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Spectroelectrochemical sensing in an optically transparent thin layer electrode (OTTLE) cell was used for detecting the polycyclic aromatic hydrocarbon (PAH) biomarkers 1-hydroxypyrene (1-pyOH) and 1-hydroxypyrene-glucuronide (1-pyOglu) in phosphate buffer and artificial urine. This approach uses selective electrochemical modulation of a fluorescence signal by sequentially oxidizing the analytes in an OTTLE cell to distinguish between their overlapping fluorescence spectra. This technique allows for complete oxidation and signal modulation in approximately 15 min for each analyte; a mixture of 1-pyOH and its glucuronic acid conjugate can be analyzed in 30 min. Calibration curves consisting of the fluorescence change vs analyte concentration for 1-pyOH and 1-pyOglu yielded linear ranges from 10 nM to 1 μM and from 1 nM to 1 μM, respectively. With the use of these results, the calculated limits of detection were determined to be 1 × 10(-8) M for 1-pyOH and 9 × 10(-11) M for 1-pyOglu.

The association between fluid intake and bladder cancer risk remains controversial. Very little is known about to which extent the amount of water intake influences the action of excreting toxics upon the urinary system. This proof of concept trial investigates the effect of water intake on mutagenesis in smokers, a high risk population for bladder cancer.Methods. Monocentric randomized controlled trial.Inclusion Criteria.Male subjects aged 2045–45 y/o, smokers, and small drinkers (24-hour urinary volume 700 mOsmol/kg).Outcomes.4-ABP DNA adducts formation in exfoliated bladder cells in 24-hour urine collection and urinary mutagenicity in 24-hour urine.Test Group.Subjects consumed 1.5 L daily of the study product (EVIAN) on top of their usual water intake for 50 days.Control Group.Subjects continued their usual lifestyle habits.Results.65 subjects were randomized. Mean age was 30 y/o and mean cigarettes per day were 20. A slight decrease in adducts formation was observed between baseline and last visit but no statistically significant difference was demonstrated between the groups. Urinary mutagenicity significantly decreased. The study shows that increasing water intake decreases urinary mutagenicity. It is not confirmed by urinary adducts formation. Further research would be necessary.

Urinary bladder cancer is the fourth most commonly diagnosed malignancy in men and the tenth most commonly diagnosed malignancy in women in the US. Arylamines have long been associated with bladder cancer and several studies documented that men exposed to arylamines (cigarette smokers, hairdressers, and workers of dye and textile industries) have several times increased risk compared to women. N-glucuronidation is an important phase II conjugation reaction that delivers the active metabolites of arylamines from the liver to the urinary bladder. In the current study, we found that male mice are more active in 4-ABP N-glucuronidation than female mice and this difference was statistically significant. In the in vivo experiments, male and female mice (strain C57BL/6) were treated with 4-ABP after modulating their 4-ABP N-glucuronidation using the plant steroid, hecogenin. The distribution of 4-ABP adducts in liver and bladder was then determined. In animals treated with 4-ABP only, males had statistically significant higher levels of DNA adducts in the bladder (p-value 0.0004) while females had statistically significant higher levels in the liver (p-value

Exposure to inorganic arsenic in drinking water is linked to cancer in humans, but the mechanism of arsenic-induced cancer is not clear. Arsenic is not a powerful point mutagen, but can cause chromosome malsegregation and mitotic recombination, two events that can cause loss of tumor suppressor alleles and thereby contribute to the evolution of cancerous cells. To determine whether arsenic increases the frequency of allele loss due to either malsegregation or mitotic recombination in vivo, Aprt+/− hybrid mice were exposed to sodium arsenite (10 mg/L) in their drinking water for 10 weeks. To determine whether arsenic enhances the action of a known mutagen, half of the arsenic-treated mice were exposed to benzo[a]pyrene (BaP) for 8 weeks by skin painting (500 nmoles/week). Cells were taken from painted dorsal skin and cultured in the presence of 2,6-diaminopurine (DAP), to select colonies lacking adenosine phosphoribosyl transferase (Aprt) activity. The frequency of DAP-resistant (DAPr) colonies varied substantially within the treatment groups, but there was no significant difference between the groups. Analysis of DNA from DAPr colonies suggested that mitotic recombination contributed to the loss of wild-type Aprt allele. Whether arsenic or BaP enhanced or diminished the frequency of this process could not be deduced from these data. Environ. Mol. Mutagen., 2006. © 2006 Wiley-Liss, Inc.

Exposure to inorganic arsenic in drinking water is linked to skin, lung and bladder cancer in humans. The mechanism of arsenic-induced cancer is not clear, but exposure to arsenic and polycyclic arylhydrocarbons (PAH) is more carcinogenic than exposure to either type of carcinogen alone. Arsenic can also generate reactive oxygen species, suggesting that oxidation of DNA may play a role in carcinogenesis. Oxidization of guanosines in polyG tracts is known to cause frameshift mutations, and such events can be detected in situ using the G11 placental alkaline phosphatase (PLAP) transgenic mouse model, which reports frameshift mutations in a run of 11 G:C basepairs by generating cells containing heat-resistant alkaline phosphatase activity. PAH can also induce frameshift mutations. In the study described here, FVB/N mice carrying the G11 PLAP transgene were crossed to C57Bl/6 mice. Half of the hybrid mice were given drinking water with sodium arsenite (10 mg/L) for 10 weeks. Half of the arsenic treated mice were also exposed to benzo[a]pyrene (BaP) by skin painting (500 nmol/week) for 8 weeks. Another group of mice was exposed to BaP but not arsenic. The effect on frameshift mutation was assessed by staining sections of skin tissue to detect cells with PLAP activity. Arsenic alone had no significant effect. On average, mice given BaP alone had approximately three times more PLAP-positive (PLAP+) cells. By contrast, mice exposed to both arsenic and BaP exhibited 10-fold more PLAP+ cells in the skin, and these cells were often arranged in large clusters, suggesting derivation from stem cells. Whereas combined treatment produced more PLAP+ cells, stable BaP adduct levels and arsenic burdens were not higher in mice exposed to both agents compared to mice exposed to either one agent or the other.

The dermal route is important in occupational exposure to polycyclic aromatic compounds (PACs), but other organs may be affected. We reported that kerosene-cleaning following treatment with used engine oil increased DNA adducts in the lungs of mice viz. animals treated with used oil alone. To determine the mechanism we topically applied 3 H-BAP(100 nmol in 25 μL acetone) and washed half the mice with 25 μL kerosene 1 h after carcinogen application. Groups of four mice were sacrificed from 1 to 72 h after treatment. Lung, liver, and skin were harvested. The fraction of the radiolabel remaining in the skin of animals treated with benzo[a]pyrene (BAP) and washed with kerosene was significantly less than those not washed, beginning at 24 h (p < .05). Fractional distribution to the lungs and livers of these animals became significantly elevated. Kerosene increased transdermal water loss. Kerosene treatment compromises dermal barrier function, enhances carcinogen absorption, and alters organ distribution.

