Influence of GSTM1 and NAT2 genotypes on the relationship between personal exposure to PAH and biomarkers of internal dose

This study examined the interaction of glutathione S-transferase (GSTM1) and N acetyltransferase (NAT2) genotypes and personal exposure to carcinogenic polycyclic aromatic hydrocarbons (PAH) with biomarkers of exposure in a cohort of 51 non-smoking women from Bohemia, CZ. The biomarkers included urinary PAH metabolities and white blood cell DNA adducts. Personal PAH exposure was significantly correlated with urinary PAH metabolites for all individuals (r 0.36, p 0.01, n 46). After stratifying by genetic polymorphism the correlation between personal PAH exposure and urinary PAH metabolites increased for individuals with NAT2 slow acetylators (r 0.58, p 0.001, n 29) and the combination of GSTM1 null and NAT2 slow acetylators (r 0.60, p 0.01, n 16). DNA adduct levels were not significantly correlated with personal PAH exposure (r 0.16, p 0.32, n 51), unless restricted to individuals with the GSTM1 gene (r 0.59, p 0.005, n 21). Personal exposure data were essential for elucidating the possible effect of genotypes on the relationship between PAH exposure and these two classes of internal biomarkers. \[This abstract does not necessarily reflect EPA policy.]     

Urinary 1-hydroxypyrene and mutagenicity in bus drivers and mail carriers exposed to urban air pollution in Denmark

BACKGROUND: Previous studies in Denmark have shown that bus drivers and tramway employees were at an increased risk for developing several types of cancer and that bus drives from central Copenhagen have high levels of biomarkers of DNA damage.AIMS: The present study evaluates 1-hydroxypyrene concentrations and mutagenic activity in urine as biomarkers of exposure in non-smoking bus drivers in city and rural areas on a work day and a day off and in non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). METHODS: Twenty-four hour urine samples were collected on a working day and a day off from 60 non-smoking bus drivers in city and rural areas and from 88 non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). The concentration of 1-hydroxypyrene was measured by means of HPLC and the mutagenic activity was assessed by the Ames assay with Salmonella tester strain YG1021 and S9 mix. The N-acetyltransferase (NAT2) phenotype was used as a biomarker for susceptibility to mutagenic/carcinogenic compounds. RESULTS: Bus drivers excreted more 1-hydroxypyrene in urine than did mail carriers. The differences were slightly smaller when NAT2 phenotype, cooking at home, exposure to vehicle exhaust, and performing physical exercise after work were included. The NAT2 slow acetylators had 29% (1.29 [CI: 1.15-1.98]) higher 1-hydroxypyrene concentrations in urine than the fast acetylators. Male bus drivers had 0.92 revertants/mol creatinine [CI: 0.37-1.47] and female bus drivers 1.90 revertants/mol creatinine [CI: 1.01-2.79] higher mutagenic activity in urine than mail carriers. CONCLUSION: The present study indicates that bus drivers are more exposed to polycyclic aromatic hydrocarbons (PAH) and mutagens than mail carriers. Mail carriers who worked outdoors had higher urinary concentration of 1-hydroxypyrene, a marker of exposure to PAH, than those working indoors. The individual levels of urinary mutagenic activity were not correlated to excretion of 1-hydroxypyrene. This might be due to the fact that the most potent mutagenic compounds in diesel exhaust are not PAH but dinitro-pyrenes. Among bus drivers, fast NAT2 acetylators had higher mutagenic activity in urine than slow NAT2 acetylators and female bus drivers had higher mutagenic activity than male bus drivers.     

