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While tobacco smoke has been conclusively identified as a lung carcinogen, there is much debate over which smoke constituent(s) are primarily responsible for its carcinogenicity. Previous studies in our laboratory suggested that highly lipophilic carcinogens are slowly absorbed in the thicker epithelium of the conducting airways, potentially allowing for substantial local metabolism. The bioactivation of polycyclic aromatic hydrocarbons in airway epithelium may, hence, be important in tobacco smoke-induced carcinogenesis. In the present study, the hypothesis of slow absorption and substantial local metabolic activation of highly lipophilic carcinogen in airway epithelium was tested in dogs. A single dose of tritiated benzo[a]pyrene (BaP) dissolved in a saline/phospholipid suspension was instilled in the trachea, just anterior to the carina. At intervals of a few minutes up to 30 min over a 3-h period, blood samples were drawn from the azygous vein, which drains the area around the point of instillation, and from the systemic circulation. Tissue samples were taken at the end of the experiment. The concentration of BaP with depth into the tracheal mucosa was determined with autoradiography. BaP was slowly absorbed into the trachea with a half-time of approximately 73 min, which is consistent with diffusion-limited passage through the epithelium and lead to local doses in the tracheal epithelium that were more than a 1000-fold those of other tissues. The long retention of BaP in the epithelium provided the local metabolizing enzymes with high substrate levels over a long period, resulting in extensive metabolism. At 3 h after the exposure, 23% of the BaP-equivalent activity remained in the tracheal mucosa. Of this fraction, 13% was parent compound, 28% was organic extractable, 31% was water-soluble, and 28-7% of the instilled dose was bound to tracheal tissues. These results explain the tendency of highly lipophilic carcinogens, such as BaP, to induce tumors at the site of entry and, furthermore, indicate that the highly lipophilic components of tobacco smoke and polluted air may be the most important contributors to lung tumors of the conducting airways. [ABSTRACT FROM PUBLISHER]

Trans- and cis-1,2-dichloroethylene (DCE) isomers inhibit their own metabolism in vivo by inactivation of the metabolizing enzyme, presumably the cytochrome P450 isoform, CYP2E1. In this study, we examined cytochrome P450 isoform-specific inhibition by three chloroethylenes, cis-DCE, trans-DCE, and trichloroethylene (TCE), and evaluated several kinetic mechanisms of enzyme inhibition with physiological models of inhibition. Trans-DCE was more potent than cis-DCE, and both were much more effective than TCE in inhibiting CYP2E1. The kinetics of in vitro loss of p-nitrophenol hydroxylase (pNP-OH) activity (a marker of CYP2E1) in microsomal incubations and of the in vivo gas uptake results were most consistent with a mechanism in which inhibition of the metabolizing enzyme (CYP2E1) was presumed to be related to interaction of a reactive DCE metabolite with remaining substrate-bound, active CYP2E1. The kinetics of inhibition by TCE, a weak inhibitor in vitro, were very different from that of the dichloroethylenes. With TCE, parent compound concentrations influenced enzyme loss. Trans-DCE was a more potent inhibitor of CYP2E1 than cis-DCE based on both in vivo and in vitro studies. Quantitative differences in the inhibitory properties of the 1,2-DCE isomers may be due to the different stability of epoxides formed from bioactivation by CYP2E1. Epoxide intermediates of DCE metabolism, reacting by water addition, would yield dialdehyde, a potent cross-linking reagent.