Background: Metabolites of the potent human carcinogen 4-aminobiphenyl (ABP) induce oxidative stress and form DNA adducts that are associated with hepatic and urinary bladder toxicity and bladder tumorigenesis. Results of in vitro and cell culture studies have suggested that cytochrome P450 1A2 (CYP1A2) is the major metabolic activator of ABP. We used Cyp1a2(–/–) knockout mice to examine the role of CYP1A2 in ABP–DNA adduct formation in the liver and the bladder. Methods: Cyp1a2(+/+) wild-type and Cyp1a2(–/–) mice (total of four mice per group) were treated topically with 10 mg/kg ABP for various times, with or without pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an inducer of CYP1A2 activity. We evaluated ABP-induced toxicity by carrying out quantitative histology (of the liver, skin, and bladder), oxidative stress by measuring hepatic thiol levels, and liver and bladder DNA adduct formation by using 32 P-postlabeling. Data were analyzed by general linear models and analysis of variance. All statistical tests were two-sided. Results: At the experimental times selected, we observed no histologic evidence of toxicity in the liver, skin, or bladder. Overall, Cyp1a2(+/+) mice had fewer DNA adducts 24 hours after ABP treatment than similarly treated Cyp1a2(–/–) mice. Compared with male mice, female mice had more DNA adducts in the liver but fewer adducts in the bladder, regardless of Cyp1a2 genotype. TCDD pretreatment was associated with a decrease in ABP–DNA adduct levels overall. After 2 hours of ABP treatment, hepatic thiol levels underwent statistically significant declines of severalfold in Cyp1a2(+/+) and Cyp1a2(–/–) males and in Cyp1a2(–/–) females. Conclusions: Contrary to our expectations, CYP1A2 expression was not associated with ABP-induced hepatic oxidative stress or with ABP–DNA adduct formation. Either CYP1A2 is not the major metabolic activator of ABP or other enzymes metabolically activate ABP in mice in the absence of CYP1A2. [J Natl Cancer Inst 2003;95:1227–37]

7H-dibenzo[c,g]carbazole (DBC) is a potent multispecies, multisite carcinogen present in the environment. The metabolic activation pathways of DBC are not completely known. It is hypothesized that DBC may be metabolically activated by oxidation to the reactive Michael acceptor o-quinones, which can form stable and depurinating DNA adducts. The synthesis of DBC-3,4-dione has been previously reported by this research group. In the present article, we describe the synthesis and chemical structural elucidation of nine DBC-nucleic acid adducts produced from reactions of DBC-3,4-dione with Ade, Cyt, 2'-deoxyguanosine (dGuo), 2'-deoxycytidine (dCyd), and Guo. Adducts were isolated from reaction mixtures by HPLC and analyzed using MS including elemental compositions and collision-activated dissociation (CAD), (1)H NMR, and two-dimensional chemical shift correlation spectroscopy (COSY) NMR. The adducts, 7-[3,4-dione-DBC-1-yl]-Ade, N4-[3,4-dione-DBC-1-yl]-Cyt, 5-[3,4-dione-DBC-1-yl]-Cyt, two conformational isomers of N2-[3,4-dihydroxy-DBC-1-yl]-dGuo, and two conformational isomers of N2-[3,4-dihydroxy-DBC-1-yl]-Guo, were characterized. Two adducts from reactions of DBC-3,4-dione with dCyd were identified by MS but not fully characterized by NMR due to instability of the adducts. Under similar conditions, the reactions of DBC-3,4-dione with Gua and 2'-deoxyadenosine (dAdo) did not result in an identifiable adduct. Liver DNA adducts from mice treated topically with DBC-3,4-dione (100 microg) in dimethyl sulfoxide/acetone (15/85, 100 microL) were identified with 32P-postlabeling. The major adduct chromatographically matched one of the adducts formed from livers of DBC-treated mouse (adduct 3) using identical conditions.

Tamoxifen (TAM), an antiestrogenic compound, has been approved for the treatment of breast cancer in high risk women. TAM has been shown to be an effective agent for prevention of breast cancer in women of varying degrees of risk and has been proposed to be used prophylactically in women whose genetic background suggests a high risk for breast cancer. However, it is not known whether TAM given prophylactically will alter the response of women to carcinogens from common environmental exposures such as tobacco smoke. Therefore, we studied the effects of TAM pretreatment on mammary DNA adducts of the model carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), in the female rats to assess whether TAM would alter the adduct pattern of DMBA. TAM (0.3 mg/day) was given for 7 days prior to a single 20 mg DMBA gavage treatment that is considered a carcinogenic dose in the rat. At 7 days post-DMBA, there was a reduction in the major DMBA–DNA adduct and a significant reduction in the minor DMBA–DNA adduct in mammary glands (P=0.002) of TAM pretreated rats compared to control rats. These data indicate that TAM may alter either metabolic steps in the formation of DNA binding species and/or enhance adduct removal. These data suggest that TAM given to women prior to the development of breast cancer may modulate the impact of environmental exposures, for example, tobacco smoke. Furthermore, research is needed to determine if modulation will be positive, or negative as in the current study.

Methemoglobin formation, as well as hemoglobin or DNA adducts, are useful biomarkers of occupational exposure to certain arylamines. It has been suggested that, in liver from animals not treated with a cytochrome P450 (CYP) inducer, hepatic CYP1A2 is the major P450 involved in N-hydroxylation. This is the first step in the metabolic activation of many arylamines, such as the human urinary bladder carcinogen 4-aminobiphenyl (ABP). The product of this catalytic step, N-hydroxy-4-ABP, reacts in the blood with oxyhemoglobin to form methemoglobin and nitrosobiphenyl. We therefore examined the role of CYP1A2 in causing methemoglobinemia in ABP-treated Cyp1a2(-/-) knockout mice. Application of ABP (100 micromol/kg body wt) to the skin resulted in a marked depletion in the levels of the hepatic thiols (reduced glutathione and cysteine) after 2 h, which rebounded to basal levels 24 h later, and we found no differences between the Cyp1a2(-/-) and wild-type Cyp1a2(+/+) animals. Unexpectedly, the methemoglobin levels were significantly (p < 0.05) higher in Cyp1a2(-/-) than Cyp1a2(+/+) mice at 2, 7, and 24 h following topical ABP. Treatment with dioxin, 24 h prior to ABP, decreased methemoglobin levels by about half at each of the time points in both the Cyp1a2(-/-) and Cyp1a2(+/+) mice. These data suggest that CYP1A2 does not play a positive role in methemoglobin formation via the activation of ABP; rather, the absence of CYP1A2 enhances ABP-induced methemoglobinemia. Because liver CYP1A2 levels are known to vary more than 60-fold between humans, our findings may be relevant to patients who are exposed to arylamines in the workplace.

Item does not contain fulltext Workers employed in the rubber industry appear to have a significant excess cancer risk in a variety of sites, including cancer of the urinary bladder. In this cross-sectional study, we investigated the occurrence of DNA adducts in exfoliated bladder cells of currently exposed, nonsmoking rubber workers (n = 52) and their relationship with occupational exposure estimates and acetylation phenotype (NAT2). Four DNA adducts were identified, with the proportion of positive samples (e.g., DNA samples with quantifiable levels of a specific DNA adduct) ranging from 3.8 to 79%. The highest proportion of positive samples and the highest relative adduct labeling levels were in workers involved in the production functions "mixing" and "curing," areas with potential for substantial exposure to a wide range of chemical compounds used in rubber manufacturing (P < 0.05 for adducts 2 and/or 3, compared to all other departments). No statistically significant relationships were found between identified DNA adducts and urinary mutagenicity or personal inhalable and dermal exposure estimates. Interestingly, subjects with a fast NAT2 acetylation phenotype tended to have higher levels of DNA adducts. This study suggests that rubber workers engaged in mixing and curing may be exposed to compounds that can form DNA adducts in urothelial cells. Larger studies among rubber workers should be conducted to study in more detail the potential carcinogenicity of exposures encountered in these work areas.

We compared two protocols for 7,12-dimethylbenz[ a ]anthracene (DMBA)-induced mammary carcinogenesis. DMBA (20 mg/ml) was prepared at room temperature or at 60°C and administered by gavage to 50-day-old Sprague-Dawley rats. Two days post-DMBA, mammary epithelial DNA was 32 P-postlabeled. At 12 weeks post-DMBA, tumor DNA was screened for H- ras codon 61 mutations using an enriched polymerase chain reaction (EPCR). The number of in situ carcinomas (3.0 - 2.7 vs. 2.5 - 2.7), invasive carcinomas (1.1 - 1.6 vs. 0.6 - 0.8), and H- ras codon 61 mutations (34 - 13% vs. 23 - 13%) were statistically not different ( p = 0.05) between the groups. The relative adduct levels in mammary organoids were significantly ( p h 0.001) greater in the 60°C (568 - 158) than the room temperature (150 - 130) group. While DMBA-DNA adduct levels differed significantly between these protocols, this did not correlate with significant differences in tumor pathologies or H- ras codon 61 mutations.