Biological indicators of genotoxic risk and metabolic polymorphisms

International scientific publications on the influence of metabolic genotypes on biological indicators of genotoxic risk in environmental or occupational exposure are reviewed. Biomarkers of exposure (substance or its metabolites in biological fluids, urinary mutagenicity, protein and DNA adducts) and of effects (chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (Mn), COMET assay, HPRT mutants) have been evaluated according to different genotypes (or phenotypes) of several activating/detoxifying metabolic activities. In less than half the studies (43 out of 95), the influence of genotype on the examined biological indicator was found, of which four report poorly reliable results (i.e., with scarce biological plausibility, because of the inconsistency of modulated effect with the type of enzymatic activity expressed). As regards urinary metabolites, the excretion of mercapturic acids (MA) is greater in subjects with high GST activity, that of 1-pyrenol and other PAH metabolites turns out to be significantly influenced by genotypes CYP1A1 or GSTM1 null, and that of exposure indicators to aromatic amines (AA) (acetylated and non-acetylated metabolites) is modulated by NAT2. In benzene exposure, preliminary results suggest an increase in urinary t, t-muconic acid (t,t-MA) in subjects with some genotypes. On urinary mutagenicity of PAH-exposed subjects, the effects of genotype GSTM1 null, alone or combined with NAT2 slow are reported. When DNA adduct levels are clearly increased in PAH-exposed group (18 out of 22), 7 out of 18 studies report the influence of GSTM1 null on this biomarker, and of the five studies which also examined genotype CYP1A1, four report the influence of genotype CYP1A1, alone or in combination with GSTM1 null. A total of 25 out of 41 publications (61%) evaluating the influence of metabolic polymorphisms on biomarkers of effect (cytogenetic markers, COMET assay, HPRT mutants) do not record any increase in the indicator due to exposure to the genotoxic agents studied, confirming the scarce sensitivity of these indicators (mainly HPRT mutants, Mn, COMET assay) for assessing environmental or occupational exposure to genotoxic substances. Concluding, in determining urinary metabolites for monitoring exposure to genotoxic substances, there is sufficient evidence that genetically-based metabolic polymorphisms must be taken into account in the future. The unfavourable association for the activating/detoxifying metabolism of PAH is also confirmed as a risk factor due to the formation of PAH-DNA adducts. The clearly protective role played by GSTT1 on DEB (and/or related compound)-induced sister chromatid exchanges (SCEs) should be noted. The modulating effects of genotypes on protein adduct levels in environmental and occupational exposure have not yet been documented, and most studies on the influence of genotype on biological indicators of early genotoxic effects report negative results.     

Comparison of biomarkers in workers exposed to 2,4,6-trinitrotoluene

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT-exposed humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly, and liver cancer. Therefore, methods were developed to biomonitor workers exposed to TNT. The workers were employed in a typical ammunition factory in China. The external dose (air levels and skin exposure), the internal dose (urinary metabolites), the biologically effective dose (haemoglobin adducts, urinary mutagenicity), biological effects (chromosomal aberrations and health effects), and individual susceptibility (genotypes of xenobiotic-metabolizing enzymes) were determined. Haemoglobin-adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urinary metabolites of TNT, 4ADNT and 2ADNT, were found in all workers and in some controls. The levels of the haemoglobin-adducts or the urinary metabolites correlated weakly with the skin or air levels of TNT. The urinary mutagenicity determined in a subset of workers correlated strongly with the levels of 4ADNT and 2ADNT in urine. The haemoglobin-adducts correlated moderately with the urinary metabolites and with the urinary mutagenicity. The genotypes of glutathione S-transferases (GSTM1, GSTT1, GSTP1) and N-acetyltransferases (NAT1, NAT2) were determined. In general, the genotypes did not significantly influence the haemoglobin-adduct levels and the urine metabolite levels. However, TNT-exposed workers who carried the NAT1 rapid acetylator genotype showed an increase in urinary mutagenicity and chromosomal aberrations as compared with slow acetylators. The haemoglobin adduct 4ADNT was significantly associated with a risk of hepatomegaly, splenomegaly and cataract; urine metabolites and genotypes were not associated with health effects. These results indicate that a set of well-selected biomarkers may be more informative regarding exposure and effect than routinely performed chemical measurements of pollutants in the air or on the skin.     

Influence of CYP2C9, GSTM1, GSTT1 and NAT2 genetic polymorphisms on DNA damage in workers occupationally exposed to organophosphate pesticides

