Application Of Biomarkers For Human Risk Assessment

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A. Complex Mixture Exposures

Many studies have used DNA and protein adducts to assess potential sources of environmental carcinogen exposure. One important study examined a variety of molecular biomarkers to assess human exposure to complex mixtures of environmental pollution in Poland (125). Measurement of genotoxic damage in peripheral blood samples from residents of high-exposure regions indicated that environmental pollution was associated with significant increases in carcinogen-DNA adducts (polynuclear aromatic hydrocarbon [PAH]-DNA and other aromatic adducts), SCEs, chromosomal aberrations, and frequency of increased ras oncogene expression. Perera and colleagues (125) found that the presence of aromatic adducts on DNA was significantly correlated with chromosomal mutation, providing a possible link between environmental exposure and genetic alterations relevant to disease.

Tobacco smoke, the primary cause of lung cancer, contains several types of known carcinogens. The most abundant are PAHs, arylamines, and the tobacco-specific nitrosamines, including the lung-specific carcinogen NNK. These carcinogens are metabolically activated to reactive species that form specific DNA adducts. Smokers usually have significantly elevated levels of aromatic or hydro-phobic adducts, as compared with nonsmokers, and some studies found that DNA-adduct levels are linearly related to total smoking exposure (126). One investigation measured the level of bulky, hydrophobic DNA adducts in lung parenchyma of smokers and exsmokers by the 32P-postlabeling method. Smokers had fivefold higher levels of DNA adducts than exsmokers. A positive linear correlation between bulky adduct levels and CYP1A1 (AHH) activity was found in smokers. A statistically significant correlation was determined when comparing pulmonary microsomal AHH activity and the level of BPDE-DNA adducts (r = 0.91; P < 0.01) (34). Additionally, BPDE-DNA adducts have been detected in oral mucosa cells of smokers and nonsmokers, and levels of DNA damage were elevated in each of 16 smokers compared with 16 age-, race-, and sex-matched nonsmokers. There was about a threefold range between smokers and non-smokers (127).

One study (128) detected PAH-DNA adducts in specific subsets of peripheral white blood cells (WBCs) and found that DNA combined from lymphocyte and monocyte fractions of smokers exhibited elevated levels of DNA adducts when compared with nonsmokers. The elevated levels of PAH-DNA adducts in DNA obtained from the WBCs of smokers compared with nonsmokers suggest that only certain subsets of WBCs are a valid, readily accessible source for monitoring genotoxicity from cigarette smoke.

There was a decline of PAH-DNA adducts and 4-aminobiphenyl-hemoglobin (4-ABP-Hb) adducts in peripheral blood after smoking cessation, based on serial samples from 40 heavy smokers (> 1 pack/day for > 1 year). The substantial reduction (50% to 75%) of PAH-DNA and 4-ABP-Hb adduct levels after quitting indicates these carcinogen adducts are reflective of smoking exposure (129). This is essential information in the validation of biomarkers. The estimated half-life of the PAH-DNA adducts in leukocytes was 9 to 13 weeks; the estimated half-life of the 4-ABP-Hb adducts was 7 to 9 weeks. Women had higher levels of 4-ABP-Hb adducts at baseline and after smoking cessation. These results show that PAH-DNA and 4-ABP-Hb adducts can be useful as intermediate biomarkers verifying smoking cessation and possibly for identifying persons who are at increased risk of cancer from exposure to cigarette smoke because of high levels of carcinogen binding.

In another study (130) immunohistochemical quantitation of 4-ABP-DNA adducts and p53 nuclear overexpression in T1 bladder cancer of smokers and nonsmokers was described. Mean relative staining intensity for 4-ABP-DNA ad-ducts was significantly higher in current smokers compared with nonsmokers. There was a linear relationship between the mean level of relative staining and the number of cigarettes smoked, with lower levels in the 1 to 19 cigarette/day group, compared with the 20 to 40 and the more than 40 cigarette/day group. Nuclear overexpression of p53 was observed in 27 (59%) of the 45 stage T1 tumors analyzed. Nuclear staining of p53 was correlated with smoking status, cigarettes/day, and 4-ABP-DNA adducts. The 4-ABP-DNA adducts in 11 human lung and 8 urinary bladder mucosa specimens were analyzed by alkaline hydrolysis and negative chemical ionization GC/MS. Adduct levels were found to be 0.32 to 49.5 adducts/108 nucleotides in the lung and 0.32 to 3.94 adducts/108 nu-cleotides in the bladder samples (131).