Complex mixtures consist of homocyclic and heterocyclic polycyclic aromatic compounds (PACs) represented by benzo[ a ]pyrene (B a P) and 7 H -dibenzo[ c,g ]carbazole (DBC), respectively. To exert their biological effects, PACs are metabolized into reactive intermediates, which can form DNA adducts. In this preliminary report, male A/J mice were given a single intraperitoneal injection. Groups of three animals were treated with DBC (2 or 10 mg/kg) or B a P (10 or 100 mg/kg). Mixtures of DBC:B a P were given at doses of 2:10, 2:100, 10:10, or 10:100 mg/kg. DNA adduct levels in lungs collected three days posttreatment were determined by the 32 P-postlabeling method. The results indicate that, in the lungs, exposure to mixtures containing more B a P than DBC resulted in the absence of adduct 3 (DBC) and significantly higher total adduct levels. This suggests that B a P is being preferentially metabolized, resulting in less DBC adduction.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.

N-Heterocyclic aromatics, such as carbazole and acridine derivatives, are environmental carcinogenic pollutants. Examples of these compounds are 7H-dibenzo[c,g]carbazole (DBC) and dibenz[a,j]acridine (DBA). The ionization potential (IP) for DBC is lower than for DBA. DBC is metabolized in lung and liver by way of phenols or directly through radical cations. DBC-induced liver and lung tumors have mutations in the 61st codon of ras. DBA is metabolized in skin by way of a diol-epoxide of DBA. DBA-induced skin tumors have mutations in 12th, 13th and 61st codons of ras. In summary, the metabolic activation of DBC proceeds through different adduction pattern pathways than does DBA and leads to different ras mutational spectra.

7H-Dibenzo[C, G]carbazole (DBC) is a potent liver, lung and skin carcinogen while dibenz[A, J]acridine (DBA) is a moderate skin carcinogen. DBC is metabolized to phenols and DBC-DNA adducts are formed through the 2-, 3-, and 4-phenols or directly through radical cations. Mutations in ras as a result of DBC, activation are found exclusively in codon 61 in liver, lung and skin. DBA is metabolized to dihydrodiols, and phenols and DBA-DNA adducts are formed through the diol-epoxides and possibly through bis-diol-epoxides. Mutations in ras, as a result of DBA activation, are found in codons 12, 13, and 61. These results indicate that although the two compounds are structurally similar, differing by one carbon in the middle ring, there are differences in their metabolism, DNA binding, mutational spectra and target-organ carcinogenesis.

7H-Dibenzo[c,g]carbazole (DBC) is a potent multi-site, multi-species carcinogen present in a variety of complex mixtures derived from the incomplete combustion of organic matter. Like many carcinogens, DBC requires metabolic activation to an electrophilic species to exert its mutagenic and carcinogenic effects. One-electron oxidation, leading to the formation of radical cation intermediates, has been proposed as a mechanism of metabolic activation for DBC in vitro resulting in unstable DNA adducts. The purpose of this research was to determine whether one-electron oxidation is a mechanism of activation and DNA binding for DBC in vivo. Specific depurinating DBC-DNA adducts formed by one-electron oxidation were analyzed in mouse liver at 4 h following a single i.p. dose of 40 mg/kg of 11 microCi [(14)C]DBC. In addition to five previously published adduct standards, two newly identified adduct standards were characterized by mass spectrometry and NMR, namely DBC-6-N7-Ade and DBC-6-N1-Ade; however, neither was observed in mouse liver. Only the DBC-5-N7-Gua adduct was observed in mouse liver extracts at a level of 6.5 +/- 1. 8 adducts/10(6) nucleotides. In addition, the formation of AP sites and stable DBC-DNA adducts was analyzed in mouse liver and lung at 4, 12 and 24 h following a single i.p. dose of 0.4, 4 or 40 mg/kg DBC (n = 3/group). There was a distinct time- and dose-response of stable DBC-DNA adducts detected by (32)P-post-labeling. There was not a clear dose-response for formation of AP sites; however, a significant increase over control levels was observed at the 4 and 40 mg/kg dose groups at 4 and 12 h post dosing, respectively. Quantitative comparisons indicate that the depurinating DBC-5-N7-Gua adduct constitutes approximately 0.4% of total adducts measured whereas the stable adducts detected by (32)P-post-labeling constitute 99.6% of total adducts measured following a 40 mg/kg dose and a 4 h time-point. The data indicate that one-electron oxidation does occur in mouse liver in vivo. However, one-electron oxidation is a minor mechanism of activation in that the percentage of total adducts formed through this route constitutes a minor percentage of the total adducts formed.

The Big Blue mouse was used to investigate the role of cell proliferation in mutation fixation in the mouse back skin model of carcinogenesis. Phorbol 12-myristate 13 acetate (TPA) was applied to the dorsum of Big Blue mice to manipulate cell proliferation, and benzo[a]pyrene (BaP) or BaP-diolepoxide (BPDE) was applied to produce premutagenic DNA damage. Mutations in the lacI transgene of skin DNA were measured. BaP and BPDE elevated mutant frequency, DNA adducts, and cell damage over untreated and acetone-treated mice. BPDE-DNA adducts peaked within 30 min of exposure and DNA adducts, formed after application of both BaP and BPDE, declined rapidly with time. As the dose of BaP increased (4 to 64 microg), DNA adducts, mutant frequency, and cell damage increased in a dose-dependent manner. TPA applied after BaP and BPDE further increased mutant frequency, DNA adducts, and cell damage, while variably affecting mitotic index and other measures of cell proliferation. TPA became less effective at increasing mitotic index as the dose of BaP increased, although all measures of cell proliferation, taken together, increased. The most effective production of DNA adducts and mutations occurred when the carcinogen was applied simultaneously with or within 1 hr of TPA. Mutations induced by BPDE were predominantly base substitutions: of these base substitutions, 35% were G:C --> A:T transitions, and 36% were G:C --> T:A and 29% G:C --> C:G transversions. Approximately 88% of all mutations and 100% of base substitutions were at G:C sites; 60% of all mutations and 70% of the base substitution mutations occurred at CpG sites. A:T --> G:C transitions were not found. All of the single-base deletions were at G:C base pairs.

In a cross-sectional study of 33 workers exposed to benzidine and benzidine dyes and 15 non-exposed controls, we previously reported that exposure status and internal dose of benzidine metabolites were strongly correlated with the levels of specific benzidine-DNA adducts in exfoliated urothelial cells. We also evaluated DNA adduct levels in peripheral white blood cells (WBC) of a subset of 18 exposed workers and 7 controls selected to represent a wide range of adducts in exfoliated urothelial cells. Samples were coded and then DNA was analyzed using 32P-postlabeling, along with n-butanol extraction. One adduct, which co-chromatographed with a synthetic N-(3'-phospho-deoxyguanosin-8-yl)-N'-acetylbenzidine standard, predominated in those samples with adducts present. The median level (range) of this adduct in WBC DNA was 194.4 (3.2-975) RAL x 10(9) in exposed workers and 1.4 (0.1-6.4) in the control subjects (p = 0.0002, Wilcoxon Rank Sum Test). There was a striking correlation between WBC and exfoliated urothelial cell adduct levels (Pearson r = 0.84, p < 0.001) among exposed subjects. In addition, the sum of urinary benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine correlated with the levels of this adduct in both tissues. This is the first study in humans to show a relationship for a specific carcinogen adduct in a surrogate tissue and in urothelial cells, the target for urinary bladder cancer.