Previous studies have revealed that organophosphate pesticides (OPs) are primarily metabolized by xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticides-exposed workers. Present study was designed to determine the influence of CYP2C9, GSTM1, GSTT1 and NAT2 genetic polymorphisms on DNA damage in workers occupationally exposed to OPs. We examined 268 subjects including 134 workers occupationally exposed to OPs and an equal number of normal healthy controls. The DNA damage was evaluated using alkaline comet assay and genotyping was done using individual polymerase chain reaction (PCR) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Acetylcholinesterase and paraoxonase activity were found to be significantly lowered in workers as compared to control subjects which were analyzed as biomarkers of toxicity due to OPs exposure (p<0.001). Workers showed significantly higher DNA tail moment (TM) compared to control subjects (14.32±2.17 vs. 6.24±1.37 tail % DNA, p<0.001). GSTM1 null genotype was found to influence DNA TM in workers (p<0.05). DNA TM was also found to be increased with concomitant presence of NAT2 slow acetylation and CYP2C9*3/*3 or GSTM1 null genotypes (p<0.05). DNA TM was found increased in NAT2 slow acetylators with mild and heavy smoking habits in control subjects and workers, respectively (p<0.05). The results of this study suggest that GSTM1 null genotypes, and an association of NAT2 slow acetylation genotypes with CYP2C9*3/*3 or GSTM1 null genotypes may modulate DNA damage in workers occupationally exposed to OPs.     

Influence of GSTM1 genotype on association between aromatic DNA adducts and urinary PAH metabolites in incineration workers

Waste incinerating workers are exposed to various pyrolysis products including polycyclic aromatic hydrocarbons (PAHs). We examined their PAH exposure by assessing urinary 1-hydroxypyrene glucuronide (1-OHPG), as a measure of internal dose, and aromatic DNA adducts in peripheral white blood cells (WBCs), as a measure of biological effect dose. The potential effect of genetic polymorphisms of three enzymes involved in PAH metabolisms (i.e., CYP1A1, GSTM1, and GSTT1) on these exposure markers was also investigated.Twenty-nine employees including workers incinerating industrial wastes and 21 non-exposed on-site controls were recruited from a company handling industrial wastes in South Korea. Sixteen ambient PAHs were determined by GC/MSD (NIOSH method) from personal breathing zone samples of nine subjects working near incinerators. Urinary 1-OHPG was assayed by synchronous fluorescence spectroscopy (SFS) after immunoaffinity purification using monoclonal antibody 8E11. Aromatic DNA adducts in peripheral WBC were measured by the nuclease P1-enhanced post-labelling assay. Genotypes were assessed by PCR-based methods. Information on smoking habits and use of personal protective equipment were collected by self-administered questionnaire.Urinary 1-OHPG levels were significantly higher in workers handling industrial wastes than in those with presumed lower exposure to PAHs (P=0.006, by Kruskal-Wallis test). A statistically significant dose-response increase in 1-OHPG levels was seen with the number of cigarettes consumed per day (r=0.686, P<0.001). Smoking and GSTM1 genotype were significant predictors for log-transformed 1-OHPG by multiple regression analysis (overall model R(2)=0.565, P<0.001), whereas smoking was the only significant predictor for log-transformed aromatic DNA adducts (overall model R(2)=0.249, P=0.201). Aromatic DNA adducts were significantly correlated with log-transformed urinary 1-OHPG level (r=0.31, P=0.04). However, the partial correlation coefficient adjusting for age, sex, and cigarette consumption was not significant (r=0.15, P=0.17). The significant association exists only in individuals with the GSTM1 null genotype (Pearson's correlation coefficient, r=0.52, P=0.01; partial correlation coefficient adjusting for age, sex, and cigarette consumption, r=0.36, P=0.04).Our results suggest that the significant increase in urinary 1-OHPG in the exposed workers is due to higher prevalence of smokers among them, and that the association between urinary PAH metabolites and aromatic DNA adducts in workers of industrial waste handling may be modulated by GSTM1 genotype. These results remain to be confirmed in future larger studies.     

Comparison of biomarkers in workers exposed to 2,4,6-trinitrotoluene

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT-exposed humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly, and liver cancer. Therefore, methods were developed to biomonitor workers exposed to TNT. The workers were employed in a typical ammunition factory in China. The external dose (air levels and skin exposure), the internal dose (urinary metabolites), the biologically effective dose (haemoglobin adducts, urinary mutagenicity), biological effects (chromosomal aberrations and health effects), and individual susceptibility (genotypes of xenobiotic-metabolizing enzymes) were determined. Haemoglobin-adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urinary metabolites of TNT, 4ADNT and 2ADNT, were found in all workers and in some controls. The levels of the haemoglobin-adducts or the urinary metabolites correlated weakly with the skin or air levels of TNT. The urinary mutagenicity determined in a subset of workers correlated strongly with the levels of 4ADNT and 2ADNT in urine. The haemoglobin-adducts correlated moderately with the urinary metabolites and with the urinary mutagenicity. The genotypes of glutathione S-transferases (GSTM1, GSTT1, GSTP1) and N-acetyltransferases (NAT1, NAT2) were determined. In general, the genotypes did not significantly influence the haemoglobin-adduct levels and the urine metabolite levels. However, TNT-exposed workers who carried the NAT1 rapid acetylator genotype showed an increase in urinary mutagenicity and chromosomal aberrations as compared with slow acetylators. The haemoglobin adduct 4ADNT was significantly associated with a risk of hepatomegaly, splenomegaly and cataract; urine metabolites and genotypes were not associated with health effects. These results indicate that a set of well-selected biomarkers may be more informative regarding exposure and effect than routinely performed chemical measurements of pollutants in the air or on the skin.     

