Aromatic DNA adducts in human white blood cells and skin after dermal application of coal tar

A group of eczema patients topically treated with coal tar (CT) ointments was used as a model population to examine the applicability of DNA adducts in WBC subpopulations as a measure of dermal exposure to polycyclic aromatic hydrocarbons (PAHs). Aromatic DNA adducts were examined by 32P-postlabeling in exposed skin and WBC subsets, and urinary excretion of PAH metabolites was determined to assess the whole-body burden. The median urinary excretion of 1-hydroxypyrene and 3-hydroxybenzo(a)pyrene was 0.39 (range, 0.12-1.57 micromol/mol creatinine) and 0.01 micromol/mol creatinine (range, <0.01-0.04 micromol/mol creatinine), respectively, before the dermal application of CT ointments. After treatment for 1 week, these levels increased to 139.7 (range, 26.0-510.5 micromol/mol creatinine) and 1.18 micromol/mol creatinine (range, <0.01-2.14 micromol/mol creatinine), respectively, indicating that considerable amounts of PAHs were absorbed. Median aromatic DNA adduct levels were significantly increased in skin from 2.9 adducts/10(8) nucleotides (nt; range, 0.7-10.0 adducts/10(8) nt) before treatment to 63.3 adducts/10(8) nt (range, 10.9-276.2 adducts/10(8) nt) after treatment with CT, in monocytes from 0.28 (range, 0.25-0.81 adducts/10(8) nt) to 0.86 adducts/10(8) nt (range, 0.56-1.90 adducts/10(8) nt), in lymphocytes from 0.33 (range, 0.25-0.89 adducts/10(8) nt) to 0.89 adducts/10(8) nt (range, 0.25-3.01 adducts/10(8) nt), and in granulocytes from 0.28 (range, 0.25-0.67 adducts/10(8) nt) to 0.54 adducts/10(8) nt (range, 0.25-1.58 adducts/10(8) nt). A week after stopping the CT treatment, the DNA adduct levels in monocytes and granulocytes were reduced to 0.38 (range, 0.25-0.71 adducts/10(8) nt) and 0.38 adducts/10(8) nt (range, 0.25-1.01 adducts/10(8) nt), respectively, whereas the adduct levels in lymphocytes remained enhanced [1.59 adducts/10(8) nt (range, 0.25-2.40 adducts/10(8) nt)]. Although the adduct profiles in skin and WBC subsets were not identical, and the adduct levels in WBCs were significantly lower as compared with those in skin, the total DNA adduct levels in skin correlated significantly with the adduct levels in monocytes and lymphocytes, but not with those in granulocytes. Excretion of urinary metabolites during the first week of treatment was correlated with the percentage of the skin surface treated with CT ointment and decreased to background levels within a week after the cessation of treatment. 3-Hydroxybenzo(a)pyrene excretion, but not that of 1-hydroxypyrene, correlated significantly with the levels of DNA adducts in skin that comigrated with benzo(a)pyrene-diol-epoxide-DNA. This study indicates that the DNA adduct levels in mononuclear WBCs can possibly be used as a surrogate for skin DNA after dermal exposure to PAHs.     

The interaction between alcohol consumption and GSTM1 genotype on polycyclic aromatic hydrocarbon-DNA adduct levels in breast tissue

We investigated the association between alcohol consumption, GSTM1 genotype, and polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels in breast tissue. Women referred for breast surgery were enrolled prior to surgery, responded to an interview, and gave a blood sample. Women diagnosed with ductal carcinoma in situ and invasive ductal or lobular cancer were defined as cases, and women with benign conditions without atypia were defined as controls. Paraffin-embedded tumor and nontumor tissue from cases and benign tissue from controls were retrieved from the pathology samples. GSTM1 genotype status was determined by PCR using WBC DNA, and PAH-DNA adduct levels were measured in breast tissue using immunohistochemistry. In tumor and nontumor tissue from cases, the GSTM1-null genotype was associated with increased adduct levels among current alcohol consumers but not among nondrinkers. In nontumor tissue, the interaction between genotype and alcohol consumption was significant (P=0.02), but in tumor tissue, the interaction did not achieve statistical significance (P=0.10). In benign tissue from controls, there was no association between genotype and adducts, regardless of drinking status. Among subjects with the null genotype who drank alcohol, adduct levels were significantly higher in tumor and nontumor tissue from cases than in benign tissue from controls. These results indicate the presence of a novel gene-lifestyle interaction that influences PAH-DNA adduct levels in breast tissue from cases but not controls. This apparent difference in PAH metabolism in response to alcohol may be an important clue as to how alcohol influences breast cancer risk.     

