The choice of controls in a case-control study on WBC-DNA adducts and metabolic polymorphisms

The choice of the control group is a key issue in case-control studies, particularly in studies of molecular epidemiology. We discuss the potential bias introduced by different options. To exemplify the consequences of different choices, we have analysed two sets of controls in the context of a case-control study on bladder cancer: 55 were patients with urological conditions (cystitis, prostate hypertrophy), while 49 had a miscellany of medical or surgical conditions. We measured DNA adducts in white blood cells (WBC) by ³²P-postlabelling and a series of metabolic polymorphisms (GSTM1, GSTT1, GSTP1, NAT2, NQO1). While no statistically significant differences were found for metabolic polymorphisms, the two series of controls showed different concentrations of DNA adducts, suggesting that conditions related to bladder cancer or intermediate steps leading to bladder cancer, such as chronic cystitis, may be associated with higher adduct levels. An association between DNA adduct levels and infection has been noted before in experimental animals: both in lung and in the skin, an inflammatory response increased the biologically effective doses of polycyclic aromatic hydrocarbons. An alternative explanation is confounding; in fact, after adjustment for the level of consumption of fruit and vegetables (but not for smoking) the difference between the two control groups was no longer statistically significant. In conclusion, the choice of controls in studies of molecular epidemiology has subtle methodological implications, including confounding of metabolic/molecular measurements by complex exposures such as diet.     

Effect of genetic polymorphism for metabolic enzymes on the relationship between smoking dose and DNA adducts in lymphocytes

The genetic polymorphism of metabolic enzymes on the relationship between smoking dose and DNA adduct levels in lymphocytes were evaluated in 51 smokers. The genetic polymorphisms of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) were analysed by a PCR method. Lymphocyte DNA adducts were measured by two analytical versions of a 32P-postlabelling method; nuclease P1 digested method and butanol extracted method. Mean adduct levels obtained with the nuclease P1 method (1.21 +/- 0.74 per 108 nucleotides) were higher than those obtained with the butanol extracted method (0.82 +/- 0.47, p < 0.01). There was a significant correlation between adduct levels by the nuclease P1 method and those by the butanol extracted method (r = 0.49, p < 0.01). A significant correlation was not found between smoking dose and DNA adduct levels obtained using both methods in lymphocytes of all subjects. When subjects were divided into two groups by CYP1A1 genotypes, significant correlations between smoking indices, such as number of cigarettes per day years or tar intake per day years, and DNA adduct levels measured by the butanol extracted method was found in heterozygous or miner homozygous for CYP1A1 exon 7 polymorphism. We could not get a significant effect of GSTM1 on the relationship between smoking dose and DNA adducts.     

Lymphocytes,DNA adducts and genetic polymorphism for metabolic enzymes in low dose cigarette smokers

The aim of this study was to investigate the relationship between genetic polymorphism of metabolic enzymes and DNA adduct levels in lymphocytes of low dose cigarette smokers (less than 20 cigarettes per day). We previously reported the effects of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) on lymphocyte DNA adducts. This time we considered not only CYP1A1 and GSTM1 but also cytochrome P4502E1 (CYP2E1) and glutathione S-transferase T1 (GSTT1). DNA adducts in lymphocytes obtained from low dose cigarette smokers (n = 41) and nonsmokers (n = 56) were measured by the 32P-postlabelling method. The adduct levels were compared regarding smoking status and polymorphic genotypes of these four enzymes. The mean SD of DNA adduct levels in all low dose cigarette smokers and non-smokers was 1 05 0 83 per 108 nucleotidesand 0 85 0 35 per 108 nucleotides, respectively. In low dose cigarette smokers, adduct levels were higher in the rare homozygous (MM) for CYP1A1-exon 7 polymorphism compared with the other types such as common homozygous (WW) and heterozygous (WM). CYP1A1-WM, MM in combination with GSTM1 null showed highest adduct levelamong low smokers. The low smokers with rare homozygous for CYP2E1 Dra1 polymorphism tended to have lower adduct levels than wild types. Low dose cigarette smokers with combined GSTM1 null and T1 null had a higher tendency for adduct levels than others. However none of the differences reached statistical significance.     