Genetic polymorphisms of NAT2, CYP2E1 and GST enzymes and the occurrence of antituberculosis drug-induced hepatitis in Brazilian TB patients

Isoniazid (INH), one of the most important drugs used in antituberculosis (anti-TB) treatment, is also the major drug involved in hepatotoxicity. Differences in INH-induced toxicity have been attributed to genetic variability at several loci, such as NAT2, CYP2E1, GSTM1 and GSTT1, that code for drug-metabolising enzymes. Our goal was to examine the polymorphisms in these enzymes as susceptibility factors to anti-TB drug-induced hepatitis in Brazilian individuals. In a case-control design, 167 unrelated active tuberculosis patients from the University Hospital of the Federal University of Rio de Janeiro, Brazil, were enrolled in this study. Patients with a history of anti-TB drug-induced acute hepatitis (cases with an increase to 3 times the upper limit of normal serum transaminases and symptoms of hepatitis) and patients with no evidence of anti-TB hepatic side effects (controls) were genotyped for NAT2, CYP2E1, GSTM1 and GSTT1 polymorphisms. Slow acetylators had a higher incidence of hepatitis than intermediate/rapid acetylators [22% (18/82) vs. 9.8% (6/61), odds ratio (OR), 2.86, 95% confidence interval (CI), 1.06-7.68, p = 0.04). Logistic regression showed that slow acetylation status was the only independent risk factor (OR 3.59, 95% CI, 2.53-4.64, p = 0.02) for the occurrence of anti-TB drug-induced hepatitis during anti-TB treatment with INH-containing schemes in Brazilian individuals.     

Influence of metabolic genotype GSTM1 on levels of urinary mutagens in patients treated topically with coal tar

Fifteen hospitalized, non-smoking, dermatological patients were treated with ointment containing 2% coal tar (CT) in order to assess the influence of metabolic genotype GSTM1 on urinary mutagen levels. Urinary 1-pyrenol, the main metabolite of pyrene, was used to check the high exposure to PAH of this population. The mean levels of urinary 1-pyrenol found in the 24-h urine of our patients were 467. 8+/-211.0 nmoles-24 h (range 94.6-890.1 nmoles-24 h). Mutagenicity was assessed on urine samples collected over a period of 24 h, after three consecutive days of topical application, using the bacterial mutagenesis test on Salmonella typhimurium strains TA98 and YG1024 in the presence of microsomal enzymes. The latter strain turned out to be more sensitive than the former in revealing urinary mutagens in these patients (42 693+/-30 867 vs. 6877+/-6040 net revertants-24 h). The mutagenicity on YG1024 strain and 1-pyrenol levels of urine samples were correlated (Spearman's rank correlation coefficient=0. 6678, P<0.01, z=2.795). The influence of genotype GSTM1 on urinary mutagen levels was assessed on strain YG1024. The values of urinary mutagenicity of subjects with genotype GSTM1-null (n=6) were on average higher than those of GSTM1-positive subjects (n=9) (55 498+/-45 957 vs. 34 156+/-11 933 net rev.-24 h), a non-significant statistical difference. The mean total excretion of mutagens corrected for PAH exposure (net rev./nmoles of urinary 1-pyrenol) in GSTM1-null patients was double that of GSTM1-positive ones (136. 8+/-34.7 vs. 70.8+/-23.3 net rev./nmoles of urinary 1-pyrenol; one-tailed Mann-Whitney U-test, U=11.5, P<0.05). These results indicate a greater body burden of promutagens, resulting from skin application of CT, in GSTM1-null subjects.     

