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Vinyl chloride (VC) is both a known carcinogen and a regulated chemical, and its production capacity has almost doubled over the last 20 years, currently 27 million tons/year worldwide. According to recent reports it is still a cause for concern. VC has been found as a degradation product of chloroethylene solvents (perchloroethylene and trichloroethylene) and in landfill gas and groundwater at concentrations up to 200 mg/m(3) and 10 mg/L, respectively. Worldwide occupational exposure to VC still seems to be high in some countries (e.g., averages of approximately 1,300 mg/m(3) until 1987 in one factory), and exposure may also be high in others where VC is not regulated. By combining the most relevant epidemiologic studies from several countries, we observed a 5-fold excess of liver cancer, primarily because of a 45-fold excess risk from angiosarcoma of the liver (ASL). The number of ASL cases reported up to the end of 1998 was 197 worldwide. The average latency for ASL is 22 years. Some studies show a small excess risk for hepatocellular carcinoma, and others suggest a possible risk of brain tumors among highly exposed workers. Lung cancer, lymphomas, or leukemia do not seem to be related to VC exposure according to recent results. The mutation spectra observed in rat and human liver tumors (ASL and/or hepatocellular carcinoma) that are associated with exposure to VC are clearly distinct from those observed in sporadic liver tumors or hepatic tumors that are associated with other exposures. In rats, the substitution mutations found at A:T base pairs in the ras and p53 genes are consistent with the promutagenic properties of the DNA adduct 1,N(6)-ethenoadenine formed from VC metabolites. Risk assessments derived from animal studies seem to overestimate the actual risk of cancer when comparing estimated and reported cases of ASL. Images Figure 1 Figure 2

The methods applied for DNA adduct determination in humans have become more reliable. Yet there is a need to characterize the adducts studied better and when possible, to identify them with the help of the available standard compounds. Use of standard compounds also allows quantification of adduct levels. There is a lack of knowledge on the adduct levels and their half-lives in target and surrogate tissues. Most adduct studies have been carried out on occupational populations exposed to complex mixtures. White blood cells have been the most common source of DNA. Other exposures and tissues should be a subject of study. Notably, dietary exposures have been largely neglected. Biomonitoring of mutations is a relatively new field and a few exposures have so far been investigated. The results have been promising but logistics of the studies have to be improved to make large field studies possible. Future biomonitoring studies should make an effort to combine many end points, with emphasis on adducts, mutations, and constitutional metabolic factors.

Quantitation of DNA adducts in human tissues has been achieved with highly sensitive techniques based on adduct radiolabeling, antisera specific for DNA adducts or modified DNA, and/or adduct structural characterization using chemical instrumentation. Combinations of these approaches now promise to elucidate specific adduct structures and provide detection limits in the range of 1 adduct/10(9) nucleotides. Documentation of human exposure and biologically effective dose (i.e., chemical bound to DNA) has been achieved for a wide variety of chemical carcinogens, including polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic amines, aflatoxins, nitrosamines, cancer chemotherapeutic agents, styrene, and malondialdehyde. Due to difficulties in exposure documentation, dosimetry has not been precise with most environmental and occupational exposures, even though increases in human blood cell DNA adduct levels may correlate approximately with dose. Perhaps more significant are observations that lowering exposure results in decreasing DNA adduct levels. DNA adduct dosimetry for environmental agents has been achieved with dietary contaminants. For example, blood cell polycyclic aromatic hydrocarbon-DNA adduct levels were shown to correlate with frequency of charbroiled meat consumption in California firefighters. In addition, in China urinary excretion of the aflatoxin B1-N7-guanine (AFB1-N7-G) adduct was shown to increase linearly with the aflatoxin content of ingested food. Assessment of DNA adduct formation as an indicator of human cancer risk requires a prospective nested case-control study design. This has been achieved in one investigation of hepatocellular carcinoma and urinary aflatoxin adducts using subjects followed by a Shanghai liver cancer registry. Individuals who excreted the AFB1-N7-G adduct had a 9.1-fold adjusted increased relative risk of hepatocellular carcinoma compared to individuals with no adducts. Future advances in this field will be dependent on chemical characterization of specific DNA adducts formed in human tissues, more-precise molecular dosimetry, efforts to correlate DNA adducts with cancer risk, and elucidation of opportunities to reduce human DNA adduct levels.