1,3-Butadiene (BD), a compound used extensively in the rubber industry, is weakly carcinogenic in Sprague-Dawley rats after chronic exposures to concentrations of 1000 and 8000 ppm. Conversely, in B6C3F1 mice, tumors occur after chronic exposures to concentrations as low as 6.25 ppm. Previously, we have shown that tissue concentrations of the mutagenic BD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), are present in greater concentrations in mice than in rats following acute exposures to low levels (100 ppm or less). This disparity was particularly significant for the diepoxide. We hypothesized that if these epoxides are involved in the carcinogenic response of BD, then they will also be present in rat tissues at relatively high concentrations following exposures to 8000 ppm BD. In the present study, concentrations of the BD epoxides, BDO and BDO2, were determined in blood of female Sprague-Dawley rats following a single 6-h exposure and 10 repeated exposures to a target concentration of 8000 ppm BD. Concentrations of these epoxides were also determined in a number of other tissues, including the primary rat target organ-mammary gland-following 10 repeated exposures. Blood concentrations of BDO were 4030 pmol/g +/- 191 following a 6-h exposure and were 18% lower following the 10-day exposure. Blood concentrations of BDO2, following the 8000 ppm exposures, were very similar to those previously observed after exposures to 62.5 ppm BD (11 +/- 1 and 17 +/- 1 pmol/g following exposures of 6h and 6h/day for 10 days, respectively.) Concentrations of BDO ranged from 740 +/- 110 (femur) to 8990 +/- 1150 (fat) pmol/g tissue. Concentrations of BDO2 were similar among eight tissues analyzed, ranging from 5 +/- 1 (femur) to 17 +/- 3 (heart) pmol/g tissue. Tissue concentrations of butadiene monoepoxide were increased by 17- to 50-fold in tissues from rats exposed by inhalation to 8000 ppm BD as compared to tissues from rats exposed to 62.5 ppm BD. Based on earlier studies at our institute the internal dose of BD increases approximately 14-fold in the 8000 ppm-exposed rats compared to rats exposed to 62.5 ppm BD. Concentrations of butadiene diepoxide in rat tissues following an exposure to 8000 ppm BD were similar to those observed in rat tissues following exposures to 62.5 ppm BD. This study shows that pathways responsible for the accumulation of BDO2 in rats are saturated following low-level BD exposures. This suggests that the primary determinant of BD tumorigenicity in rats is not butadiene diepoxide. The high levels of BDO observed in rat mammary tissue suggest that this metabolite may be a more important determinant of BD carcinogenesis in the rat., (Copyright 1998 Academic Press.)

Two members of the cytochrome P450 2A subfamily, CYP2A10 and 2A11, are abundant nasal enzymes previously characterized in rabbit olfactory microsomes. Rabbit CYP2A is active toward a number of nasal toxicants, including the rat nasal procarcinogen hexamethylphosphoramide (HMPA). While P450s immunochemically related to the rabbit CYP2As have been detected in rat and human nasal mucosa, confirmation of these enzymes as members of the CYP2A subfamily and efforts to characterize their ability to bioactivate toxicants have been limited. In the present study, the regional distribution and cell-specific expression of CYP2A in the rat nasal cavity were examined using an antibody to rabbit CYP2A10/11. In sections of the anterior nose, immunoreactive CYP2A was present in ciliated cells of the nasal respiratory epithelium and cuboidal epithelial cells of the nasal transitional epithelium, but was absent in squamous epithelial cells. The most intense immunostaining was observed in the posterior nose. Olfactory sustentacular cells and Bowman's gland cells in sections posterior to the nasal papilla stained most intensely. Western blot analysis revealed that anti-CYP2A10/11 recognized a sharp band of approximately 50 kDa in nasal respiratory and olfactory microsomes, supporting the premise that the antibody is reacting with a cytochrome P450 enzyme. The nasal expression of CYP2A6 mRNA--a member of the human CYP2A subfamily having a high degree of homology to rabbit 2A10 and 2A11--was examined in human surgical patients. Middle turbinectomy tissues--largely composed of nasal respiratory epithelia--from 11 patients were analyzed for the presence of CYP2A6 using reverse transcription-polymerase chain reaction (RT-PCR). Identification of CYP2A6 was confirmed by DNA sequencing of RT-PCR products. CYP2A6 mRNA was detected in all of the human samples analyzed. In additional experiments, human CYP2A6 metabolized HMPA to formaldehyde, suggesting that this compound might cause nasal toxicity in humans. The identification of CYP2A cytochromes in rat and human nasal tissues may have important implications for risk assessment of inhaled xenobiotics.