Design issues in cross-sectional biomarkers studies: urinary biomarkers of PAH exposure and oxidative stress

Cross-sectional biomarker studies can provide a snapshot of the frequency and characteristics of exposure/disease in a population at a particular point in time and, as a result, valuable insights for delineating the multi-step association between exposure and disease occurrence. Three major issues should be considered when designing biomarker studies: selection of appropriate biomarkers, the assay (laboratory validity), and the population validity of the selected biomarkers. Factors related to biomarker selection include biological relevance, specificity, sensitivity, biological half-life, stability, and so on. The assay attributes include limit of detection, reproducibility/reliability, inter-laboratory variation, specificity, time, and cost. Factors related to the population validity include the frequency or prevalence of markers, greater inter-individual variation than intra-individual variation, intra-class correlation coefficients (ICC), association with potential confounders, invasiveness of specimen collection, and subject selection. Three studies are selected to demonstrate different features of cross-sectional biomarker studies: (1) characterizing the determinants of the biomarkers (study I: urinary PAH metabolites and environmental particulate exposure), (2) relationship of multiple biomarkers of exposure and effect (study II: relationship between urinary PAH metabolites and oxidative stress), and (3) evaluating gene-environmental interaction (study III: effect of genetic polymorphisms of GSTM1 on the association of green tea consumption and urinary 1-OHPG levels in shipbuilding workers).     

The association between N-acetyltransferase 2 gene polymorphisms and pancreatic cancer

Pancreatic cancer has been linked with exposure to environmental chemicals, which generally require metabolic activation to highly reactive toxic or carcinogenic intermediates. N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) are expressed primarily in extrahepatic and hepatic tissues, respectively. Both enzymes catalyze N- and O-acetylation of aromatic and heterocyclic amines. It is believed that these compounds are activated via O-acetylation and detoxified by N-acetylation. Several polymorphisms of these two genes have been associated with an increased risk of cancer. Twenty-seven cases of pancreatic cancer and 104 controls were included in this study. Blood was collected in EDTA-containing tubes, and genomic DNA was extracted from the white blood cells by using a high pure PCR template preparation kit. Genotyping of NAT2 polymorphisms was detected by a real time PCR instrument. There was a significant difference in the distribution of the NAT2*6A acetylators phenotype between cases and the controls. The odds ratio of pancreatic cancer for the NAT2*6A slow phenotype was 5.7 (95% CI = 1.27-25.55; p = 0.023) compared with the fast type. Our results suggest that slow acetylators have higher risk of developing pancreatic cancer than fast acetylators. NAT2 gene polymorphisms may be associated with genetic susceptibility to pancreatic cancer.     

Antituberculosis drug-induced hepatitis: risk factors, prevention and management

Apart from infectious or viral hepatitis, other most common non-infectious causes of hepatitis are alcohol, cholestatic, drugs and toxic materials. The most common mode that leads to liver injuries is antituberculosis drug-induced hepatitis. The severity of drug-induced liver injury varies from minor nonspecific changes in hepatic structure to fulminant hepatic failure, cirrhosis and liver cancer. Patients receiving antitubercular drug frequently develop acute or chronic hepatitis. The time required for the metabolites to reach hepatotoxic levels is much earlier with isoniazid plus rifampicin treatment than isoniazid alone and this has been shown to be synergistic rather than additive. Antituberculosis drug (ATT)-inducible cytochrome P-4502E1 (CYP2E1) is constitutively expressed in the liver. Recent studies show that polymorphism of the N-acetyltransferase 2 (NAT2) genes and glutathione-S-transferase (GST) are the major susceptibility risk factors for ATT-induced hepatitis. The hepatic NAT and GST are involved in the metabolism of several carcinogenic arylamines and drugs. The NAT2 enzyme has a genetic polymorphism in human. N-acetyltransferase 2 genes (NAT2) have been identified to be responsible for genetic polymorphism of slow and rapid acetylation in humans. Slow acetylators of NAT2 prove to develop more severe hepatotoxicity than rapid acetylators making it a significant risk factor. Deficiency of GST activity, because of homozygous null mutations at GSTM1 and GSTT1 loci, may modulate susceptibility to drug and xenobiotic-induced hepatotoxicity. Polymorphisms at GSTM1, GSTT1 and NAT2 loci had been linked to various forms of liver injury, including hepatocellular carcinoma.     