7H-Dibenzo[c,g]carbazole, DBC, is a potent environmental liver carcinogen. Liver DNA from mice treated with DBC exhibited seven distinct DBC-DNA adducts as detected by 32P-postlabeling using multidimensional TLC. To improve quantitation and chemically characterize the adducts, DNA samples were hydrolyzed, 32P-postlabeled and the adducts were separated from the unadducted normal nucleotides on TLC using a D1 solvent, 0.65 M sodium phosphate (pH 6.8). Adducts were eluted from the TLC plates with 4.0 M pyridinium formate, concentrated, resuspended in 50% aqueous methanol and injected onto the HPLC; five individual adduct peaks were resolved and collected by this method. This approach will prove useful to decrease analysis time and improve chemical characterization of tightly clustered DNA adducts generated in vivo.

Jet fuel vapor exposures were measured using industrial hygiene sampling techniques at three domestic U.S. Air Force installations. Breathing zone samples were collected from within three general job categories at each location: aircraft maintenance, fuel handling, and flightline positions. These samples were analyzed using gas chromatography and flame ionization detector techniques. Mean exposure concentrations for all samples collected were 0.01 ppm benzene [standard deviation (SD) = 0.01] and 1.33 ppm naphthas/jet fuels (SD = 1.95) for the aircraft maintenance positions (n = 63), 0.01 ppm benzene (SD = 0.01) and 0.61 ppm naphthas/jet fuels (SD = 0.90) for fuel handling positions (n = 16), and 0.004 ppm benzene (SD = 0.005) and 0.33 ppm naphthas/jet fuels (SD = 0.40) for flightline positions (n = 8).

4-Aminobiphenyl (ABP) is a recognized human bladder carcinogen, whose presence in cigarette smoke results in DNA adduct formation in the human urothelium. Since preliminary studies indicated that even higher levels of ABP–DNA adducts may be present in human peripheral lung, we utilized a sensitive immunochemical assay, in combination with 32 P-postlabeling, to quantify the major 4-aminobiphenyl (ABP)–DNA adduct, N -(guan-8-yl)-ABP, in surgical samples of peripheral lung tissue from smokers and ex-smokers. No differences in adduct levels were detected between smokers and ex-smokers by immunoassay. In contrast, the 32 P-postlabeling method showed statistically significant differences between adduct levels in smokers and ex-smokers; however, a relatively high background of smoking-related adducts chromatograph near the major ABP adducts and may compromise estimation of the level of ABP–DNA adducts in smokers. Furthermore, the levels measured by 32 P-postlabeling were 20- to 60-fold lower than that measured by immunoassay. Since 32 P-postlabeling may underestimate and immunochemical assays may overestimate adduct levels in the lung, selected samples were also evaluated by GC/MS. The immunochemical and GC/MS data were concordant, leading us to conclude that N -(guan-8-yl)-ABP adducts were not related to smoking status. Since ABP–DNA adduct levels in human lung did not correlate with smoking status as measured by immunoassay and GC/MS, the metabolic activation capacity of human lung microsomes and cytosols was examined to determine if another exposure (e.g., 4-nitrobiphenyl) might be responsible for the adduct. The rates of microsomal ABP N -oxidation were below the limit of detection, which was consistent with a lack of detectable cytochrome P4501A2 in human lung. N -Hydroxy-ABP O -acetyltransferase (but not sulfotransferase) activity was detected in cytosols and comparative measurements of N -acetyltransferase (NAT) using p -aminobenzoic acid and sulfamethazine indicated that NAT1 and NAT2 contributed to this activity. 4-Nitrobiphenyl reductase activity was found in lung microsomes and cytosols, with the reaction yielding ABP and N -hydroxy-ABP. Lung microsomes also demonstrated high peroxidative activation of ABP, benzidine, 4,4′-methylene- bis (2-chloroaniline), 2-aminofluorene, and 2-naphthylamine. The preferred co-oxidant was hydrogen peroxide and the reaction was strongly inhibited by sodium azide but not by indomethacin or eicosatetraynoic acid, which suggested the primary involvement of myeloperoxidase rather than prostaglandin H synthase or lipoxygenase. This was confirmed by immunoinhibition and immunoprecipitation studies using solubilized human lung microsomes and antisera specific for myeloperoxidase. These data suggest that ABP–DNA adducts in human lung result from some environmental exposure to 4-nitrobiphenyl. The bioactivation pathways appear to involve: (1) metabolic reduction to N -hydroxy-ABP and subsequent O -acetylation by NAT1 and/or NAT2; and (2) metabolic reduction to ABP and subsequent peroxidation by myeloperoxidase. The myeloperoxidase activity appears to be the highest peroxidase activity measured in mammalian tissue and is consistent with the presence of neutrophils and polymorphonuclear leukocytes surrounding particulate matter derived from cigarette smoking.

Ferric oxide (Fe 2 O 3 ) and aluminum oxide (Al 2 O 3 ) particles are widely encountered in occupational settings. Benzo[a] pyrene (B[a]P), a well-characterized environmental carcinogen, is frequently adsorbed onto particles. It has been shown that B[a]P-coated Fe 2 O 3 particles (B[a]P-Fe 2 O 3 ) significantly increased lung tumors in the hamster in contrast to B[a]P-coated Al 2 O 3 (B[a]P-Al 2 O 3 ) or B[a]P alone. In order to determine the genotoxic effects of these particles on the metabolism of B[a]P, pulmonary alveolar macrophages (AM) from male Syrian golden hamsters were incubated with 5 μg (19.8 nmol) B[a]P-coated respirable size (99%

Carcinogen-DNA adducts appear to be biomarkers of effective dose and/or early effect. The relationship between these biomarker levels in target and non-target organs under acute and chronic exposures is important. 7H-Dibenzo(c,g)carbazole(DBC) has been shown to induce tumors in experimental animals. The time courses of DBC-DNA adduct formation in skin, liver and lung following both single (i.p.) and repeated (topical) administration of DBC were investigated. In the acute study, DNA binding levels increased rapidly in the lung over 3–7 days at all doses and then decreased slowly over 21 days; levels in the liver were 10 times that in the lung with similar increases in levels over time. In the chronic study, levels increased with time over 15 weeks with the liver binding levels 8–10 times that seen in skin and lung. In both studies the major adduct in liver was adduct 6, and in the lung adduct 3, while in skin adducts 2 and 3 were the major adducts. These data indicate that the adduct pattern is ta...