Short Communication: The effect of CYP1A1 induction on the formation of benzo a pyrene adducts in liver and lung DNA and plasma albumin in rats exposed to benzo a pyrene:adduct quantitation by immunoassay and an HPLC method

Induction of cytochrome P450 enzymes by exposure to polycyclic aromatic hydrocarbons (PAH) can result in both decreased or increased PAH adduct levels. The lung is a main target site for PAH-carcinogenesis. By HPLC determination of B [a]P-r-7, t-8-dihydrodiol, t-9, 10-epoxide (BPDE-I)-DNA adducts in rat, the level of the ultimate carcinogenic B[a]P-metabolite was higher in lungs than in liver. However, measured by immunoassay, the total benzo[a]pyrene (B[a]P)-DNA adduct levels were higher in liver than in lungs. Induction of CYP1A1 in vivo in rat by repeated i.p. doses of methylcholanthrene (MC) prior to a single dose of B[a]P resulted in a 2.4 times increase in CYP1A1 activity in liver tissue and 1.5 times higher levelsof total B[a]P-DNA adducts in lung and liver compared with controls which only received B[a]P. Increased levels of BPDE-I-DNA adducts were significantly correlated to increased CYP1A1 activity in induced lung tissue but not in liver. The times to reach maximum adduct levels were similar for both controls and MC-induced rats in both lung and liver,and plasma albumin. The BPDE-I-albumin adducts reached a maximum level around 1 day after B[a]P exposure and could not be used as a reliable marker of the short term PAH exposure in this study.     

A cross-sectional study of polycyclic aromatic hydrocarbon-DNA adducts and polymorphism of glutathione S-transferases among heavy smokers by race/ethnicity

Differences in lung cancer risk by race/ethnicity have been observed among smokers. To determine whether these observations might reflect differences in the formation of carcinogen-DNA adducts, we analysed blood specimens (n =151) collected from smokers who were recruited for possible participation in an antioxidant vitamin intervention study. Mononuclear cells were analysed for polycyclic aromatic hydrocarbon (PAH)-DNA adducts by competitive enzyme-linked immunosorbent assay. Genotypes of glutathione S-transferase M1 and P1 (GSTM1 and GSTP1), enzymes involved in the detoxification of PAH metabolites, were determined by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism, respectively. GSTM1 was present in 65 out of 88 (73.4%), 16 out of 32 (50.0%) and 16 out of 29 (54.8%) of African-Americans, Caucasians and Latinos, respectively (p =0.022). Homozygosity for the GSTP1 codon 105 variant was found in 25.6%, 6.3% and 10.0% of African-Americans, Caucasians and Latinos, respectively (p =0.023). Regression analysis of the log-transformed adduct levels confirmed that Caucasian and Latino subjects had lower PAH-DNA adduct levels than African-American subjects, after adjustment for gender, education,α -tocopherol and β-carotene levels, and GSTM1 status. Further adjustment for age and current smoking habits had no impact on these findings. Although crude analysis suggested that the GSTM1-positive genotype may be associated with lower PAH-DNA levels in Caucasians (but not in African-Americans or Latinos), a formal test for interaction between GSTM1 and ethnicity was not significant. We found no association between adduct levels and GSTP1 genotype. Although the mechanism is unclear, ethnic differences in DNA damage levels may in part explain why African-Americans have higher lung cancer incidence rates than other ethnic groups.     

32P-postlabelling analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro

Butadiene monoepoxide (BMO), epoxybutanediol (EBD) and diepoxybutane (DEB) are reactive metabolites of 1,3-butadiene (BD), an important industrial chemical classified as a probable human carcinogen. The covalent interactions of these metabolites with DNA lead to the formation of DNA adducts which may induce mutations or other types of DNA damage, resulting in tumour formation. In the present study, two pairs of diastereomeric N-1-BMO-adenine adducts were identified in the reaction of BMO with 2´-deoxyadenosine-5´-monophosphate (5´-dAMP). The major products formed by reacting EBD with 2´-deoxyguanosine-5´-monophosphate (5´-dGMP) were characterized as diastereomeric N-7-(2´,3´,4´-trihydroxybut-1´-yl)-5´-dGMP by UV and electrospray mass spectrometry. The formation of N-7-BMO-guanine adducts (1´-carbon, 60; 2´carbon, 54/10⁴ nucleotides) in BMO-treated DNA was about four times higher than that of N-1-BMO-adenine adducts (1´-carbon, 20; 2´-carbon, 8.7/10⁴ nucleotides). However, the recovery of N-1-BMO-adenine adducts in DNA (45 ± 5%) was two times higher than that of N-7-guanine adducts (20 ± 4%) by 32P-postlabelling analysis. Using the 32P-postlabelling/ HPLC assay, N-1-BMO-adenine, N-7-BMO-guanine and N-7-EBDguanine adducts were detected in BMO- or DEB-treated DNA and in liver DNA of rats exposed to BD by inhalation. The amount of N-7-EBD-guanine adducts (11/10⁸ nucleotides) in rat liver was about three-fold higher than N-7-BMO-guanine adducts (4.0/10⁸ nucleotides). The novel finding of N-1-BMO-adenine adducts formed in vivo may contribute to the understanding of the mechanisms of BD carcinogenic action.     