Concentration-dependent block of sodium current in guinea pig ventricular myocytes by a class III antiarrhythmic agent, MS-551

The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 microM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7 +/- 2.9 adducts/10(8) nucleotides, n = 8), followed by lymphocytes (5.9 +/- 1.7, n = 8), and granulocytes (0.5 +/- 0.2, n = 8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7 +/- 1.0, n = 5) > monocytes (2.0 +/- 0.5, n = 8) > or = lymphocytes (1.6 +/- 0.4, n = 8) > granulocytes (0.8 +/- 0.2, n = 8) by NP1-enrichment and monocytes (9.0 +/- 3.2, n = 5) > or = lymphocytes (8.0 +/- 2.1, n = 6) > granulocytes (2.1 +/- 0.3, n = 7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non-smokers, except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n = 78). Adduct levels in MNC were related to daily exposure to cigarette-tar (r = 0.31, P < 0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6-fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r = 0.86, P < 0.001, n = 12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts.     

Polycyclic aromatic hydrocarbon-DNA adducts in human lung and cancer susceptibility genes

Molecular dosimetry for polycyclic aromatic hydrocarbon-DNA adducts, genetic predisposition to cancer, and their interrelationships are under study in numerous laboratories. This report describes a modified 32P-postlabeling assay for the detection of polycyclic aromatic hydrocarbon-DNA adducts that uses immunoaffinity chromatography to enhance chemical specificity and quantitative reliability. The assay incorporates internal standards to determine direct molar ratios of adducts to unmodified nucleotides and to assess T4 polynucleotide kinase labeling efficiency. High performance liquid chromatography is used to assure adequacy of DNA enzymatic digestion. The assay was validated using radiolabeled benzo(a)pyrene-diol-epoxide modified DNA (r = 0.76, P < 0.05) thereby assessing all variables from enzymatic digestion to detection. Thirty-eight human lung samples were examined and adducts were detected in seven. A subset of samples also was examined for benzo(a)pyrene-diol-epoxide-DNA adducts by immunoaffinity chromatography, high performance liquid chromatography, and synchronous fluorescence spectroscopy. A high correlation between the two assays was found (P = 0.006). The lung samples were then analyzed by the polymerase chain reaction for the presence of mutations in the cytochrome P-450 (CYP) 1A1 and glutathione S-transferase mu (GST mu) genes. A positive association was identified for adduct levels and GST mu null genotypes (P = 0.038). No correlation was found between polycyclic aromatic hydrocarbon-adduct levels and CYP1A1 exon 7 mutations. Age, race, and serum cotinine were not related to adduct levels. Multivariate analysis indicated that only the GST mu genotype was associated with polycyclic aromatic hydrocarbon-DNA adduct levels. This work demonstrates that the 32P-postlabeling assay can be modified for chemically specific adduct detection and that it can be used in the assessment of potentially important genetic factors for cancer risk. The absence of a functional GST mu gene in humans is likely one such factor.     

Putative susceptibility markers of coronary artery disease: association between VDR genotype, smoking, and aromatic DNA adduct levels in human right atrial tissue

Cancer and cardiovascular diseases share risk factors such as smoking, and the onset of both diseases have been suggested to have a common mechanistic basis. The binding of carcinogens to DNA (carcinogen-DNA adducts), genetic polymorphisms in carcinogen-detoxifying enzymes glutathione S-transferases (GSTs), and genetic polymorphisms in the vitamin D receptor (VDR) are among the candidates for modifiers of cancer risk. We determined whether these biomarkers could be related to individual characteristics of patients suffering from cardiovascular diseases. For that purpose, DNA from the right atrial appendage of 41 patients who underwent open heart surgery was analyzed for smoking-related DNA adducts and polymorphisms in GSTM1, GSTT1, and VDR genes. Statistical analysis was used to identify any patient's characteristics associated with these molecular markers. Our results showed that heart tissue of cigarette smokers contained a variety of aromatic DNA adducts in significantly elevated levels compared to ex-smokers (P<0.01) or nonsmokers (P<0.001). A linear relationship was observed between DNA adduct levels and daily cigarette smoking (rs=0.73; P=0.0003). Since cardiac myocytes are terminally differentiated cells that have lost their ability to divide and seemingly have limited DNA repair capacities, their levels might accumulate with time and thereby affect heart cell function or viability. Substantial interindividual differences between DNA adduct levels were observed, and persons with severe coronary artery disease (CAD), as assessed by coronary angiography, had higher DNA adduct levels than persons with no or mild CAD (P=0.04). As polymorphisms in GST genes have been shown to modulate DNA adduct levels and risk for lung cancer in smokers, we explored for the first time whether the GST polymorphisms could also explain deviating heart DNA adduct levels and CAD risk. However, no relation could be found between these covariants. In contrast, a VDR genotype, which has been associated with decreased serum levels of the active hormonal form of vitamin D and increased risk for certain cancers, seemed to be related to severity of CAD (P=0.025). Our findings support the hypothesis that smoking-related DNA damage may be involved in the onset of cardiovascular diseases and suggest that VDR genotype may be a useful susceptibility marker of CAD.     