Wild city pigeons were caught at four different locations in the Netherlands to represent areas of high (Amsterdam-high), moderate (Amsterdam-medium), and low (Maastricht and Assen) traffic density. It is assumed that local ambient air pollution decreases as a function of traffic density. In these pigeons levels of polycyclic aromatic hydrocarbon (PAH)-DNA adducts, oxidative DNA damage, and heavy metal residues were determined in kidney, lung, liver, and blood (no adduct analysis in blood). The contribution of leaded gasoline to total body lead content was estimated by measuring concentrations of Pb and its isotopes in blood. We also analyzed samples of ambient air particulate matter for PAH and heavy metal concentrations at the four different locations. Interregional differences in heavy metals in ambient air particulate matter were reflected relatively well by pigeon body loads. The higher lead and cadmium concentrations in blood, kidney, liver, and lung were found in the Amsterdam high traffic density area, followed by Amsterdam medium, Assen, and Maastricht. A high Pb concentration in blood coincided with relatively low 206Pb/207Pb values, indicating a high contribution of leaded gasoline to total blood Pb concentrations in pigeons from the Amsterdam high traffic density area. Significantly enhanced blood zinc values were found in pigeons from both locations in Amsterdam compared to pigeons from the other two areas. However, no differences in Zn tissue levels between the four different groups were found. Oxidative DNA damage, determined as the ratio of 7-Hydro-8-oxo-2'-deoxyguanosine/ deoxyguanosine, in pigeon liver was highest in Amsterdam-high, followed by Assen (low traffic density). Pb content, but not the Cd content, was positively associated with oxidative DNA damage in liver tissue. In lung tissue, a negative correlation was found between oxidative DNA damage and Zn content. These results indicate that the carcinogenic potential of Pb might be ascribed to oxygen radical formation, whereas Zn plays a protective role against oxidative DNA damage. Places with high and medium traffic density could be clearly discriminated on the basis of PAH levels in the ambient air. The PAH content in particulate air samples was not, however, reflected in total PAH-related DNA adduct levels because no differences could be observed in tissue adduct levels in pigeons from the four different locations. Our results indicate that wild city pigeons can be used as biological indicators of exposure to heavy metal pollution in outdoor air. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. A Figure 5. B Figure 5. C Figure 5. D

We have investigated the formation of DNA adducts in the bone marrow and white blood cells of male B6C3F1 mice treated with benzene using P1-enhanced 32P-postlabeling. No adducts were detected in the bone marrow of controls or mice treated with various doses of benzene once a day. After twice-daily treatment for 1 to 7 days with benzene, 440 mg/kg, one major (no. 1) and up to two minor DNA adducts were detected in both the bone marrow and white blood cells. The relative adduct levels in these cells ranged from 0.06 to 1.46 x 10(-7). a significant correlation (r2 = 0.95) between levels of adducts in bone marrow and white blood cells was observed. After a 7-day treatment with benzene, 440 mg/kg twice a day, the number of cells per femur decreased from 1.6 x 10(7) to 0.85 x 10(7), indicating myelotoxicity. In contrast, administration of benzene once a day produced only a small decrease in bone marrow cellularity. The observed induction of toxicity in bone marrow was paralleled by formation of DNA adducts. In vitro treatment of bone marrow with hydroquinone (HQ) for 24 hr produced the same DNA adducts as found after treatment of mice with benzene, suggesting that HQ is the principal metabolite of benzene leading to DNA adduct formation in vivo. Using P-postlabeling the principal DNA adduct formed in vivo was compared with N2-(4-hydroxyphenyl)-2'-deoxyguanosine-3'-phosphate. The results of this comparison demonstrated that the DNA adduct formed in vivo co-chromatographs with N2-(4-hydroxyphenyl)-2'-deoxyguanosine-3'-phosphate. These studies indicate that metabolic activation of benzene leads to the formation of DNA adducts in bone marrow and white blood cells and suggest that measurement of DNA adducts in white blood cells may be an indicator of biological effect following benzene exposure. Images Figure 1. A Figure 1. B Figure 1. C Figure 1. D Figure 2. Figure 3. A Figure 3. B Figure 4. A Figure 4. B