DNA adducts in marine and freshwater fish as biomarkers of environmental contamination

The analysis of DNA modifications in aquatic animals may serve as a sensitive marker of exposure to genotoxic contaminants. This is of importance in assessing water quality regarding pollution with genotoxic compounds, food safety analysing edible aquatic animals and in terms of ecotoxicology. Covalent modification of DNA is considered a crucial event in chemical carcinogenesis and thus may be considered a biomarker of an early genotoxic effect. Measuring DNA adducts is unique in that these lesions may be considered a biomarker of both exposure and effect. A number of studies have described the analysis of the DNA isolated from the liver of both freshwater and marine fish. Considerable levels of DNA adducts have been observed in some animals from contaminated lakes or rivers. Low levels were observed in DNA from the liver of marine fish. The background levels of DNA adducts have to be determined in animals from appropriate uncontaminated control sites that are matched for species, gender, age and season of the year. It is of crucial importance to consider the species analysed since there have been reports of the non responsiveness of some species.     

Short Communication: The effect of CYP1A1 induction on the formation of benzo a pyrene adducts in liver and lung DNA and plasma albumin in rats exposed to benzo a pyrene:adduct quantitation by immunoassay and an HPLC method

Induction of cytochrome P450 enzymes by exposure to polycyclic aromatic hydrocarbons (PAH) can result in both decreased or increased PAH adduct levels. The lung is a main target site for PAH-carcinogenesis. By HPLC determination of B[ a]P-r-7, t-8-dihydrodiol, t-9, 10-epoxide (BPDE-I)-DNA adducts in rat, the level of the ultimate carcinogenic B[a]P-metabolite was higher in lungs than in liver. However, measured by immunoassay, the total benzo[a]pyrene (B[a]P)-DNA adduct levels were higher in liver than in lungs. Induction of CYP1A1 in vivo in rat by repeated i.p. doses of methylcholanthrene (MC) prior to a single dose of B[a]P resulted in a 2.4 times increase in CYP1A1 activity in liver tissue and 1.5 times higher levelsof total B[a]P-DNA adducts in lung and liver compared with controls which only received B[a]P. Increased levels of BPDE-I-DNA adducts were significantly correlated to increased CYP1A1 activity in induced lung tissue but not in liver. The times to reach maximum adduct levels were similar for both controls and MC-induced rats in both lung and liver,and plasma albumin. The BPDE-I-albumin adducts reached a maximum level around 1 day after B[a]P exposure and could not be used as a reliable marker of the short term PAH exposure in this study.     

DNA adducts in marine and freshwater fish as biomarkers of environmental contamination

The analysis of DNA modifications in aquatic animals may serve as a sensitive marker of exposure to genotoxic contaminants. This is of importance in assessing water quality regarding pollution with genotoxic compounds, food safety analysing edible aquatic animals and in terms of ecotoxicology. Covalent modification of DNA is considered a crucial event in chemical carcinogenesis and thus may be considered a biomarker of an early genotoxic effect. Measuring DNA adducts is unique in that these lesions may be considered a biomarker of both exposure and effect. A number of studies have described the analysis of the DNA isolated from the liver of both freshwater and marine fish. Considerable levels of DNA adducts have been observed in some animals from contaminated lakes or rivers. Low levels were observed in DNA from the liver of marine fish. The background levels of DNA adducts have to be determined in animals from appropriate uncontaminated control sites that are matched for species, gender, age and season of the year. It is of crucial importance to consider the species analysed since there have been reports of the non responsiveness of some species.     

Capillary zone electrophoresis with on-line blotting for separation and detection of 32P-postlabelled DNA adducts

A new technique for the detection of ³²P-postlabelled DNA adducts separated by capillary electrophoresis was developed. By direct transfer from the capillary outlet to a positively charged moving filter paper, eluted radioactive peaks can be quantified using a phosphor imaging detector. With this method it is possible to separate DNA adducts from different carcinogens after ³²P-postlabelling of the modified and unmodified nucleotides with high sensitivity approaching 1 adduct per 10⁹ nucleotides.     