Species differences in the metabolism of 1,3-butadiene (BD) have been studied in an effort to explain the major differences observed in the responses of mice, the sensitive species, and rats, the resistant species, to the toxicity of inhaled BD. BD is metabolized by the same metabolic pathways in all species studied, but there are major species differences in the quantitative aspects of those pathways. Of the species studied, mice are the most efficient at metabolizing BD to the initial metabolite, the monoepoxide (BDO). Mice either convert most of the BDO to the diepoxide (BDO2), the most mutagenic of the BD metabolites, or form conjugates of the BDO with glutathione (GSH). Rats, on the other hand, are less active at forming BDO, oxidize very little of the BDO to BDO2, and form GSH conjugates with either the BDO or its hydrolysis product, butenediol. Primates convert even less of inhaled BD to BDO and hydrolyze most of the BDO to the butenediol. The extent to which primates form BDO2 is unknown. Because of the association of high levels of the highly mutagenic BDO2 with the sensitive rodent strain, it is important to determine the production of this metabolite in primates, particularly humans.

Levels of butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2) were compared in tissues of male Sprague-Dawley rats and male B6C3F1 mice and in tissues of male and female Sprague-Dawley rats following inhalation exposures to 62.5 ppm 1,3-butadiene (BD). In male rats, BDO2 levels were highest in blood and were present at a concentration of only 5 +/- 1 pmol/g. Following a 6-h exposure, the concentration of BDO2 in the blood, femurs, lung and fat of female rats was 3 to 7-fold that of male rats. Levels of BDO were similar in tissues of female and male rats. Generally, levels of BDO were approximately 3 to 8-fold greater in mouse tissues as compared with rat tissues following 4-h exposures to BD. In blood, 204 +/- 15 pmol/g BDO2 was detected in male mice, while in rats, blood BDO2 levels were 5 +/- 1 pmol/g. This study shows marked species differences in tissue levels of BD epoxides, particularly BDO2, in rats and mice, and is the first to show gender differences in BD metabolism.

Bromodichloromethane (BDCM), a carcinogenic water disinfection by-product, has been shown to be metabolized to intermediates that covalently bind to lipids and proteins, and this binding has been associated with trihalomethane-induced renal and hepatic toxicity. In this study, the effects of glutathione (GSH) on in vivo BDCM toxicity and in vitro BDCM macromolecular binding were evaluated. The in vivo toxicity of BDCM in animals pretreated with buthionine sulfoximine (BSO, a glutathione synthesis inhibitor) and in untreated male Fischer 344 rats was investigated. In another experiment, covalent binding to protein and lipid was quantified after [14C]BDCM was incubated with hepatic microsomal and S9 fractions and renal microsomes from F344 rats, under aerobic and anaerobic conditions, with and without added GSH. After oral dosing with BDCM, the BSO-pretreated animals had greatly increased levels of serum indicators of hepatotoxicity and serum and urinary indicators of nephrotoxicity compared to those in animals dosed solely with BDCM. Histopathological examination revealed that hepatic necrosis was more severe than renal necrosis in the BSO-treated rats. When GSH was added to an aerobic incubation, protein binding was decreased in hepatic microsomal and S9 fractions by 92 and 83%, respectively. GSH also decreased lipid binding by 55% in hepatic microsomal incubations carried out under anaerobic conditions. Addition of GSH decreased renal microsomal protein (aerobic) and lipid binding (anaerobic) by 20 and 43%, respectively. These data indicate that GSH is an important protective factor in the toxicity associated with BDCM.