We report here a compilation of p53 mutation results from two smoking-related cancers, lung cancer and bladder cancer. The overall mutation frequencies reported for these two types of cancer were relatively similar--50% in lung cancer and 40% in bladder cancer. The compiled data from lung cancer and bladder cancer suggest an increasing proportion of patients with p53 mutations in nonsmokers, former smokers, and current smokers, in that order, in both cancer groups. Taken together, more than half (55% and 56% for lung cancer and bladder cancer, respectively) of the patients who continued smoking (CS), less than 40% (38% and 38%) of those who had stopped smoking before > or = 1 or > or = 5 years) clinical diagnosis (ES), and less than 30% (25% and 29%) of those who were nonsmokers (NS) had a p53 mutation. The differences seen in the mutation frequencies between the three smoking groups did not, however, reach statistical significance (lung cancer--CS vs ES: odds ratio [OR] = 2.0, 95% CI 0.7-5.4; CS vs NS: OR = 3.7, 95% CI 0.4-37; bladder cancer--CS vs ES: OR = 2.1, 95% CI 0.6-7.9; CS vs NS: OR = 3.1, 95% CI 0.7-13). Guanine to thymine transversions were the most common type in lung cancer followed by guanine to adenine transitions. In bladder cancer, on the contrary, G:C to A:T transitions at cytosine-guanine dinucleotide sites were the most frequently detected base substitutions. Analysis of the compiled p53 mutation data suggest that, in addition to lifetime cumulative exposure to cigarette smoke, also stopping smoking for years prior to clinical manifestation of the disease may affect the incidence of p53 mutations. The differences in the mutation profiles appear to support the view that the main genotoxic agents from cigarette smoke exposure may be different in bladder cancer as compared to lung cancer, as suggested previously by DNA adduct studies.

The effect of interactions between urban air particulates (UAP) and carcinogens on bioavailability, metabolism, and DNA binding was studied in the isolated perfused and ventilated rat lung. The availability of benzo[a]pyrene (B[a]P) varied from 29 to 60% after intratracheal doses of carcinogen particulates dissolving extremely slow and fast, respectively. Several cytochrome P450 enzyme (P450) inducers acting as 2,3,7,8-tetrachlorodibenzo[p]dioxin-receptor ligands have been identified in UAP extracts. beta-Naphthoflavone (BNF) was used to study how P450 induction alters the lung metabolism of carcinogens. Pretreatment increased the lung clearance for B[a]P 8-fold and for 2-nitrofluorene (2NF) by a factor of four from 0.55 +/- 0.06 ml/min to 2.37 +/- 0.62 ml/min. Studies with the intact lung and with isolated lung cells show that carcinogen metabolism and pharmacokinetics depend both on the route of exposure and dosage and on the distribution of specific enzymes. A cytochrome P450IIB1 enzyme was detected in lung epithelial cells where it catalyzes 9-hydroxylation of 2NF. This rat lung 2NF-9-hydroxylation capacity increases in parallel with the age dependent up-regulation of lung P450IIB1 expression. Both human and rat lung tissue have the capacity to form 9-hydroxy-2-nitrofluorene (9-OH-2NF) that is mutagenic. A BNF-inducible P450IA1 was detected in endothelial and alveolar type II cells. Consequently, aromatic hydroxylation dominated when 2NF was dosed directly into the lung circulation. Pretreatment of rats with BNF before intratracheal B[a]P dosage induced lung cytochrome P450IA1. The 7,8-dihydroxy-9,10-oxybenzo[a]pyrene-deoxyguanosine adduct and the total lung DNA adduct levels increased significantly from a peak level of 75 +/- 8 to 151 +/- 19 fmole/mg DNA in lungs from control and BNF pretreated rats, respectively. 3-Amino-1,4-dimethyl-5H-pyridol-(4,3)-indole (TRP-P1) is a potent mutagen and carcinogen identified in UAP extracts. Dietary BNF pretreatment of rats altered the [14C]TRP-P1 distribution as analyzed by whole-body autoradiography. An enhanced retention was observed in the small intestine, forestomach, esophagus, and lung. UAP catalyzes oxygen radical formation and deoxyguanosine-8-hydroxylation, which were inhibited when the UAP samples were extracted with organic solvents or when they were incubated in the presence of desferroxamine. We therefore postulate that a polycyclic aromatic hydrocarbon autooxidation pathway may be responsible for generation of hydrogen peroxide, which may be further converted to hydroxyl radicals through an iron-dependent reaction. Images Figure 3.

