Influence of cigarette smoking on the levels of DNA adducts in human bronchial epithelium and white blood cells

The presence of carcinogen-DNA adducts in human tissues is evidence of exposure to carcinogens and may be an indicator of cancer risk. DNA was isolated from non-tumorous bronchial tissue of 37 cigarette smokers, 8 former smokers and 8 non-smokers and analyzed for the presence of aromatic and/or hydrophobic DNA adducts in the 32P-post-labelling assay. Adducts were detected as bands of radioactive material when 5'-32P-labelled deoxyribonucleoside 3',5'-bisphosphates were chromatographed on polyethyleneimine-cellulose tlc plates, and the patterns indicated the formation of adducts by a large number of compounds. Adduct levels detected in DNA from non-smokers, former smokers and current smokers were 3.45 +/- 1.62, 3.93 +/- 1.92 and 5.53 +/- 2.13 adducts/10(8) nucleotides, respectively. The differences in adduct levels between smokers and former and non-smokers were statistically significant (p less than 0.01); and among the smokers, significant correlations were found between adduct levels and both daily cigarette consumption and total cigarette consumption (daily consumption X number of years smoked). DNA was also isolated from the peripheral-blood leukocytes of 31 heavy smokers (greater than 20 cigarettes/day) and 20 non-smokers and analyzed by 32P-post-labelling. Adduct levels in the smokers' samples were not significantly different from levels in the non-smokers' samples (2.53 +/- 1.31 and 2.12 +/- 1.44 adducts/10(8) nucleotides, respectively). Thus, evidence for carcinogen exposure was found in human bronchial epithelium, a target tissue for tobacco-induced tumour formation, but not in peripheral-blood cells, indicating possible limitations in the use of the latter as a surrogate, non-target tissue source of DNA for monitoring human exposure to inhaled carcinogens.     

Lack of evidence for tamoxifen- and toremifene-DNA adducts in lymphocytes of treated patients

Tamoxifen (TAM) is used for the adjuvant treatment of women with breast cancer and has also been recommended as a chemopreventive agent. Among unwanted side effects, TAM was shown to increase endometrial cancer in treated women by mechanisms that are not yet clearly understood. We studied DNA adducts in lymphocytes of female breast cancer patients treated with TAM or toremifene (TOR), a TAM analogue and compared them with adducts formed by TAM in rat liver, where the drug induces tumours. DNA adducts were measured by TLC-(32)P-post-labelling assays. After TLC, all DNA samples including DNA from untreated healthy women showed a faint radioactive zone, where the positive control DNA adducts isolated from the liver of rats treated with TAM migrated. The relative adduct levels were calculated from the radioactivity present in this zone. Means +/- SD of adduct levels per 10(8) nucleotides (associated with this area) were for untreated volunteers (control) 1.83 +/- 1.41 (n = 13), for TAM treatment 2.17 +/- 3.04 (n = 25) and for TOR treatment 1.18 +/- 1.05 (n = 8). Most of the human samples were further analysed by HPLC after labelling with (32)P in order to compare adducts in human DNA with those in liver DNA isolated from TAM-treated rats. None of the human samples showed any peaks at retention times where putative TAM-DNA adducts were eluted. In conclusion, lymphocyte DNA from female patients treated at therapeutic levels did not show evidence of the formation of TAM- or TOR-DNA adducts.     

Cigarette smoking related polycyclic aromatic hydrocarbon-DNA adducts in peripheral mononuclear cells.