Carcinogen-DNA adducts in human breast tissue samples have been reported by Perera et al (132), who analyzed a total of 31 breast tissue samples, which included tumor and tumor-adjacent tissues from 15 women with breast cancer and normal tissue samples from four women undergoing breast reduction. Among the breast cancer cases, the mean aromatic/hydrophobic-DNA adduct level assayed was roughly twofold higher, compared with the noncancer patients. Five of 15 tissues from the cases displayed a pattern of adducts associated with tobacco smoke exposure, and all these positive samples were from current smokers. Tissue samples from the eight nonsmoking cases did not exhibit this pattern. This study indicated that biomarkers may be useful in investigating specific environmental exposures that could contribute to breast cancer.

Alkylating agents such as N-nitroso compounds are potential human carcinogens. Humans are known to be exposed to N-nitrosoamines from diet, work place, cigarette smoke, and through endogenous formation. These compounds alkylate DNA, which leads to formation of various types of DNA adducts.

Among them are 7-alkyl-2-deoxyguanosine (dG) adducts, such as 7-methyl-dGp and 7-ethyl-dGp. Several investigations have focused on the levels of 7-methyl-dG adducts in human lung tissue (133). Higher levels have been found in smokers compared with nonsmokers (134). Separately, 7-methyl-dG levels in lung tissues have been associated with cytochrome P4502D6 and 2E1 genetic polymorphisms (135). One study analyzed N7-alkylguanine adducts levels in DNA in a group of 46 patients with larynx tumors by the 32P-postlabeling method. The average level of N7-alkylguanines was 26.2/107 nucleotides in tumor cells, 22.7/107 in nontumor cells, and 13.1/107 in blood leukocytes. Men and smokers had significantly higher levels of adducts than women and nonsmokers (136). In another study, 7-alkyl-2-dG adducts were measured in eight separate lung segments of ten autopsy specimens; 7-methyl-dGp levels were detected in all eight samples. 7-Ethyl-dGp was detected in all but five of the samples (ranging from < 0.1 to 7.1 adducts/107 dG). 7-Methyl-dG levels were approximately 1.5-fold higher than 7-ethyl-dG levels and were positively correlated with each other in most individuals. There was no consistent pattern of adduct distribution in the different lung lobar segments (137).

A wide variety of aromatic amines and PAHs have been found to bind at high levels to hemoglobin (138). One of the carcinogen-Hb adducts that has been well characterized is formed by the potent urinary bladder carcinogen, 4-ABP. Several studies have reported 4-ABP-Hb adducts in human blood specimens

(139). The results of these studies indicate that the presence of 4-ABP-Hb ad-ducts is closely associated with three major risk factors for bladder cancer: cigarette smoking, the type of tobacco smoked, and acetylator phenotype. One report

(140) described the relation between exposure to environmental tobacco smoke (ETS) and levels of 4-ABP-Hb adducts in nonsmoking pregnant women, compared with adduct levels in those women who smoked during pregnancy. A questionnaire on smoking and exposure to ETS was administered to pregnant women. Samples of maternal blood and cord blood were collected during delivery and analyzed for 4-ABP-Hb adducts by GC/MS. The mean adduct level in smokers was approximately ninefold higher than that in nonsmokers. Among nonsmokers, the levels of 4-ABP-Hb adducts increased with increasing ETS level. This relationship between ETS exposure and 4-ABP-Hb adduct levels supports the concept that ETS is a probable hazard during pregnancy.