Background: In April 1991, an excess of bladder cancer cases among workers employed at a chemical manufacturing facility in Niagara Falls, NY, was reported. This excess was primarily confined to 708 workers who had ever been employed in the rubber chemicals manufacturing area of the plant, where the aromatic amines aniline and o-toluidine have historically been used. Purpose: An environmental and biological monitoring survey was conducted to evaluate current exposures to aniline and o-toluidine in the rubber chemicals department. Methods: Personal air sampling for aniline and o-toluidine was conducted with the use of a modified Occupational Safety and Health Administration (OSHA) 73 method. Urine samples were collected before and after work (i.e., pre-shift and post-shift, respectively) and stored at -70 °C. Base hydrolysis was used to convert acetanilide and A'-acetyl-o-toluidine, metabolites of aniline and o-toluidine present in the urine, to the parent compounds. The parent compounds were extracted from the alkaline urine into butyl chloride and then back-extracted from the butyl chloride into aqueous hydrochloric acid. An aliquot of each acidic extract was subjected to ion-interaction reversed-phase liquid chromatography with coulometric electrochemical detection. Hemoglobin (Hb) was extracted from blood and stored at -70 °C. For the measurement of adducts of aniline, o-toluidine, and 4-aminobiphenyl (4-ABP), precipitated Hb was dissolved in 0.1 M sodium hydroxide in the presence of recovery standards, and the hydrolysate was extracted with hexane, derivatized with pentafluoropropionic anhydride, and analyzed by gas chromatography-mass spectrometry with negative chemical ionization. Results: A total of 73 workers, including 46 of 64 exposed workers who were employed in the rubber chemicals department and had the potential for exposure to aniline and o-toluidine and 27 of 52 unexposed workers employed in other departments where aniline and otoluidine were not used or produced, had data available for both aniline and o-toluidine and Hb adducts; 28 of the workers in the former group also had personal air-sampling data. PersonaJ air sample measurements showed that airborne concentrations of aniline and o-toluidine were well within the limits allowed in the workplace by OSHA. Urinary aniline and o-toluidine levels, however, were substantially higher among exposed workers than among unexposed control subjects. The most striking differential was for postshift urinary o-toluidine levels, which averaged (± standard deviation) 2.8 |.ig/L (±1.4 ng/L) in unexposed subjects and 98.7 ng/L (±119.4 ng/L) in exposed subjects {P = .0001). Average aniline-Hb and o-toluidine-Hb adduct levels were also significantly higher (P = .0001) among exposed workers than among unexposed control subjects. Average levels of adducts to 4-ABP, a potential contaminant of process chemicals, were not significantly different (P = .48), although three exposed workers had 4-ABP levels above the range in unexposed workers. Conclusions: The adduct data suggest that, among current workers, o-toluidine exposure substantially exceeds aniline exposure and that 4-ABP exposure, if it occurs at all, is not widespread. These data support the conclusion that occupational exposure to o-toluidine is the most likely causal agent of the bladder cancer excess observed among workers in the rubber chemicals department of the plant under study, although exposures to aniline and 4-ABP cannot be ruled out. [J Natl Cancer Inst 1996;88:1046-52]

Carcinogen-DNA adduct measurements may become useful biomarkers of effective dose and/or early effect. However, validation of this biomarker is required at several levels to ensure that human exposure and response are accurately reflected. Important in this regard is an understanding of the relative biomarker levels in target and nontarget organs and the response of the biomarker under the chronic, low-dose conditions to which humans are exposed. We studied the differences between single and chronic topical application of benzo[a]pyrene (BAP) on the accumulation and removal of BAP-DNA adducts in skin, lung, and liver. Animals were treated with BAP at 10, 25, or 50 nMol topically once or twice per week for as long as 15 weeks. Animals were sacrificed either at 24, 48, or 72 hr after the last dose at 1 and 30 treatments, and after 24 hr for all other treatment groups. Adduct levels increased with increasing dose, but the slope of the dose-response was different in each organ. At low doses, accumulation was linear in skin and lung, but at high doses the adduct levels in the lung increased dramatically at the same time when the levels in the skin reached apparent steady state. In the liver adduct, levels were lower than in target tissues and apparent steady-state adduct levels were reached rapidly, the maxima being independent of dose, suggesting that activating metabolism was saturated in this organ. Removal of adducts from skin, the target organ, was more rapid following single treatment than with chronic exposure. This finding is consistent with earlier data, indicating that some areas of the genome are more resistant to repair. Thus, repeated exposure and repair cycles would be more likely to cause an increase in the proportion of carcinogen-DNA adducts in repair-resistant areas of the genome. These findings indicate that single-dose experiments may underestimate the potential for carcinogenicity for compounds that follow this pattern.

N-Heterocyclic polynuclear aromatic hydrocarbons (NHA) are environmental pollutants formed during the combustion of organic materials. 7-H-Dibenzo[c,g]carbazole (DBC) is a potent carcinogen in lung, liver and skin. We undertook these studies to determine whether tissue specificity for DBC lung carcinogenicity in the strain A/J mouse is mirrored by formation of DBC-DNA adducts in lung tissue and whether these adducts are consistent with mutation patterns in the K-ras gene. Strain A/J mice were given a single i.p. injection of DBC at doses of 0, 5, 10, 20 or 40 mg/kg and levels of DNA adducts in the lung were monitored by 32P-postlabeling on days 1, 3, 5, 7, 14 and 21. The remaining animals were sacrificed 8 months after DBC treatment and lung tumor multiplicity and K-ras mutation patterns in the tumors were determined. The lung tumor response to DBC was dose related, with an average of 4.7 +/- 1.2 tumors/mouse at 5 mg/kg and 48.1 +/- 5.5 tumors/mouse at 40 mg/kg. As many as seven DBC-DNA adducts were observed in the lung. DNA binding levels in the lung were highest at 40 mg/kg, with maximum binding at 5-7 days. At lower dose levels the maximum binding to DNA decreased and shifted to earlier time points. The DBC-DNA adduct in the lung with the highest level of binding at all dose levels was DBC-DNA adduct 3. The majority of DBC-induced mutations in the K-ras gene in the lung were A-->T (80%) transversions in the third base of codon 61, a mutation that has not been previously observed in chemically induced lung tumors in strain A/J mice.

Complex mixtures that are produced from the combustion of organic materials have been associated with increased cancer mortality. These mixtures contain homocyclic and heterocyclic polycyclic aromatic hydrocarbons (PAHs), many of which are known carcinogens. In particular, N-heterocyclic aromatic compounds (NHA) are present in these mixtures. Studies to determine the metabolic activation of these compounds have been undertaken. The purpose of this review is to compare and contrast the metabolic activation and biological effects of two NHA, 7H-dibenzo[c,g]carbazole (DBC) and dibenz[a,j]acridine (DBA), in order to better assess the contribution of NHA to the carcinogenic potency of complex mixtures and to develop biomarkers of the carcinogenic process. DBC has both local and systemic effects in the mouse; it is a potent skin and liver carcinogen following topical application and a lung carcinogen following i.p. application. On the other hand, DBA is a moderate mouse skin carcinogen following topical application and a lung carcinogen following subcutaneous injection. The biological differences for DBC and DBA are reflected in target organ-specific proximate and mutagenic metabolites and DNA adduct patterns.

Few studies have investigated the chronic cytokinetic effects of carcinogen exposure in the mouse skin. We report two experiments involving the repeated application of benzo[a]pyrene (BaP) to the dorsal skin of female Ha/ICR mice. In the first experiment, the cytokinetic, inflammatory, and DNA adduct responses were studied daily over a 9-day period encompassing the fourth and fifth weekly applications of BaP at doses of 16, 32, and 64 micrograms. The second experiment involved the same cytokinetic measurements at 1, 3, 5, and 8 months, and the weekly BaP doses were 4, 8, and 16 micrograms. The first study showed that after each application of 32 or 64 micrograms BaP, there was a wave of slow DNA synthesis in the epidermis which peaked at 24 hr, in coincidence with a wave of BaP-DNA adducts, followed by the appearance of dead and damaged keratinocytes. For the first few days after BaP application there was a depression in the mitotic rate which recovered several days before the next BaP application. There was a predominantly monocytic dermal inflammation throughout the observation period. In the second experiment, at the lower BaP doses, there was proliferative depression at 1 month, without dermal inflammation. With continued exposure, the proliferative depression changed to a dose-dependent increase in the rate of proliferation and dermal inflammation. The level of BaP-DNA adducts was followed in the 4 micrograms/week dose group, which showed a threefold increase after 4 months with the appearance of inflammation and heightened cell proliferation. These results suggest that the delayed inflammatory reaction, possibly based on a cell-mediated immune reaction to BaP, might have been responsible for the late cytokinetic responses and the associated increase in the level of BaP-DNA adducts.