1,3-Butadiene (BD), a compound used extensively in the rubber industry, is carcinogenic in the male and female Sprague-Dawley rats after chronic exposures to 1000 and 8000 p.p.m. In terms of incidence of tumors the majority were in mammary tissue, thus the incidence of tumors in female rats exceeded that in males in chronic carcinogenicity studies. In the present study the production and disposition of butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), mutagenic BD metabolites, were examined in male and female Sprague-Dawley rats following a low level inhalation exposure to BD. The rats were exposed to a target concentration of 62.5 p.p.m. BD by nasal inhalation for 6 h. Immediately after exposure blood, bone marrow, lung and fat samples were removed from all the animals and mammary tissue was removed from females. The samples were prepared by cryogenic-vacuum line distillation and analyzed for the epoxides using multidimensional gas chromatography-mass spectrometry. Levels of BDO in the blood were 25.9 +/- 2.9 and 29.4 +/- 2 pmol/g in male and females respectively. The levels of this metabolite were also similar in males and females in the other tissues examined. The greatest amounts of BDO were in fat (175 +/- 21 and 203 +/- 13 pmol/g in males and females respectively). Levels of BDO2 were approximately 5-fold greater in the blood of female rats compared with male rats. In the other tissues examined BDO2 was also consistently greater in tissues from females. In fat BDO2 was present at a concentration of 7.7 +/- 1.3 and 1.1 +/- 0.1 pmol/g tissue in females and males respectively. Mammary tissue from female rats contained 10.5 +/- 2.4 pmol/g BDO2, a level slightly lower than that observed in blood. The ratios of the two epoxides differed markedly between males and females in all tissues examined. Differences were most pronounced in lung and fat tissues, where BDO/BDO2 ratios were 9 and 0.6 (lung) and 159 and 26 (fat) for males and females respectively. This study is the first to describe a gender difference in the metabolism of BD. The greater production of the highly mutagenic BDO2 in females may play a role in the increased incidence of mammary tumors after chronic exposure to BD.

1,3-Butadiene (BD), a chemical used extensively in the production of styrene-butadiene rubber, is carcinogenic in Sprague-Dawley rats and B6C3F1 mice. Chronic inhalation studies revealed profound species differences in the potency and organ-site specificity of BD carcinogenesis between rats and mice. BD is a potent carcinogen in mice and a weak carcinogen in rats. Previous studies from our laboratory and others have shown marked differences between rats and mice in the metabolism of BD, which may account for species differences in carcinogenicity. The purpose of the present study was to examine the production and disposition of two mutagenic BD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), in blood and other tissues of rats and mice during and following inhalation exposures to a target concentration of 62.5 p.p.m. BD. BDO was increased above background in blood, bone marrow, heart, lung, fat, spleen and thymus tissues of mice after 2 h and 4 h exposures to BD. In rats, levels of BDO were increased in blood, fat, spleen and thymus tissues. No increases in BDO were observed in rat lungs. BDO2, the more mutagenic of the two epoxides, was increased in the blood of rats and mice at 2 and 4 h after initiation of exposure to BD. In mice, BDO2 was detected in all tissues examined immediately following the 4 h exposure. This metabolite was detected in heart, lung, fat, spleen and thymus of rats, but at levels 40- to 160-fold lower than those seen in mice. Immediately after the 4 h exposure, blood levels of BDO2 were 204 +/- 15 pmol/g for mice but were 41-fold lower for rats. In the sensitive mouse target organs, heart and lungs, levels of BDO2 exceeded BDO levels immediately after the exposure. This study shows that the levels of BD epoxides are markedly greater in the mouse BD target organs. The high concentrations of BDO2 in these organs suggest that this compound may be particularly important in BD-induced carcinogenesis. Thus, although BD is oxidatively metabolized by similar metabolic pathways in rats and mice, the substantial quantitative differences in tissue levels of mutagenic epoxides between species may be responsible for the increased sensitivity of mice to BD-induced carcinogenicity.