Studies on cigarette smoking related polycyclic aromatic hydrocarbon-DNA adducts in blood have produced conflicting results. To determine whether a subset of specific white blood cells is a useful marker for monitoring exposure to cigarette smoke, blood was obtained from 63 heavy smokers and 27 non-smokers. Adduct levels were determined by competitive enzyme-linked immunosorbent assay with a polyclonal antiserum recognizing benzo[a]pyrene and structurally related diolepoxide-DNA adducts. Analysis of the lymphocyte plus monocyte fraction from smokers indicated 70% had detectable adducts with a mean of 4.38 +/- 4.29 adducts/10(8) nucleotides, while in non-smokers the corresponding values were 22% and 1.35 +/- 0.78/10(8) (P < 0.001). Plasma cotinine levels differed significantly in smokers (286 +/- 90 micrograms/l) compared to non-smokers (4.4 +/- 3.3 micrograms/l) (P < 0.001). However, cotinine was not correlated with self-reported smoking history in these heavy smokers. Nor were DNA adducts in smokers correlated with cigarettes per day, pack-years and plasma cotinine, indicating large interindividual variation in DNA adduct formation. These data demonstrate lymphocytes plus monocytes from smokers have elevated levels of polycyclic aromatic hydrocarbon diolepoxide-DNA adduct levels compared to non-smokers.     

DNA adducts in lymphocytes and granulocytes of smokers and nonsmokers detected by the 32P-postlabelling assay

The effects of smoking and DNA adduct formation were analysed in isolated human white blood cell populations. As the white cells are composed mainly of granulocytes with a short half-life and T-lymphocytes with a half-life of several years, we isolated the lymphocytes and granulocytes of 11 smokers and 10 nonsmokers to determine any smoking-related DNA adducts by the nuclease-P1-enhanced 32P-postlabelling assay. The differences between the mean lymphocyte DNA adducts/10(8) nucleotides of 31 +/- 5.7 (SE) of smokers were significantly higher (P less than 0.05) than those in the lymphocytes 13 +/- 1.6 (SE) of nonsmokers. The total DNA adducts/10(8) nucleotides obtained from the granulocytes of smokers and nonsmokers was 9.6 +/- 1.9 and 7.6 +/- 1.9 respectively. The plasma cotinine concentrations were in good agreement with the smoking information given by the individual smokers (r = 0.847, P less than 0.001). The DNA adduct levels of the lymphocytes of the 10 smokers correlated with the plasma cotinine concentrations (r = 0.639, P less than 0.05). The variation between the results was explained by the variation among the individuals and the samples, but not by the variation in the parallel determinations. More detailed studies are needed to analyse the source of the individual variations between the smokers' adduct levels, DNA repair, and differences in the metabolism of the compounds in cigarette smoke.     

Detection of DNA adducts of acetaldehyde in peripheral white blood cells of alcohol abusers

The effect of alcohol drinking on the formation of DNA adducts of acetaldehyde, the primary oxidative metabolite of ethanol, was investigated in humans. DNA was isolated from granulocytes and lymphocytes from 24 alcoholic patients and 12 control subjects. DNA adduct levels were measured by 32P-postlabelling using reversed-phase HPLC with on-line detection of radioactivity. A large interindividual variation in adduct levels was observed. The average adduct levels in granulocyte and lymphocyte DNA from alcoholic patients were 3.4 +/- 3.8 and 2.1 +/- 0.8 adducts/10(7) nucleotides (n = 24), respectively. These levels were 13- and 7-fold higher than the corresponding levels in control subjects (P<0.001). The average adduct level in granulocyte DNA from alcoholic patients was 60% higher than in lymphocyte DNA (P<0.01). Our results, in conjunction with the genotoxicity of acetaldehyde, thus suggest the formation of DNA adducts of acetaldehyde as a plausible mechanism explaining the involvement of alcohol drinking in carcinogenesis.      