The outcome of contact tracing for gonorrhoea in the United Kingdom

BACKGROUND: The p53 mutation spectrum of prostate cancers developing in Japan indicates a role for environmental factors. This suggests there might be differences in susceptibility due to genetic polymorphisms in metabolic activation enzyme genes. We analyzed genetic polymorphisms of the xenobiotic-metabolizing enzymes, CYP1A1 and GSTM1. METHOD: Genotyping of CYP1A1 and GSTM1 was investigated by using allele-specific PCR in 115 prostate cancer (PCa) patients and 204 control patients. RESULTS: The CYP1A1 Val/Val genotype significantly increased the risk for PCa (OR = 2.6; 95% CI = 1.11-6.25) and the Ile/Val genotype showed a similar tendency (OR = 1.4; CI = 0.86-2.29). Individuals with the GSTM1 (0/0) genotype demonstrated a slightly increased risk (OR = 1.3; CI = 0.82-2.04). The combination of the CYP1A1 Val allele and GSTM1 (0/0) genotype was associated with a higher risk (OR = 2.3; CI = 1.18-4.48) than the CYP1A1 Val allele alone. When cases were analyzed by age at initial diagnosis, the relative risks with both the CYP1A1 Val allele and the GSTM1 (0/0) genotype were higher in the young group than in the old group (CYP1A1; OR = 1.7, CI = 0.89-3.17: GSTM1; OR = 1.6, CI = 0.84-2.99). The frequency of the GSTM1 (0/0) genotype was also higher in patients with advanced stage disease. In stage D, the OR was 1.7 with a CI of 0.93-3.17 and in stages A and B, the OR was 0.8 with a CI of 0.40-1.62. CONCLUSIONS: These results suggest that CYP1A1 and GSTM1 polymorphisms are linked to a propensity for PCa development.     

Accumulation of DNA adducts of 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) in tissues and white blood cells of the Fischer-344 rat after multiple oral dosing

The genotoxic effect of an environmental chemical may be estimated from the concentration of its DNA adducts in peripheral white blood cells (WBCs). The food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is carcinogenic in the Fischer-344 rat, affecting principally the liver, small intestine and large intestine. In the present study we have determined whether DNA adducts of IQ are present in circulating WBCs of rats after single or multiple oral doses of IQ and how these adducts are related to those in internal organs. Male Fischer-344 rats received IQ as an oral dose (5 or 50 mg/kg, starting on day 0) by daily gavage (1, 8 or 15 days of treatment). Using 32P-postlabeling assays, IQ-DNA adducts were isolated and quantitated in organs and WBCs on days 1, 8 and 15. Adduct patterns in WBCs were qualitatively similar to those in the organs and adduct formation was highest in the liver, followed by the lungs, kidneys, stomach, large intestine, WBC and small intestine. Accumulation of adducts occurred in all organs and in WBCs in a dose- and time-dependent manner. For all organs, IQ-DNA adduct formation was strongly correlated with those in WBCs. It is concluded that IQ-DNA adducts in WBCs are qualitatively and quantitatively directly related to those in internal organs, independent of the target organ specificity of the carcinogenic effect of IQ.     

Absence of the glutathione S-transferase M1 gene increases cytochrome P4501A2 activity among frequent consumers of cruciferous vegetables in a Caucasian population

