Detection of benzo[a]pyrene-DNA adducts in leukocytes of coke oven workers

Coke oven workers are often heavily exposed to polynuclear aromatic hydrocarbons (PAHs) and particularly to benzo[a]pyrene (B[a]P), which has been associated with a high incidence of cancer. B[a]P is metabolically activated to its diol-epoxide derivative, benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE-I), a potent carcinogen which binds covalently to DNA, thereby producing BPDE-I-DNA adducts. In this study, an investigation was made of the exposure of coke oven workers to PAH via the measurement of urinary 1-hydroxypyrene (1-OHP) levels, and of exposure to B[a]P by the analysis of BPDE-I-DNA adducts in leukocytes using an ELISA competitive immunoassay. 1-OHP levels measured in post-shift samples correlated with those of DNA adducts detected in leukocytes, with a mean value (140.11 fM/50 micrograms of DNA) which differed significantly from the control value (P < 0.001). It is concluded that measurement of BPDE-I-DNA adduct levels in coke plant workers is essential in determining cancer risk due to high exposure to PAHs, and in particular of B[a]P.     

Gene‐diet interactions in exposure to heterocyclic aromatic amines and bulky DNA adduct levels in blood leukocytes

Heterocyclic aromatic amines (HAAs), carcinogens produced in meat when cooked at high temperatures, are an emerging biologic explanation for the meat‐colorectal cancer relationship. HAAs form DNA adducts; left unrepaired, adducts can induce mutations, which may initiate/promote carcinogenesis. The purpose of this research was to investigate the relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct levels. Least squares regression was used to examine the relationship between dietary HAA exposure and bulky DNA adduct levels in blood measured using ³²P‐postlabeling among 99 healthy volunteers. Gene‐diet interactions between dietary HAAs and genetic factors relevant to the biotransformation of HAAs and DNA repair were also examined. No main effects of dietary HAAs on bulky DNA adduct levels was found. However, those with the putative NAT1 rapid acetylator phenotype had lower adduct levels than those with the slow acetylator phenotype (P = 0.02). Furthermore, having five or more 'at‐risk' genotypes was associated with higher bulky DNA adduct levels (P = 0.03). Gene‐diet interactions were observed between NAT1 polymorphisms and dietary HAAs (P < 0.05); among the slow acetylator phenotype, higher intakes of HAAs were associated with an increase in DNA adduct levels compared to lower intakes. This study provides evidence of a biologic relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct formation. However, the lack of a strong main effect of HAAs suggests that dietary HAAs are not a large contributor to bulky DNA adducts in this population; future studies should consider relevant gene‐diet interactions to clarify the role of HAAs in carcinogenesis. Environ. Mol. Mutagen. 56:609–620, 2015. © 2015 Wiley Periodicals, Inc.     

DNA adducts in normal bladder tissue and bladder cancer risk

Cigarette smoking is an established cause of bladder cancer. The direct relationship between smoking-induced DNA adducts in bladder cells and cancer risk at that site has, however, been poorly assessed. We therefore investigated the relationship between bladder cancer risk and levels of DNA adducts measured in normal bladder biopsies by 32P-post-labeling in a hospital-based case-control study of 59 bladder cancer patients and 45 controls submitted to surgery for prostatic hyperplasia or urinary incontinence. An approximately 2-fold risk for bladder cancer was found in individuals with an adduct level >14.8 (median among controls) compared with those with an adduct level < or =14.8 (OR = 1.9, 95% CI 0.8-4.3, P = 0.13). A dose-response relationship was also suggested (trend test, P = 0.13): compared with adduct levels below 13.5, the OR for bladder cancer was 1.7 (95% CI 0.6-4.6) for adduct levels between 13.5 and 18.5 and 2.2 (95% CI 0.8-6.1) for adduct levels >18.5. These findings provide some evidence that DNA adducts in bladder tissue might predict smoking-induced bladder cancer. Larger studies are still warranted to confirm these results.     