Personal exposure to different levels of benzene and its relationships to the urinary metabolites S-phenylmercapturic acid and trans,trans-muconic acid

This report is part of an extensive study to verify the validity, specificity, and sensitivity of biomarkers of benzene at low exposures and assess their relationships with personal exposure and genetic damage. The study population was selected from benzene-exposed workers in Tianjin, China, based on historical exposure data. The recruitment of 130 exposed workers from glue-making or shoe-making plants and 51 unexposed subjects from nearby food factories was based on personal exposure measurements conducted for 3-4 weeks prior to collection of biological samples. In this report we investigated correlation of urinary benzene metabolites, S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA) with personal exposure levels on the day of urine collection and studied the effect of dose on the biotransformation of benzene to these key metabolites. Urinary S-PMA and t,t-MA were determined simultaneously by liquid chromatography-tandem mass spectrometry analyses. Both S-PMA and t,t-MA, but specifically the former, correlated well with personal benzene exposure over a broad range of exposure (0.06-122 ppm). There was good correlation in the subgroup that had been exposed to <1 ppm benzene with both metabolites (P-trend <0.0001 for S-PMA and 0.006 for t,t-MA). Furthermore, the levels of S-PMA were significantly higher in the subgroup exposed to <0.25 ppm than that in unexposed subjects (n=17; P=0.001). There is inter-individual variation in the rate of conversion of benzene into urinary metabolites. The percentage of biotransformation of benzene to urinary S-PMA ranged from 0.005 to 0.3% and that to urinary t,t-MA ranged from 0.6 to approximately 20%. The percentage of benzene biotransformed into S-PMA and t,t-MA decreased with increasing concentration of benzene, especially conversion of benzene into t,t-MA. It appears that women excreted more metabolites than men for the same levels of benzene exposures. Our data suggest that S-PMA is superior to t,t-MA as a biomarker for low levels of benzene exposure.     

Linking exposure to environmental pollutants with biological effects

Exposure to ambient air pollution has been associated with cancer. Ambient air contains a complex mixture of toxics, including particulate matter (PM) and benzene. Carcinogenic effects of PM may relate both to the content of PAH and to oxidative DNA damage generated by transition metals, benzene, metabolism and inflammation. By means of personal monitoring and biomarkers of internal dose, biologically effective dose and susceptibility, it should be possible to characterize individual exposure and identify air pollution sources with relevant biological effects. In a series of studies, individual exposure to PM(2.5), nitrogen dioxide (NO(2)) and benzene has been measured in groups of 40-50 subjects. Measured biomarkers included 1-hydroxypyrene, benzene metabolites (phenylmercapturic acid (PMA) and trans-trans-muconic acid (ttMA)), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in urine, DNA strand breaks, base oxidation, 8-oxodG and PAH bulky adducts in lymphocytes, markers of oxidative stress in plasma and genotypes of glutathione transferases (GSTs) and NADPH:quinone reductase (NQO1). With respect to benzene, the main result indicates that DNA base oxidation is correlated with PMA excretion. With respect to exposure to PM, biomarkers of oxidative damage showed significant positive association with the individual exposure. Thus, 8-oxodG in lymphocyte DNA and markers of oxidative damage to lipids and protein in plasma associated with PM(2.5) exposure. Several types of DNA damage showed seasonal variation. PAH adduct levels, DNA strand breaks and 8-oxodG in lymphocytes increased significantly in the summer period, requiring control of confounders. Similar seasonal effects on strand breaks and expression of the relevant DNA repair genes ERCC1 and OGG1 have been reported.In the present setting, biological effects of air pollutants appear mainly related to oxidative stress via personal exposure and not to urban background levels. Future developments include personal time-resolved monitors for exposure to ultrafine PM and PM(2.5,) use of GPS, as well as genomics and proteomics based biomarkers.     

Is there an influence of enzyme polymorphisms on ochratoxin A genotoxicity?