Comparison between smoking-related DNA adduct analysis in induced sputum and peripheral blood lymphocytes

We investigated the applicability of induced sputum (IS), a non-invasive derivative from the lower respiratory tract, for smoking-related DNA adduct analysis and its comparability with peripheral blood lymphocytes (PBL). Lipophilic DNA adducts were quantified by the (32)P-post-labeling assay in IS and PBL of smokers (n = 9) with stable smoking status at three time points (one week intervals) and non-smokers (n = 9) at one time point. The success rate for sputum induction was 100% at all time points. There was no significant difference in total cell count, cell viability, squamous cell count and DNA yield between smokers and non-smokers. Within the smokers, there was no significant difference in IS cytology at the three time points: overall (mean of three measurements) total cell count, 9.0 +/- 2.4 x 10(6); cell viability, 77 +/- 4%; squamous cell count, 28 +/- 5%; non-squamous cell count, 72 +/- 4% (bronchoalveolar macrophages, 75 +/- 6%; neutrophils, 17 +/- 3%; bronchoepithelial cells, 7 +/- 2%; lymphocytes, 0.7 +/- 0.2%; metachromatic cells, 0.3 +/- 0.2%). IS DNA yield did not differ significantly at the three time points [overall (mean of three extractions) DNA yield, 66 +/- 20 microg]. A typical smoking-associated diagonal radioactive zone was observed in the adduct maps of IS and PBL of all and five smokers, respectively, and of none of the non-smokers. Lipophilic DNA adduct levels in both IS and PBL of smokers were higher than those of non-smokers (3.7 +/- 0. 9 versus 0.7 +/- 0.2/10(8) nt, P = 0.0005, and 2.1 +/- 0.3 versus 0. 6 +/- 0.1/10(8) nt, P = 0.0001, respectively). In smokers the level of adducts in IS was non-significantly higher than that in PBL (3.7 +/- 0.9 versus 2.1 +/- 0.3/10(8) nt, P = 0.1), whilst in non-smokers the difference was not appreciable (0.7 +/- 0.2 versus 0.6 +/- 0. 1/10(8) nt). Within the smokers there was no significant change in the level of adducts at the three time points either in IS or in PBL (coefficients of variation 34 and 29%, respectively). Adduct levels in IS at each time point were higher than those in PBL, leading to a significantly higher overall (mean of three quantifications) level of adducts in IS than PBL (3.3 +/- 0.2 versus 2.1 +/- 0.1/10(8) nt, P = 0.02). The overall levels of adducts in both IS and PBL were dose-dependently related to smoking indices. We conclude that IS is a preferable matrix as compared with PBL for molecular dosimetry of (current) exposure to inhalatory carcinogens as its analysis reveals both the existence and the magnitude of exposure more explicitly.     

Analyses of bronchial bulky DNA adduct levels and CYP2C9, GSTP1 and NQO1 genotypes in a Hungarian study population with pulmonary diseases.

Carcinogen-DNA adducts may represent an intermediate end-point in the carcinogenic cascade and may reflect exposure to chemical carcinogens, as well as susceptibility and, ultimately, cancer risk. Interindividual variability in activity of enzymes involved in the metabolism of polycyclic aromatic hydrocarbons to mutagenic diol epoxides may predict adduct levels and, indirectly, lung cancer risk. Using 32P-postlabeling methods, the levels of bulky DNA adducts were determined in macroscopically normal bronchial tissues obtained from resected lobes of 143 Hungarian patients with lung malignancy and other pulmonary conditions. DNA from normal tissue was also evaluated for polymorphisms in cytochrome P450 2C9 (CYP2C9) at two sites, codons 144 (Arg/Cys) and 359 (Ile/Leu), for glutathione S-transferase P1 (GSTP1) at codon 105 and for NAD(P)H:quinone oxidoreductase (NQO1) at codon 187 (Pro/Ser). Using the Mann-Whitney U-test and analysis of variance, levels of adducts were evaluated in relation to variant genotypes, separately for smokers and non-smokers. As previously reported, bulky DNA adduct levels in smokers (n = 104) were estimated to be 54% higher than in non-smokers (n = 39) (8.6 +/- 4.2 versus 5.6 +/- 3.3 per 10(8) nucleotides, respectively, P < 0.01). Adduct levels were 16-29% higher in individuals with the homozygous Ile359/Ile359 CYP2C9 allele than in those heterozygous for the variant allele (Ile359/Leu359) [8.8 +/- 4.3 (n = 84) versus 7.6 +/- 3.5 (n = 20) for smokers and 5.8 +/- 3.5 (n = 32) versus 4.5 +/- 1.3 (n = 7) for non-smokers], although differences were not statistically significant. There were no clear differences in adduct levels in relation to genotypes of NQO1 or GSTP1. Although numbers of patients in this study are large in relation to many studies of carcinogen-DNA adducts, it is still possible that significant differences were not noted for polymorphisms in xenobiotic metabolizing enzymes due to relatively small numbers in stratified data.     

