the southern part of Thailand. Full-shift inhalable dust personal air sampling was conducted using IOM sampling heads equipped with polytetrafluoroethylene filters at a 2.0 l min(-1) flowrate. After weighing to determine inhalable dust levels, filters were extracted and analysed for Hev b 6.02 using an enzyme immunometric assay. RESULTS: Two hundred and seventy-five workers agreed to participate, resulting in a total of 292 measurements. Geometric mean (GM) personal exposure to inhalable dust was 0.88 mg m(-3), but individual exposures up to 12.34 mg m(-3) were measured. The pattern of exposure was similar across factories, with highest exposures in the stripping (GM 2.08-4.05 mg m(-3) for the 3 factories) and tumbling departments (1.11-2.17 mg m(-3)). Within-worker (day-to-day) variability contributed 92% to total variability. The Hev b 6.02 exposure pattern was similar with time-weighted average GM exposure levels in the oldest factory ranging from 8.7 mg m(-3) in the laboratory to 30.2mg m(-3) in the stripping department. In contrast to inhalable dust exposure, total exposure variability was primary driven by variability between workers (67%). CONCLUSIONS: Workers in these latex product factories get routinely exposed to measurable Hev b 6.02 levels, which may give rise to increased incidence of allergic symptoms and occupational asthma. Also, in this measurement campaign a 10mg m(-3), but not 15 mg m(-3), occupational exposure limit for inhalable dust was occasionally exceeded. Highest Hev b 6.02 exposures were found in the stripping and tumbling departments, which would be natural targets for interventions aimed at reducing exposure.

Bayesian inference supports a location and neighbour-dependent model of DNA methylation propagation at the MGMT gene promoter in lung tumours.

We exploit model-based Bayesian inference methodologies to analyse lung tumour-derived methylation data from a CpG island in the O6-methylguanine-DNA methyltransferase (MGMT) promoter. Interest is in modelling the changes in methylation patterns in a CpG island in the first exon of the promoter during lung tumour development. We propose four competils of methylation state propagation based on two mechanisms. The first is the location-dependence mechanism in which the probability of a gain or loss of methylation at a CpG within the promoter depends upon its location in the CpG sequence. The second mechanism is that of neighbour-dependence in which gain or loss of methylation at a CpG depends upon the methylation status of the immediately preceding CpG. Our data comprises the methylation status at 12 CpGs near the 5' end of the CpG island in two lung tumour samples for both alleles of a nearby polymorphism. We use approximate Bayesian computation, a computationally intensive rejection-sampling algorithm to infer model parameters and compare models without the need to evaluate the likelihood function. We compare the four proposed models using two criteria: the approximate Bayes factors and the distribution of the Euclidean distance between the summary statistics of the observed and simulated datasets. Our model-based analysis demonstrates compelling evidence for both location and neighbour dependence in the process of aberrant DNA methylation of this MGMT promoter CpG island in lung tumours. We find equivocal evidence to support the hypothesis that the methylation patterns of the two alleles evolve independently.

Chlorination by-products in tap water and semen quality in England and Wales.

OBJECTIVES: Disinfection by-products (DBPs) have been associated with adverse semen outcomes in laboratory animals, although the evidence for trihalomethanes (THMs) is limited. Three small epidemiological studies found little evidence for an association between DBPs and adverse semen outcomes in humans. Using data from a large case-referent study (Chemicals and Pregnancy Study, Chaps-UK), we investigated the association between total THM (TTHM), chloroform and total brominated THMs and sperm concentration, percent motile sperm and motile sperm concentration (MSC). METHODS: Chaps-UK recruited men from 13 fertility clinics in nine urban centres across England and Wales between 1999 and 2002. We linked modelled THM concentrations in water zones to semen quality data for 642 cases (men with low MSC) and 926 referents (other men investigated for infertility), based on the men's residence during semen sampling. We assessed risk of low MSC in relation to DBP exposure using continuous THM concentrations. A secondary analysis investigated continuous outcomes (MSC, sperm concentration and percent motile sperm). RESULTS: In the case-referent analysis there was little evidence of elevated risk associated with chloroform, total brominated THM or TTHM concentration after adjustment (OR per 10 µg/L TTHM 1.01; 95% CI 0.91 to 1.12). Similarly, there was no significant effect of THMs on the continuous outcomes. CONCLUSIONS: In the largest study to date on DBPs in public water supplies, and semen quality we found that concentrations of THMs were not associated with poor semen quality. Large-scale investigation of other DBPs (eg, haloacetic acids) and other semen quality parameters (eg, sperm morphology and/or sperm DNA integrity) is recommended.

Synthesis of oligodeoxyribonucleotides containing a tricyclic thio analogue of O6-methylguanine and their recognition by MGMT and Atl1.

Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic.

Meta- and pooled analysis of GSTP1 polymorphism and lung cancer: a HuGE-GSEC review.

