Interindividual variations in DNA adduct levels assessed by analysis of multiple genetic polymorphisms in smokers.

Genetic polymorphisms in genes involved in processes that affect DNA damage may explain part of the large interindividual variation in DNA adduct levels in smokers. We investigated the effect of 19 polymorphisms in 12 genes involved in carcinogen metabolism, DNA repair, and oxidant metabolism on DNA adduct levels (determined by (32)P post-labeling) in lymphocytes of 63 healthy Caucasian smokers. The total number of alleles that were categorized as putatively high-risk alleles seemed associated with bulky DNA adduct levels (P = 0.001). Subsequently, to investigate which polymorphisms may have the highest contribution to DNA adduct levels in these smokers, discriminant analysis was done. In the investigated set of polymorphisms, GSTM1*0 (P < 0.001), mEH*2 (P = 0.001), and GPX1*1 (P < 0.001) in combination with the level of exposure (P < 0.001) were found to be key effectors. DNA adduct levels in subjects with a relatively high number of risk alleles of these three genes were >2-fold higher than in individuals not having these risk alleles. Noteworthy, all three genes are involved in deactivation of reactive carcinogenic metabolites. This study shows that analysis of multiple genetic polymorphisms may predict the interindividual variation in DNA adduct levels upon exposure to cigarette smoke. It is concluded that discriminant analysis presents an important statistical tool for analyzing the effect of multiple genotypes on molecular biomarkers.     

Tissue doses of ethylene oxide in cigarette smokers determined from adduct levels in hemoglobin

Determination of adducts to hemoglobin (Hb) is a useful approachfor monitoring tissue doses of ultimate carcinogens. This approachprovides a basis for both risk estimation and for the identificationof a priori unknown environmental carcinogens. This paper describesthe application of a new method for the analyses of Hb adductsto cigarette smokers and non-smokers. The results demonstratea raised level of hydroxyethylation of N-terminal valine ofHb of smokers that is quantitatively compatible with ethenein the smoke being the source. The magnitude of the tissue dosesof ethylene oxide originating from inhaled ethene suggests thatthis factor is a major contributor to smoking-associated cancerrisk.     

Monocyte chemotaxis in bronchial carcinoma and cigarette smokers

Chemotaxis of blood monocytes was measured in 31 patients with bronchial carcinoma and 19 cigarette smokers. Thirteen patients with metastatic bronchial carcinoma had significantly less (P less than 0.005) chemotactic response than matched controls. Those with disease confined to the chest, or with recurrent or operable bronchial carcinoma, had no significant depression of monocyte chemotaxis. There was also no significant difference in monocyte chemotaxis between cigarette smokers and matched controls. These results support the concept that in human cancer there is a defect in monocyte chemotaxis, but in bronchial carcinoma significant depression was only apparent in those with advanced disease.     

Formation of cigarette smoke-induced DNA adducts in the rat lung and nasal mucosa

The formation of DNA adducts in the nasal, lung, and liver tissues of rats exposed daily to fresh smoke from a University of Kentucky reference cigarette (2R1) for up to 40 weeks was examined. The amount of smoke total particulate matter (TPM) inhaled and the blood carboxyhemoglobin (COHb) values averaged 5-5.5 mg smoke TPM/day/rat and 5.5%, respectively. The pulmonary AHH activity measured at the termination of each experiment showed an average increase of about two- to threefold in the smoke-exposed groups. These observations suggested that animals effectively inhaled both gaseous and particulate phase constituents of cigarette smoke. DNAs from nasal, lung, and liver tissue were extracted and analyzed by an improved 32P-postlabeling procedure. The results showed that the mainstream cigarette smoke induced a spectrum of at least four new DNA adducts in the nasal mucosa of the exposed rats and the magnitude of these adducts increased with the duration of exposure. In the lung tissue, the smoke exposure induced an accumulation of one DNA adduct, which upon cessation of exposure for 19 weeks was reduced by about 75%. Smoke-related adducts were not detected in the liver, a nontarget tissue. Selective chromatography and butanol extractability suggested that the nasal and lung DNA adducts are aromatic and/or hydrophobic in nature and that the smoke-related lung DNA-adduct may contain polar group(s). These data demonstrate the DNA-damaging potential of long term fresh cigarette smoke exposure and suggest the ability of the tissue to partially recover from such damage following cessation of the exposure.     