Primary cultured human urothelial cells derived from ureter specimens of urological patients were used to evaluate induction of DNA-damage by OTA in the alkaline single-cell gel electrophoresis (comet) assay. With the cultured cells from each donor a separate comet assay was performed and tail length of the damaged DNA was measured. A broad spectrum of effects was detected between the individual cell cultures with effects reaching from tail lengths on control level up to strongly enhanced tail lengths.All donors of urothelial tissue were additionally genotyped for several xenobiotic metabolising enzymes (cytochrome P450 1A2, glutathione S-transferases T1, M1, and P1, N-acetyltransferase 2) in lymphocyte DNA. The genotype was then correlated with the genotoxic effects obtained in the comet assay.No correlation was found with CYP1A2, GSTT1, and GSTM1 genotypes whereas for GSTP1 stronger genotoxic effects were found in cells from donors with hetero-and homozygously mutated (w/m, m/m) genotypes compared to homozygous wildtypes. The strongest hint for a correlation was found for NAT2, as cells from donors with homozygous mutated alleles (m/m), known as slow acetylators, displayed a higher susceptibility to OTA in the comet assay than cells from donors with the heterozygously mutated or wildtype alleles (rapid acetylators).     

GSTM1 null genotype as a risk factor for anti-BPDE-DNA adduct formation in mononuclear white blood cells of coke-oven workers

The influence of the genetic deletion polymorphism of glutathione S-transferase micro 1 (GSTM1 *0/*0) on levels of anti (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-DNA) adduct in the peripheral blood lymphocyte plus monocyte fraction (LMF) of coke-oven workers was investigated. A total of 95 male Polish coke-oven workers (60% current smokers) from two different plants comprised the sample population. Polycyclic aromatic hydrocarbons (PAH) exposure was assessed by means of the individual post-shift urinary excretion of 1-pyrenol (mean +/- S.D.: 6.93 +/- 7.20 micromol/mol creatinine; 70% of the subjects exceeded the proposed biological exposure index (BEI) 2.28 micromol/mol creatinine). Anti-BPDE-DNA adduct levels were detected by high performance liquid chromatography (HPLC)/fluorescence analysis of the anti-BPDE tetrol I-1 released after acid hydrolysis of DNA samples. Genotypes were determined by polymerase chain reaction (PCR) on the genomic DNA of each subject. Coke-oven workers without active GSTM1 (GSTM1 *0/*0, 33%) had significantly higher adduct levels than those with active GSTM1 (GSTM1*1/*1 and *1/*0) (5.90 +/- 5.59 versus 3.25 +/- 2.01 adducts/10(8) bases, Mann-Whitney U-test, z = 2.53, P = 0.011), PAH exposure in the two subgroups being similar (7.06 +/- 6.83 versus 6.67 +/- 8.00 1-pyrenol micromol/mol creatinine). The highest number of GSTM1 null subjects (12/23, 39%) belonged to the quartile with the highest adduct levels (i.e., >4.67 adducts/10(8) nucleotides). That is, coke-oven workers with GSTM1 *0/*0 genotype had a significantly higher risk of having high adduct levels than individuals with active GSTM1 genotype (Fisher exact test P = 0.0355; odds ratio (OR) = 4.145, 95% CI 1.0-18.8). Multiple linear regression analysis showed that the increase in anti-BPDE-DNA adduct levels in LMF was significantly related to the high occupational exposure to PAHs (benzo[a]pyrene (BaP)) of coke-oven workers (t = 3.087, P < 0.01) and to the lack of GSTM1 activity (t = 3.512, P < 0.001), rather than to the two other confounding factors of PAH intake, i.e. charcoal-broiled meat consumption and smoking habits. In conclusion, our results indicate the clear influence of the GSTM1 detoxifying genotype on anti-BPDE-DNA adduct formation in the LMF of coke-oven workers. This is invaluable for future environmental-occupational studies using this biomarker of PAH exposure.     

Characterization of human lymphocyte N-acetyltransferase and its relationship to the isoniazid acetylator polymorphism

Characterization of human lymphocyte N-acetyltransferase (NAT) for specific activity, substrate specificity, inhibition, pH optimum, apparent K_m, kinetic mechanism, trypsin stability, freezing stability, and heat stability was carried out in rapid and slow isoniazid (INH) acetylators. There is a statistically significant difference in the heat stability of lymphocyte NAT from rapid and slow INH phenotypes. The lymphocyte enzyme from rapid INH acetylators is less heat stable than the lymphocyte enzyme from slow INH acetylators. This is an indication of a structural, possibly polymorphic, difference in lymphocyte NAT from the two acetylator phenotypes.     

