Prenatal expression of N-acetyltransferases in C57Bl/6 mice

Exposure to carcinogens such as 4-aminobiphenyl (4ABP), found in tobacco smoke and other combustion products, results in the formation of detectable levels of 4ABP-hemoglobin adducts in cord blood and 4ABP-DNA adducts in conceptal tissue. The presence of these adducts requires that the parent compound undergo biotransformation. When exposure occurs in utero, the maternal, placental and conceptal tissues are all possible sites for the formation of DNA-reactive products. One step in the activation of 4ABP is catalyzed by N-acetyltransferases (NAT). The expression of NAT was evaluated in gestational day (GD) 10-18 conceptal tissues from C57Bl/6 mice. There was a quantitative increase in NAT1 and NAT2 mRNAs with increasing gestational age that was also reflected in age-related changes in functional protein measured as 4ABP-NAT activity. The ability to acetylate 4ABP increased from GD10 to 18 and was lower in conceptal tissue than in adult liver. The potential toxicologic significance of prenatal NAT expression was assessed by formation of 4ABP-DNA adducts. At GD 15 and 18, 4ABP-DNA adducts were detected by immunohistochemistry 24 h following a single oral dose of 120 mg 4ABP/kg. Based on nuclear fluorescence, conceptual 4ABP-DNA adducts were present at similar levels at GD15 and 18. Levels of 4ABP-DNA adducts were significantly higher in maternal liver compared with the conceptus. Results from this study show that both NAT genes were expressed prenatally and that functional enzymes were present. These data support the possible in situ generation of reactive products by the conceptus. The relative contributions of maternal activation of 4ABP and that by the conceptus remain to be determined.     

The effects of genetic variation in N-acetyltransferases on 4-aminobiphenyl genotoxicity in mouse liver

Inbred, congenic and transgenic strains of mice were characterized for acetylation of p-aminobenzoic (PABA) and the carcinogen 4-aminobiphenyl (4ABP). C57Bl/6 mice have the NAT2*8 allele, A/J mice have NAT2*9 and congenic B6.A mice have NAT2*9 on the C57Bl/6 background. The first transgenic strain with human NAT1, the functional equivalent of murine NAT2, was also tested. The murine NAT2*9 allele correlated with a slow phenotype measured with the murine NAT2 selective substrate PABA. The two strains having this allele also had a lower capacity to acetylate 4ABP. A line with five copies of the human NAT1 transgene was bred for at least five generations with either C57Bl/6 or A/J mice. There was no significant change in PABA NAT activity on the C57Bl/6 background but a 2.5-fold increase was seen in hNAT1:A/J compared with A/J. The effect of variation in NATs on 4ABP genotoxicity was assessed in these strains. Twenty-four hours after exposure to a single oral dose of 120 mg 4ABP/kg, hepatic 4ABP-DNA adducts were detected by immunofluoresence in all strains. Nuclear fluorescence intensities (mean+/-S.D.) were 41.1+/-3.6 for C57Bl/6, 37.9+/-1.11 for A/J and 36.3+/-2.44 for B6.A. There was no correlation between murine NAT2 alleles and 4ABP-DNA adduct levels. Similar results were seen with the transgenic strains. The data indicate that the range of variation present in these strains of mice was insufficient to alter susceptibility to 4ABP genotoxicity. The impact of these relatively modest differences in the acetylation of the activation of 4ABP may be masked by other competing biotransformation reactions since 4ABP is a substrate for both NAT1 and NAT2. Mouse models with variation in both isoforms are needed to adequately assess the role of variation in NATs in susceptibility to 4ABP genotoxicity.     

Comparison of DNA adduct levels associated with oxidative stress in human pancreas