In an attempt to elucidate the relationship between DNA adduct formation and tumorigenesis, DNA adducts were measured in the livers and bladders of mice during chronic exposure to several different doses of 2-acetylaminofluorene (2-AAF) and 4-aminobiphenyl (4-ABP). Continuous oral administration of these compounds for 4 weeks produced an increase in DNA adduct formation during the first 2 weeks, followed by a plateau, which presumably occurred because the rate of adduct removal offset the rate of adduct formation. The quantity of DNA adducts present at equilibrium correlated directly with the carcinogen concentration; therefore, when exposure was continued for 4 weeks, DNA adducts that reflected the plateau level at each dose could be expressed as a function of dose. Liver and bladder DNA adduct profiles thus obtained during administration of multiple doses of 2-AAF (to female mice) and 4-ABP (to male and female mice) were compared to profiles for tumor incidences obtained during lifetime exposures to the same doses. These experiments demonstrated similar profiles for DNA adduct formation and tumorigenesis in liver. In the bladder, DNA adducts were linear, but tumors only appeared at the higher doses in conjunction with cell proliferation. In addition to these aromatic amines, similar data are available for aflatoxin B1, diethylnitrosamine, and (methylnitrosamino)-1-(3-pyridyl)-1-butanone (also known as nicotine-derived nitrosoketone). Of the nine different biological situations (carcinogen/species/sex/organ) for which data are available, correlations between steady-state DNA adduct levels and tumorigenic response at the different doses were linear in five of the nine biological models.(ABSTRACT TRUNCATED AT 250 WORDS)

Certain aromatic amines carcinogenic for the human urinary bladder, such as 4-aminobiphenyl, undergo hepatic metabolic activation to N-hydroxylamines, which are transported to the bladder. During the transport process, these reactive species come in contact with hemoglobin and react with this blood protein. The principal hemoglobin adduct formed is a cysteine sulfinamide, and quantitative methods have been developed for the analysis of sulfinamide adducts at the levels present in ordinary human blood specimens. N-acetylation is an alternative metabolic fate to N-hydroxylation. The amount of hemoglobin adduct is decreased to the extent that this pathway is increased relative to N-hydroxylation. Thus, the hemoglobin adduct is sensitive to dose, cytochrome P-450-mediated activation, and N-acetyltransferase-mediated detoxification. In addition, it has been shown that DNA adduct concentration of 4-aminobiphenyl present in human bladder epithelial cells is significantly associated with hemoglobin adduct levels. Thus, the hemoglobin adduct of 4-aminobiphenyl, and perhaps several other aromatic amines, is a good dosimeter for the target tissue dose of the ultimate carcinogenic metabolite of these amines. Several studies have been undertaken in which the hemoglobin adducts of aminobiphenyls in human blood specimens were determined quantitatively. Information concerning exposure status and acetylator phenotype of the same individuals was obtained simultaneously. The results of these studies indicate that the hemoglobin adduct of 4-aminobiphenyl is closely associated with three major risk factors for bladder cancer: cigarette smoking, type of tobacco smoked, and acetylator phenotype. They also support a major etiologic role for aromatic amines in much of human bladder cancer.

Development of methods to evaluate certain classes of polycyclic aromatic compounds (PAC) detected in complex mixtures to which humans are exposed would greatly improve the diagnostic potential of 32P-postlabeling analysis. Identification of DNA adduct patterns or specific exposure-related marker adducts would strengthen associations between observed DNA adducts and exposures to different environmental pollutants (e.g., kerosene, cigarette smoke, coke oven, and diesel). We have compared diesel-modified DNA adduct patterns in various in vitro and in vivo rodent model systems and compared them to DNA reactive oxidative and reductive metabolites of 1-nitropyrene. The formation of nitrated polycyclic aromatic hydrocarbon (nitrated PAH) DNA adducts, derived from the metabolism of diesel extract constituents, was enhanced relative to other PAH-derived DNA adducts via xanthine oxidase-catalyzed nitroreduction. These adducts were detectable only by the butanol extraction version of the postlabeling analysis. Five major DNA adducts were detected in human lymphocytes treated in vitro with diesel extract. A major adduct detected in human lymphocytes treated in vitro with diesel extract comigrated with a major adduct detected in lymphocyte DNA treated with benzo[a]pyrene (BaP) alone. Other adducts that co-migrated with the major BaP-derived adducts were detected in skin and lung DNA isolated from rodents topically treated with (50 mg) diesel extract and the major adduct detected in calf thymus DNA treated with rat liver S9 and diesel particle extract. Postlabeling of lung DNA isolated from rodents exposed via lung inhalation for 24 months to diesel combustion emissions resulted in the formation of a major nuclease-P1-sensitive DNA adduct that did not co-migrate with the major BaP-diol epoxide adduct.(ABSTRACT TRUNCATED AT 250 WORDS) Images FIGURE 1. a FIGURE 1. b FIGURE 1. c FIGURE 2. FIGURE 3.

