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.     

Smoking-reIated DNA adducts and genetic polymorphism for metabolic enzymes in human lymphocytes

Smoking-related aromatic DNA adducts in lymphocytes were measured from smokers (n = 76), ex-smokers (n = 25) and non-smokers (n = 56) by the ³²P-postlabelling method, to clarify whether a genetic polymorphism for metabolic enzymes could explain the inter-individual variation of DNA adduct levels. Adduct levels were compared with respect to smoking status and polymorphic genotypes of cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GTSM1). The mean adduct level (1.24 per 10⁸ nucleotides) in smokers was significantly higher than that (0.85 per 10⁸) in non-smokers. Although we expected higher adduct levels in the CYP1A1 variant or GSTM1 null subjects, the adduct level in 'GSN1 nulls' was significantly lower than that in 'GSTM1 presents' among smokers. DNA adduct levels had significant positive correlations with smoking indices such as number of cigarettes or smoking years in all subjects. In smokers only, however, no correlation was found, because there were negative correlations between adduct levels and smoking dose in GSTM1 null genotypes. CYP1A1 genotypes had no effects on adduct levels.     

Postlabelling HPLC analysis of lipophilic DNA adducts from human lung

A new modification of the 32P postlabelling method was described for the analysis of lipophilic DNA in human tissues. To isolate these DNA adducts the method applied nuclease P1 enrichment before labelling and butanol extraction after labelling, followed by high performance liquid chromatography HPLC separation and flow through radioactivity detection. These enrichment methods are known to work for many adducts of polycyclic aromatic hydrocarbons PAHs . In human peripheral lung tissue fro m smokers the apparent level of the DNA adducts observed was 25-244 adducts per 108 nucelotides; in two alleged non smokers the level of adducts was 17 and 109 adducts per 108 nucleotides. When the same samples were analysed by thin layer chromatography TLC , as in the conventional postlabelling assay, the recovery was 5 of that of the HPLC method. Nevertheless, the results from the two methods correlated. In TLC the adducts were lost because they did not remain in the origin in D1 of the TLC development. There was no large difference in recovery between the two techniques for the PAH-DNA adduct standards used. The present results are underestimates of the true adduct levels because there is no way to correct for labelling efficiency and recovery of unknown adducts. Yet they give a lower estimate of the level of lipophilic DNA adducts in human lung tissue.     

Cigarette smoking during pregnancy: comparison of biomarkers for inclusion in epidemiological studies

Using proteomic approach in cerebrospinal fluid (CSF) we identified pigment epithelium-derived factor (PEDF) and Haptoglobin (Hp) as putative markers that could discriminate between AD and other dementias. ELISA assays were developed to measure the levels of PEDF and Hp in CSF from patients with AD (AD, n?=?27), non-AD (NAD, n?=?30) and in non-demented patients (ND, n?=?27). The combined assessment of PEDF, Hp and Tau levels, using Iterative Marginal Optimization, improved the differential diagnosis of AD, especially in patients with moderate to severe dementia (p<0.002). This pilot study highlights the probable different contribution of oxidative mechanisms in dementia.     

Postlabelling HPLC analysis of lipophilic DNA adducts from human lung

A new modification of the 32P postlabelling method was described for the analysis of lipophilic DNA in human tissues. To isolate these DNA adducts the method applied nuclease P1 enrichment before labelling and butanol extraction after labelling, followed by high performance liquid chromatography HPLC separation and flow through radioactivity detection. These enrichment methods are known to work for many adducts of polycyclic aromatic hydrocarbons PAHs. In human peripheral lung tissue fro m smokers the apparent level of the DNA adducts observed was 25-244 adducts per 108 nucelotides; in two alleged non smokers the level of adducts was 17 and 109 adducts per 108 nucleotides. When the same samples were analysed by thin layer chromatography TLC, as in the conventional postlabelling assay, the recovery was 5 of that of the HPLC method. Nevertheless, the results from the two methods correlated. In TLC the adducts were lost because they did not remain in the origin in D1 of the TLC development. There was no large difference in recovery between the two techniques for the PAH-DNA adduct standards used. The present results are underestimates of the true adduct levels because there is no way to correct for labelling efficiency and recovery of unknown adducts. Yet they give a lower estimate of the level of lipophilic DNA adducts in human lung tissue.     

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.     

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.     

Lymphocyte DNA adducts and polymorphism in the DNA repair enzyme XPD

The effect of genetic polymorphism of DNA repair enzyme on the DNA adduct levels was evaluated in this study. We explored the relationship between polymorphism in the nucleotide excision repair enzyme XPD and DNA adduct levels in lymphocytes. Lymphocyte DNA adducts were measured by a ³²     

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.     

Lymphocyte DNA adducts and polymorphism in the DNA repair enzyme XPD

The effect of genetic polymorphism of DNA repair enzyme on the DNA adduct levels was evaluated in this study. We explored the relationship between polymorphism in the nucleotide excision repair enzyme XPD and DNA adduct levels in lymphocytes. Lymphocyte DNA adducts were measured by a ³²     

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.     

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.     

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.     

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.     

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.     

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.