Persistence of 7-(2-hydroxyethyl) guanine-DNA adducts in rats exposed to ethene by inhalation

Quantification of 7 2 hydroxyethyl guanine 7 HEG adduct in DNA of livers and lymphocytes of male Sprague-Dawley rats exposed to 300 ppm ethene by inhalation 12 h a day for three consecutive days was performed to evaluate the potential of ethene to produce DNA adducts in these tissues. The persistence of 7 HEG in livers and lymphocytes was studied in rats sacrificed 0, 1, 5, and 20 days after the last exposure. DNA samples from control and treated animals were analysed for 7 HEG and 7 methylguanine 7 MG adducts by thin layer chromatography TLC combined with a high pressure liquid chromatography HPLC assay. After a 3 day exposure to ethene, 7 HEG accumulated to a similar extent in liver and lymphocytes, with the mean adduct level of 7.0 0.7 adducts per 107 nucleotides in liver and 7.4 0.7 adducts per 107 nucleotides in lymphocytes of rats sacrificed immediately after cessation of exposure. The approximate half life of 7 HEG was 5 days in liver and 3 days in lymphocytes, which is consistent with the loss of adduct primarily by spontaneous depurination. In addition, the background levels of 7 HEG and 7 MG were determined in the liver and lymphocytes from the control rats.     

Measurement of 7 methyl and 7 2 hydroxyethyl guanine DNA adducts in white blood cells of smokers and non smokers

The goal of the present study was to measure the levels of 7-methylguanine and 7-(2- hydroxyethyl)guanine DNA adducts in human white blood cells in relation to smoking. DNA was isolated from samples of 11 smokers and eight non-smokers. The 32P-postlabelled 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts were analysed by thin-layer chromatography (TLC) combined with a high pressure liquid chromatography (HPLC) assay. In smokers the mean 7-methylguanine and 7-(2-hydroxyethyl)guanine levels were 32.3 +/- 7.1 and 6.6 +/- 2.3 adducts per 108 nucleotides respectively. The corresponding values in non-smokers were 25.0 +/- 7.0 and 3.7 +/- 2.4 adducts per 108 nucleotides. There were significantly higher levels of 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts in WBC in smokers than in non-smokers (p = 0.041; p = 0.018), respectively. A positive correlation between 7-methylguanine and 7-(2-hydroxyethyl)guanine levels was observed.     

DNA adducts in relation to lung tumour outcome are not markers of susceptibility following a single dose treatment of SWR,BALB/c and C57BL/6J mice with N-nitrosodiethylamine

The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a valuable marker for assessing exposure to carcinogens but as yet the role of DNA adducts as a biomarker of carcinogenic susceptibility still needs to be clearly ascertained. To address this question an animal study was instigated using mice (SWR (high), BALB/c (intermediate) and C57BL/6J (low)) varying in their susceptibility to lung carcinogenesis. Groups of animals from each strain were dosed with a single intraperitoneal injection of saline or N -nitrosodiethylamine (NDEA) at 15 or 90 mg kg^-1 body weight. Lung and liver tissues were removed at different time points following dosing. Further groups of mice dosed with the same regime had urine samples collected 24 h post dosing and were then left up to 18 months to allow for the development of tumours. Immunoslot-blot analysis was used for the determination of N-7 ethylguanine (N-7EtG) and O⁶ ethylguanine (O⁶EtG) adduct levels in the DNA from the tissues and gas chromatography-mass spectrometry (GC-MS) was used to determine N-3 ethyladenine (N-3EtA) adduct levels in the urine samples. Levels of alkyltransferase (ATase) were also determined in the tissues. The results showed that the DNA adduct levels and persistence were similar across the three strains of mice following dosing with 15 and 90 mg kg^-1 NDEA. High levels of adducts were observed in the urine of the BALB/c strain, implying an increased metabolic or repair capacity in this strain. However there were no differences in the levels of ATase in the lung and liver of the three strains of mice following dosing with 15 mg kg^-1 NDEA. The incidence of tumours in C57BL/6J mice was lower compared with the other two strains and showed a dose dependent increase. The results from this study show that the differences in susceptibility to lung carcinogenesis between the three strains of mice do not appear to be linked to the formation of the two adducts detected. These results imply that dosing with NDEA resulted in toxicity which may have led to cell death and induction of tumours by compensatory cell proliferation. Although these results do not allow decisive conclusions to be drawn concerning the relationship between total levels of DNA adducts and differences in carcinogenic susceptibility for the three strains of mice it is clear that the increased presence of a DNA adduct in the target tissue increases the likelihood of tumour development.     