DNA adducts of 1,3-butadiene in humans: relationships to exposure, GST genotypes, single-strand breaks, and cytogenetic end points

The modulation of 1,3-butadiene (BD)-induced DNA adducts by occupational exposure, glutathione S-transferase (GST) genotypes, single-strand breaks, and cytogenetic end points was studied in 15 workers and 11 controls. The exposed group consisted of 8 smokers and 7 nonsmokers, whereas the control group consisted of 7 nonsmokers and 4 smokers. Among all subjects, the adduct levels in workers lacking GSTM1 were significantly higher than in those who were GSTM1 positive (P = 0.026), and individuals with combined GSTM1(-) and GSTT1(+) genotype had elevated level of adducts compared to that of persons with GSTM1(+) and GSTT1(+) (P = 0.049). The increase in BD-DNA adduct levels in all subjects was significantly related to BD exposure and GSTM1 genotype (linear multiple regression analysis, P = 0.001; P = 0.035). The results suggest that DNA adducts serve as a sensitive and specific biomarker, integrating exposure and host metabolic capacity, although the data are limited to a small number of subjects.     

Correlation between biomarkers of human exposure to genotoxins with focus on carcinogen-DNA adducts

Correlations among biomarkers, an important issue in biomarker research, provide enhanced insight and understanding of the complexity of molecular mechanisms initiated by environmental genotoxic agents in the human organism. Occupational and environmental exposures mostly represent mixtures of genotoxic agents, whereas the specificity of biomarker measurements varies widely. Here, we give an overview of the correlation studies with particular emphasis on DNA adduct biomarker analysis of exposure to polycyclic aromatic hydrocarbons (PAHs) and/or tobacco smoke. We have collected data on correlations between different DNA adduct detection methods, DNA adduct structures and DNA adduct levels in human tissues. Data are also presented on the correlation between DNA adducts and other biomarkers of exposure and of early biological effects, including protein adducts, urinary metabolites and cytogenetic end points. In numerous studies, 32P-postlabelling and immunoassay measurements of DNA adducts recognized the difference between exposure groups similarly; however, at the individual level, there was, in general, not a statistically significant correlation between the two determinations. Inconsistency was found regarding the correlation between the levels of total bulky adducts and specific single DNA adduct structures. A number of studies found a positive correlation between DNA adduct levels in target and surrogate tissues, although stratification for exposure level may have influenced the results. Characteristically, there was a positive correlation between DNA adduct levels in tumour and normal tissue pairs. In general, there was a lack of correlation between DNA adducts and urinary PAH metabolites, but after stratification for particular genetic polymorphisms correlation may have emerged between the two biomarkers of exposure. The correlations with cytogenetic biomarkers were very complex, with examples of both positive correlation and lack of correlation. Exploration of correlations among biomarkers contributes to the further progress of molecular cancer epidemiology and to the selection of the optimal biomarkers for the investigation of human exposure to carcinogens.     

Time- and dose-dependent DNA binding of PAHs derived from diesel particle extracts, benzo[a]pyrene and 5-methychrysene in a human mammary carcinoma cell line (MCF-7).

Cultures of a human mammary carcinoma cell line (MCF-7) were exposed to the soluble organic fraction of diesel particle emissions, benzo[a]pyrene (B[a]P) and 5-methylchrysene (5-MeCHR) to study time- and dose-related PAH-DNA binding. The concentrations of 14 PAHs in three extracts were analyzed by HPLC and PAH-DNA adducts were measured by (32)P post-labeling assay. Time-dependent DNA adducts formation of 2.5 microM B[a]P was lower than that of 2.5 microM 5-MeCHR. In comparison with B[a]P, 2-fold higher adduct formation by 5-MeCHR was observed at 12 h exposure, after which BPDE adducts decreased and 5-MeCHR continued to form adducts linearly during 48 h exposure. The data for these two PAH compounds demonstrate a large variation in adduct-forming potency, which should be taken into account when estimating DNA adducts formed by mixtures of unknown PAHs. A clear dose-response effect on formation of DNA adducts was obtained for B[a]P and a Standard Reference Material (SRM) of diesel particulate matter. The amount of B[a]P contributed more to total DNA adduct formation by SRM than by three diesel extracts. Thus, no conclusions can be drawn from diesel particle-derived B[a]P as to the adduct-forming potency of other carcinogenic PAHs. There was little change in adduct levels formed by three diesel extracts from 0 to 12 h exposure. Thereafter, the number of adducts formed by RD2 increased more rapidly than those formed by RD1 and EN97. The concentrations of 14 PAHs and adduct levels analyzed at 24 and 48 h did not change in the same proportion between the extracts. Neither could PAH-DNA adduct levels be explained by the sum of strong and weak adduct-forming PAHs analyzed in the extracts. This indicates that other PAHs in the extracts RD1, RD2 and EN97 contributed to adduct formation more than the carcinogenic adduct-forming PAHs analyzed in this study.     