DNA adducts associated with oxidative stress are believed to involve the formation of endogenous reactive species generated by oxidative damage and lipid peroxidation. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been carried out. In this study, we isolated DNA from the pancreas of 15 smokers and 15 non-smokers, and measured the levels of 1,N⁶-etheno(2′-deoxy)guanosine (edA), 3,N⁴-etheno(2′-deoxy)cytidine (edC), 8-oxo-2′-deoxyguanosine (8-oxo-dG), and pyrimido[1,2-]purin-10(3H)-one (m₁G). Using the same DNA, the glutathione S-transferase (GST) M1, GSTT1, and NAD(P)H quinone reductase-1 (NQO₁) genotypes were determined in order to assess the role of their gene products in modulating adduct levels through their involvement in detoxification of lipid peroxidation products and redox cycling, respectively. The highest adduct levels observed were for m₁G, followed by 8-oxo-dG, edA, and edC, but there were no differences in adduct levels between smokers and non-smokers and no correlation with the age, sex or body mass index of the subject. Moreover, there was no correlation in adduct levels between edA and eC, or between edA or edC and m₁G or 8-oxo-dG. However, there was a significant correlation (r=0.76; p<0.01) between the levels of 8-oxo-dG and m₁G in human pancreas DNA. Neither GSTM1 nor NQO₁ genotypes were associated with differences in any of the adduct levels. Although the sample set was limited, the data suggest that endogenous DNA adduct formation in human pancreas is not clearly derived from cigarette smoking or from (NQO₁)-mediated redox cycling. Further, it appears that neither GSTM1 nor GSTT1 appreciably protects against endogenous adduct formation. Together with the lack of correlation between m₁G and edA or edC, these data indicate that the malondialdehyde derived from lipid peroxidation may not contribute significantly to m₁G adduct formation. On the other hand, the apparent correlation between m₁G and 8-oxo-dG and their comparable high levels are consistent with the hypothesis that m₁G is formed primarily by reaction of DNA with a base propenal, which, like 8-oxo-dG, is thought to be derived from hydroxyl radical attack on the DNA.     

Identification of new DNA adducts in human bladder epithelia exposed to the proximate metabolite of 4-aminobiphenyl using 32P-postlabeling method

The DNA adducts were analyzed by 32P-postlabeling method following exposure of human uroepithelial cells (HUC) to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). TLC of the postlabeled products on the first dimension revealed several products, the majority of which stayed close to the origin and were earlier identified as the 3',5' -bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (Carcinogenesis 13 (1993) 955; Carcinogenesis 16 (1995) 295). Here we report characterization of two additional adducts that amounted to less than 5% of the total adducts. Autoradiography of D1 chromatogram of the postlabeled products of calf thymus DNA chemically interacted with N-OH-ABP under acidic conditions revealed two adducts, #1 and #2, with R(f) values of about 0.2 and 0.3, respectively. Two adducts with D1 thin layer chromatographic properties similar to those of adducts #1 and #2 were obtained on postlabeling analyses of products generated by chemical interaction of N-acetoxy-4-aminobiphenyl (N-OAc-ABP) with deoxyguanosine-3' -monophosphate (dGp). Based on proton NMR and mass spectroscopic analyses of the synthetic products derived from N-OAc-ABP, the chemical structures of adducts #1 and #2 have been identified as 3-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, and N-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, respectively. Both of these adducts were insensitive to digestion with nuclease P1. 32P-Postlabeling analysis of the nuclease P1 enriched DNA hydrolysate of HUC cells treated with N-OH-ABP showed the presence of adduct #2 but not adduct #1. Adduct #2 was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl CoA. These results suggest that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP is bioactivated by acetyl CoA-dependent acyltransferases to reactive arylnitrenium ions that covalently interact at N(2)-position of deoxyguanosine in DNA.     

Complex Relationships between Occupation,Environment, DNA Adducts,Genetic Polymorphisms and Bladder Cancer in a Case-Control Study Using a Structural Equation Modeling

DNA adducts are considered an integrate measure of carcinogen exposure and the initial step of carcinogenesis. Their levels in more accessible peripheral blood lymphocytes (PBLs) mirror that in the bladder tissue. In this study we explore whether the formation of PBL DNA adducts may be associated with bladder cancer (BC) risk, and how this relationship is modulated by genetic polymorphisms, environmental and occupational risk factors for BC. These complex interrelationships, including direct and indirect effects of each variable, were appraised using the structural equation modeling (SEM) analysis. Within the framework of a hospital-based case/control study, study population included 199 BC cases and 213 non-cancer controls, all Caucasian males. Data were collected on lifetime smoking, coffee drinking, dietary habits and lifetime occupation, with particular reference to exposure to aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs). No indirect paths were found, disproving hypothesis on association between PBL DNA adducts and BC risk. DNA adducts were instead positively associated with occupational cumulative exposure to AAs (p = 0.028), whereas XRCC1 Arg 399 (p<0.006) was related with a decreased adduct levels, but with no impact on BC risk. Previous findings on increased BC risk by packyears (p<0.001), coffee (p<0.001), cumulative AAs exposure (p = 0.041) and MnSOD (p = 0.009) and a decreased risk by MPO (p<0.008) were also confirmed by SEM analysis. Our results for the first time make evident an association between occupational cumulative exposure to AAs with DNA adducts and BC risk, strengthening the central role of AAs in bladder carcinogenesis. However the lack of an association between PBL DNA adducts and BC risk advises that these snapshot measurements are not representative of relevant exposures. This would envisage new scenarios for biomarker discovery and new challenges such as repeated measurements at different critical life stages.     