Tobacco smoking is associated with an increased risk of cancer in a number of organs, including bladder and lung. Tobacco smoke contains at least 50 known chemical carcinogens that exert their biological effects through their covalent binding to cellular DNA. Examining human DNA for the presence of altered nucleotides is a means of monitoring exposure to genotoxic chemicals. DNA isolated from 73 human bladder biopsies has been analyzed by 32P-postlabeling for the presence of aromatic/hydrophobic adducts. Butanol extraction of DNA digests resulted in up to a 3-fold greater recovery of adducts than nuclease P1 digestion. Among 16 nonsmokers, adduct levels were in the range 3.2-20.8/10(8) nucleotides (mean 9.7). Eight ex-smokers had values in the range 2.6-12.3 (mean 7.1). Thirteen smokers had adduct levels between 1.3 and 26.7 adducts/10(8) nucleotides (mean 9.5, not different from nonsmokers). Six cigar smokers had higher levels of adducts (mean 12.1, range 7.3-15.0), but pipe smokers did not (five samples, mean 8.6, range 2.9-12.7). A further 8 samples from nonsmokers and 17 from smokers were examined in more detail. Although most of the DNA binding appears not to be smoking related, the levels of one adduct were found to be on average 2-fold higher in smokers (p < 0.005, one-tailed t test). Studies on tissues of the respiratory tract demonstrate a correlation between DNA adduct levels and exposure to tobacco smoke. Evidence to date on the influence of smoking on adducts in peripheral blood cells is equivocal; some studies demonstrate a significant effect, whereas others do not.(ABSTRACT TRUNCATED AT 250 WORDS) Images FIGURE 1. a FIGURE 1. b FIGURE 1. c FIGURE 1. d

The high lung cancer rate in Xuan Wei, China, is associated with smoky coal use in unvented homes, but not with wood or smokeless coal use. Smoky coal combustion emits higher polycyclic aromatic hydrocarbon (PAH) concentrations than wood combustion. This study used DNA adducts as biomarkers for human exposure to PAH from combustion emissions. DNA adducts were determined by enzyme-linked immunosorbent assays (ELISA) in placentas and peripheral and cord white blood cells (WBC) from Xuan Wei women burning smoky coal or wood and from Beijing women using natural gas. Color ELISA gave positive results in 58, 47, and 5% of the placentas from Xuan Wei women burning smoky coal without and with chimneys, and from Beijing women, respectively. Fluorescence ELISA indicated that 46, 65, 56, and 25% of placentas were positive from Xuan Wei women who lived in houses without and with chimneys, Xuan Wei women burning wood, and Beijing controls, respectively. Peripheral WBC samples were positive in 7/9, 8/9, and 3/9 for the Xuan Wei women who lived in houses without and with chimneys and Beijing women, respectively. PAH-DNA adducts were detected in a higher percentage of placentas from Xuan Wei women living in houses exposed to smoky coal or wood emissions than from those of the Beijing controls. No dose-response relationship was observed between the air benzo[alpha]pyrene concentrations and DNA adduct levels or percentage of detectable samples. The results suggest that DNA adducts can be used as a qualitative biomarker to assess human exposure to combustion emissions.