Assessment of environmental and occupational exposures to butadiene as a model for risk estimation of petrochemical emissions

1,3-Butadiene (BD) is an important industrial chemical and environmentalcontaminant, e.g. in urban air, traffic exhausts and tobaccosmoke. It has been shown to be genotoxic in vitro and in vivoand carcinogenic in rodents, mice being more sensitive thanrats. The present study confirmed this species difference. Usingmicronuclei in erythrocytes or bone marrow as a marker, miceresponded at an effective level of 50 p.p.m., while the highestineffective level in rats was 500 p.p.m. (inhalation of BD for5 days). A dose-dependent increase in N-terminal valine haemoglobinadducts was seen in both rats and mice, but the adduct levelsin the latter species were on average five times higher. Forthe first time, specific N⁶-alkyldeoxyadenosine adducts wereidentified in lung and liver DNA of rats exposed to BD by inhalation.No significant difference in DNA adduct level was seen in lungtissue of rats and mice at similar exposure levels. Occupationalexposure levels to BD in the European Process industry are variable,but generally <1 p.p.m. Haemoglobin adduct levels were seento be increased among the worker groups with higher potentialexposure to BD (process work, bomb voiding and repair duties)as compared with adduct levels in less exposed workers in maintenanceand the laboratory or control personnel. However, the N-terminalvaline haemoglobin adducts measured in the workers were oneto two orders of magnitude lower than extrapolated for the sameexposure dose in mice. In the same workers no exposurerelatedeffects were seen in the cytogenetic parametres studied, i.e.chromosomal aberrations, sister chromatid exchanges or micronucleiin peripheral blood lymphocytes, or in the Ras oncoprotein levelsof plasma samples. The studies so far conducted suggest thathuman exposure at the levels seen in the present day processindustry can be documented at the biological dose level usinghaemoglobin adduct measurement, but not at the biological effectlevel using cytogenetic biomarkers. In order to quantitate thehuman genotoxic risk of BD exposure more work needs to be doneon the role of other active BD metabolites than l,2-epoxy-3-buteneand on the genetic polymorphisms controlling the variabilityof individual responses. ⁶To whom correspondence should be addressed     

Influence of GSTM1 and NAT2 genotypes on placental DNA adducts in an environmentally exposed population