DNA adducts in human nasal mucosa and white blood cells from smokers and non-smokers

The goal of the present study was to measure the levels of DNA adducts in human nasal mucosa cells and in total white blood cells in relation to smoking. DNA was isolated from samples of 20 healthy volunteers (six smokers and 14 non-smokers). The levels of DNA adducts were measured by 32P-postlabelling assay. In smokers the mean DNA adduct levels were 3.3 and 17.0 adducts/10(8) nucleotides in total white blood cells and nasal mucosa cells respectively. The corresponding values in non-smokers were 2.0 and 6.8 adducts/10(8) nucleotides. The mean adduct level was significantly higher in nasal mucosa cells than in total white blood cells both in smokers and non-smokers. The mean adduct levels in smokers' nasal mucosa cells were significantly higher than those in non- smokers. Thus the nasal mucosa cells constituted a sensitive tissue for the determination of cigarette smoking induced DNA adducts. Combining the sensitivity of the 32P-postlabelling assay with the specificity of the nasal mucosa to the airborne chemical exposures, the DNA adduct analysis from human nasal mucosa cells represents a method of choice in the assessment of exposure to airborne carcinogens.      

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   

Comparison of pulmonary DNA adduct levels, measured by 32P-postlabelling and aryl hydrocarbon hydroxylase activity in lung parenchyma of smokers and ex-smokers

In order to compare pulmonary DNA adducts and aryl hydrocarbon hydroxylase (AHH) activity, we have measured these two parameters in non-neoplastic surgical lung parenchymal samples from four ex-smokers and 19 smokers, out of 20 patients operated for lung cancer, and three for nonmalignant lung diseases. DNA adducts were determined by scintillation counting after 32P-postlabelling analysis. The microsomal fractions of the same lung specimen were assayed for AHH activity by a fluorometric method. Autoradiograms of DNA adducts found in lungs of smokers revealed two distinct diagonal radioactive zones that were absent in ex-smokers. The smokers had significantly higher levels (1.68-13.4 DNA adducts/10(8) nucleotides; mean +/- SD 5.38 +/- 3.19) than ex-smokers (0.23-2.21; 1.09 +/- 0.84). AHH activity in smokers ranged from 0.01 to 0.69 pmol/min/mg. This activity was significantly (P less than 0.05) higher in smokers (0.26 +/- 0.26) who had smoked until 1 week before surgery than in those who had stopped smoking for greater than 7 days (0.11 +/- 0.11). A positive linear correlation between DNA adduct levels and AHH activity (r = 0.69; P less than 0.001; n = 19) was found in smokers. This relationship could explain why AHH inducibility appears to be a crude marker for lung cancer risk in smokers.     

DNA adduct levels and DNA repair polymorphisms in traffic-exposed workers and a general population sample.

Peripheral blood DNA adducts have been considered an acceptable surrogate for target tissues and possibly predictive of cancer risk. A group of 114 workers exposed to traffic pollution and a random sample of 100 residents were drawn from the EPIC cohort in Florence, a population recently shown to present increased DNA adduct levels (Palli et al., Int J Cancer 2000;87:444-51). DNA bulky adducts and 3 DNA repair gene polymorphisms were analyzed in peripheral leukocytes donated at enrollment, by using (32)P-postlabeling and PCR methods, respectively. Adduct levels were significantly higher for traffic workers among never smokers (p = 0.03) and light current smokers (p = 0.003). In both groups, urban residents tended to show higher levels than those living in suburban areas, and a seasonal trend emerged with adduct levels being highest in summer and lowest in winter. Traffic workers with at least 1 variant allele for XPD-Lys751Gln polymorphism had significantly higher levels in comparison to workers with 2 common alleles (p = 0.02). A multivariate analysis (after adjustment for age, season, area of residence, smoking, XPD-Lys751Gln genotype and antioxidant intake) showed a significant 2-fold association between occupational exposure and higher levels of adducts (odds ratio 2.1; 95% confidence interval 1.1-4.2), in agreement with recent pooled estimates of increased lung cancer risk for similar job titles. Our results suggest that traffic workers and the general population in Florence are exposed to high levels of genotoxic agents related to vehicle emissions. Photochemical pollution in warmer months might be responsible for the seasonal trend of genotoxic damage in this Mediterranean urbanized area.     

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

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.     