A new method was developed to quantify the levels of 1,3-butadiene (BD), butadiene monoxide (BDO), and butadiene diepoxide (BDO2) in blood. The method was based on vacuum distillation of tissues followed by analysis of the distillates using multidimensional GC/MS. Metabolites isolated from blood by vacuum distillation were condensed into a cold trap. After warming the traps to room temperature, BD and BDO were sampled from the trap vapor phase. BDO2 was extracted from the codistilled water phase using ethyl acetate. Samples were analyzed using a multidimensional GC system equipped with a custom-built interface. The method was validated by analysis of 0.75-mL aliquots of mouse blood spiked with 5.0, 3.4, and 0.55 nmol of BD, BDO, and BDO2, respectively. The recoveries of analytes were 96 +/- 18%, 125 +/- 15%, and 98 +/- 12%, respectively (mean +/- SD, n = 6). Kinetic studies indicated no loss of BDO and BDO2 in blood held at room temperature in closed containers for up to 1 h. The method was applied to blood samples from B6C3F1 mice and Sprague-Dawley rats exposed by inhalation (nose-only) to 100 ppm BD for 4 h. Blood levels of BD and BDO in exposed rats were 4.1 +/- 1.0 and 0.10 +/- 0.06 microM, respectively (mean +/- SD, n = 6). Levels of BDO2 were below the limits of detection (0.01 nmol/mL). Blood levels of BD, BDO, and BDO2 in mice exposed to 100 ppm BD for 4 h were 2.9 +/- 1.3, 0.38 +/- 0.14, and 0.33 +/- 0.19 microM, respectively (mean +/- SD, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

The carcinogenic water disinfection byproduct, bromodichloromethane (BDCM), produces renal and hepatic toxicity in rodents in acute and subchronic studies. In the present investigation, female rats and mice (n = 6) were dosed daily for 5 consecutive days with BDCM (dissolved in an aqueous, 10% Emulphor solution) by gavage. Rats received 75, 150 and 300 mg BDCM/kg body weight/day and mice received 75 and 150 mg BDCM/kg body weight/day. Two rats in the 300 mg/kg/day treatment group died on day 5. On day 6, the animals were sacrificed and serum samples were taken for analysis of indicators of hepatic and renal toxicity. Livers and kidneys were excised and samples taken for histopathological evaluation. Portions of the livers were also utilized to produce microsomes for analysis of cytochrome P450 enzyme activities and total P450 content. Total hepatic cytochrome P450 was decreased in rats dosed with 150 and 300 mg BDCM/kg body weight/day, but was not significantly affected in BDCM-treated mice. Serum lactate (LDH) and sorbitol (SDH) dehydrogenase, aspartate aminotransferase (AST), creatinine and blood urea nitrogen were increased above those of controls in rats dosed with 300 mg BDCM/kg/day. These data suggested that hepatic and renal damage had occurred in this treatment group. This was confirmed by histopathological analyses which revealed that lesions occurred in both hepatic and renal tissues from rats dosed with 150 and 300 mg BDCM/kg/day. The hepatic lesions were centrilobular and primarily consisted of vacuolar degeneration. The hepatotoxicity indicators alanine aminotransferase (ALT) and SDH were increased in mice dosed with 150 mg BDCM/kg/day. However, no histopathological lesions were observed in these animals. This study shows that BDCM is both hepatotoxic and nephrotoxic to female rats after repeated dosing, but is only weakly hepatotoxic to female mice at the administered doses. Also, reduced activities of hepatic cytochrome P450 were observed in rats, but not mice. These species differences in toxicity and xenobiotic metabolizing enzyme inhibition caused by BDCM suggest that an understanding of the mechanism of toxicity of this compound will be critical when extrapolating rodent toxicity data to humans for this environmental pollutant.