N-Heterocyclic aromatics are environmental carcinogenic pollutants. Of these compounds, 7H-dibenzo[c,g]carbazole (DBC) is a liver and skin carcinogen in the mouse by topical application and a lung carcinogen by IP injection, whereas dibenz[a,j]acridine (DBA) is a skin carcinogen by topical application. Using 32P-postlabeling techniques, it was found that DBC-DNA adduct 6 is the major adduct in the liver, consistent with the metabolism of DBC to the 3-OH-DBC; adducts 2 and 3 are major adducts in the skin and adduct 3 is the major adduct in the lung. On the other hand, DBA adduction is apparent only in the skin and is consistent with the initial metabolism to DBA-3, 4-dihydrodiol. These data indicate that the adduct pattern, as well as the proximate metabolites, are target organ specific; these data will have an impact on the development of biomarkers of the carcinogenic process.

4,4'-Methylenebis(2-chloroaniline) (MOCA) has considerable human occupational exposure and it induces urinary bladder tumors in the dog, a species that has been often used as a model for aromatic amine-induced urinary bladder carcinogenesis in humans. Metabolic activation and formation of DNA adducts are considered to be critical steps in this process; and two major C8-adenine adducts have been shown to be formed in vitro by reaction with the proximate carcinogenic metabolite N-hydroxy-MOCA. MOCA-DNA adducts have also been detected in vivo in treated rats and in exfoliated urothelium of a worker accidentally exposed to MOCA. Thus, the aim of this study was to detect and quantify DNA adducts in the urinary bladder of dogs exposed to MOCA and thereby provide data that could be useful for risk assessment after human exposure to MOCA. Beagle dogs were treated with single and multiple doses of MOCA and DNA adduct levels were determined in liver and bladder epithelium. After a single dose, adduct levels in the liver were 1.5-fold higher than that in the bladder epithelium. Adduct levels in these two organs increased 3- to 5-fold after 10 doses and adducts in the liver were then 2.8-fold higher than that in the bladder epithelium. The levels found in these two organs after single exposures were compared, per unit exposure dose, with that reported for other carcinogenic aromatic amines. The comparison showed that MOCA was as effective in DNA adduct formation as most other potent urinary bladder carcinogens. These results suggest that MOCA may have high carcinogenic potential in humans and are consistent with the recent classification of MOCA as a probable human carcinogen.

Purpose: Characterize ethylbenzene and xylene air concentrations, and explore the biological exposure markers (urinary t,t-muconic acid (t,t-MA) and unmetabolized toluene) among petroleum workers offshore. Offshore workers have increased health risks due to simultaneous exposures to several hydrocarbons present in crude oil. We discuss the pooled benzene exposure results from our previous and current studies and possible co-exposure interactions. Methods: BTEX air concentrations were measured during three consecutive 12-h work shifts among 10 tank workers, 15 process operators, and 18 controls. Biological samples were collected pre-shift on the first day of study and post-shift on the third day of the study. Results: The geometric mean exposure over the three work shifts were 0.02ppm benzene, 0.05ppm toluene, 0.03ppm ethylbenzene, and 0.06ppm xylene. Benzene in air was significantly correlated with unmetabolized benzene in blood (r=0.69, p

Purpose Exposure to environmental tobacco smoke (ETS) leads to molecular damage in the form of DNA adducts. While lung cancer risk is higher among African Americans compared to White Americans, a few studies have tested for racial differences in DNA adducts among children exposed to ETS. The purpose of this study was to test whether African American children have higher DNA adducts levels compared to White children adjusted for ETS exposure. Methods Data and biologic specimens were drawn from an existing cohort of 212 asthmatic children. These subjects participated in a 12-month ETS-reduction trial that employed HEPA air cleaners with active filter cartridges and sham filter cartridges. White blood cell (WBC) DNA was analyzed for DNA adducts using 32P-postlabeling. We assessed ETS exposure using a validated air nicotine dosimeter. We determined the independent relationship between African American race and DNA adduct levels adjusted for ETS exposure and air cleaner use. Results The mean age of the subjects was 8.4 years; 55% were African American. There was no difference in DNA adduct levels between African American and White children (11.8 vs. 11.2 adducts per 109 nucleotides, p = 0.86), despite slightly higher levels of air nicotine exposure (3.4 vs. 2.2 μg/m3, p = 0.14). African American children used their air cleaners less often than White children. We found that the best predictor of DNA adduct levels was the duration of air cleaner use (r = −0.133, p = 0.056). This association was independent of cartridge type. Conclusions We did not see differences in adduct levels by race even after accounting for the level of ETS exposure. However, there was a marginal inverse association between air cleaner use and adducts. Additional research is required to understand this phenomenon.

Exposure monitoring programs have been used in the aluminum smelter industry for decades to decrease the risk of cancer from exposure to polycyclic aromatic hydrocarbons (PAHs). Biological monitoring of PAHs incorporates all routes of exposure. Measuring postshift urinary 1-hydroxypyrene (1OHP), a metabolite of pyrene, determines worker's daily PAH exposures, while measuring DNA adducts reflect chronic exposures to PAHs. We reviewed the scientific literature to identify changes over time in (1) 1OHP levels, (2) DNA adduct levels, and (3) other contributing factors associated with 1OHP and DNA adduct levels in the aluminum smelter industry. No trends were observed in 1OHP and DNA adduct levels. This could be due to variable selection of study populations and poorly identified job tasks that prevent comparison of jobs across plants and times, unassessed worker exposure variability, and the impact of cumulative exposures. Thus, it cannot be demonstrated that the use of biological monitoring to estimate PAH exposures has brought about an exposure reduction in the industry. Future studies should be aimed at follow-up in workplaces where dermal and inhalation exposure interventions have been employed. Inconsistent findings were also observed in the analysis of CYP1A1, GSTM1, and GSTP1 polymorphisms and their effect on biomarker levels.

The prevalence of covalent modifications to DNA (carcinogen-DNA adducts) in 42 human urinary bladder biopsy samples was investigated by 32P-postlabeling methods, with enhancement by both nuclease P1 treatment and 1-butanol extraction. Total mean carcinogen-DNA adduct levels and the mean levels of several specific adducts were significantly elevated in DNA samples of 13 current smokers, as opposed to 9 never smokers or 20 ex-smokers (5 years abstinence). There was no significant difference between the latter two groups. Several DNA adducts enhanced by nuclease P1 treatment were chromatographically similar to putative hydrocarbon DNA adducts reported earlier for placenta and lung DNA samples obtained from cigarette smokers. Putative aromatic amine adducts were detected by 1-butanol extraction that were not present when the samples were treated with nuclease P1. One of these displayed chromatographic behavior identical to the predominant adduct induced by the human urinary bladder carcinogen, 4-aminobiphenyl, which is present in cigarette smoke. This adduct comigrated in several thin-layer chromatographic systems with a synthetic N-(deoxyguanosin-8-yl)-4-amino[2,2'-3H]biphenyl-3',5'-bisphosphate marker. Moreover, when this adduct was eluted from the thin-layer chromatograms of several individuals and injected onto an HPLC system, the 32P from the human bladder DNA samples coeluted in the same fraction as the tritiated synthetic N-(deoxyguanosin-8-yl)-4-aminobiphenyl marker. These data reinforce an association between cigarette smoking and DNA damage and suggest a molecular basis for the initiation of human urinary bladder cancer by cigarette smoke.

The antihistamine methapyrilene hydrochloride has been shown to be a potent hepatocarcinogen in Fischer 344 rats. It has also been evaluated in a number of short-term in vivo and in vitro genotoxicity assays with conflicting results. We studied its ability to form DNA adducts in the L5178Y/TK +/- mouse lymphoma cells, an assay system in which methapyrilene is a moderately active mutagen and appears to induce mutations predominantly of chromosomal origin. Methapyrilene failed to induce formation of DNA adducts in L5178Y cell DNA at doses which induced mutations at the thymidine kinase locus. These data suggest that methapyrilene induces mutations in this system through an indirect genotoxic mechanism; e.g., via an oxidative mechanism or interaction with chromosomal proteins.