Variation in PAH‐related DNA adduct levels among non‐smokers: The role of multiple genetic polymorphisms and nucleotide excision repair phenotype

Polycyclic aromatic hydrocarbons (PAHs) likely play a role in many cancers even in never‐smokers. We tried to find a model to explain the relationship between variation in PAH‐related DNA adduct levels among people with similar exposures, multiple genetic polymorphisms in genes related to metabolic and repair pathways, and nucleotide excision repair (NER) capacity. In 111 randomly selected female never‐smokers from the Golestan Cohort Study in Iran, we evaluated 21 SNPs in 14 genes related to xenobiotic metabolism and 12 SNPs in eight DNA repair genes. NER capacity was evaluated by a modified comet assay, and aromatic DNA adduct levels were measured in blood by 32 P‐postlabeling. Multivariable regression models were compared by Akaike's information criterion (AIC). Aromatic DNA adduct levels ranged between 1.7 and 18.6 per 10⁸ nucleotides (mean: 5.8 ± 3.1). DNA adduct level was significantly lower in homozygotes for NAT2 slow alleles and ERCC5 non‐risk‐allele genotype, and was higher in the MPO homozygote risk‐allele genotype. The sum of risk alleles in these genes significantly correlated with the log‐adduct level (r = 0.4, p < 0.001). Compared with the environmental model, adding Phase I SNPs and NER capacity provided the best fit, and could explain 17% more of the variation in adduct levels. NER capacity was affected by polymorphisms in the MTHFR and ERCC1 genes. Female non‐smokers in this population had PAH‐related DNA adduct levels three to four times higher than smokers and occupationally‐exposed groups in previous studies, with large inter‐individual variation which could best be explained by a combination of Phase I genes and NER capacity.     

Carcinogen hemoglobin adducts, urinary mutagenicity, and metabolic phenotype in active and passive cigarette smokers

In 100 healthy volunteers, we have studied the relationship between the type (air- or flue-cured) and number of cigarettes smoked and different biomarkers relevant to the risk of bladder cancer, including the levels of 4-aminobiphenyl (ABP) hemoglobin adduct (a marker of internal dose), urinary mutagenicity in Salmonella typhimurium TA98, and the N-acetylation phenotype (a marker of susceptibility). ABP is a potent bladder carcinogen that is N-acetylated as an overall detoxification step. Levels of the ABP hemoglobin adduct were higher in smokers of black tobacco (air-cured) than in smokers of blond tobacco (flue-cured), confirming our earlier study. In addition, "slow" acetylators had higher levels of the ABP hemoglobin adduct for the same type and quantity of cigarettes smoked. Urinary mutagenicity was also associated with quantity of cigarettes but not with the acetylation phenotype. Convex dose-response relationships were found between the amount smoked and ABP hemoglobin adduct levels or urinary mutagenicity. In 15 nonsmokers who reported exposure to environmental tobacco smoke, ABP hemoglobin adduct levels, unlike urinary mutagenicity, were found to be an aspecific exposure indicator.     

Elevation of serum placental alkaline phosphatase levels in cigarette smokers

In an ongoing longitudinal study for tumor markers in breast cancer patients, levels of placental alkaline phos-phatase (PAP) measured by RIA were found to be elevated in 34% of patients who smoked cigarettes as opposed to 5 % of non-smoking subjects. A normal range was established (0-2.14 ng/ml) using healthy non-smoking females. In an expanded analysis by disease state, PAP levels were found to be elevated significantly (p<0.02) in the smoking groups of each category. This isoenzyme, when detected at elevated serum levels by RIA, was enzymatically active in 48% of the patients as analyzed by immunoenzymatic assay. One smoker with high PAP levels discontinued smoking following a myocardial infarction. Her PAP levels returned to normal within 2 months.     

DNA adducts, protein adducts, and sister chromatid exchange in cigarette smokers and nonsmokers