3-Methylindole (3MI), a fermentation product of tryptophan produced by intestinal and ruminal microflora, has been shown to cause pneumotoxicity in several species subsequent to cytochrome P450-mediated biotransformation. Among several species studied, rabbits are comparatively resistant to 3MI-induced pneumotoxicity, especially when compared to goats or mice. In this study, rabbit pulmonary cells and subcellular fractions were used to examine the metabolism and bioactivation of 3MI. A covalent-binding metabolite was produced in 3MI incubations by both Clara cells and macrophages. The addition of the cytochrome P450 inhibitor, 1-aminobenzotriazole, to these incubations inhibited the production of covalent-binding metabolite(s) by 94% in Clara cells and only 24% in macrophages. In incubations of Clara cells or macrophages with 3MI and N-acetylcysteine (NAC), a polar conjugate was detected and tentatively identified as an adduct of 3-hydroxy-3-methylindolenine (3H3MIN). Also identified were 3[(N-glutathione-S-yl)-methyl]-indole (3MI-GSH) and 3-methyloxindole (3MOI). In rabbit lung microsomal incubations with 3MI and glutathione (GSH), 3MI-GSH, 3MOI, indole-3-carbinol, and a GSH adduct of 3H3MIN were identified. The addition of cytosol to the microsomal incubations with GSH did not increase the rate of formation of the GSH adducts, indicating that cytosolic GSH-S-transferases are not essential in the formation of these metabolites. GSH significantly decreased the covalent binding of an electrophilic metabolite in microsomal incubations. These data suggest that GSH may be important in the mitigation of 3MI toxicity. Furthermore, the comparison of 3MI bioactivation to electrophilic intermediates in Clara cells and alveolar macrophages suggests that 3MI is metabolized by different oxidative pathways in the two different cell types, although the same metabolites were produced by the two cell types. This study shows that rabbit pulmonary enzymes are capable of bioactivating 3MI to reactive intermediates which become covalently bound to cellular macromolecules. This indicates that the relative resistance of rabbits to 3MI-induced pneumotoxicity is probably not due to differences in metabolic enzymes which convert 3MI to reactive intermediates.

Both nitroreduction and ring-oxidation appear to be important pathways for the in vivo metabolic activation of 1-nitropyrene (1-NP), a tumorigenic environmental contaminant. Previous studies, however, suggest that ring-oxidation is primarily responsible for the S9-mediated mutagenicity of 1-NP in Chinese hamster ovary (CHO) cells. In this study, an anaerobic rat liver S9 metabolizing system was used to facilitate the nitroreduction of 1-NP to a mutagen in repair-proficient CHO-K1-BH4 cells and excision-repair-deficient CHO-UV5 cells, and results using this system were compared with those obtained from the aerobic S9 metabolism of 1-NP. Anaerobic S9 metabolism of 2.5-15 micrograms/ml of 1-NP produced 15 +/- 3 mutants/10(6) cells/microgram 1-NP/ml with CHO-UV5 cells and 3 +/- 1 mutants/10(6) cells/microgram 1-NP/ml with CHO-K1-BH4 cells (different at P less than 0.001). When the assays were conducted with CHO-K1-BH4 cells, the number of mutants produced by 1-NP using aerobic treatment conditions was similar to that found using anaerobic conditions. In contrast, the aerobic incubations resulted in significantly fewer 1-NP-induced mutants than the anaerobic treatments when the assays were conducted with CHO-UV5 cells. Examination of the metabolites produced during these incubations indicated that under anaerobic conditions 1-NP was efficiently converted to 1-aminopyrene, while aerobic metabolism resulted in the formation of 1-NP phenols and dihydrodiols. DNA adduct analysis by 32P-postlabeling revealed that 1-NP treatment using the anaerobic procedure produced CHO-cell adducts by the reduction of 1-NP to N-hydroxy-1-aminopyrene, while aerobic incubations resulted in adducts produced by other metabolic pathways, probably involving ring-oxidation. These findings indicate that the S9-mediated metabolism of 1-NP under anaerobic conditions produces mutations and DNA adducts in CHO cells that are the result of nitroreductive metabolism. The results with aerobic S9 metabolism were consistent with the previous conclusion that this system mediated the mutagenicity of 1-NP in CHO cells mainly through the generation of ring-oxidized metabolites. The combination of the anaerobic and aerobic S9 metabolism procedures provides a new approach for evaluating the mutagenicity of nitropolycyclic aromatic hydrocarbons in mammalian cells.