As previously described, metabolic polymorphism, both in NAT and in CYP1A2, is also expected to affect the formation of 4-ABP-DNA- and Hb-adducts. One study (141) determined levels of DNA adducts in bladder cells and 4-ABP-Hb adducts in 79 individuals, together with the acetylator phenotype and genotype. Among the slow acetylators, levels of 4-ABP-Hb adducts were significantly higher compared with those present in rapid acetylators. This study indicated that clearance of low-dose carcinogen is decreased in the slow acetylator phenotype. Because the highest levels of adducts were found in individuals with rapid N-oxidation (CYP1A2) and slow N-acetylation (NAT2) phenotype, determination of phenotypes and genotypes may provide a better prediction and assessment of human cancer risk.

Yu et al (142) found that mean 3- and 4-ABP-Hb adduct levels in 151 subjects were statistically significantly higher in cigarette smokers compared with nonsmokers and that the level increased with increasing number of cigarettes smoked/day. Again, slow acetylators consistently exhibited higher mean levels of ABP-Hb adducts compared with rapid acetylators. The mean level of 4-ABP-Hb adducts was higher in subjects possessing the GSTM1-null versus GSTM1-non-null genotype (46.5 vs 36.0 pg/g Hb; P = 0.037). In another study (143), the polymorphic distribution of the CYP1A2 and NAT2 phenotypes was examined in relation to ABP-Hb adduct formation in 97 healthy men. Rapid oxidizers and subjects with the combined slow acetylator-rapid oxidizer phenotype showed the highest ABP-Hb adduct levels at a low smoking dose. However, in a subset of 45 available samples, no association was seen between ABP-Hb adduct levels and GSTM1 genotype.

B. Occupational Carcinogen Exposures

Occupational exposure to many chemical carcinogens has been well documented. Since the initial reports of Rehn in aniline dye workers, many laboratories have applied technologies for measuring these carcinogens and their metabolites to confirm exposure in the workplace (9). The occurrence of DNA adducts in exfoliated urothelial cells of a worker exposed to the aromatic amine, 4,4-methyl-ene-bis(2-chloroaniline) (MOCA), an agent that induces lung and liver tumors in rodents and urinary bladder tumors in dogs, has been reported (144). 32P-postlabeling analysis revealed the presence of a single, major DNA adduct that cochroma-tographed with the major N-hydroxy-MOCA-DNA adduct, N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, formed in vitro. In a recent study, PAH-DNA adducts in WBC and 1-hydroxypyrene in urine were examined in a group of 105 workers from an aluminum plant with different PAH exposures. The exposure to PAH was measured by personal monitoring, and ranged from 0.4 to 150 |g/m3. High exposure to PAH in the work atmosphere was associated with increased concentration of 1-hydroxypyrene in the urine. Polynuclear aromatic hydrocarbon-DNA adducts were detected in 93% of the worker samples. Workers with a high PAH exposure had significantly higher adduct levels than those with a low PAH exposure. A good correlation was found between PAH exposure and the average PAH-adduct values in blood. A statistically significant correlation was also found between the average adduct values and the concentration of 1-hydroxypyrene in the urine of smokers (145).

In another report (146), PAH-DNA adduct levels were examined in workers exposed to ambient air pollution. Significantly higher adduct levels were found in bus drivers working in central Copenhagen, compared with those driving in suburban areas. The urban drivers had higher adduct levels than rural controls. No significant influence on adduct level by potential confounders, including smoking and diet, was observed. A separate study (147) measured BPDE-DNA ad-ducts in 39 coke oven workers (exposed to PAH) and 39 nonexposed controls (each group consisting of smokers and nonsmokers). Adducts were detected in 51% of workers and in 18% of controls. The mean level in workers (15.7 x 108 nucleotides) was 15 times higher than in nonexposed controls. Although large interindividual variations were noted, smoking workers had 3.5 times more adducts than nonsmokers.