Human DNA adducts of 1,3-butadiene, an important environmental carcinogen

The N-1-(2,3,4-trihydroxybutyl)adenine (N-1-THB-Ade) adducts induced by 1,3-butadiene (BD) were analysed from lymphocytes of 15 workers occupationally exposed to BD and 11 controls by (32)P-post-labelling using HPLC with radioactivity detection. The difference in the adduct levels between the BD-exposed workers (4.5 +/- 7.7 adducts/10(9) nucleotides) and the controls (0.8 +/- 1.2 adducts/10(9) nucleotides) was statistically significant (Wilcoxon rank sum test, P = 0.038). This study shows for the first time BD-induced DNA adducts in humans and suggests that N-1-THB-Ade adducts may be used to biomonitor human exposure to BD.     

Body mass index modulates aromatic DNA adduct levels and their persistence in smokers.

Smokers with a low body mass index (BMI; weight/height(2)) have a higher risk for developing lung malignancies as compared with smokers of average weight, but there is no mechanistic explanation for this observation. Carcinogens in cigarette smoke are thought to elicit cancer by the formation of DNA adducts, which give the opportunity to additionally investigate the biological link between BMI and lung cancer. DNA adduct levels in peripheral blood lymphocytes of 24 healthy smoking volunteers (0.76 +/- 0.41 adducts per 10(8) nucleotides) positively correlated with cigarette consumption (r = 0.51; P = 0.01) and were inversely related with BMI (r = -0.48; P = 0.02). A significant overall relationship was observed when both parameters were included in multiple regression analysis (r = 0.63; P = 0.007). Moreover, body composition may affect DNA adduct persistence, because lipophilic tobacco smoke-derived carcinogens accumulate in adipose tissue and can be mobilized once exposure ceases. Therefore, DNA adduct levels and BMI were reassessed in all of the subjects after a nonsmoking period of 22 weeks. Adduct levels declined to 0.44 +/- 0.23 per 10(8) nucleotides (P = 0.002), and the estimated half-life was 11 weeks on the basis of exponential decay to background levels in never-smoking controls (0.33 +/- 0.18 per 10(8) nucleotides). Overweight subjects (BMI >25) with little weight gain after smoking cessation (相似文献   

7-Methylguanine levels in DNA of smokers' and non-smokers' total white blood cells, granulocytes and lymphocytes.

The effect of smoking was investigated on the formation of 7-methylguanines in human peripheral white blood cells. DNA was isolated from total white blood cells, granulocytes and lymphocytes from 10 smokers and 10 non-smokers. 32P-Postlabeling was performed by using anion-exchange chromatography enrichment of adducts. In smokers the mean DNA adduct levels were 6.9, 4.7 and 23.6 7-methylguanine residues/10(7) nucleotides in total white blood cells, granulocytes and lymphocytes respectively. The corresponding values in non-smokers were 3.4, 2.8 and 13.5 adducts/10(7) nucleotides. The mean adduct level was significantly higher in lymphocytes than in total white blood cells or granulocytes both in smokers and in non-smokers. The mean adduct levels differed significantly between smokers and non-smokers.     