The cancer protective effect of cruciferous vegetables has been attributed to induction of phase II enzymes. But cruciferous vegetables also induce cytochrome P4501A2 (CYP1A2), which catalyzes the metabolic activation of various procarcinogens, including aromatic amines in tobacco. Thus, frequent intake of cruciferous vegetables could also result in cancer-enhancing effects. GSTM1 is involved in the detoxification of various carcinogens, but it also enhances the excretion of isothiocyanates and possibly other enzyme inducers in cruciferous vegetables. We, therefore, hypothesized that GSTM1 null genotype might be associated with increased CYP1A2 activity among frequent consumers of cruciferous vegetables because the excretion of CYP1A2 inducers contained in these vegetables may be partially inhibited in the absence of the GSTM1 enzyme. Three hundred twenty-eight non-Hispanic white residents of Los Angeles County (265 males and 63 females) were genotyped for the presence or absence of GSTM1 alleles and phenotyped for CYP1A2 activity. Information on usual dietary habits was obtained from these subjects through in-person interviews. Among frequent (at least once a week) consumers of broccoli, GSTM1 null individuals exhibited a 21% higher geometric mean level of CYP1A2 activity relative to GSTM1 non-null individuals (5.24 versus 4.32, two-sided P = 0.01). No such difference was observed in subjects who consumed broccoli less frequently (two-sided P = 0.39). This interactive effect of GSTM1 genotype and vegetable intake on CYP1A2 activity also was observed when overall intake of the five cruciferous vegetables under study (broccoli, cabbage, cauliflower, Brussels sprouts, and mustard greens) was examined. Among weekly consumers of cruciferous vegetables, GSTM1 null individuals showed a 16% higher geometric mean level of CYP1A2 activity relative to GSTM1 non-null individuals (5.03 versus 4.33, two-sided P = 0.02), whereas no difference was evident among those who consumed cruciferous vegetables less frequently (two-sided P = 0.35). Our results suggest that cruciferous vegetables contain CYP1A2 inducers, which are deactivated in the presence of GSTM1.     

Aromatic DNA adducts in foundry workers in relation to exposure, life style and CYP1A1 and glutathione transferase M1 genotype

Levels of aromatic DNA adducts in foundry workers and controls were followed at four annual samplings. During this time exposure to polycyclic aromatic hydrocarbons (PAH) decreased and the level of DNA adducts decreased accordingly. In the total group exposure was related to the level of adducts. Adduct levels correlated with urinary 1-hydroxypyrene (LOGU1OH), air benzo[a]pyrene, weekly working hours and daily cigarette consumption. In a multivariate model 1-hydroxypyrene had a consistent effect. Neither glutathione transferase M1 (GSTM1) nor cytochrome P450 1A1 (CYP1A1) genotypes had clear effects. Yet the individuals lacking GSTM1 had a stronger effect of LOGU1OH and some effect by other sources of PAH, such as charcoal broiled food, although all these variables were not significant in the multivariate model. The rare individuals with a CYP1A1 polymorphism MspI containing an amino acid change at isoleucine had an increased level of adducts. The results showed that the postlabelling method used was able to detect an increase in aromatic DNA adducts in leukocytes when exposure to benzo[a]pyrene in air was approximately 5 ng/m3. At such low levels smoking and charcoal broiled food may be important contributors to adducts.     

Impact of inherited polymorphisms in glutathione S-transferase M1, microsomal epoxide hydrolase, cytochrome P450 enzymes on DNA, and blood protein adducts of benzo(a)pyrene-diolepoxide

The benzo(a)pyrene (BaP) metabolite benzo(a)pyrenediolepoxide (BPDE) is strongly implicated as a causative agent of lung cancer. To assess the risk of exposure to BaP, we made a combined analysis of levels of BPDE adducts to hemoglobin (Hb), serum albumin (SA), and lymphocyte DNA in 44 patients with incident lung cancer, as a prototype of a population mainly exposed to tobacco-derived BaP. We also investigated whether genetic polymorphisms of cytochrome P450IA1 (CYPIA1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase M1 (GSTM1), which are involved in BaP metabolism, can be determinants of adduct formation. BPDE-Hb, BPDE-SA, and BPDE-DNA adducts were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry to achieve high specificity and sensitivity. Individuals with detectable Hb adducts were positive for SA adducts but not vice versa, suggesting that BPDE-Hb adducts are less informative indicators of BaP exposure. Using PCR methods on DNA, we characterized GSTM1 deletion, CYPIA1 MspI and exon 7 valine variants, and mEH polymorphisms at amino acid positions 113 (EH3) and 139 (EH4). Levels of BPDE adducts were no different among CYPIA1, mEH, and GSTM1 genotypes. However, individuals with measurable BPDE-SA adducts were CYPIA1 variant carriers more frequently (P = 0.03). There was a slightly higher percentage of DNA detectable adducts in subjects with CYPIA1 exon 7 valine polymorphism. When subjects were classified by both polymorphisms on the mEH gene, those with two slow alleles (EH3 homozygous mutated) and no fast alleles (EH4 homozygous wild type) had a lower frequency of BPDE-SA adducts and no DNA adducts (P = 0.06). These results are based on a small number of observations thus far, but this exploratory study suggests that CYPIA1 and mEH variants might have an impact on BPDE exposure markers such as BPDE-SA adducts. Chemical specificity in adduct measurements is important to identify the biomarkers that reflect BaP exposure more accurately.     