Twelve human cytochrome P450s and one mouse P450 were produced in HepG2 cells using vaccinia virus cDNA expression and analyzed for their ability to bioactivate the pneumotoxin, 3-methylindole (3MI), to an electrophilic metabolite(s) which alkylated cellular macromolecules. Cell lysates containing CYP2C8, CYP3A4, CYP2A6 and CYP2F1 metabolized 3MI to an intermediate(s) that became covalently bound to lysate material. A control lysate produced from cells which had been infected with a wild-type vaccinia virus was not able to bioactivate 3MI. The mouse 1A2 enzyme metabolized 3MI at a rate of 75.4 pmol/mg protein/minute, while the rate of metabolism in the lysate containing the human 1A2 P450 enzyme was not different from that in the control lysate. Therefore, the catalytic capabilities of orthologous P450 enzymes to activate 3MI cannot be extrapolated among different species. These results indicate that human P450s are capable of bioactivating 3MI to a metabolite which binds to cellular macromolecules suggesting that this compound may be toxic to humans.

The direct-acting mutagenicity of 1-nitropyrene (1-NP), a tumorigenic environmental pollutant and model nitropolycyclic aromatic hydrocarbon, was studied in Chinese hamster ovary (CHO) cells. Previous reports indicated that 1-NP, a potent direct-acting mutagen in Salmonella typhimurium, was mutagenic in CHO cells only in the presence of an exogenous activation system. In this study, a DNA-repair-deficient CHO cell line, CHO-UV5, and the repair-proficient CHO-K1-BH4 cell line were used to measure the direct-acting mutagenicity of highly purified samples of 1-NP. Exposure of CHO-K1-BH4 cells for 5, 12, and 24 hr did not increase mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (p greater than 0.1; paired t test; n = 5-6), while treatment of CHO-UV5 cells for 24 hr produced a statistically significant induction of mutants (22 +/- 6 mutants per 10(6) clonable cells vs a solvent control value of 9 +/- 3 per 10(6) cells; p less than 0.01; n = 6). 32P-Postlabeling analysis of the DNA adducts formed in CHO-UV5 cells following 1-NP exposure revealed the presence of a single major adduct. Based on its chromatographic properties, its sensitivity to nuclease P1 digestion, and its resistance to hydrazine treatment, the adduct appeared to be N-(deoxyguanosin-8-yl)-1-aminopyrene, which was presumably produced by nitroreduction of 1-NP to N-hydroxy-1-aminopyrene. These results suggest that the use of extended treatment times and DNA-repair-deficient cells may be required to assess adequately the mutagenicity of nitropolycyclic aromatic hydrocarbons in CHO cells.