Evaluation of 32P-postlabeling analysis of DNA from exfoliated oral mucosa cells as a means of monitoring exposure of the oral cavity to genotoxic agents

Development of oral cavity cancer in man has been linked to alcohol consumption and use of tobacco products. In order to understand the underlying carcinogenic mechanisms in the oral cavity a method is needed to monitor exposure of this site to various environmental insults. In this pilot study we evaluate the use of the 32P-postlabeling assay to detect adducts in DNA from exfoliated oral mucosa cells. Exfoliated cells were collected from the cheek and tongue of 27 men aged 35-69 years. DNA was extracted from the cells and analyzed by the enhanced 32P-postlabeling technique using butanol extraction. A variety of adduct spots were detected but none was consistently associated with exposure to alcohol or tobacco products. Some of the adducts detected had migration patterns in TLC very similar to the major deoxyguanosine adducts formed by the diol epoxides of benzo[a]pyrene and 5-methylchrysene, suggesting that they may have been formed from polynuclear aromatic hydrocarbons. Adduct spots with migration patterns similar to polynuclear hydrocarbon adducts accounted for only about one third of the total adduct spots observed. Relative adduct labeling (RAL) values were determined for samples from 12 of the 27 individuals. RAL values ranged from 1.6 X 10(-6) to 7.7 X 10(-11) adducts per nucleotide. The RAL values for adducts from the cheek or tongue were not significantly different. Adduct levels in smokers (median RAL of 4.8 X 10(-8) were significantly higher (P less than 0.001) than adduct levels in non-smokers (median RAL of 2.9 X 10(-9). Adduct levels in drinkers (median RAL of 9.1 X 10(-10) were significantly lower (P less than 0.001) than adduct levels in non-drinkers (median RAL of 3.7 X 10(-8). Four of the subjects in this study have subsequently developed squamous cell carcinoma of the oral cavity. 32P-Postlabeling analysis of DNA from the oral cavity of these subjects did not demonstrate unique patterns or RAL values. Lack of information on the structure of the majority of adducts observed in this study was a serious limitation. Further improvements in adduct identification will be needed before 32P-postlabeling can be a useful tool for monitoring exposure of the oral cavity to carcinogens.     

Smoking-related DNA adducts in human gastric cancers.

DNA was extracted from the tumour tissue of 26 patients (18 smokers, 8 non-smokers) undergoing surgery for gastric cancer, and analyzed for the presence of DNA adducts by the 32P-post-labelling method. Adducts were detected in all samples tested, and adduct levels ranged from 2 adducts/10(8) nucleotides to 60 adducts/10(8) nucleotides. In male subjects, adduct levels were significantly greater in the DNA of smokers than in that of non-smokers. These results support epidemiological data suggesting that smoking is a risk factor for gastric cancer.     

DNA adducts in tumour, normal peripheral lung and bronchus, and peripheral blood lymphocytes from smoking and non-smoking lung cancer patients: correlations between tissues and detection by 32P-postlabelling and immunoassay

Smoking is a major risk factor for lung cancer. This comparative study of smoking-related carcinogen-DNA adducts in pulmonary tissues and peripheral blood lymphocytes aims to further explore the primary DNA damaging processes by cigarette smoke in target and surrogate tissues. Samples of tumour and normal peripheral lung tissue, normal bronchial tissue and peripheral blood lymphocytes were obtained from a total of 85 lung cancer patients who underwent lung resection. Bulky DNA adducts were determined by 32P-postlabelling, and polycyclic aromatic hydrocarbon (PAH)-DNA adducts were detected by (+/-)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-DNA chemiluminescence immunoassay (BPDE-DNA CIA) in smaller subsets of tissue samples subject to availability of DNA. Bulky DNA adduct levels ranged between 0.3 and 27.8 adducts/10(8) nucleotides (nt) with mean adduct levels between 2.8 and 11.5 adducts/10(8) nt. Mean PAH-DNA adduct levels were 2.6-6.2 adducts/10(8) nt. Significantly higher bulky DNA adduct levels were detected in smokers' lungs as compared with non-smokers' (P < 0.02). PAH-DNA adduct levels appeared higher in the lungs of smokers compared with non-smokers but the difference was not significant. Lung tumour contained on average a 50% lower DNA adduct level compared with normal lung tissue. A statistically significant positive correlation was found between the DNA adduct levels of the corresponding tumour and normal lung tissue samples in both smokers and non-smokers using both methodologies. Bulky DNA adduct levels in normal lung and blood lymphocytes correlated significantly in non-smokers only (r = 0.55, P = 0.023). In lung tumour DNA samples there was a weak correlation between values obtained by 32P-postlabelling and by the BPDE-DNA immunoassay (r = 0.27, P = 0.054). However, with normal lung DNA samples, values obtained by the two assays did not correlate.     