Biomonitoring of tobacco smoke carcinogenicity by dosimetry of DNA adducts and genotyping and phenotyping of biotransformational enzymes: a review on polycyclic aromatic hydrocarbons

In this review article, we summarize the data on tobacco smoke carcinogenicity in relation to DNA adduct dosimetry and genotyping and phenotyping of biotransformational enzymes. A major class of carcinogens, polycyclic aromatic hydrocarbons, present in substantial quantities in tobacco smoke, is discussed. The historical background and an overview of the metabolic pathways are given. The epidemiological and biological data in particular on dosimetry of the representative DNA adducts and genotyping and phenotyping of the respective activating and detoxifying enzymes are presented. The salient findings are highlighted, the uncertainties are underlined and, finally, recommendations for future research are made.     

Biomonitoring of tobacco smoke carcinogenicity by dosimetry of DNA adducts and genotyping and phenotyping of biotransformational enzymes: a review on polycyclic aromatic hydrocarbons

In this review article, we summarize the data on tobacco smoke carcinogenicity in relation to DNA adduct dosimetry and genotyping and phenotyping of biotransformational enzymes. A major class of carcinogens, polycyclic aromatic hydrocarbons, present in substantial quantities in tobacco smoke, is discussed. The historical background and an overview of the metabolic pathways are given. The epidemiological and biological data in particular on dosimetry of the representative DNA adducts and genotyping and phenotyping of the respective activating and detoxifying enzymes are presented. The salient findings are highlighted, the uncertainties are underlined and, finally, recommendations for future research are made.     

Measurement of 7 methyl and 7 2 hydroxyethyl guanine DNA adducts in white blood cells of smokers and non smokers

The goal of the present study was to measure the levels of 7-methylguanine and 7-(2- hydroxyethyl)guanine DNA adducts in human white blood cells in relation to smoking. DNA was isolated from samples of 11 smokers and eight non-smokers. The 32P-postlabelled 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts were analysed by thin-layer chromatography (TLC) combined with a high pressure liquid chromatography (HPLC) assay. In smokers the mean 7-methylguanine and 7-(2-hydroxyethyl)guanine levels were 32.3 +/- 7.1 and 6.6 +/- 2.3 adducts per 108 nucleotides respectively. The corresponding values in non-smokers were 25.0 +/- 7.0 and 3.7 +/- 2.4 adducts per 108 nucleotides. There were significantly higher levels of 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts in WBC in smokers than in non-smokers ( p = 0.041; p = 0.018), respectively. A positive correlation between 7-methylguanine and 7-(2-hydroxyethyl)guanine levels was observed.     

Conversion of 1 nitropyrene by Brown trout Salmo trutta and turbot Scophthalamus maximus to DNA adducts detected by 32P postlabelling

The ability of the common aquatic contaminant 1-nitropyrene to form DNA adducts in fish was investigated in vitro and in vivo using Brown trout (Salmo trutta) and turbot (Scophthalmus maximus)in comparison to the Wistar rat. In vitro studies used Brown trout (control and induced (50 mg kg-1-naphthoflavone (NF), i.p. 3 day pre-treatment single injection)) and induced rat (PB; 0 1% w/v for 7 days in drinking water, NF; 80 mg kg-1, single injection 2 days prior to sacrifice). Hepatic 9000 g supernatant (S9 fractions) were incubated for 2 hours (at 25 C for fish and 37 C for rat) with calf thymus DNA (1mg) and 1-NP (100 M). With all S9 fractions the presence of three distinct 1-NP-related DNA adducts was detected using the butanol enrichment procedure of the 32Ppostlabelling assay. A greater level of DNA adducts was observed with the uninduced compared to the induced trout S9 (37, 12 and 8 fold greaterfor adducts in chromatograph areas 1-3 respectively) suggesting the enhancement of detoxification pathways with respect to bulky adducts following NF pre-treatment. DNA adduct levels in the induced rat consistently demonstrated approximately two-fold higher levels as compared to the induced fish, reflecting the lower protein levels in the S9 fraction of Brown trout (42 and 22 mg ml-1 for rat and fish respectively). Turbot, rat and Brown trout (uninduced and induced (NF; 50 mg kg-1; i.p. single injection 3 days prior)) were dosed with 100 mg kg-1 1-NP (i.p. single injection, 24 hours). Liver DNA from both turbot and rat exhibited a 1- NP related adduct spot which was similar in position to that of area 1 in the incubations with S9 from rat and Brown trout. However, in contrast to the in vitro studies no 1-NPrelated adducts were found in liver DNA from induced and uninduced Brown trout. This study highlights the potential, in a marine and a freshwater fish, for 1-NP metabolism to reactive of binding to DNA. However, activation of 1-NP was more optimal in the S9-mediated system, possibly reflecting the influence of detoxification systems.     