C. Cancer Risks For Environmental Carcinogen Exposures

The DNA and protein adducts can serve as biomarkers for both exposure and for cancer risk. This approach has been applied in several published human epidemi-ological studies. A nested case-control study initiated in 1986 in Shanghai was started to examine the relationship between biomarkers for aflatoxin and HBV and the development of liver cancer (148, 149). In this study, over 18,000 urine samples were collected from healthy men between the ages of 45 and 64. In the subsequent 7 years, 50 of these individuals had liver cancer develop. The urine samples for cases were age-matched and residence-matched with controls and analyzed for both aflatoxin biomarkers and hepatitis B surface antigen (HBsAg) status. A highly significant increase in the relative risk (RR) (3.5) was observed for those liver cancer cases where urinary aflatoxin biomarkers (AFBrN7-guanine + other AFBX metabolites) were detected. The RR for people who tested positive for the HBsAg was about 8, but individuals with both urinary aflatoxin biomarkers and positive HBsAg status had an RR for developing liver cancer of 57. These results showed for the first time a relationship between the presence of carcinogen-specific biomarkers and cancer risk. Moreover, these findings provided the first demonstration of a multiplicative interaction between these two major risk factors for liver cancer. Also, when individual aflatoxin metabolites were stratified for liver cancer outcome, the presence of the AFB1-N7-guanine in urine always resulted in a two- to threefold elevation in risk of having liver cancer develop (149).

Another study investigated PAH-DNA adducts in peripheral leukocytes from 119 non-small-cell lung cancer patients and 98 controls. Among them, 31 cases had adduct measurements in leukocytes, lung tumor, and nontumor specimens collected at surgery and 34 had paired leukocyte and tumor specimens. After adjustment for age, gender, ethnicity, season, and smoking, DNA adducts in leukocytes were significantly higher in cases than controls; the OR was 7.7 (95% Cl: 1.7-34; P < 0.01). The DNA adducts in leukocytes were increased significantly in smokers and exsmokers compared with nonsmokers among cases and controls (separately and combined) after adjusting for age, gender, ethnicity, and season (150).

Two studies used aflatoxin-albumin adducts as biomarkers for monitoring liver cancer risks. One nested case-control study (151) was carried out in Taiwan. A cohort of 8,068 men was followed up for 3 years, and 27 cases of HCC were identified and matched with 120 healthy controls. Serum samples were analyzed for AFB1-albumin adducts by ELISA. The proportion of subjects with detectable serum AFBralbumin adducts was higher for HCC cases (74%) than matched controls (66%), giving an OR of 1.5. There was a statistically significant association between detectable level of AFB1-albumin adduct and HCC risk among men younger than age 52, showing a multivariant-adjusted OR of 5.3, although no association was observed between AFB1-albumin adduct level and HBsAg carrier status. Another prospective nested case-control study (152) was instituted in Qidong County, Jiangsu Province, China, where liver cancer accounts for 10% of all adult deaths, and both HBV and aflatoxin exposures are common. Serum samples from 804 healthy HBsAg-positive individuals (728 men, 76 women, aged 30 to 65, were obtained and stored frozen in 1991. Between the years 1993 to 1995, 38 of these individuals developed liver cancer. The serum samples for 34 of these patients were matched by age, gender, residence, and time of sampling to 170 controls. Serum AFB1-albumin adduct levels were determined by RIA. The RR for HCC among AFB1-albumin positive individuals was 2.4.

McGlynn et al (153) have reported that mutant alleles at two AFBj detoxification gene loci (epoxide hydrolase [EPHX] and GSTM1) were significantly overrepresented in individuals with detectable serum AFB1-albumin adducts in a cross-sectional study. Mutant alleles of EPHX were also significantly overrepre-sented in persons with HCC in a case-control study. The relationship of EPHX to HCC varied with HBsAg status and suggested that a synergistic effect may exist. Mutations of p53 at codon 249 were only observed among HCC patients with one or both mutated EPHX and GSTM1 genotypes. These results indicate that individuals with mutant genotypes at EPHX and GSTM1 may be at greater risk of developing AFB1 adducts, p53 mutations, and HCC when exposed to AFB1. Hepatitis B surface antigen carriers with these high-risk genotypes may be an even greater risk than carriers with low-risk genotypes. These findings further support the existence of genetic susceptibility in humans to AFB1 exposure and indicate that there is a synergistic increase in risk of HCC with the combination of HBV infection and susceptible genotype.

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