High DNA damage by benzo[a]pyrene 7,8-diol-9,10-epoxide in bronchial epithelial cells from patients with lung cancer: comparison with lung parenchyma

In the present study, the level of benzo[a]pyrene 7,8-diol-9,10-epoxide-N(2)-deoxyguanosine (BPDE-N(2)-dG) in normal bronchial epithelial cells from non-cancerous bronchus of 22 lung cancer subjects was evaluated and compared to the lung parenchyma. We found very high formation of BPDE-N(2)-dG adduct in samples corresponding to a pure preparation of bronchial epithelial cells with 4-fold interindividual differences in the DNA adduct levels in the range of 36.5-175.4 BPDE-N(2)-dG adducts/10(8) nucleotides in smokers (mean: 84.7+/-38.4; n = 13) and 3-fold differences in the range of 19.7-62.4 in non-smokers (mean: 37.6+/-22.2; n = 3). DNA isolated from the bronchial tissue consisting of bronchial lining epithelium with adjacent lamina propria showed significantly lower BPDE-N(2)-dG formation (P < 0.001) in the range of 0.4-4.2 BPDE-N(2)-dG adducts/10(8) nucleotides (mean: 1.8+/-0.56; n = 6). This difference is clearly related to the procedure used to prepare the bronchial tissue samples leading to the presence of different types of cells. Eight samples from the normal parenchyma did not show measurable adducts, the other 14 samples showed 50-fold variation (mean: 1.7+/-1.5; range 0.1-5.2 adducts/10(8) nucleotides; n = 14). There were considerably higher adduct levels in pure bronchial epithelial cells than in parenchymal tissue (75.8+/-38.8 vs 0.9+/-1.5 adducts/10(8) nucleotides) (P < 0.0002) BPDE-N(2)-dG adduct concentrate almost exclusively in bronchial epithelial cells. The adduct values obtained in bronchial epithelial cells could be considered as 'critical' for the initiation of human lung cancer. The high formation of BPDE-N(2)-dG adducts in bronchial epithelial cells and investigations showing that the profile of mutations induced by BPDE in these cells is similar to that seen in the p53 gene isolated from human lung tumors implicates benzo[a]pyrene as important carcinogen in tobacco-induced lung cancer in human beings.     

Smoking-associated bulky DNA adducts in bronchial tissue related to CYP1A1 MspI and GSTM1 genotypes in lung patients

Relationships between smoking status and levels of bulky DNA adducts were investigated in bronchial tissue of lung patients in relation to their GSTM1 and CYP1A1 MspI genotypes. A total of 150 Hungarian patients undergoing pulmonary surgery were included in the study, 124 with lung malignancies and 26 with non-malignant lung conditions. There were significant relationships between smoking status and bulky DNA adduct levels, as determined by 32P-post-labelling analysis, in macroscopically normal bronchial tissues. There was a highly significant difference in the adduct levels of a combined group consisting of current smokers and short-term ex-smokers (< or = 1 year abstinence) compared with life-time non-smokers and long-term ex- smokers (> 1 year abstinence) (P = 0.0001). The apparent half-life was estimated to be 1.7 years for bulky DNA adducts in the bronchial tissue from ex-smokers. There were no statistically significant correlations between (i) daily cigarette dose and DNA adduct levels in current smokers, (ii) DNA adduct level and histological type of lung cancer, or (iii) GSTM1 and CYP1A1 MspI genotypes and DNA adduct levels after adjustment for either smoking status or malignancy. By multiple logistic regression analysis, smoking and GSTM1 null genotype were found to be risk factors for squamous cell carcinoma. However, bulky DNA adduct levels in bronchial tissue did not appear to be a statistically-significant risk factor for the major histological types of lung cancer.      