Objectives: Several studies have suggested that genotoxic risks might still be present in the contemporary rubber manufacturing industry. Previously, we observed elevated levels of urinary mutagenicity and bladder DNA adducts in rubber workers. Presently, we investigated whether DNA adducts in peripheral blood mononuclear cells (PBMC) and/or urothelial cells may be caused by polycyclic aromatic hydrocarbons or other genotoxic compounds. Methods: Spot urine samples from 116 rubber manufacturing workers were collected on Sunday and during the workweek (post-shift) to determine 1-hydroxypyrene and mutagenicity levels. For 52 nonsmokers, urothelial cell DNA adducts and PBMC DNA adducts were measured additionally. Results: Urinary 1-hydroxypyrene levels were significantly higher in workweek samples compared with Sunday (P = 0.0001). This increase was not uniform across tasks and only reached statistical significance for the curing department (+99%; P = 0.003). Weekday urinary mutagenicity was significantly increased for mixing (+56%) and curing (+21%) workers when compared with that for Sunday. Total urothelial cell DNA adducts were related to urinary 1-hydroxypyrene (P = 0.021) and mutagenicity (P = 0.027). No significant relationship was found between the adduct levels in PBMC and urothelial cells or between the former and urinary 1-hydroxypyrene or mutagenicity. Conclusions: Workers in the compounding, mixing, and curing departments were at highest genotoxic risk among rubber manufacturing workers. Increased levels of urinary 1-hydroxypyrene, mutagenicity, and urothelial cell DNA adducts were found in these workers. Urothelial cell and PBMC DNA adducts were not related, hinting possibly to the presence of specific bladder carcinogens in the rubber manufacturing industry. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1452–9)

In order to characterize the tobacco-derived mutagens excreted in the urine of tobacco smokers, 32P-postlabelling techniques were used to examine DNA adducts formed from these mutagens with calf thymus DNA in the presence of a metabolic activation system (rat liver S9, Aroclor 1254-induced, with or without acetyl coenzyme A). Using either nuclease P1 or butanol extraction procedures, four-six and three spots, respectively, were reproducibly found on the autoradiograms in the case of the urine extract from two smokers of black tobacco. Using the urinary extract from a non-smoker, only three faint spots were detected after nuclease P1 enrichment. DNA adducts produced in smokers' urine were then compared with those formed by four N-hydroxyarylamines, N-hydroxy-2-amino-3,8-dimethyl-3H-imidazo[4,5-f]quinoxaline, N-hydroxy-2-amino-3-methyl-imidazo[4,5-f]quinoxaline, N-hydroxy-2-naphthylamine and N-hydroxy-4-aminobiphenyl. Visual inspection revealed that none of the reference aromatic amines contributed to the adduct pattern produced by the urinary mutagen(s). However, primary aromatic amines are mainly implicated as urinary mutagens because: (i) they produce frameshift mutations in Salmonella typhimurium strains, (ii) they are easily extractable with blue cotton and (iii) their mutagenicity is abolished by a nitrite treatment procedure for deamination.

Found in tobacco smoke, fossil fuel and other organic combustion products, 7H-dibenzo[c,g]carbazole (DBC) is a potent mouse lung carcinogen and potential human carcinogen. Although the first hydroxylation is critical for determining activation versus detoxication, the enzymes responsible for site-specific hydroxylation of DBC are not known. We found that DBC-DNA adduct levels are significantly higher in aromatic hydrocarbon receptor null Ahr(-/-) mice, suggesting that the induction of Aromatic hydrocarbon receptor (AHR)-regulated genes, such as those in the CYP1 family, decrease DBC genotoxicity. Using knockout mice for Cyp1a1, Cyp1a2 and Cyp1b1, we showed that the major CYP1 enzymes that metabolize DBC are CYP1A1 in beta-naphthoflavone (BNF)-induced liver, CYP1A2 in non-induced liver, CYP1B1 and CYP1A1 in induced lung and none in non-induced lung. DBC metabolism by the human CYP1 enzymes was examined in vitro using Supersomestrade mark. Each mouse CYP1, as well as each human CYP1, has a unique DBC metabolite profile. Comparison of the metabolite profile in BNF-induced mice suggested that CYP1A1 primarily generates 1-OH, 2-OH and (5 + 6)-OH-DBC, whereas CYP1A2 generates primarily (5 + 6)-OH-DBC and CYP1B1 primarily generates 4-OH-DBC. This was similar to that observed in the human CYP1 enzymes. Most importantly, lung CYP1B1 is associated with forming 4-OH-DBC, the most potent metabolite leading to DBC-DNA adducts. These studies suggest that for non-pulmonary routes of exposure (i.e. skin, gastric, i.p.), low hepatic expression of CYP1A2 and CYP1A1, together with high expression levels of lung CYP1B1 and CYP1A1, may define a phenotype for high susceptibility to carcinogens such as DBC.

The mammalian inner ear loses its sensory cells with advancing age, accompanied by a functional decrease in balance and hearing. This study investigates oxidant stress in the cochlea of aging male CBA/J mice. Glutathione-conjugated proteins, markers of H2O2-mediated oxidation, began to increase at 12 months of age; 4-hydroxynonenal and 3-nitrotyrosine, products of hydroxyl radical and peroxynitrite action, respectively, were elevated by 18 months. Immunoreactivity to these markers was stronger in the supporting cells (Deiters and pillar cells) than the sensory cells. It appeared later (23 months) in the nerve fibers of the spiral ganglion and in the stria vascularis and spiral ligament. Conversely, antioxidant proteins (AIF) and enzymes (SOD2) decreased by 18 months in the organ of Corti (including the sensory cells) and nerve fibers but not in the stria vascularis. These results suggest the presence of different reactive oxygen species and differential time courses of oxidative changes in individual tissues of the aging cochlea. An imbalance of redox status may be a component of age-related hearing loss.

Markers of exposure to polycyclic aromatic hydrocarbons (urinary 1-hydroxypyrene-glucuronide) and aromatic amines (4-aminobiphenyl-hemoglobin adducts), as well as urinary mutagenicity, were measured in 47 healthy smokers and 50 non-smokers. DNA adducts were determined by P32-postlabeling in the exfoliated bladder cells of 39 healthy subjects. Both 1-hydroxypyrene-glucuronide (1-OHPG) and 4-aminobiphenyl adducts (4-ABP-Hb) were associated with smoking habits, but only 4-ABP-Hb adducts were associated with consumption of black, air-cured tobacco. The levels of 2 DNA adducts (numbers 2 and 4) in urothelial cells were clearly associated with 4-ABP-Hb adducts, in all subjects and in smokers. Levels of one of these DNA adducts (number 2) were also associated with 1-hydroxypyrene-glucuronide in urines, but in smokers the association was not statistically significant. Overall, these observations constitute further evidence of a role of arylamines in tobacco-induced bladder cancer. © 1996 Wiley-Liss, Inc.

A simple and sensitive method for determination of the N-glucuronidation activity of mouse, rat, and human liver microsomes toward the carcinogenic arylamine 4-aminobiphenyl (4-ABP) using high-performance liquid chromatography with ultraviolet detection has been developed. The method uses chemically synthesized 4-ABP- N -glucuronide (4-ABP-G) as a standard for method validation. Validation was done with respect to specificity, linearity, precision, accuracy, and lower limits of detection. The method was specific since there were no interference peaks from the reaction matrix. The calibration curve for 4-ABP-G was linear from 50 to 5000 pmol/200 μl with R 2 = 0.999. The newly developed method has good precision and accuracy. The intra- and interday precisions were less than 5 and 10%, respectively, and the highest values for intra- and interday accuracies were −4.6 and −12%, respectively. The lower limit of detection was 10 pmol/200 μl. The developed method was used to determine the glucuronidation activity of mouse, rat, and human liver microsomes. Human liver microsomes were the most active in 4-ABP glucuronidation (344.1 pmol/min/mg) followed by rats (30.6 pmol/min/mg) and then mice (12.3 pmol/min/mg). Human UGT1A4 supersomes were much more active than UGT1A9 (184.4 mol/min/mg versus 25.2 mol/min/mg). These results are consistent with those of earlier studies that used the radioactive [C 14 ]UDPGA.