Lung cancer is the most common cancer worldwide. Polymorphisms in genes associated with carcinogen metabolism may modulate risk of disease. Glutathione S-transferase pi (GSTP1) detoxifies polycyclic aromatic hydrocarbons found in cigarette smoke and is the most highly expressed glutathione S-transferase in lung tissue. A polymorphism in the GSTP1 gene, an A-to-G transition in exon 5 (Ile105Val, 313A --> 313G), results in lower activity among individuals who carry the valine allele. The authors present a meta- and a pooled analysis of case-control studies that examined the association between this polymorphism in GSTP1 and lung cancer risk (27 studies, 8,322 cases and 8,844 controls and 15 studies, 4,282 cases and 5,032 controls, respectively). Overall, the meta-analysis found no significant association between lung cancer risk and the GSTP1 exon 5 polymorphism. In the pooled analysis, there was an overall association (odds ratio = 1.11, 95% confidence interval: 1.03, 1.21) between lung cancer and carriage of the GSTP1 Val/Val or Ile/Val genotype compared with those carrying the Ile/Ile genotype. Increased risk varied by histologic type in Asians. There appears to be evidence for interaction between amount of smoking, the GSTP1 exon 5 polymorphism, and risk of lung cancer in whites.

GSTM1 copy number and lung cancer risk.

The GSTM1 null genotype is associated with a small increased lung cancer risk when compared to controls with at least one copy of the GSTM1 gene. As two copies of the GSTM1 gene might provide more protection than a single copy, we have determined GSTM1 copy number in a lung cancer case-control study. Cases with incident lung cancer were identified through a Bronchoscopy Unit and two separate hospital based control groups with non-malignant disease were selected with one from the same Bronchoscopy Unit and the other from a chest clinic at the same hospital. Subjects with at least one GSTM1 copy had a decreased lung cancer risk whatever the control group: the odds ratio (95% CI), after adjustment for age, gender and smoking duration, was 0.64 (0.41-0.98) and 0.54 (0.32-0.91) with bronchoscopy and chest clinic controls, respectively. Lung cancer risk varied with GSTM1 copy number with chest clinic controls only: the OR was 0.56 (0.32-0.97) for one copy of the GSTM1 gene and with two copies 0.43 (0.15-1.22), a trend that was significant (p=0.02): with bronchoscopy controls the trend was not significant (p=0.07). Results then confirm that the presence of GSTM1 provides protection against the risk of lung cancer. In addition there is equivocal evidence that this protection varies with the number of gene copies.

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.

Association between lung cancer risk and single nucleotide polymorphisms in the first intron and codon 178 of the DNA repair gene, O6-alkylguanine-DNA alkyltransferase.

The association between lung cancer risk and 2 polymorphisms, rs12268840 and rs2308327 (codon K178R), in the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase, which are associated with interindividual differences in activity, have been investigated in 3 hospital-based case-control studies. Genotyping was carried out on 617 subjects of whom 255 had lung cancer. In 2 of the 3 series, there was a significant inverse association between the 178R allele and case status (p < 0.05). In a meta-analysis, the odds ratio (95% CI) associated with the 178R allele relative to the 178K allele was 0.64 (0.45-0.92, p = 0.01) and 0.51 (0.24-1.11, p = 0.09) in fixed effects and random effects models, respectively. In a pooled analysis, after adjustment for sex, age, pack years and series, the OR (95% CI) for a heterozygote was 0.67 (0.45-1.01) and for a 178R homozygote was 0.10 (0.01-0.94); the trend for a decreased risk with the number of R alleles was significant (p = 0.008). This trend was particularly pronounced in heavy smokers (trend test p = 0.003), but not significant in light smokers (p = 0.73). There was no evidence of an association between rs12268840 and lung cancer risk. These results suggest that the R allele may protect against lung cancer, specifically in heavy smokers, an effect that may result from this polymorphism affecting the function of the MGMT protein and/or levels in MGMT activity.

Bladder tumor contains higher N7-methylguanine levels in DNA than adjacent normal bladder epithelium.

Schistosoma haematobium-infected patients are more likely to develop bladder cancer and be more exposed to carcinogenic N-nitroso compounds than uninfected patients. As N7-methylguanine is a marker of exposure to methylating agents of this type, we have measured N7-methyldeoxyguanosine 3'-monophosphate (N7-MedGp) by (32)P postlabeling. DNA was isolated from 42 paired normal and tumor tissue of Egyptians with bladder cancer. N7-MedGp was detected in DNA from 93% of the tumors and 74% of the normal bladder tissue samples. Adduct levels were highly variable and ranged from 0.04 to 6.4 and from 0.02 to 0.72 micromol/mol deoxyguanosine 3'-monophosphate (dGp) in tumor and normal DNA, respectively. N7-MedGp levels in normal and tumor DNA were highly correlated with one another (P = 0.007). The mean difference (95% confidence interval) in adduct levels between tumor and normal DNA was 0.21 (0.13-0.32) micromol/mol dGp and this was statistically significant (P < 0.001). The adduct ratio (tumor DNA/normal DNA) varied between 0.2 and 136 (median, 4.6). N7-MedGp levels were not associated with gender, age, or the presence of schistosomiasis. However, lower N7-MedGp levels were found in normal DNA from individuals lacking the GSTM1 gene (P = 0.03) but not the GSTT1 gene or in subjects with the Ile105Val GSTP1 polymorphism. These results show that exposure to methylating agents is widespread and suggest that such exposure may play a role both in tumor initiation and progression.