Applicability of induced sputum for molecular dosimetry of exposure to inhalatory carcinogens: 32P-postlabeling of lipophilic DNA adducts in smokers and nonsmokers.

The lung is a major target organ for smoking-associated cancer. We examined the applicability of induced sputum for molecular dosimetry of exposure to tobacco smoke-related carcinogens. Sputum induction was performed by inhalation of 4.5% saline delivered from an ultrasonic nebulizer for a period of up to 21 min in a group of smoking (n = 20) and nonsmoking (n = 24) healthy individuals. Samples were analyzed for total and differential cell counts and cell viability. Subsequently, DNA contents of the samples were isolated, and measurement of lipophilic DNA adducts was done by the 32P-postlabeling assay using nuclease P1 (NP1) and butanol enrichment methods. All subjects tolerated the induction procedure without experiencing any troublesome symptoms, and 90% of smokers (18 of 20) and 88% of nonsmokers (21 of 24) succeeded in producing sufficient amounts of sputum. Total cell counts and percentages of viable cells in smokers were higher than those in nonsmokers (6.7+/-6.0 versus 4.7+/-6.0 x 10(6), P = 0.40 and 80+/-15 versus 63+/-17, P = 0.01, respectively). In cell differentials, smokers had lower percentages of bronchoalveolar macrophages and higher percentages of neutrophils (69+/-24 versus 92+/-5, P = 0.002 and 26+/-26 versus 4+/-4, P = 0.008, respectively). Using the NP1 digestion method, all smokers and only one nonsmoker showed a diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (3.1+/-1.4 versus 0.6+/-0.8/10(8) nucleotides; P = 0.0007), and also, adduct levels were significantly related to smoking indices. Applying the butanol extraction method, however, only half of the smokers and three nonsmokers showed the diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (4.6+/-3.7 versus 1.0+/-1.9/10(8) nucleotides; P = 0.02), and the levels of adducts were significantly related to the smoking indices. There was a correlation between the levels of adducts determined by the two enrichment methods (r = 0.7; P = 0.02). Paired comparison showed no differences between the levels of adducts measured by the two methods (P = 0.55). We conclude that induced sputum can serve for molecular dosimetry of inhalatory exposure to carcinogens and that the NP1 version of the 32P-postlabeling assay is a choice of preference for studying smoking-induced DNA adducts in the lower respiratory tract.     

32P-post-labelling of 7-(3-chloro-2-hydroxypropyl)guanine in white blood cells of workers occupationally exposed to epichlorohydrin

Epichlorohydrin (ECH) is a simple 3-carbon epoxide of industrial importance. It has been shown to be genotoxic in several systems and carcinogenic in experimental animals. The aim of the present investigation was to study DNA adducts of ECH as a biomarker of occupational exposure to this chemical. 7-(3-Chloro-2-hydroxypropyl)guanine (7-CHP-guanine) was analysed in DNA from white blood cells using an anion exchange-based adduct enrichment protocol of the (32)P-post-labelling/HPLC-based assay. Blood samples were collected from seven workers handling ECH (exposed), nine workers not handling ECH but normally present in the premises where this chemical is used (potentially exposed) and 13 office and factory workers from locations in the plant where ECH is not handled (controls). 7-CHP-guanine was detected in five of the seven workers exposed to ECH (1.6-7.1 mol/10(9) mol nucleotides) and in two of the nine workers potentially exposed to ECH (0.8-1.5 mol/10(9) mol nucleotides). This adduct was not detected in any of the 13 controls. The difference in adduct levels between exposed workers and controls was statistically significant (Mann-Whitney test, P < 0.001), as was the difference between exposed workers and potentially exposed workers (P = 0.017). The recovery of 7-CHP-guanine in the (32)P-post-labelling assay was on average 48 +/- 7%, which is considerably higher than previously reported for other 7-alkylguanines. The method used had a limit of detection of approximately 0.4 mol adduct/10(9) mol nucleotides using 20 microg DNA. This study shows for the first time ECH-induced DNA adducts in humans and suggests that 7-CHP-guanine may be used as a biomarker of occupational exposure to ECH.     