Human epidemiology and experimental animal data have provided the statistical association and biological information necessary to propose that aflatoxins are risk factors for human liver cancer. As liver cancer causes at least 200,000 deaths per year, prevention measures must be developed to ameliorate this nearly always fatal disease. Preventive strategies will be facilitated by the identification of individuals at high risk. It is the goal of the molecular dosimetry field to provide facile and accurate biomarkers to identify people at high risk for carcinogen exposure and consequent adverse health effects. We have developed methods to defect the major aflatoxin DNA adduct, aflatoxin N7-guanine (AFB-N7-guanine), in urine, examined the dose-response characteristics in people living in China and The Gambia, and have found an excellent association of this biomarker with exposure. In addition to exposure studies in people, our laboratories have monitored AFB-N7-guanine excretion in the urine of rats whose risk for developing cancer has been modulated with dietary chemoprotective agents such that independent groups of animals receiving the same dosage of aflatoxin B1 were at either high or low risk for tumorigenesis. The production of DNA damage by aflatoxins is not the exclusive mechanism for liver cancer. Many other factors, including hepatitis B virus, cell proliferation, and nutritional status, can exert strong modification effects in human disease. Thus, molecular epidemiological investigations that examine only one biomarker may greatly underestimate or overestimate the risk for an individual.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined DNA adduct formation in HL-60 cells and human bone marrow treated with either hydroquinone or p-benzoquinone and have found that these treatments produce the same DNA adduct in both cell types. The DNA adduct level from these treatments varied from 0.05 to 7.5 adducts per 10(7) nucleotides as a function of treatment time and concentration for both compounds. Reaction of calf thymus DNA with p-benzoquinone produced three adducts as detected by 32P-postlabeling. These adducts have been identified as (3'-hydroxy)-3,N4-benzetheno-2'-deoxycytidine-3'-phosphate; (3'-hydroxy)-1,N6-benzetheno-2'-deoxyadenosine-3'-phosphate; and (3'-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine-3'-phosphate. The DNA adduct formed in HL-60 cells did not correspond to any of the principal adducts formed in DNA reacted with p-benzoquinone, suggesting that cellular environment modifies DNA adduct production by p-benzoquinone. These studies demonstrate that DNA adduct formation occurs in human bone marrow treated with benzene metabolites and suggest that P1-enhanced 32P-postlabeling may be used to detect DNA adducts resulting from benzene exposure. Images FIGURE 1. FIGURE 3. A FIGURE 3. B FIGURE 3. C FIGURE 4. A FIGURE 4. B FIGURE 6.

Human cancer risk assessment at a genetic level involves the investigation of carcinogen metabolism and DNA adduct formation. Wide interindividual differences in metabolism result in different DNA adduct levels. For this and other reasons, many laboratories have considered DNA adducts to be a measure of the biologically effective dose of a carcinogen. Techniques for studying DNA adducts using chemically specific assays are becoming available. A modification of the 32P-postlabeling assay for polycyclic aromatic hydrocarbon DNA adducts described here provides potential improvements in quantification. DNA adducts, however, reflect only recent exposure to carcinogens; in contrast, genetic testing for metabolic capacity indicates the extent to which carcinogens can be activated and exert genotoxic effects. Such studies may reflect both separate and integrated risk factors together with DNA adduct levels. A recently described restriction fragment length polymorphism for the CYP1A1, which codes for the cytochrome P450 enzyme primarily responsible for the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, has been found to be associated with lung cancer risk in a Japanese population. In a subset of individuals enrolled in a U.S. lung cancer case-control study, no association with lung cancer was found. Images FIGURE 1. FIGURE 2.

Cigarette smoking is the strongest risk factor for lung cancer, but genetically determined variations in the activities of pulmonary enzyme that metabolize tobacco-derived carcinogens may affect individual risk. To investigate whether these enzymes (e.g., CYP1A-related) can serve as markers for carcinogen-DNA damage, lung tissue specimens were taken during surgery from middle-aged men with either lung cancer or non-neoplastic lung disease. Phase I [aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECOD)] and phase II (epoxide hydrolase, UDP-glucuronosyltransferase, glutathione S-transferase) enzyme activities, glutathione and malondialdehyde contents were determined in lung parenchyma and/or bronchial tissues; some samples were also analyzed for DNA adducts, using 32P-postlabeling. The data were then analyzed for the following: a) differences in metabolic profiles between bronchial and parenchymal lung tissue; b) the effect of recent exposure to tobacco smoke on enzyme inducibility and benzo[a]pyrene metabolism; c) differences in enzyme inducibility between lung cancer and non-lung cancer patients; d) the effect of smoking on metabolism of mutagens in vitro; e) pulmonary DNA adduct levels and AHH activity in lung parenchyma of smokers and ex-smokers; f) lipid peroxidation products in lung tissue from lung cancer and non-lung cancer patients, as related to smoking habits and degree of airway obstruction; and g) prognostic value of AHH pulmonary activity in lung cancer patients. The results demonstrate a pronounced effect of tobacco smoke on pulmonary metabolism of xenobiotics and prooxidant state and suggest the existence of a metabolic phenotype at higher risk for tobacco-associated lung cancer.