DNA adduct formation from tobacco-specific N-nitrosamines

Tobacco-specific N-nitrosamines are a group of carcinogens derived from the tobacco alkaloids. They are likely causative factors for cancers of the lung, esophagus, pancreas, and oral cavity in people who use tobacco products. The most carcinogenic tobacco-specific nitrosamines in laboratory animals are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N'-nitrosonornicotine (NNN). DNA adduct formation from NNK and NNN has been studied extensively and is reviewed here. NNK is metabolically activated by cytochromes P450 to intermediates which methylate and pyridyloxobutylate DNA. The resulting adducts have been detected in cells and tissues susceptible to NNK carcinogenesis in rodents. The methylation and pyridyloxobutylation pathways are both important in carcinogenesis by NNK. NNK also induces single strand breaks and increases levels of 8-oxodeoxyguanosine in DNA of treated animals. NNAL, which like NNK is a potent pulmonary carcinogen, is also metabolically activated to methylating and pyridyloxobutylating intermediates. NNN pyridyloxobutylates DNA in its rat target tissues, esophagus and nasal mucosa. Methyl and pyridyloxobutyl DNA adducts are detected in human tissues. The methyl adducts most likely result in part from exposure of smokers to NNK, but these adducts are also detected in non-smokers. Some of the methyl adducts detected in non-smokers may be due to environmental tobacco smoke exposure. There are also potential dietary and endogenous sources of these adducts. Pyridyloxobutyl DNA adducts in human tissues result mainly from exposure to tobacco-specific N-nitrosamines. In laboratory animals, DNA adduct formation and carcinogenicity of tobacco-specific N-nitrosamines are closely correlated in many instances, and it is likely that similar relationships will hold in humans.     

A fluorescence-based analysis of aristolochic acid-derived DNA adducts

Aristolochic acids (AAs), major components of plant extracts from Aristolochia species, form (after metabolic activation) pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present here a fast, nonisotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition and analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.     

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.]     

Peroxynitrite irreversibly inactivates the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) in human breast cancer cells: a cellular and mechanistic study

Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations, and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7 cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses using recombinant NAT1 showed that the enzyme is rapidly (k(inact) = 5 x 10(4) m(-1).s(-1)) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine. We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as NAT1.     

32P-postlabelling analysis of DNA from human tissues.

DNA extracted from human lung, bladder, liver, pancreas, cervix and breast tissue samples taken at autopsy (37 sample sets) was analysed by the nuclease P1 enhancement modification of the 32P-postlabelling assay for levels of aromatic carcinogen DNA adducts. Results were combined with those from a previous study for statistical analysis of 56 sample sets (32 male+24 female). A strong trend was seen for increased adduct levels in the lung DNA of smokers and a weak association for the bladder DNA of smokers compared to non-smokers. Aromatic adducts were also detected in other tissues.     

Adverse reproductive outcomes from exposure to environmental mutagens.