1-Nitropyrene, a tumorigenic environmental pollutant, is mutagenic in Chinese hamster ovary (CHO) cells in the presence of a liver homogenate 9000 g supernatant fraction (S9). The metabolic pathways involved in this response were studied by comparing the mutagenicities at the hypoxanthine-guanine phosphoribosyl transferase locus of 1-nitropyrene, some oxidized microsomal metabolites of 1-nitropyrene, and related compounds. In the absence of S9, pyrene 4,5-oxide and 6-hydroxy-1-nitropyrene displayed the highest mutagenicities, followed by 1-nitropyrene 9,10-oxide and 1-nitropyrene 4,5-oxide; 3- and 8-hydroxy-1-nitropyrene were weaker mutagens, while pyrene and 1-nitropyrene were essentially without activity. With S9, the order of mutagenic potency was 1-nitropyrene 4,5-oxide greater than 6-hydroxy-1-nitropyrene approximately 1-nitropyrene 9,10-oxide greater than 1-nitropyrene approximately 3-hydroxy-1-nitropyrene approximately 8-hydroxy-1-nitropyrene greater than pyrene approximately pyrene 4,5-oxide, with the latter two compounds being essentially inactive. Inclusion of the epoxide hydrolase inhibitor 1,2-epoxy-3,3,3-trichloropropane during the S9-mediated treatment of CHO cells with 1-nitropyrene increased mutation induction 5-fold. Also, liver microsomes prepared from guinea-pigs treated with Aroclor 1254 mediated a stronger mutagenic response with 1-nitropyrene than microsomes from Aroclor-treated rats. 1-Nitropyrene was essentially non-mutagenic in the presence of microsomes from untreated and phenobarbital-treated rats. Examination of the 1-nitropyrene metabolites produced during the microsomal incubations indicated that Aroclor-induced guinea-pig microsomes yielded substantial amounts of 1-nitropyrene 4,5-dihydrodiol, while Aroclor-induced rat microsomes produced 6-fold more 6- and 8-hydroxy-1-nitropyrene than phenobarbital microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are environmental pollutants and are S9-mediated mutagens in the Chinese hamster ovary (CHO) cell/hypoxanthine-guanine phosphoribosyl transferase assay. In this study, we examined the pathways leading to the mutagenic activation of these compounds in CHO cells. The microsomal metabolites of 1- and 3-nitro-BaP, the 1- and 3-nitro-BaP trans-7,8-dihydroxy-7,8-dihydrodiols (trans-7,8-dihydrodiols) and the 1- and 3-nitro-BaP trans-9,10-dihydrodiols, were isolated and tested for mutagenicity. At the concentrations assayed, both trans-9,10-dihydrodiols were non-mutagenic with and without S9 activation. In contrast, the trans-7,8-dihydrodiols of 1- and 3-nitro-BaP were direct-acting mutagens and these responses were similar in magnitude to the S9-mediated mutagenicities of the parent nitro-BaPs. S9 increased the mutagenic responses of the trans-7,8-dihydrodiols approximately 20-fold. Inhibition of epoxide hydrolase decreased the S9-mediated mutagenicity of 1-nitro-BaP by half, but doubled the S9-mediated mutagenicity of 3-nitro-BaP. These results suggest that in CHO cells: (i) the major route of mutagenic activation of 1- and 3-nitro-BaP involves S9-generated derivatives of the trans-7,8-dihydrodiols, e.g. bay-region diol epoxides; (ii) reactive nitroarene oxides may contribute to mutation induction by 3-nitro-BaP; and (iii) metabolic routes involving trans-9,10-dihydrodiol formation result in detoxification.

Previous studies showed that binary mixtures of the environmental pollutants 1- and 3-nitrobenzo[a]pyrene produced a synergistic mutational response in the Salmonella reversion assay. Since nitroreduction is believed to mediate the direct-acting mutagenicity of the individual compounds, we have examined the role of nitroreduction in the mutagenicity of mixtures of 1- and 3-nitrobenzo[a]pyrene in the Salmonella plate incorporation assay. While mixtures of 1- and 3-nitrobenzo[a]pyrene induced up to 183% more revertants in strain TA98 than produced by equivalent amounts of the individual compounds, in the nitroreductase-deficient strain TA98NR the same mixtures only induced up to 57% more revertants than the individual compounds. Analysis of mixtures of 1- and 3-nitrosobenzo[a]pyrene (the two-electron reduction products of 1- and 3-nitrobenzo[a]pyrene) for mutation induction in TA98 yielded no evidence of a synergistic effect between the compounds. The mutagenicity of the mixtures was dependent upon the amount of the more mutagenic component. Salmonella cultures were also incubated with mixtures of 1- and 3-nitrobenzo[a]pyrene, as well as with equivalent amounts of the individual compounds. In two experiments, nitroreductive ability, as measured by the amount of 1-nitropyrene metabolized to 1-aminopyrene in 1 hr, was increased 9 to 105% in cultures pretreated with the mixtures as compared with cultures pretreated with the individual compounds. The results of this study support the hypothesis that nitroreduction is a major factor in the synergistic mutational response induced by 1- and 3-nitrobenzo[a]pyrene in Salmonella typhimurium.