Autistic disorder: a review for the pediatric dentist

A combined analysis of two polymorphic enzymes, glutathione S-transferase mu (GST M1) and q (GST T1) and their implication as cancer risk factors was performed in a case-control study of lung and bladder cancers. Using a multiplex polymerase chain reaction (PCR) based method, the frequency of the homozygous deleted GSTM1 and GSTT1 genotypes was examined in 117 lung cancer patients, 67 urinary bladder cancer patients, and in a community-based sample of 248 healthy, unrelated individuals. In both cancer groups the frequency of the GSTM1 null genotype was higher in comparison with that of the control group (59% and 59.7% vs. 49.6%), but this increase did not reach statistical significance (p > 0.05). After grouping by the smoking status, among smokers in both cancer groups (62.1% in lung cancer and 71.4% in the bladder cancer group, respectively) there were statistically significantly (p < 0.05) increased frequencies of the GSTM1 deletion genotype as compared to the control group (49.6%). Smokers with absence of the GSTM1 gene were at an approximately 1.7-fold higher risk for lung cancer (odds ratio--OR = 1.67, 95% confidence interval--CI 95% = 1.0-2.7, p = 0.04) and an approximately 2.5-fold higher risk for bladder cancer (OR = 2.54, CI 95% = 1.2-5.5, p = 0.02). As related to GSTT1, our study demonstrated an overall GSTT1 effect on bladder cancer risk. Individuals with absence of the GSTT1 gene were at an approximately 2.5-fold higher risk of developing bladder cancer. In the lung cancer cases, the frequency of the putatively high risk GSTT1 null genotype was not increased as compared with controls. No effect of smoking was found on risk of lung and bladder cancer associated with the GSTT1 0/0 genotype. In combined analysis, the obtained results suggested that individuals who were both GSTM1 null and GSTT1 null may be at increased risk because they lack both enzymes. The findings suggest that the GSTM1 null genotype may be associated with susceptibility to lung and urinary bladder cancer in dependence on the exposure to carcinogens in cigarette smoke and that the GSTT1 null genotype is not a critical factor in mediating the risk of lung cancer, but may be associated with an increased susceptibility to bladder cancer.     

Higher frequency of glutathione S-transferase deletions in black children with acute lymphoblastic leukemia

The genetic polymorphisms in human glutathione S-transferases (GST) M1 and T1 have been associated with race, disease risk, and outcome of some adult cancers. Also, there are racial differences in the incidence and characteristics of childhood acute lymphoblastic leukemia (ALL). Our objectives were to compare the frequency of the null genotype for GSTM1, GSTT1, or both in children with ALL to that in healthy controls, and to determine whether GST genotype was associated with treatment outcome and prognostic factors. We studied GSTM1 and GSTT1 genotypes in somatic cell DNA from black children and white children with ALL and in 416 healthy controls, using a polymerase chain reaction technique. Ninety of 163 (55.2%) white ALL patients and 14 of 34 (41.2%) black patients were GSTM1 null, frequencies not significantly different (P = .19) than healthy controls (53.5% in whites and 27.6% in blacks), although there was a trend toward more null genotypes in black ALL patients. Twenty-three of 163 (14.1%) white ALL patients and 12 of 34 (35.3%) black ALL patients were GSTT1 null, not different (P = .34) than the frequencies in healthy controls (15.0% in whites and 24.1% in blacks). However, the frequency of the "double-null" genotype, lacking both GSTM1 and GSTT1, was higher in black patients with ALL (8 of 34 or 23.5%) than in black controls (3.9%) (P = .0005), but this was not the case in white patients with ALL (10 of 163 or 6.1%) compared to white controls (8.0%) (P = .68). In stratified analyses, the GST double-null genotype was not associated with other characteristics that might differ between whites and blacks with ALL, such as age, T-lineage immunophenotype, presenting white blood cell count, DNA index, or insurance status. The null genotype for GSTM1, GSTT1, or both was not found to be a prognostic factor for disease-free survival or probability of hematologic remission; central nervous system relapse tended to be less common in those with the GSTM1 null genotype (P = .054). The double-null genotype for GSTM1 and GSTT1 is more common among blacks but not whites with childhood ALL. These data suggest that GST genotype, coupled with unidentified additional risk factors, may play a role in risk of childhood ALL in American blacks.     