In order to validate markers of internal dose and biologically effective dose of carcinogens, a battery of measurements was made on blood samples from 22 smokers and 24 nonsmokers. The markers included immunoreactivity in an enzyme-linked immunosorbent assay (ELISA) quantified in white blood cells with the use of a polyclonal anti-benzo[a]pyrene diol epoxide-I-DNA antibody, 4-aminobiphenyl hemoglobin (4-ABP-Hb) adducts measured by negative chemical ionization mass spectrometry, sister chromatid exchange (SCE) in cultured lymphocytes, and cotinine in plasma measured by radioimmunoassay. Several blood samples were drawn from each subject. In blood samples 1 and 3 having detectable levels of DNA adducts, mean femtomole-per-microgram levels were consistently higher among smokers compared to nonsmokers. The borderline significance of this difference may be attributable to the small numbers of subjects. Consistently higher adduct levels were seen in females compared to males. In sample 3, adduct levels were significantly correlated with measurements of active smoking in smokers and with passive smoking in nonsmokers. By contrast to the ELISA data, which may reflect cumulative exposure from multiple background sources, the 4-ABP-Hb assay was able to distinguish clearly between smokers and nonsmokers. SCEs were significantly elevated in the smokers compared to nonsmokers. Also observed were significant correlations between 4-ABP-Hb and both cotinine and SCEs, as well as a positive correlation between the 4-ABP-Hb and DNA adduct levels (sample 3) that was highly significant. The correlation between DNA and 4-ABP-Hb adducts was significant in smokers but not nonsmokers (sample 3). These results support the need for batteries of markers to detect and to quantify the carcinogenic dose to humans resulting from both specific and "background" environmental exposures.     

An aromatic DNA adduct in colonic mucosa from patients with colorectal cancer

The incidence of colorectal cancer in the western hemisphere is though to be the result, in part, of environmental agents, and many studies strongly implicate diet as a determining factor. It is conceivable that the ingestion of genotoxic chemicals present in food or the endogenous formation of such substances in the gut may initiate colorectal cancer in humans. In the present study, 32P-postlabelling has been used to examine DNA from normal-appearing colonic mucosa obtained from (i) patients undergoing surgery for colorectal cancer and (ii) adult and fetal controls for the presence of aromatic DNA adducts.     

Effect of DNA repair gene polymorphisms on BPDE-DNA adducts in human lymphocytes

To determine whether variations in DNA repair genes are related to host DNA damage, we investigated the association between polymorphism in the XPD gene (codon 199, 312, 751) and the XRCC1 gene (codon 194, 399) and the presence of benzo(a)pyrene diolepoxide adducts to lymphocyte DNA (BPDE-DNA) in a group of male patients with incident lung cancer, all current smokers. BPDE-DNA adducts were analyzed by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry. XPD and XRCC1 genotypes were identified by PCR-RFLP. XRCC1 and XPD genotypes did not affect the levels and proportion of detectable BPDE-DNA adducts. The patients were also genotyped for the GSTM1 polymorphism, given its role in the detoxification of BPDE. Individuals with the GSTM1 deletion had significantly higher levels of BPDE-DNA adducts when they were XPD-Asp312Asp+Lys751Lys than carriers of at least one variant allele. No such association was found with the XRCC1 genotypes. Because of the small study population (n = 60), further statistical analysis of possible gene-gene and gene-environment would not be informative. This is the first study analysing the specific BPDE-DNA adduct in vivo with regard to polymorphic repair genes (XPD, XRCC1) and xenobiotic metabolizing gene (GSTM1). Our results raise the possibility that the XPD-Asp312Asp+Lys751Lys genotype may increase BPDE-DNA damage; this effect might be evident in individuals who are especially likely to have accumulated damage, probably because of lower detoxification capacity and high environmental exposure.     

Comparison of multiple DNA adduct types in tumor adjacent human lung tissue: effect of cigarette smoking

Cigarette smokers inhale a broad range of carcinogens derived from tobacco and its pyrolysis products, including free radicals, which induce oxidative stress and subsequent lipid peroxidation (LPO). Miscoding carcinogen-DNA adducts are formed by cigarette smoke constituents and are thought to initiate lung carcinogenesis. The presence of various types of DNA damage was therefore analyzed in tumor adjacent uninvolved lung tissues of 13 smoking and 11 non-smoking operated lung cancer patients. O(4)-ethylthymidine (O(4)etT), 1,N(6)-ethenodeoxyadenosine ( epsilon dA) and 3,N(4)-ethenodeoxycytidine ( epsilon dC) were determined by immuno-enriched (32)P-postlabeling. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were measured as diagonal radioactive zones after nuclease P1 enriched (32)P-postlabeling. Mean O(4)etT and PAH-DNA adduct levels were higher in lung DNA of smokers than of non-smokers (O(4)etT/10(8) thymidine: 3.8 versus 1.6, P < 0.01; PAH-DNA adducts/10(8) nucleotides: 11.2 versus 2.2, P < 0.01). Pulmonary etheno-DNA adduct levels did not differ between smokers and non-smokers, but large inter-individual variations were observed (80- and 250-fold differences for epsilon dA and epsilon dC, respectively). As all smokers (except one) refrained from smoking at least for 1 week before surgery, our results demonstrate the persistence of O(4)etT and PAH-DNA adducts in human lung. A positive correlation obtained between O(4)etT and PAH-DNA adducts (R = 0.65, P < 0.01) suggests that both adducts are formed from cigarette smoke as the main exposure source. We conclude that in addition to the DNA adducts derived from PAH and tobacco-specific nitrosamines, miscoding O(4)etT lesions are formed by cigarette smoke that contribute to the increased genomic instability and increased lung cancer risk in smokers.     