Formation and persistence of DNA adducts in different target tissues of rats after multiple administration of benzo[a]pyrene

Exposure of humans to poly cyclic aromatic hydrocarbons is anongoing concern because of the carcinogenicity of these substances.DNA adducts are being increasingly used as indicators of carcinogenexposure. While considerable experimental evidence exists tosupport their use there are aspects that require further attention,especially after repeated exposure, which has led to this seriesof experiments. Male Sprague-Dawley rats were dosed with 10mg/kg benzo[a]pyrene (B[a]P) i.p., 3 times/week for 2 weeks.At 1, 3, 7, 14, 28 and 56 days after the last treatment liver,lung, spleen and peripheral blood mononuclear cells (PBMNs)were sampled. The DNA adduct levels, as measured by the ³²P-postlabellingtechnique, were significantlyincreased in all tissues, withlung having the highest levels. At day 14 total DNA adductsin lung, spleen and PBMNs were still >50% of the level atday 1. The removal of total DNA adducts was found to be fastestin liver > spleen < PBMNs > lung. A consistent correlationof total adducts between the lung and PBMNs was observed. Amajor adduct, designated adduct 1, was detected in all tissues,while adduct 4 was only found in liver and lung. Adduct 5 wasdetected only in lung, where it constituted     

Formation and persistence of nucleotide alterations in rats exposed whole-body to environmental cigarette smoke.

The assessment of pathological effects produced by environmental tobacco smoke in humans is controversial in epidemiological studies. On the other hand, animal models are poorly sensitive to smoke carcinogenicity. We designed an experimental study assessing the tissueselective formation and persistence of DNA adducts in smoke-exposed rats. Sprague-Dawley rats were exposed for 6 h per day, 5 days per week, to environmental smoke resulting from a mixture of sidestream and mainstream smoke generated from Kentucky 2R1 reference cigarettes. The total particulate matter was in the range of 73-93 mg/m(3). DNA adducts were measured by (32)P-post-labelling in rat organs (lung, heart, liver, bladder and testis), tissues (dissected tracheal epithelium) and cells [isolated bronchoalveolar lavage (BAL) cells]. A time-related increase of (32)P-post-labelled DNA modifications was detectable by autoradiography, in the form of massive diagonal radioactive zones and individual spots. Top levels were reached after 4-5 weeks of exposure. The ratio of smoke-induced DNA adducts to the background levels detected in sham-exposed rats was 11.2 in the tracheal epithelium, 10.4 in BAL cells, 7.3 in the heart, 6.3 in the lung, 5.1 in the bladder, 1.9 in the testis and 1. 1 in the liver. Appearance of DNA adducts in the lung was also revealed by synchronous fluorescence spectrophotometry. Smoke-related oxidative damage was demonstrated by a significant enhancement of 8-hydroxy-2'-deoxyguanosine in lung DNA. In parallel, there was a time-related induction of lung microsomal arylhydrocarbon hydroxylase activity, an elevation in cytosolic glutathione S-transferase activity, and a moderate but progressive and significant depletion of reduced glutathione. After discontinuing exposure to environmental cigarette smoke for 1 week, DNA adduct levels significantly dropped in the lung, tracheal epithelium, heart and bladder. The decrease was evident but not statistically significant in BAL cells, and was negligible in the heart. The selective localization and the differential persistence of these promutagenic nucleotide modifications in rat organs, tissues and cells suggest that exposure to environmental cigarette smoke, at least under the high exposure regimens used in experimental studies, may pose a potential risk of developing mutation-related diseases.