Acetylator genotype-dependent formation of 2-aminofluorene-hemoglobin adducts in rapid and slow acetylator Syrian hamsters congenic at the NAT2 locus

Arylamine-hemoglobin adducts are a valuable dosimeter for assessing arylamine exposures and carcinogenic risk. The effects of age, sex, time-course, dose, and acetylator genotype on levels of 2-aminofluorene-hemoglobin adducts were investigated in homozygous rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/H-Pats) acetylator hamsters congenic at the polymorphic (NAT2) acetylator locus. Following administration of a single ip dose of [3H]2-aminofluorene, peak 2-aminofluorene-hemoglobin adduct levels were achieved at 12-18 hr and retained a plateau up to 72 hr postinjection in both rapid and slow acetylator congenic hamsters. 2-Aminofluorene-hemoglobin adduct levels did not differ significantly between young (5-6 weeks) and old (32-49 weeks) hamsters or between male and female hamsters within either acetylator genotype. 2-Aminofluorene-hemoglobin adduct levels increased in a dose-dependent manner (r = 0.95, p = 0.0001) and were consistently higher in slow versus rapid acetylator congenic hamsters in studies of both time-course and dose-effect. The magnitude of the acetylator genotype-dependent difference was a function of dose; 2-aminofluorene-hemoglobin adduct levels were 1.5-fold higher in slow acetylator congenic hamsters following a 60 mg/kg 2-aminofluorene dose (p = 0.0013) but 2-fold higher following a 100 mg/kg 2-aminofluorene dose (p < 0.0001). These results show a specific and significant role for NAT2 acetylator genotype in formation of arylamine-hemoglobin adducts, which may reflect the relationship between acetylator genotype and the incidence of different cancers from arylamine exposures.     

Polycyclic aromatic hydrocarbon-DNA adducts in human placenta and modulation by CYP1A1 induction and genotype

This study investigated the relationship in human placenta between polycyclic aromatic hydrocabon (PAH)-DNA adduct levels and two biomarkers of cytochrome P4501A1 (CYP1A1): gene induction evidenced by CYP1A1 mRNA, and a genetic polymorphism, the CYP1A1 MspI RFLP. CYP1A1 codes for an inducible enzyme system that catalyzes the bioactivation of PAHs. Prior research found a high correlation in human lung tissue between CYP1A1 activity and DNA damage from PAHs. The CYP1A1 Mspi RFLP has been linked in some studies to risk of lung cancer. The relationships in human placenta between DNA damage, CYP1A1 activity and genotype have not been well characterized and may be relevant to risks from transplacental PAH exposure. The study cohort consisted of 70 newborns from Krakow, Poland, a city with elevated air pollution, and 90 newborns from nearby Limanowa, an area with lower air pollution but greater indoor coal use. Contrary to results seen previously in lung tissue, CYP1A1 mRNA was not significantly correlated with PAH-DNA adduct levels in the placenta. Smoking (self-reported maternal and infant plasma cotinine) was significantly associated with CYP1A1 mRNA levels (P < 0.01), but not with PAH-DNA adduct levels. Placental PAH-DNA adduct levels were significantly higher in infants with the CYP1A1 MspI restriction site compared with infants without the restriction site (P < 0.01), implicating a genetic factor in inter-individual variation in DNA damage in human placenta. Further studies are needed to determine the relevance of this finding to risk of transplacental carcinogenesis.     

Glutathione S-transferase M1 and T1 and cytochrome P4501A1 polymorphisms in relation to the risk for benign and malignant head and neck lesions

BACKGROUND: Susceptibility to head and neck cancer in a particular individual may depend in part on the metabolic balance between Phase 1 enzymes, such as cytochromes P450 (CYPs), and Phase II enzymes, such as glutathione S-transferases (GSTs). Genetic variability in CYP and GST isoenzymes may contribute to individual differences in susceptibility to chemical carcinogenesis. GSTM1 and GSTT1 null genotypes as well as polymorphic variants in the CYP1A1 gene that may help determine the risk for head and neck cancer have been described in previous reports. METHODS: Polymorphisms of GSTM1, GSTT1, and CYP1A1 in whole blood were detected by polymerase chain reaction (PCR) in 185 patients with head and neck squamous cell carcinoma (HNSCC), 78 patients with benign head and neck lesions (BHNL), and 207 blood donors. RESULTS: GSTM1 null genotype was demonstrated to be equally frequent in patients with HNSCC (50.8%), patients with BHNL (47.4%), and blood donors (51.7%). GSTT1 null genotype occurred significantly more often in patients with BHNL (33.3%) as compared with blood donors (20.3%), demonstrating that lack of GSTT1 may be a risk factor for BHNL. Presence of the rare valine in the CYP1A1/Nco1 site was found in 33 patients with HNSCC (17.8%), in 20 patients with BHNL (25.6%), and in 34 blood donors (16.4%). The frequencies with which the presence of the rare cytosine nucleotide in the CYP1A1/Msp1 site was detected were 17.8%, 15.4%, and 15.9%, respectively. CONCLUSIONS: The occurrence of polymorphic variants in the GSTM1, GSTT1, and CYP1A1 genes did not differ between the groups investigated, therefore indicating no significant contribution to the development of head and neck cancer.     