Genetic polymorphisms of DNA repair and xenobiotic-metabolizing enzymes: effects on levels of sister chromatid exchanges and chromosomal aberrations

Elevated levels of chromosomal aberrations (CAs) in peripheral blood lymphocytes, widely used as a cytogenetic biomarker of genotoxic effects, have been linked to cancer predisposition. However, tobacco smoking, occupational carcinogen exposure, or time since CA analysis do not appear to explain the cancer predictivity of CAs. Alternatively, the observed CA-cancer association could reflect unidentified exposures or individual susceptibility. We assessed the effects of genetic polymorphisms of DNA repair proteins and xenobiotic-metabolizing enzymes (XMEs) on the levels of CAs and sister chromatid exchanges (SCEs) in peripheral lymphocytes of 145 (CAs) and 60 (SCEs) healthy Caucasians. Genotypes of DNA repair genes X-ray repair cross-complementation group 1 (XRCC1 codons 194, 280, 399) and 3 (XRCC3 codon 241 [corrected]), and XME genes glutathione-S-transferase (GST) M1 and T1 and N-acetyl transferase 2 (NAT2) were determined using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)-based methods. After Poisson regression adjustment for age, sex, smoking, country, and genotypes, a higher frequency of chromosome-type breaks was observed for NAT2 slow acetylators (in nonsmokers) and GSTT1 null subjects (in smokers). Individuals carrying variant alleles for XRCC1 codons 280 and 194 showed a decreased level of chromosome-type breaks. The effect of GSTM1 null and XRCC1 codon 399 genotypes on the frequency of CAs was modified by smoking. In linear regression models adjusting for age, sex, smoking, and genotypes, none of the polymorphisms significantly affected SCE frequency, although GSTT1 null subjects had a slightly elevated SCE level. Our results are in line with earlier findings on the influence of NAT2, GSTT1, and GSTM1 polymorphisms on the level of lymphocyte chromosome damage and suggest that also XRCC1 polymorphism affects CA frequencies, thus apparently influencing DNA repair phenotype. It remains to be examined whether these or other genetic polymorphisms could explain the observed cancer risk predictivity of high CA frequency.     

Analysis of the N-acetyltransferase 2 gene polymorphism in the patients with chronic obstructive pulmonary disease and in populations of the Volga-Ural region

Restriction fragment-length polymorphism of the gene coding for N-acetyltransferase 2 (NAT2) was typed in populations of the Volga-Ural region (Bashkirs, Tatars, Chuvashes, Udmurts, and Russians) as well as in patients with chronic obstructive pulmonary disease (COPD) and in healthy individuals. Rapid and slow acetylator phenotypes were determined based on the presence or absence of the KpnI, TaqI, and BamHI restriction endonuclease recognition sites. The proportion of slow acetylators in the populations examined varied from 40.00% in Bashkirs to 64.15% in Chuvashes with statistically significant difference between these two ethnic groups (chi 2 = 5.7; p = 0.02). Overall, in the Volga-Ural populations slow acetylators represented 56.25% of the subjects examined. This value was similar to those presented in other studies of Caucasoid populations. In the COPD patients a statistically significant decrease of the slow acetylator frequency to 48.28% compared to healthy individuals (62.18%) was observed (chi 2 = 4.60; p = 0.036). The data obtained suggest a possible association between the drug resistance in the COPD patients with the rapid acetylator phenotype, which can lead to the development of the chronic form of the disease.     

Comparative analysis of N-acetylation polymorphism in humans as determined by phenotyping and genotyping

The N-acetylation polymorphisms of volunteers from the Moscow population analyzed by phenotyping and genotyping have been compared. The ratios between the proportions of fast acetylators (FAs) and slow acetylators (SAs) estimated by phenotyping and genotyping do not differ significantly from each other (47 and 44%, respectively). The absolute acetylation rate widely varies in both FAs and SAs. The NAT2 genotype and allele frequencies in the population sample have been calculated. The most frequent alleles are NAT2*4 (a "fast" allele), NAT2*5, and NAT2*6 ("slow" alleles); the most frequent genotypes are NAT2*5/*5, NAT2*4/*6, and NAT2*4/*5. Comparative analysis of N-acetylation polymorphism estimated by phenotyping and genotyping in the same subjects has shown a complete concordance between the phenotype and genotype in only 62 out of 75 subjects (87%). Comparative characteristics and presumed applications of the two approaches (quantitative estimation of acetylation rate and qualitative determination of the acetylator genotype) to the identification of individual acetylation status are presented.