The placenta bulky DNA adducts have been studied in relation to metabolic genotypes for glutathione S-transferase M1 (GSTM1) and N-acetyl transferase 2 (NAT2) in 158 mothers (113 nonsmokers and 45 smokers) living in two regions with different annual average air pollution levels of sulphur dioxide, nitrogen oxides, particulate matter <10 μm, and polycyclic aromatic hydrocarbons. One region was the district of Teplice as the polluted industrial region with mines and brown coal power plants, and the other was the district of Prachatice, an agricultural region without heavy industry. DNA adduct levels were determined by using a butanol extraction enrichment procedure of ³²P-postlabeling. GSTM1 and NAT2 genotypes were studied by using polymerase chain reaction. The total DNA adduct levels included a diagonal radioactive zone (DRZ) and one distinct spot outside DRZ (termed X), which was detected in almost all placenta samples and correlated with DRZ (r = .682; P < .001). We found the total DNA adduct levels 2.12 ± 1.46 (0.04–7.70) and 1.48 ± 1.09 (0.11–4.98) adducts per 10⁸ nucleotides for Teplice and Prachatice districts, respectively, indicating significant differences between both regions studied (P = .004). Elevated DNA adduct levels were found in smoking mothers (10 or more cigarettes per day) by comparison with nonsmoking mothers (3.21 ± 1.39 versus 1.32 ± 0.88 adducts per 10⁸ nucleotides; P < .001). Placental DNA adduct levels in smokers correlated with cotinine measured in plasma (r = .432; P = .003). This relation indicates that cigarette smoking could be predominantly responsible for DNA adduct formation in placentas of smoking mothers. DNA adduct levels were evaluated separately for nonsmokers (1.50 ± 1.00 vs. 1.09 ± 0.66 adducts/10⁸ nucleotides for the Teplice and Prachatice districts, respectively; P = .046) and smokers (3.35 ± 1.47 vs. 2.91 ± 1.20 adducts/10⁸ nucleotides for Teplice and Prachatice districts, respectively; P = .384) to exclude the effect of active cigarette smoking on the district variation. These findings indicate that the effect of the environmental pollution in cigarette smokers is practically overlapped by tobacco exposure. No seasonal variation was observed for DNA adduct levels in the overall population studied and no relation between total DNA adduct levels in placenta and levels of vitamins A, C, and E in venous and cord blood was found. A positive GSTM1 genotype was detected in 78 subjects, while negative GSTM1 genotype was found in 80 subjects. Higher DNA adduct levels were detected in the group with GSTM1-negative genotype by comparison with GSTM1-positive genotype (2.05 ± 1.30 vs. 1.66 ± 1.39 adducts/10⁸ nucleotides; P = .018). This finding is more pronounced in the Teplice district (2.33 ± 1.36 vs. 1.88 ± 1.56 adducts/10⁸ nucleotides; P = .053) than for the Prachatice district (1.61 ± 1.09 vs. 1.36 ± 1.10 adducts/10⁸ nucleotides; P = .248) and for nonsmokers (1.45 ± 0.82 vs. 1.18 ± 0.93 adducts/10⁸ nucleotides; P = .029) more than for smokers (3.45 ± 1.14 vs. 2.95 ± 1.62 adducts/10⁸ nucleotides; P = .085). Significant district and seasonal differences were found in subgroups with GSTM1-negative genotype. DNA adduct levels in placentas of the GSTM1-negative subgroup were higher in mothers living in the polluted district of Teplice than in Prachatice (P = .012). The adduct levels in placentas sampled in the summer period were higher than in the winter period in the GSTM1-negative population (P = .006). No effect of the NAT2 genotype on DNA adduct levels was observed. Environ. Mol. Mutagen. 30:184–195, 1997. © 1997 Wiley-Liss, Inc.     

Influence of arylamine n‐acetyltransferase,sex, and age on 4‐aminobiphenyl‐induced in vivo mutant frequencies and spectra in mouse liver

One model for cancer initiation by 4‐aminobiphenyl (ABP) involves N‐oxidation by cytochrome P450 CYP1A2 followed by O‐conjugation by N‐acetyltransferase(s) NAT1 and/or NAT2 and decomposition to a DNA‐binding nitrenium ion. We recently observed that neonatal ABP exposure produced liver tumors in male but not in female mice, and that NAT deficiency reduced liver tumor incidence. However, ABP‐induced liver tumor incidence did not correlate with liver levels of N‐(deoxyguanosin‐8‐yl)‐ABP adducts 24 hr after exposure. In this study, we compared in vivo ABP‐induced DNA mutant frequencies and spectra between male and female wild‐type and NAT‐deficient Muta?Mouse using both the tumor‐inducing neonatal exposure protocol and a 28‐day repetitive dosing adult exposure protocol. ABP produced an increase in liver DNA mutant frequencies in both neonates and adults. However, we observed no sex or strain differences in mutant frequencies in neonatally exposed mice, and higher frequencies in adult females than males. Neonatal ABP exposure of wild‐type mice increased the proportion of G‐T transversions in both males and females, while exposure of Nat1/2(‐/‐) mice produced increased G‐T transversions in males and a decrease in females, even though females had higher levels of N‐(deoxyguanosin‐8‐yl)‐4‐ABP adducts. There was no correlation of mutant frequencies or spectra between mice dosed as neonates or as adults. These results suggest that observed sex‐ and NAT‐dependent differences in ABP‐induced liver tumor incidence in mice are not due to differences in either mutation rates or mutational spectra, and that mechanisms independent of carcinogen bioactivation, covalent DNA binding and mutation may be responsible for these differences. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

Influences of DNA isolation and RNA contamination on carcinogen-DNA adduct analysis by 32P-postlabeling