Aflatoxin B1-adduct formation in rat and human small bowel enterocytes

BACKGROUND/AIMS: Hepatic CYP3A enzymes have been implicated in the bioactivation of aflatoxin B1 (AFB1) to DNA binding metabolites. CYP3A enzymes are also abundant in the small bowel, and we therefore examined the ability of this tissue to form intracellular AFB1 adducts. METHODS: Immunohistochemistry using a antibody to the stable AFB1-DNA adduct was performed on small bowel sections obtained from rats orally gavaged with AFB1 and on human small bowel biopsy specimens maintained in explant culture. 3H-AFB1 was instilled into a loop of small bowel of untreated rats and rats pretreated with the CYP3A inducer dexamethasone during vivisection. DNA was isolated from the loop 2 hours later and assayed for specific activity. RESULTS: In both rats and humans, AFB1-adducts were detected exclusively in mature enterocytes in a pattern similar to the distribution of CYP3A enzymes. Induction of enterocyte CYP3A in rats resulted in an increase in enterocyte immunoreactive AFB1 adducts and in a 1.8-fold increase in 3H-AFB1-nucleic acid adducts (P = 0.01). CONCLUSIONS: Intracellular AFB1 adducts are formed in the small intestine, and this reflects, at least in part, the catalytic activity of CYP3A enzymes. Because these AFB1 adducts should ultimately pass in stool, enterocyte CYP3A may represent a regulatable barrier to dietary aflatoxins.     

Association of glutathione S-transferase GSTM1 and GSTT1 null genotypes with clinical outcome in epithelial ovarian cancer

Epithelial ovarian cancer is generally associated with a poor outcome, although the mechanisms that determine survival and progression-free interval (PFI) are unclear. Data from ovarian tumors showing associations between (a) null genotypes at the glutathione S-transferase GSTM1 and GSTT1 loci and expression of p53 protein and (b) outcome and expression of p53 suggest that polymorphism at these loci is a factor determining outcome. Accordingly, we have studied the association between the GSTM1 null and GSTT1 null genotypes and survival and PFI in 148 women with epithelial ovarian cancer. Although we did not find an association between individual genotypes and outcome, women with both GSTM1 null and GSTT1 null genotypes demonstrated poorer survival (P = 0.001) and reduced PFI (P = 0.003). Thus, no cases with both these genotypes survived past 42 months postdiagnosis. In contrast, 43% of the women without this combination survived beyond this time. Because response to chemotherapy is a major factor determining outcome in ovarian cancer, we also examined the data for associations between the glutathione S-transferase genotypes and response to such treatment. Thus, in 78 patients treated with chemotherapy, the combination of GSTM1 null and GSTT1 null was associated with unresponsiveness to primary chemotherapy (P = 0.004); none of the eight patients with both these genotypes responded, compared with 38 of 70 (54%) of patients with other genotype combinations. The effect of the combination of genotypes on survival and PFI was lost in a multivariate model that included response to chemotherapy as a confounding factor. This suggests that the combination of GSTM1 null/GSTT1 null is associated with outcome because of its influence on response to chemotherapy. These preliminary findings may provide a basis for the selection of patients for treatment with chemotherapeutic agents.     

Glutathione S-transferase M1 null genotype as a risk modifier for solvent-induced chronic toxic encephalopathy