In their article, Tarantini et al. (2009) focused on the basic question of mutual relationships between environmental and genetic factors. From a more general point of view, this also involves the question concerning the relative role of “intrinsic toxicity” of xenobiotics and individual susceptibility or host response. In particular, data from epidemiologic and in vitro studies must cope with pathologic evidence for pathologists, as well as cause-and effect-relationships for pathophysiologists. In between are inferences and extrapolations on the basis of plausibility. Tarantini et al. (2009) showed, for the first time in humans, that reactive oxygen species (ROS)—which are considered one of the main cellular stressors generated by PM exposure—may produce genomic hypomethylation and increased expression and activity of inducible nitric oxide synthase (iNOS) not only in vitro, but in humans exposed to particulate matter (PM). Although this finding is expected, it is a step forward, based on DNA adduct generation produced by polycyclic aromatic hydrocarbons (PAHs) and other PM components, namely transition metals. Alterations of DNA methylation of the promoter is a common finding in environmental-related chronic or cancerous diseases. Alterations in DNA methylation and iNOS methylation, as observed by Tarantini et al. (2009) in association with exposure to PM < 10 μm in aerodynamic diameter, may represent an initial step in reproducing decreases in global DNA methylation content that are eventually observed in cardiovascular diseases and cancer. However, pathologists and pathophysiologists are required to interpret more correctly what this means. Subjects with inherited multi tumoral syndromes have a germline mutation, usually a point mutation present in all cells of the body, which determines the occurrence of multiple tumors in the same individual. The occurrence of DNA adducts or mutations in some cells or tissues due to exposure to PAHs or diesel exhaust does not necessarily induce clinically evident outcomes in the future, because each individual is endowed with a wide variety of natural defenses and repair mechanisms that usually overcome every type of DNA damage. Therefore, the first inference to avoid is that DNA adduct formation or alterations in the promoter methylation of a gene causes cancer in the absence of inherited or acquired predisposition (i.e., a point germline mutation of a tumor suppressor gene or an acquired sporadic mutation). In addition, even in patients with inherited multitumoral syndromes (i.e., in subjects with germline mutations of suppressor genes), tumor occurrence (type and severity) is also greatly influenced by epigenetic factors, environmental stimuli, or even long-distance catastrophes. We previously demonstrated an increased incidence of papillary thyroid carcinoma in three members of the same familial adenomatous polyposis (FAP) family (i.e., a kindred having a 1061 APC gene mutation). This mutation is responsible for FAP, in part as a side effect (long-term–long-distance) after the Chernobyl disaster, thus suggesting a wider than expected impact of environmental disasters in predisposed subjects (Cetta et al. 1997, 2000). Analogously, chronic exposure to toxic or carcinogenic environmental substances does not elicit the same results in all individuals. The final clinical outcome (cancer, asthma, pulmonary fibrosis, atherosclerosis, or coronary diseases) seems to be less dependent on the toxic potency of the pollutant or of the exposure dose and more on the individual susceptibility of the host (Cetta 2009a, 2009b). This approach should facilitate a better understanding of the < 5% incidence of mesotheliomas in subjects with the same chronic exposure to asbestos, or the absence of health effects in husbands with chronic occupational exposure to asbestos but the occurrence of mesotheliomas in wives with minor indirect exposure from their husbands (Cetta F, unpublished data). However, acute and chronic inflammation is the first pathological step. The final clinical outcome (e.g. cancer) does not depend on the first DNA adduct formed or a genetic mutation produced by xenobiotics. A long, automaintaining process will start, such as in liver cirrhosis, leading to cancer or chronic alterations as the final result of the interactions between host and the pathogenic agent. This process is greatly influenced by individual susceptibility or resistance. In PM-related diseases, a major role—in addition to the intrinsic toxicity of the xenobiotic—is played by individual host susceptibility and reactivity, similar to what occurs in auto immune or autoinflammatory diseases.

Human exposure to chemical compounds, often termed xenobiotics, has been linked to a number of enhanced incidences of various neoplasias. A majority of these enter the human body through inhalation. Most xenobiotics are metabolized in the body to more hydrophilic metabolites before excretion in the urine and bile. During this process, promutagens and procarcinogens could be activated and could interact with proteins as well as DNA to form adducts. DNA adducts formed by chemical carcinogens can, therefore, be used as biomarkers of exposure and other host factors. This study that DNA adduct analysis can be performed on cells from human nasal mucosa. Using the nasal lavage procedure performed on 20 healthy volunteers, 5 x 10(5) to 5 x 10(6) cells were obtained from which 5 to 40 micrograms DNA was isolated. DNA adducts were analyzed by the 32-P-postlabeling assay. The DNA adduct levels ranged between 1.4 and 6 adducts/10(8) nucleotides. In addition to its simplicity, the nasal lavage procedure is an inexpensive, noninvasive procedure that requires no anesthetics or special equipment. Moreover, the cells obtained are the first to come in contact with air pollutants. DNA adduct analysis from human nose mucosa cells could therefore be used to develop a technique suitable for the assessment of exposure to chemical carcinogens through inhalation. Images Figure 1. Figure 1. Figure 1.