³²P-Postlabeling is a widely applied assay for the analysis of carcinogen-DNA adducts. Optimization of most steps in this assay has been given attention, but influences of DNA isolation and DNA purity on adduct quantitation have not been investigated systematically. In this study, DNA was isolated from human lymphocytes exposed to benzo[a]pyrene (B[a]P, 10 μM) for 18 hr and from liver of rats i.p.-treated with B[a]P (10 mg/kg body weight) using two different DNA isolation methods: a phenol-extraction and a salting-out procedure. Subsequently, DNA was analysed by nuclease P1 (NP1) or butanol-enriched ³²P-postlabeling. Influences of RNA contamination were studied by labeling RNA isolated from in vitro exposed lymphocytes. In the in vitro experiment, DNA adduct levels were significantly higher using the salting-out procedure (63.2 ± 13.7 adducts per 10⁸ nucleotides, n = 9) as compared with the phenol-extraction (14.3 ± 0.8). RNA was ∼4 times less efficiently labeled as compared to DNA. Nonetheless, RNA contamination of DNA samples may result in an overestimation of DNA adduct levels when butanol enrichment is used, because RNA adduct levels seemed to be substantially higher than DNA adduct levels in the same cells. DNA adduct analysis by nuclease P1 enrichment is probably less affected, since RNA adducts appeared to be NP1 sensitive. In vivo, three different adducts were found by NP1 enriched ³²P-postlabeling in the liver of B[a]P-exposed rats. Again, DNA adduct levels were significantly higher using salting out as compared to phenol extraction for the adduct which comigrated with the BPDE-DNA adduct standard (adduct 1) and an unknown adduct (adduct 2). However, the results were the opposite for another B[a]P-derived DNA adduct (adduct 3). Our results suggest that differences in DNA isolation procedures as well as RNA contamination influence quantitative DNA adduct analysis by ³²P-postlabeling. Environ. Mol. Mutagen. 32: 344–350, 1998 © 1998 Wiley-Liss, Inc.     

Immunodetection of benzo[a]pyrene adducts in ovarian cells of women exposed to cigarette smoke

Benzo[a]-pyrene (B[a]P) is a potent mutagen and carcinogen present in cigarettes. We report here on immunodetection and quantification of B[a]P-DNA adducts in granulosa-lutein cells of patients undergoing in- vitro fertilization (IVF) and embryo transfer, who were exposed to cigarette smoke. Follicular fluids (FF) and granulosa-lutein cells were obtained from the same follicular aspirate from 32 women self-reported as active smokers, passive smokers, or non-smokers. Cells were immunostained with 5D11, an anti-B[a]P diolepoxide monoclonal antibody that recognizes DNA adducts. Cotinine, a reliable marker for recent smoke exposure and dose, was assessed by radioimmunoassay in 32 FF samples. Individual scores of cell immunoreactivity were highly correlated with FF cotinine concentrations. Evaluations of immunostaining intensity in 9770 granulosa-lutein cells from the 32 women revealed higher average scores in active and passive smokers, relative to non-smokers. In passive smokers the average level of cell immunostaining was 63% of that of active smokers. These relationships provide quantitative evidence that B[a]P-DNA adduct levels are related to smoke exposure and dose, both recent and long term. Immunostaining was confined to the nucleus, suggesting adduct formation by covalent binding to DNA. Presence of adducts in granulosa-lutein cells from women exposed to cigarette smoke may increase the risk for DNA damage.      

Cigarette smoke-induced DNA adducts in the respiratory and nonrespiratory tissues of rats

Formation of DNA adducts is regarded as an essential initial step in the process of chemical carcinogenesis. To determine how chronic exposure to cigarette smoke affects the distribution of DNA adducts in selected respiratory and nonrespiratory tissues, we exposed male Sprague-Dawley rats daily to fresh mainstream smoke from the University of Kentucky reference cigarettes (2R1) in a nose-only exposure system for 32 weeks. Blood carboxyhemoglobin, total particulate matter (TPM) intake, and pulmonary aryl hydrocarbon hydroxylase values indicated effective exposure of animals to cigarette smoke. DNA was extracted from three respiratory (larynx, trachea, and lung) and three nonrespiratory (liver, heart, and bladder) tissues and analyzed for DNA adducts by the 32P-postlabeling assay under conditions capable of detecting low levels of diverse aromatic/hydrophobic adducts. Data showed that the total DNA adducts in the lung, heart, trachea, and larynx were increased by 10- to 20-fold in the smoke-exposed group. Five-fold increase was observed in the bladder tissue, but differences were not present in the liver DNA of control and smoke-exposed groups. These data suggest selective formation of DNA adducts in the tissues.     