The effect of environmental pollution on reproductive outcomes has been studied in the research project 'Teplice Program' analyzing the impact of air pollution on human health. Genotoxicity of urban air particles <10 microm (PM10) in in vitro system was determined by the analysis of DNA adducts. The highest DNA binding activity was observed in aromatic fraction, identifying DNA adducts of carcinogenic polycyclic aromatic hydrocarbons (PAHs) presumably diolepoxide-derived from: 9-hydroxybenzo[a]pyrene (9-OH-B[a]P), benzo[a]pyrene-r-7,-dihydrodiol-t-9,10-epoxide[+] (anti-BPDE), benzo[b]fluoranthene (B[b]F), chrysene (CHRY), benz[a]antracene (B[a]A), indeno[1,2,3-cd]pyrene (I[cd]P). Reproductive studies were conducted in both females and males. A study of the effects of PM10 exposure on pregnancy outcomes found the relationship between the intrauterine growth retardation (IUGR) and PM10 levels over 40 microg/m(3) in the first gestational month (Odds Ratio for 40-50 microg/m(3)50 microg/m(3)=1.9). Selected biomarkers were analyzed in venous blood, cord blood (chromosomal aberrations, comet assay) and placenta (DNA adducts, genetic polymorphisms of GSTM1 and NAT2 genotypes) of women enrolled in a nested case-control study. DNA adduct levels were higher in polluted vs. control districts, in smoking vs. nonsmoking mothers, and in GSTM1 null genotype, which was more pronounced in polluted district. No effect of air pollution was observed by cytogenetic analysis of chromosomal aberrations or by comet assay. The reproductive development of young men was followed by measures of semen quality, adjusted for ambient SO(2) exposure. The analysis identified significant associations with air pollution for <13% morphologically normal sperm, <29% sperm with normal head shape, <24% motile sperm. Analysis of aneuploidy in human sperm by FISH showed, aneuploidy YY8 was associated with season of heaviest air pollution. These findings are suggestive for an influence of air pollution on YY8 disomy. All these results indicate that air pollution may increase DNA damage in human population, which may be even higher for susceptible groups. Biomarkers of exposure (DNA adducts) and susceptibility (GSTM1 and NAT2) may indicate the risk of presumable low environmental exposure. Pregnancy outcome and semen studies imply that relatively low air pollution (higher than 40 microg PM10/m(3)) can significantly increase the adverse reproductive outcomes affecting both genders.     

Association of arylamine N-acetyltransferase (NAT1 and NAT2) genotypes with urinary bladder cancer risk

Arylamines are known bladder carcinogens deriving from tobacco smoke and environmental pollution. Arylamines are metabolised by NAT1 and NAT2 polymorphic enzymes in reactions of carcinogen activation and detoxification. We analysed genetic polymorphisms in both NAT1 and NAT2 genes in 56 bladder cancer patients and 320 healthy patients. Peripheral blood lymphocytes were collected from each subject and genotyped for NAT1 (six alleles) and NAT2 (four alleles) by PCR-RFLP. A weak association between NAT1 and NAT2 genotypes and bladder cancer risk was found when the genotypes were estimated separately (odds ratio OR 1.2, 95%CI 0.7-2.0, and OR 1.3, 95%CI 0.7-1.9, respectively). Almost all NAT1 genotypes possessing at least one "risk" *10 allele were more frequent in the bladder cancer group than in the control group. There was also an increased frequency of "risk" genotypes along with increased cigarette smoking in bladder cancer patients. The coincidence of NAT1-fast/NAT2-slow appears as a potential risk factor for urinary bladder cancer (OR 1.5, 0.8-3.0), as compared with the other genotype combinations.     

Trapping of 4-hydroxynonenal by glutathione efficiently prevents formation of DNA adducts in human cells

4-hydroxynonenal (HNE), one of the main breakdown products of lipid peroxides, has been shown to react with DNA yielding a 1,N2-propano adduct to 2'-deoxyguanosine. However, HNE may also react with a wide range of biomolecules before reaching the nucleus. Glutathione (GSH), the most abundant cellular thiol-containing peptide, is likely to be a major cytosolic target for HNE because of its high reactivity and its implication in the detoxification of this aldehyde. In order to estimate the proportion of HNE actually reaching DNA in human THP1 monocytes, we designed an experimental protocol aimed at quantifying DNA adducts and HNE-GSH in the same sample of cells exposed to extracellularly added HNE. Reverse-phase HPLC associated with tandem mass spectrometry detection was used as the analytical tool. It was first observed that, once produced, the HNE-GSH conjugate was very efficiently excreted from the cells into the culture medium. More strikingly, we determined that the amount of HNE-GSH conjugate produced was 4 orders of magnitude higher than that of DNA adduct. These results emphasize the major role played by glutathione in the protection of DNA against electrophilic species.     