DNA adducts derived from complex mixtures of polycyclic aromatic compounds emitted from tobacco smoke are compared to industrial pollution sources (e.g., coke ovens and aluminum smelters), smoky coal burning, and urban air pollution. Exposures to coke oven emissions and smoky coal, both potent rodent skin tumor initiators and lung carcinogens in humans, result in high levels of DNA adducts compared to tobacco smoke in the in vitro calf thymus DNA model system, in cultured lymphocytes, and in the mouse skin assay. Using tobacco smoke as a model in human studies, we have compared relative DNA adduct levels detected in blood lymphocytes, placental tissue, bronchoalveolar lung lavage cells, sperm, and autopsy tissues of smokers and nonsmokers. Adduct levels in DNA isolated from smokers were highest in human heart and lung tissue with smaller but detectable differences in placental tissue and lung lavage cells. Comparison of the DNA adduct levels resulting from human exposure to different complex mixtures shows that emissions from coke ovens, aluminum smelters, and smoky coal result in higher DNA adduct levels than tobacco smoke exposure. These studies suggest that humans exposed to complex combustion mixtures will have higher DNA adduct levels in target cells (e.g., lung) as compared to nontarget cells (e.g., lymphocytes) and that the adduct levels will be dependent on the genotoxic and DNA adduct-forming potency of the mixture. ImagesFIGURE 1.FIGURE 1.FIGURE 2.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 4.

Dietary administration of the hepatocarcinogen 2-acetylaminofluorene (2-AAF) to rats results in the formation of a major hepatic DNA adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF). In liver DNA, dG-C8-AF reaches steady-state conditions after approximately 2 weeks of feeding and is removed in a biphasic manner. In these experiments, we have quantified adduct concentrations in a 370 base-pair repetitive DNA fragment to determine if the adduct levels and kinetics of adduct removal were similar to those found in total genomic DNA. Male F344 rats were fed 0.02% 2-AAF for 28 days and were sacrificed at intermittent times up to 56 days after being returned to the control diet. Hepatic DNA adduct levels were measured by 32P-postlabeling or radioimmunoassay (RIA) in total genomic DNA and in a 370 base-pair fragment obtained by digesting genomic DNA with Hind III. Biphasic removal of dG-C8-AF, which composed about 90% of the total adducts measured, was observed in total genomic DNA, with comparable rate constants being detected by both 32P-postlabeling and RIA. 32P-Postlabeling also showed analogous biphasic removal of dG-C8-AF in the 370 base-pair fragment. A second adduct, 3-(deoxyguanosin-N2-yl)-2-AAF (dG-N2-AAF), which accounted for about 10% of the total adducts measured, showed similar biphasic removal kinetics in the total genomic DNA and the 370 base-pair fragment; however, as compared to dG-C8-AF, little removal of dG-N2-AAF was observed during the slow phase. Images FIGURE 2.

Due to difficulties in conducting epidemiological studies, most estimates of cancer risk are based on data from animal bioassays. Extrapolation of cancer risk estimates in animals to humans requires an assumption of equal potency across species based on the average daily dose. The purpose of this paper is to examine the ability to predict tumor incidence across species from DNA adduct concentrations resulting from exposure to carcinogens. A 100-fold range of structurally diverse adduct concentrations corresponding to the same tumor incidence raises questions about quantitative predictability across chemical classes and across species. Differences in adduct structure, mutagenic efficiency, adduct repair rates, and cellular proliferation could account for some of the differences. For specific carcinogen-DNA adducts, the steady-state levels associated with a 50% tumor incidence appear to vary over a narrower range. An equal incidence of liver tumors was obtained at equal concentrations of aflatoxin B1-DNA adducts for rats and trout. A 2to 3-fold range of 4-aminobiphenyl-DNA adduct concentrations between mice and dogs appears to be associated with nearly equal bladder tumor incidence, on the basis of limited data. In humans, a 5-fold higher concentration of a 4-aminobiphenyl-DNA adduct in bladders of smokers than of nonsmokers is compatible with the relative risk of bladder cancer due to smoking. DNA adduct concentrations certainly can be used to improve quantification of chemical exposures for epidemiological studies. Although promising, more data are needed to judge the usefulness of DNA adduct concentrations to predict cancer incidence across species.