Exposure to agrochemicals and DNA adducts in Western Liguria, Italy.

Pesticides are used to control pests and improve agricultural production. Despite their selectivity of action, a number of agrochemicals have been reported to be genotoxic using the (32)P-DNA postlabeling assay. Greenhouse floriculturists are suspected of being heavily exposed to agrochemicals during loading, mixing, and application of pesticides, as well as during manual activities by continuous contact with flowers and ornamental plants. We analyzed the DNA adduct formations in the white blood cells (WBCs) of 57 nonsmoker greenhouse floriculturists and 33 nonsmoker age-matched referents residing in the Western Liguria Region, Italy-the most important Italian greenhouse floriculture area. The averages of DNA adducts, expressed as relative adduct labeling (RAL), were 8.50 x 10(9) +/- 1.98 (SE) in floriculturists and 2.17 x 10(9) +/- 1.05 (SE) in referents. DNA adducts were significantly higher in floriculturists than in controls after adjustment for age and gender (P = 0.007). A specific adduct pattern, with up to six different spots, was observed in 60% of floriculturists, while no adducts were generally detected in controls. Our study represents an important contribution to the correct evaluation of the potential health risk associated with floriculture activity and supports the adoption of measures ensuring pesticide exposure reduction in greenhouses.     

Influence of genetic polymorphisms on biomarkers of exposure and genotoxic effects in styrene-exposed workers

A study on 44 workers exposed to styrene and 44 matched referents was performed in order to examine the influence of genetic polymorphisms in biotransformation and DNA repair enzymes on the levels of N-terminal hemoglobin adducts and genotoxicity biomarkers. Urinary mandelic acid concentration averaged 201.57 mg/g creatinine +/-148.32 in exposed workers, corresponding to a calculated average airborne styrene exposure of 9.5 ppm +/-9.6. Individuals with a high level of N-terminal valine adducts had higher levels of DNA damage, as evaluated by the Comet assay (r = 0.29, P = 0.008). Frequencies of micronucleated mononucleated lymphocytes (MNMC) (0.71 +/- 0.88 vs 0.11 +/- 0.20, P<0.0001), micronucleated binucleated lymphocytes (MNBC) (3.93 +/- 2.75 vs 2.65 +/- 1.94, p = 0.02) and micronucleated nasal epithelial cells (0.52 +/- 0.49 vs 0.23 +/- 0.31, p = 0.04) differed significantly between the exposed and referent groups. In the whole group of 88 individuals, higher frequencies of MNMC were found in individuals possessing the XRCC3 Met(241) allele and those individuals with the XRCC1 Gln( (399) ) allele showed higher frequencies of MNMC and MNCB. In vitro DNA repair capacity, as measured by residual DNA strand breaks in peripheral blood leukocytes after a styrene oxide challenge, was also influenced by styrene exposure, with an apparent induction of early repair mechanisms associated with the intensity of recent exposure and a reduction of late (24 h) repair capacity that was associated with the duration of employment. After 1 h of repair, lower levels of residual DNA damage were found in individuals possessing GSTT1 (P = 0.043). After 24 h of repair, lower residual DNA damage was found in individuals homozygous for XRCC1 Arg(194) (P = 0.013). Multivariate regression analysis indicated that the duration of exposure, smoking habits and polymorphisms of XRCC1 at codon 399 were important variables affecting the genotoxic responses. Our data suggest that DNA damage is formed in workers exposed to low concentrations of styrene, and that genotypes of metabolising and DNA-repair genes are important for the assessment of individual genotoxic risk to styrene. The in vitro DNA repair phenotype assay might be a valuable method to estimate the susceptibility of workers.     

Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons

We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 μg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30-60%) than levels of bulky DNA adducts measured in the same samples by (32)P-postlabelling. The BPDE-DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE-DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between (32)P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.     

DNA adduct assay in cervical epithelium

Numerous epidemiological studies have shown that there is an association between smoking and cervical cancer. However, the essential evidence to show whether this relationship is casual or causal is lacking. The demonstration of DNA modification by tobacco components in the cervical epithelium would provide biochemical evidence to support a causal role. In this study, DNA from 39 cervical biopsies was analysed for the presence of DNA adducts using the ³²P-postlabelling technique. A questionnaire on smoking habit and a urinary cotinine assay were used to identify smokers and nonsmokers. DNA samples from smokers [identified from questionnaire] were found to have significantly higher adduct levels than nonsmokers (Mann-Whitney one-tailed U-test, 95% CI > 0.339, P = 0.024). Exclusion of the women whose urinary cotinine levels did not confirm their self-reported smoking status (smoker or nonsmoker) increased this significance (95% CI > 0.508, P = 0.01). Women who had abnormal cervical smears hadsignificantly higher DNA adduct levels than those with normal smears (95% CI > 0.439, P = 0.015). Monitoring of women with high DNA adduct levels may be a way of identifying women at risk of cervical cancer. These findings demonstrate that tobacco smoking by women leads to elevated levels of DNA adducts in cervical epithelium and provides the biochemical evidence to support the concept that smoking is a cause of cervical cancer. © 1994 Wiley-Liss, Inc. Published 1994 Wiley-Liss, Inc. © 1994 Wiley-Liss, Inc.     

Biomonitoring the human population exposed to pollution from the oil fires in Kuwait: analysis of placental tissue using (32)P-postlabeling

The placenta is a readily available source of material for molecular epidemiological investigations. As such, DNA damage in this tissue can be indicative of maternal exposure to environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs). Previous reports have demonstrated that (32)P-postlabeling (PPL) is able to detect the presence of aromatic adducts in human placenta that are associated with maternal smoking during pregnancy. Using PPL we have assayed the DNA damage in placental samples from Kuwaiti mothers who were exposed to environmental pollution during pregnancy. This pollution arose in the aftermath of the Iraqi invasion of Kuwait, which left hundreds of oil wells burning. For comparison, further Kuwaiti samples were obtained approximately 1 year after the oil well fires and, as such, are from individuals unexposed to the airborne pollution from the oil well fires during pregnancy. In addition, placental samples were obtained from subjects in the United Kingdom. Adduct levels were measured in all samples using both the nuclease P1 and butanol extraction enhancement procedures. No elevation of adduct levels was observed in the placenta of mothers exposed to the oil well fires (n = 40) with either procedure (144 +/- 30 attomol/microg DNA for nuclease P1 enrichment, 245 +/- 50 attomol/microg DNA for butanol extraction), when compared with the nonexposed Kuwaiti mothers (180 +/- 32 and 281 +/- 39 attomol/microg DNA, respectively, n = 24). Similar adduct levels were observed in UK mothers who smoked cigarettes (178 +/- 30 and 284 +/- 52 attomol/microg DNA, n = 30), which in turn were approximately twice those observed in nonsmoking mothers (90 +/- 14 and 141 +/- 15 attomol/microg DNA, n = 12), although there is no significant difference in the distribution of adduct levels when statistical analysis is performed. Comprehensive interpretation of the Kuwaiti data is difficult as precise information on PAH levels is unavailable, although the data do seem to indicate that exposure to PAHs was not biologically significant.     

DNA adducts and combinations of multiple lung cancer at‐risk alleles in environmentally exposed and smoking subjects