Molecular mechanism of genotoxicity of the environmental pollutant 3-nitrobenzanthrone

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen identified in diesel exhaust and air pollution. This article reviews the results of our laboratories showing which of the phase I and II enzymes are responsible for 3-NBA genotoxicity, participating in activation of 3-NBA and its human metabolite, 3-aminobenzanthrone (3-ABA), to species generating DNA adducts. Among the phase I enzymes, the most of the activation of 3-NBA in vitro is attributable to cytosolic NAD(P)H:quinone oxidoreductase (NQO1), while N,O-acetyltransferase (NAT), NAT2, followed by NAT1, sulfotransferase (SULT), SULT1A1 and, to a lesser extent, SULT1A2 are the major phase II enzymes activating 3- NBA. To evaluate the importance of hepatic cytosolic enzymes in relation to microsomal NADPH:cytochrome P450 (CYP) oxidoreductase (POR) in the activation of 3-NBA in vivo, we treated hepatic POR-null and wild-type C57BL/6 mice with 3-NBA or 3-ABA. The results indicate that 3-NBA is predominantly activated by cytosolic nitroreductases such as NQO1 rather than microsomal POR. In the case of 3-ABA, CYP1A1/2 enzymes are essential for the oxidative activation of 3-ABA in liver. However, cells in the extrahepatic organs have the metabolic capacity to activate 3-ABA to form DNA adducts, independently from CYP-mediated oxidation in the liver. Peroxidases such as prostaglandin H synthase, lactoperoxidase, myeloperoxidase, abundant in several extrahepatic tissues, generate DNA adducts, which are formed in vivo by 3-ABA or 3-NBA. The results suggest that both CYPs and peroxidases may play an important role in metabolism of 3-ABA to reactive species forming DNA adducts, participating in genotoxicity of this compound and its parental counterpart, 3-NBA.     

Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts

The detection of 1,N2-propanodeoxyguanosine adducts in the DNA of rodent and human tissues as endogenous lesions has raised important questions regarding the source of their formation and their roles in carcinogenesis. Both in vitro and in vivo studies have generated substantial evidence which supports the involvement of short- and long-chain enals derived from oxidized polyunsaturated fatty acids (PUFAs) in their formation. These studies show that: (1) the cyclic propano adducts are common products from reactions of enals with DNA bases; (2) they are formed specifically from linoleic acid (LA; omega-6) and docosahexaenoic acid (omega-3) under in vitro stimulated lipid peroxidation conditions; (3) the levels of propano adducts are dramatically increased in rat liver DNA upon depletion of glutathione; (4) the adduct levels are increased in the liver DNA of the CCl4-treated rats and the mutant strain of Long Evans rats which are genetically predisposed to increased lipid peroxidation; and (5) adduct levels are significantly higher in older rats than in newborn rats. These studies collectively demonstrate that tissue lipid peroxidation is a main endogenous pathway leading to propano adduction in DNA. The possible contribution from environmental sources, however, cannot be completely excluded. The mutagenicity of enals and the mutations observed in site-specific mutagenesis studies using a model 1,N2-propanodeoxyguanosine adduct suggest that these adducts are potential promutagenic lesions. The increased levels of the propano adducts in the tissue of carcinogen-treated animals also provide suggestive evidence for their roles in carcinogenesis. The involvement of these adducts in tumor promotion is speculated on the basis that oxidative condition in tissues is believed to be associated with this process.     

The effect of the genetic polymorphisms of CYP1A1,CYP2D6, GSTM1 and GSTP1 on aromatic DNA adduct levels in the population of healthy women

Aromatic DNA adduct levels and polymorphisms of two phase I enzymes - CYP1A1 and CYP2D6 and two phase II enzymes - GSTM1 and GSTP1 were analyzed in a group of 133 nonsmoking healthy women 35-45 years old and holding jobs not connected with the exposure to the combustion products of organic matter. They were office workers from the south and north-eastern parts of Poland. Blood samples were collected in winter and in summer. Aromatic DNA adduct levels were measured in all winter and summer samples. The frequencies of CYP1A1, CYP2D6, GSTM1 and GSTP1 polymorphisms in samples from the studied women did not show any differences when compared with other Caucasian populations and the Polish male population studied previously. The differences in the levels of DNA adducts among the carriers of different genotypes were statistically non-significant. Analysis of combined genotypes selected the groups of volunteers with the highest and the lowest DNA adduct levels. The highest levels of DNA adducts were observed in the carriers of GSTM1(null)/CYP1A1Ile/Val (8.00+/-13.00 adducts/10(8) nucleotides in summer samples) and GSTP1-AA/CYP1A1Ile/Val genotypes (7.00+/-4.32 in winter and 7.30+/-7. 27/10(8) nucleotides in summer). The lowest levels of DNA adducts (3. 00+/-2.30 in winter and 2.00+/-3.16/10(8) nucleotides in summer) were found in the carriers of the genotype GSTP1-AG+GG/CYP1A1Ile/Val. The levels of DNA adducts in these groups were determined by the polymorphisms of GSTM1 and GSTP1 phase II detoxifying enzymes.     