The field method for near real-time analysis of perchloroethylene (Perc) in breath is simple, fast, and reproducible for Perc breath analysis in field settings and should prove useful in industrial hygiene practice. The method allows Perc monitoring with good specificity to the sub-part per million (ppm) level within minutes of exposure. A commercially available, portable gas chromatograph with a photoionization detector was used in these analyses. Gas chromatograph settings were optimized in the laboratory for measurement of Perc in Tedlar bags. Laboratory development of the method included evaluation of the sensitivity, specificity, precision, and speed of analysis for Perc. Replicate aliquots of Perc at concentrations ranging from 0.01 to 100 ppm were used to construct a calibration curve. The mean retention time for Perc was 238 sec. The impact of potential interference by acetone, toluene, isoprene, methanol, ethanol, acetaldehyde, carbon tetrachloride, benzene, or chloroform was evaluated by mixing Perc with each compound and performing analyses. Measurements of Perc in human breath samples collected in Tedlar bags in a workplace setting were made and compared to measurements of the same samples made by an established analytical method using charcoal tubes (National Institute of Occupational Safety and Health [NIOSH] Method 1003). The accuracy, precision, and speed of the gas chromatograph method were determined. Measurements made with the new method were within a margin of +/- 8.8% (95% CI, n = 6) of measurements made according to NIOSH Method 1003 for field samples in the range of 0.9 to 6 ppm. Method precision was determined by calculating the pooled coefficient of variation for all measurements (replicates = 3) made in the field and was found to be 5.8%.

Concomitant exposures to arsenic and polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are widespread. While BaP acts by binding to and inducing mutations in critical sites on DNA, the mechanism(s) of arsenic carcinogenesis remains unknown. Data from epidemiological studies of arsenic copper smelter workers and arsenic ingestion in drinking water suggest a positive interaction for arsenic exposure and smoking and lung cancer. A previous in vitro study showed that arsenic potentiated the formation of DNA adducts at low doses of BaP and arsenic. The present study was conducted to test the effect of arsenic on BaP-DNA adduct formation in vivo. We hypothesized that arsenic co-treatment would significantly increase BaP adduct levels in C57BL/6 mouse target organs: skin and lung. Treatment groups were: five mice, -BaP/-arsenic; five mice, -BaP/+arsenic; 15 mice, +BaP/-arsenic; 15 mice, +BaP/+arsenic. Mice in the appropriate groups were provided sodium arsenite in drinking water (2.1 mg/l), ad libitum, for 13 days (starting 9 days before BaP treatment), and 200 nmol BaP/25 ml acetone (or acetone alone) was applied topically, once per day for 4 days. DNA was extracted from skin and lung and assayed by (32)P-postlabeling. Statistical comparisons were made using independent t-tests (unequal variances assumed). BaP-DNA adduct levels in the +BaP groups were significantly higher than -BaP controls. Arsenic co-treatment increased average BaP adduct levels in both lung and skin; the increase was statistically significant in the lung (P = 0.038). BaP adduct levels in the skin of individual animals were positively related to skin arsenic concentrations. These results corroborate our in vitro findings and provide a tentative explanation for arsenic and PAH interactions in lung carcinogenesis.

The role of Fenton oxidants in DNA damage, aging, and cancer is appreciated, but not well understood. Six potential iron-binding (PIB) DNA motifs were previously identified as sites of preferential strand cleavage. Since DNA–metal binding domains are a known determinant of oxidative DNA damage, and the location of strand breaks explains where oxidant attack occurs, we sought to determine whether the likelihood of base change mutations is a function of neighboring PIB motifs. We developed a sliding window function that computes the density of PIB motifs on both strands, within 4–12 bp, for each location along a target gene. This range of window sizes reflects known diffusion distances of Fenton reaction products. Using mutational databases, odds of mutation at each base were calculated relative to PIB motif density, for all PIB motif types in aggregate, or for individual PIB motifs. Using mutational data from lacI transgenic animals, we observed a non-random distribution of PIB motifs, associated with increased odds of mutation, showing a strand bias. Sensitivity analysis confirmed that the optimum association between PIB motif density and mutations occurs when a 7 bp radius is used for the window size. Randomly simulated mutations showed no association with PIB motif density. When the method was applied to human TP53 mutation data, we saw similar results, but no strand bias. As PIB motif density rises, linear trends are observed for increasing odds of mutation. Sensitivity analysis revealed associations between PIB motifs and GC → AT transitions and GC → TA transversions—the most commonly observed types of mutations arising from oxidative DNA damage. DNA–metal binding motifs are found in a wide variety of biological contexts, including many where conformational sensitivity to redox state is important. These techniques can help elucidate how DNA–iron-binding may affect lesions and subsequent mutations from multiple agents.

A multiple biomarker approach is required to integrate for metabolism, temporal response and exposure-response kinetics, biological relevance, and positive predictive value. Carcinogen DNA adduct analysis can be used in animal and in vitro studies to detect absorption permutations caused by mixture interactions, and to control metabolic variation when specific CYP450 genes (1A1 or 1A2) are knocked out. These enzymes are not critical to the metabolic activation of model Polycyclic Aromatic Compounds (PAC) and aromatic amines, respectively, as suggested by in vitro analysis. Several human studies have been carried out where multiple biomarkers have been measured. In a study of benzidine workers, the similarities in elimination kinetics between urinary metabolites and mutagenicity is likely responsible for a better correlation between these markers than to BZ-DNA adducts in exfoliated cells. In a study of rubber workers, the relationship between specific departments, urinary 1 HP and DNA adducts in exfoliated cells coincided with the historical urinary bladder cancer risk in these departments; the same relationship did not hold for urinary mutagenicity. In a study of automotive mechanics, biomarkers were used to monitor the effectiveness of exposure interventions. These data reinforce the notion that carcinogen biomarkers are useful to monitor exposure, but that a complementary approaches involving effect and perhaps susceptibility biomarkers is necessary to obtain the necessary information.

Co-exposures to complex mixtures of arsenic and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) are common in the environment. These two environmental pollutants are carcinogenic, but the nature of their molecular interactions in the induction of cancer is not well understood. Additive or synergistic interactions have been proposed to explain why arsenic, which is not a potent mutagen itself, is comutagenic with a variety of DNA-damaging agents. We have examined the genotoxicity of BaP-arsenic mixtures. We find that exposure of mouse hepatoma Hepa-1 cells to low concentrations of arsenite increases BaP-DNA adduct levels by as much as 18-fold. This effect requires the activation of BaP by cytochrome p450 1A1 (CYP1A1), although arsenite does not alter BaP-inducible CYP1A1 enzymatic activity, suggesting that arsenite acts downstream of metabolic BaP activation. Glutathione homeostasis was important in modulating the potency of arsenite. In cells depleted of reduced glutathione, arsenite increased BaP-DNA adduct formation by an even greater degree than in cells co-treated with BaP and arsenite in control medium. Although arsenic comutagenicity has been attributed to inhibition of DNA repair, arsenite treatment did not alter adduct removal kinetics in BaP-treated cells, suggesting that mechanisms upstream of DNA repair are responsible for increased adduct levels. Concentrations of arsenite and BaP that had no measurable mutagenic effect alone, increased mutation frequency at the Hprt locus by eight-fold when given in combination, demonstrating a comutagenic response between BaP and arsenite. These results provide strong support for the positive interaction between arsenic and PAH-induced cancer observed in epidemiology studies, and help to identify additional mechanistic steps likely to be involved in arsenic comutagenesis.