Interindividual variation in DNA adduct levels in individuals exposed to similar amounts of environmental carcinogens may be due to genetic variability. We analysed the influence of genes involved in determining/modifying DNA damage, including microsomal epoxide hydrolase1 (EPHX1) His139Arg, N‐acetyl‐transferase, NAD(P)H:quinone oxidoreductase1 (NQO1) Pro187Ser, manganese superoxide dismutase2 (MnSOD2) Val16Ala, and apurinic/apyrimidinic endonuclease1 (APE1) Asp148Glu polymorphisms in blood of 120 smokers. Subsequently, we examined the effects of the combinations of the variant alleles of EPHX, NQO1 and MnSOD2 together with the wild type allele of APE1 on DNA damage by calculating the “sum of at‐risk alleles.” We reviewed the studies examining the relationships of DNA adducts with at‐risk alleles in environmentally exposed subjects. Our findings showed that smokers carrying the EPHX1–139Arg and the NQO1–187Ser variants were significantly more likely to have higher adduct levels. Null associations were found with the other variants. Nevertheless, DNA adduct levels in smokers with ≥5 at‐risk alleles were significantly different from those with fewer than two alleles. A similar picture emerged from studies of DNA adducts and at‐risk alleles in environmentally exposed and smoking subjects. Certain at‐risk allele combinations may confer a greater likelihood of increased levels of adducts after environmental insults. The increase in DNA adduct levels in susceptible subjects exposed to environmental carcinogens may reflect changes in the mechanisms that protect cells from the accumulation of genetic damage. Alterations of the physiological processes designed to maintain homeostasis may reduce the individual “genotoxic tolerance” to environmental challenges and result in phenotypes characterized by high levels of DNA adducts. Environ. Mol. Mutagen. 54:375–383, 2013. © 2013 Wiley Periodicals, Inc.     

Evidence for the presence of mutagenic arylamines in human breast milk and DNA adducts in exfoliated breast ductal epithelial cells

Aromatic and heterocyclic amines are ubiquitous environmental mutagens present in combustion emissions, fried meats, and tobacco smoke, and are suspect human mammary carcinogens. To determine the presence of arylamines in breast tissue and fluid, we examined exfoliated breast ductal epithelial cells for DNA adducts and matched human milk samples for mutagenicity. Breast milk was obtained from 50 women who were 4-6 weeks postpartum, and exfoliated epithelial-cell DNA was evaluated for bulky, nonpolar DNA adducts by (32)P-postlabeling and thin-layer chromatography. Milk was processed by acid hydrolysis, and the extracted organics were examined in the standard plate-incorporation Ames Salmonella assay using primarily strain YG1024, which detects frameshift mutations and overexpresses aryl amine N-acetyltransferase. DNA adducts were identified in 66% of the specimens, and bulky adducts migrated in a pattern similar to that of 4-aminobiphenyl standards. The distribution of adducts did not vary by NAT2 genotype status. Of whole milk samples, 88% (22/25) had mutagenic activity. Among the samples for which we had both DNA adduct and mutagenicity data, 58% (14/19) of the samples with adducts were also mutagenic, and 85% (11/13) of the mutagenic samples had adducts. Quantitatively, no correlation was observed between the levels of adducts and the levels of mutagenicity. Separation of the milk showed that mutagenic activity was found in 69% of skimmed milk samples but in only 29% of the corresponding milk fat samples, suggesting that the breast milk mutagens were moderately polar molecules. Chemical fractionation showed that mutagenic activity was found in 67% (4/6) of the basic fractions but in only 33% (2/6) of acidic samples, indicating that the mutagens were primarily basic compounds, such as arylamines. Although pilot in nature, this study corroborates previous findings of significant levels of DNA adducts in breast tissue and mutagenicity in human breast milk and indicates that breast milk mutagens may be moderately polar basic compounds, such as arylamines.     

Quantitation and immunohistochemical localization of DNA adducts in rat embryos and associated yolk sac membranes exposed in vitro to N-acetoxy-2-acetylaminofluorene (N-Ac-AAF).

Specific antibodies and radioimmunoassay (RIA) were used to measure the levels of acetylated and deacetylated C-8 substituted deoxyguanosine adducts in day 11 rat embryos and their associated yolk sacs after exposure of whole rat conceptuses in vitro to the teratogen N-acetoxy-2-acetylaminofluorene (N-Ac-AAF). The deacetylated adduct predominates in both the embryo and the associated yolk sac, and a dose response for adduct formation was observed when adducts were quantitated by RIA. Immunohistochemical localization of the deacetylated adducts revealed that adducts were confined to the nuclei in all tissues examined and that the abundance of adducts varied within and between tissues. Our initial findings indicate that specific DNA adduct antibodies may be useful in the study of teratogenesis induced by a wide variety of agents that modify DNA.