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by ³²P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 μg/m³, PM2.5 27–38 μg/m³, c-PAHs 18–22 ng/m³; personal exposure to c-PAHs: 9.7 ng/m³ versus 5.8 ng/m³ (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 ± 0.28 adducts/10⁸ nucleotides versus 0.82 ± 0.23 adducts/10⁸ nucleotides, P = 0.065), whereas the level of the B[a]P-“like” adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10⁸ nucleotides versus 0.099 ± 0.035 adducts/10⁸ nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-“like” DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = −0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-“like” DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.     

Benzoapyrene-induced DNA damage in Mytilus galloprovincialis: measurement of bulky DNA adducts and DNA oxidative damage in terms of 8-oxo-7,8-dihydro-2´-deoxyguanosine formation

Bulky DNA adducts and 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodGuo) were measured in gill DNA of benzo[a]pyrene (B[a]P)-exposed mussels (50 mg kg^-1 dw day^-1), respectively by the ³²P-post-labelling technique and high performance liquid chromatography coupled to electrochemical detection assay. A time-course study was performed for both biomarkers and their potential use for marine biomonitoring discussed for the sentinel species studied. In gills, B[a]P-related DNA adducts were positively correlated with B[a     

Carcinogen mmetabolism and DNA adducts in human lung tissues as affected by tobacco smoking or metabolic phenotype: a case-control study on lung cancer patients

Individual variations in activity of pulmonary enzymes that metabolize tobacco-derived carcinogens may affect an individual's cancer risk from cigarette smoking. To investigate whether some of these enzymes (e.g., cytochrome P450IA-related) can serve as markers for carcinogen-induced DNA damage accumulating in the lungs of smokers, non-tumorous lung tissue specimens were taken during surgery from middle-aged men with either lung cancer (n = 54) or non-neoplastic lung disease (n = 20). Phase I (AHH, ECDE) and phase II (EH, UDPGT, GST) enzyme activities, glutathione and malondialdehyde contents were determined in lung parenchyma and/or bronchial tissues; some samples were analyzed for DNA adducts, using ³²P-postlabeling.

Data analysis of subsets or the whole group of patients yielded the following results. (1) Phase I and II drug-metabolizing enzyme (AHH, EH, UDPGT, GST) activities in histologically normal surgical specimens of lung parenchyma were correlated with the respective enzyme activities in bronchial tissues of the same subject. (2) In lung parenchyma, enzyme (AHH, ECDE, EH, UDPGT) activities were significantly and positively related to each other, implying a similar regulatory control of their expression. (3) Mean activities of pulmonary enzymes (AHH, ECDE) were significantly (2- and 7-fold, respectively) higher in lung cancer patients who had smoked within 30 days before surgery (except GST, which was depressed) than in cancer-free subjects with a similar smoking history. (4) In the cancer patients, the time required for AHH, EH and UDPGT activities to return to the level found in non-smoking subjects was several weeks. (5) Bronchial tree and peripheral lung parenchyma preparations exhibited a poor efficiency in activating promutagens to bacterial mutagens in Salmonella. However, they decreased the mutagenicity of several direct-acting mutagens, an effect which was more pronounced in tissue from recent smokers. GSH concentration and GST activity were positively correlated with mutagen inactivation in the same sample. (6) In recent smokers, AHH activity in lung parenchyma was positively correlated with the level of tobacco smoke-derived DNA adducts. (7) Pulmonary AHH and EH activity had prognostic value in tobacco-related lung cancer patients. (8) An enhanced level of pro-oxidant state in the lungs was associated with recent cigarette smoking. Malondialdehyde level in lung parenchyma was associated with the degree of small airway obstruction, suggesting a common free radical-mediated pathway for both lung cancer induction and small airway obstruction.

These results demonstrate the pronounced effect of recent cigarette smoke exposure on pulmonary xenobiotic metabolism and lipid peroxidation and lend further support to the hypothesis that the inducibility of pulmonary AHH activity (cytochrome P450IA1 levels) in tobacco smokers is associated with lung cancer risk. Results on DNA adducts in smokers' lung tissue may help to explain why a certain metabolic phenotype accumulates more DNA damage in lung cells.