OBJECTIVES: Exposure to organic solvents increases the risk of neuropsychiatric disability or chronic toxic encephalopathy (CTE). Polymorphisms in the biotransformation of xenobiotics and solvents may influence individual susceptibility to develop toxic effects. In this study the problem of whether there could be any association between the glutathione S-transferase M1 (GSTM1) null genotype and the risk for CTE, with regard to solvent exposure, was investigated. METHODS: Sixty patients referred to a clinic because of some degree of psychiatric or neurological symptoms, as well as exposure to solvents, were examined by means of a validated questionnaire and psychometric testing. The degree of exposure to solvents was assessed by a thorough interview. According to clinical findings, the patients were classified into three categories as those with solvent-induced CTE, those with incipient CTE, and those who were non-CTE patients. Afterwards, leukocyte DNA (deoxyribonucleic acid) was isolated and the GSTM1 null genotype was determined by an assay based on polymerase chain reaction, blindly with regard to both exposure and disease status. RESULTS: The relative proportion (RP) of GSTM1 null genotypes was significantly increased for patients with a diagnosed CTE when they were compared with non-CTE patients (RP 2.55, 95% confidence interval 1.0-6.2). Dichotomizing the patients by high and low exposure revealed an increased risk for both GSTM1 gene carriers and the GSTM1 null genotype in the high-exposure group, the relative risks (RR) being 4.5 and 7.9, respectively. The chi-square for the Mantel extension for trend was 6.2 (P = 0.025). CONCLUSIONS: The GSTM1 null genotype acts as a risk modifier for CTE among patients occupationally exposed to solvents. The risk seems to increase in a dose-dependent fashion.     

Expression in human prostate of drug- and carcinogen-metabolizing enzymes: association with prostate cancer risk

The role of two common polymorphisms of enzymes involved in the metabolism of drugs and carcinogens was studied in relation to prostate cancer. The gene encoding one of these enzymes (NAT2) is located in an area where frequent allelic loss occurs in prostate cancer. Mutations at the genes CYP2D6 and NAT2 were analysed by allele-specific polymerase chain reaction and restriction mapping in DNA from 94 subjects with prostate cancer and 160 male healthy control subjects. Eleven prostate specimens were analysed for genotype and enzymatic activities NAT2, CYP2D6 and CYP3A by using the enzyme-specific substrates sulphamethazine and dextromethorphan. Enzyme activities with substrate specificities corresponding to NAT2, CYP2D6 and CYP3A are present in human prostate tissue, with mean +/-s.d. activities of 4.8+/-4.4 pmol min(-1) mg(-1) protein, 156+/-91 and 112+/-72 nmol min(-1) mg(-1) protein respectively. The Km values for the prostate CYP2D6 and CYP3A enzyme activities corresponded to that of liver CYP2D6 and CYP3A activities, and the CYP2D6 enzyme activity is related to the CYP2D6 genotype. The N-acetyltransferase, in contrast, had a higher Km than NAT2 and was independent of the NAT2 genotype. The CYP2D6 and CYP3A enzymes, and an N-acetyltransferase activity that is independent of the regulation of the NAT2 gene, are expressed in human prostate tissue. The presence of carcinogen-metabolizing enzymes in human prostate with a high interindividual variability may be involved in the regulation of local levels of carcinogens and mutagens and may underlie interindividual differences in cancer susceptibility.     

Inhibition of cigarette smoke-related lipophilic DNA adducts in rat tissues by dietary oltipraz

The present study investigated the effects of dietary oltipraz on cigarette smoke-related lipophilic DNA adduct formation. Female Sprague-Dawley rats were exposed daily to sidestream cigarette smoke in a whole-body exposure chamber 6 h/day for 4 consecutive weeks. One group of rats was maintained on control diet while another group received the same diet supplemented with either a low (167 p.p.m.) or high (500 p.p.m.) dose of oltipraz, starting 1 week prior to initiation of smoke exposure until the end of the experiment. Analysis of lipophilic DNA adducts by the nuclease P1-mediated 32P-post-labeling showed up to five smoke-related adducts. Adduct no. 5 predominated in both the lung and the heart while adduct nos 3 and 2 predominated in the trachea and bladder, respectively. Quantitative analysis revealed that the total adduct level was the highest in lungs (270+/-68 adducts/10(10) nucleotides), followed by trachea (196+/-48 adducts/10(10) nucleotides), heart (141+/-22 adducts/10(10) nucleotides) and bladder (85+/-16 adducts/10(10) nucleotides). High dose oltipraz treatment reduced the adduct levels in lungs and bladder by >60%, while the reduction in lungs in the low-dose group was approximately 35%. In trachea, the effect of low and high dietary oltipraz on smoke DNA adduction was equivocal, while smoke-related DNA adducts in the heart were minimally inhibited by high-dose oltipraz. In a repeat experiment that employed a 3-fold lower dose of cigarette smoke, oltipraz (500 p.p.m.) was found to inhibit the formation of DNA adducts in rat lungs and trachea by 80 and 65%, respectively. These data clearly demonstrate a high efficacy of oltipraz in inhibiting the formation of cigarette smoke-induced DNA adducts in the target tissues.