Myeloperoxidase (MPO) -463G->A reduces MPO activity and DNA adduct levels in bronchoalveolar lavages of smokers.

The myeloperoxidase (MPO) -463G-->A genetic polymorphism is associated with a reduced risk for lung cancer, but the underlying mechanism is not yet elucidated. Therefore, the impact of this polymorphism on MPO activity and lipophilic DNA adducts was studied in respectively bronchoalveolar lavage (BAL) fluid and cells, from 106 smoking Caucasian lung patients. MPO activity was determined spectrophotometrically, aromatic DNA adducts by (32)P-postlabeling and MPO genotypes by RFLP analysis. Frequencies of MPO -463AA (13%), MPO -463AG (36%), and MPO -463GG (51%) were in line with earlier observations. MPO activity/neutrophil was lower in MPO -463AA (median 0.04 pU/cell) than in MPO -463AG (median 0.07 pU/cell) and MPO -463GG (median 0.14 pU/cell; P = 0.059) individuals. DNA adducts in BAL cells were measured in 11 MPO -463AA subjects and equal numbers of MPO -463AG and MPO -463GG subjects matched for smoking, age, gender, and clinical diagnosis. DNA adduct levels in MPO -463AA individuals (median 0.62 adducts/10(8) nucleotides) were lower than in MPO -463AG (median 1.51 adducts/10(8) nucleotides) and MPO -463GG (median 3.26 adducts/10(8) nucleotides; P = 0.003) subjects. Overall, no significant correlation was observed between amount of inhaled tar/day and DNA adduct levels. However, correlations improved considerably on grouping according to the MPO genotype; MPO -463AA subjects were the least responsive (R(2) = 0.73, slope = 0.4, P = 0.01) followed by MPO -463AG subjects (R(2) = 0.70, slope = 1.3, P = 0.01) and MPO -463GG patients (R(2) = 0.67, slope = 2.8, P = 0.02). These data demonstrate that MPO -463AA/AG genotypes are associated with (a) reduced MPO activity in BAL fluid and (b) reduced smoking-related DNA adduct levels in BAL cells in a gene-dose manner. These data provide a plausible biological explanation for the reduced risk for lung cancer as observed in MPO -463AA/AG compared with MPO -463GG subjects.     

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.     

Aberrant promoter methylation of multiple genes in bronchial brush samples from former cigarette smokers.

Promoter hypermethylation plays an important role in the inactivation of tumor suppressor genes during tumorigenesis. Recent data suggest that such epigenetic abnormality may occur very early in lung carcinogenesis. To determine the extent of promoter hypermethylation in early lung tumorigenesis, we analyzed promoter methylation status of the p16, death-associated protein kinase (DAPK) and glutathione S-transferase P1 (GSTP1) genes using methylation-specific PCR in bronchial brush samples obtained from 100 former smokers enrolled in a chemoprevention clinical trial. We found that 17% of the samples showed methylation for p16 and 17% the same for DAPK, whereas only 6% of the samples displayed methylation for GSTP1. A total of 32% of the samples had methylation in at least one of the three genes tested, and 8% of the samples had methylation in two genes. The methylation status of p16 was correlated with that of DAPK (P = 0.04, Fisher's exact test). p16 methylation was higher in former smokers with a history of previous cancer than in former smokers without a history of cancer (P = 0.04, Fisher's exact test), and methylation of DAPK was detected more frequently in older patients than it was in younger patients (P = 0.01, Wilcoxon rank-sum test). Surprisingly, no correlation was found between methylation in any of these genes and the smoking characteristics of the individuals analyzed (packs per day, pack-years, smoking years, quitting years). The precise meaning of methylated genes in the bronchial brush samples of former smokers must be sought by means of careful follow-up of these individuals.