O-alkylation in DNA does not correlate with the formation of chromosome breakage events in D. melanogaster

Postmeiotic cell stages of repair-proficient ring-X (RX) males were treated with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), diethylnitrosamine (DEN) or ethylnitrosourea (ENU) and then mated to either repair-defective (mei-9L1) or to repair-competent females (mei-9+). Absence of the mei-9+ function resulted in a hypermutability effect to all alkylating agents (AAs) when they were assayed for their ability to induce chromosomal aberrations (chromosome loss; CL), irrespective of marked differences in distribution of DNA adducts brought about by these AAs. This picture is different from that described previously for the induction of point mutations (Vogel et al., 1985a). There, evidence was presented indicating that reduction in DNA excision repair does not affect point mutation induction (recessive lethals) by those AAs most efficient in ring-oxygen alkylation such as ENU, DEN, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and isopropyl methanesulfonate (iPMS): the order of hypermutability of AAs with mei-9L relative to mei-9+ was MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females were plotted against those determined for mei-9+ females, straight lines of following slopes were obtained: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4, and iPMS = ENU = DEN = ENNG = 1. Those findings, together with the recent observation that AAs do not split into two groups when assayed for their ability to cause CL, point to the involvement of different DNA alkylation products in ENU- and DEN-induced chromosome loss vs. that of point mutations. It is concluded that with ENU and DEN chromosomal loss results from N-alkylation products whereas point mutations (SLRL) are the consequence of interactions with oxygen-sites in DNA. Thus, as a consequence of a very dominating role of O-ethylguanine (and possibly O4-alkylation of thymine), N-alkylation in DNA does not contribute measurably to mutation induction in the case of ENU-type mutagens while O-alkylation, very clearly, does not show a positive correlation with the formation of chromosome breakage events in Drosophila. Conversely, it appeared that with MMS-type mutagens (MMS; dimethyl sulfate, DMS; trimethyl phosphate, TMP), alkylation products such as 7-methylguanine and 3-methyladenine, if unrepaired or misrepaired, are potentially mutagenic lesions causing both mutations and chromosomal aberrations.     

Biomonitoring of exposure to aromatic amines: haemoglobin adducts in humans

Haemoglobin (Hb) adducts from aromatic amines (AAs) are well established biomarkers of exposure. Tobacco smoking and occupational exposure are major sources of AA Hb adducts. The origin of background levels in non-smokers and non-occupationally exposed humans are largely unknown. Here we examine the determination of AA Hb adducts, focussing on the analytical strategies for Hb isolation, removal of unbound AAs from Hb solutions, hydrolysis of the Hb bound AAs, extraction, preconcentration, clean-up and derivatisation of the free amines for determination by gas chromatography-mass spectrometry. Finally, a detailed summary of available results on the determination of AA Hb adducts is given.     

Increased DNA alterations in atherosclerotic lesions of individuals lacking the GSTM1 genotype.

Reduced glutathione (GSH) plays a critical role as an intracellular defense system providing detoxification of a broad spectrum of reactive species and their excretion as water-soluble conjugates. Conjugation of GSH with electrophiles is catalyzed by GSH S-transferases (GST), which constitute a broad family of phase II isoenzymes. Two of the GST encoding genes, GSTM1 (mu) and GSTT1 (theta), have a null genotype due to their homozygous deletion that results in lack of active protein. Polymorphisms within GSTT1 and especially GSTM1 have often been associated with cancer in various organs as well as with elevated levels of DNA adducts in various cell types. We recently demonstrated that DNA adducts are consistently detectable in smooth muscle cells (SMC) of human abdominal aorta affected by atherosclerotic lesions. Here we provide evidence that levels of adducts to SMC DNA from atherosclerotic lesions are consistently increased in individuals having the null GSTM1 genotype, whereas no association was established with the GSTT1 polymorphism. The influence of GSTM1 deletion was better expressed in never-smokers and ex-smokers than in current smokers. These findings bear relevance to the epidemiology of atherosclerosis and suggest that metabolic polymorphisms may contribute to the interindividual variability in susceptibility not only to carcinogens, but also to DNA binding atherogens.