Molecular analysis of lacI mutants from transgenic fibroblasts exposed to 1,2-epoxybutene

1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites: 1,2-epoxybutene (EB) and 1,2:3,4- diepoxybutane (DEB). We reported previously that lacI transgenic mice exposed to BD had an increased frequency of specific base substitution mutations in the bone marrow and spleen relative to unexposed controls. In the experiments described here, we determined the mutagenicity and mutational spectrum of EB in Rat2 lacI transgenic fibroblasts as a means of assessing the contribution of this metabolite to the lacI mutational spectrum of BD. Rat2 cells were exposed to 0, 0.4, 0.6, 0.8 or 1.0 mM EB for 24 h, resulting in a range of cell survival from 100 to 15%, respectively. Mutagenicity was assessed at 0, 0.6 and 1.0 mM EB. Unexposed controls had a background mutant frequency of 6 +/- 1 +/- 10(-5), while the mutant frequency in cells exposed to 0.6 and 1.0 mM EB was increased 2- and 3-fold, respectively. DNA sequence analysis of 154 lacI mutants recovered in these experiments revealed an increase in the frequency of specific base substitution mutations in cells exposed to 1.0 mM EB compared with controls. These included G:C-->A:T transitions at non-CpG sites, G:C-->T:A transversions and A:T-->T:A transversions, which have all been observed in lacI mutants isolated from transgenic mice exposed to BD. These results suggest that EB causes mutation primarily by base substitution and that the spectrum of these mutations closely resembles that of BD. These data, along with previous findings from our laboratory, suggest that EB is more likely than DEB to be primarily responsible for the lacI mutational spectrum observed in lacI transgenic mice exposed to BD.      

1,2-dihydro-1,2-dihydroxy-5-methylchrysene, a major activated metabolite of the environmental carcinogen 5-methylchrysene.

The metabolic activation of the environmental carcinogen 5-methylchrysene was studied by combining high-pressure liquid chromatographic analysis of metabolites formed in vitro with assays of these metabolites for mutagenic activity toward Salmonella typhimurium. Metabolites were formed by incubation of 5-methylchrysene with the 9000 x g supernatant from Aroclor-treated rat livers. With the use of reverse-phase columns, the metabolites were resolved into nine peaks, A to I. Each peak was collected and tested for mutagenicity with activiation. Significant mutagenic activity was observed primarily in peak E and to a lesser extent in peak D. None of the other metabolites showed significant mutagenic activity. The major mutagenic metabolite (peak E) was identified as 1,2-dihydro-1,2-dihydroxy-5-methylchrysene (7.0% from 5-methylchrysene); Peak D was 7,8-dihydro-7,8-dihydroxy-5-methylchrysene (2.6% from 5-methylchrysene). Other metabolites included 9,10-dihydro-9,10-dihydroxy-5-methylchrysene, 9-hydroxy-5-methylchrysene, 7-hydroxy-5-methylchrysene, 1-hydroxy-5-methylchrysene, and 5-hydroxymethylchrysene. These results indicate that 1,2-dihydro-1,2-dihydroxy-5-methylchrysene is a major proximate mutagen of 5-methylchrysene.     

Detection of mutagenicity of the colon carcinogen 1,2-dimethylhydrazine by the host-mediated assay and its correlation to carcinogenicity.

Mutagenic potential of 1,2-dimethylhydrazine (DMH) was investigated in the host-mediated assay with mice used as hosts. This assay revealed potent mutagenicity of this colon carcinogen for Salmonella typhimurium G46. The mutagenicity of DMH was inhibited by pretreatment of mice with disulfiram. In addition, mouse strain and sex differences influenced the mutation induction by DMH: Mutation induction was significantly lower in C57BL/6 mice than in outbred ICR mice of either sex and was generally higher in male than in females of either C57BL/6 or ICR mice.     

Human DNA adducts of 1,3-butadiene, an important environmental carcinogen

The N-1-(2,3,4-trihydroxybutyl)adenine (N-1-THB-Ade) adducts induced by 1,3-butadiene (BD) were analysed from lymphocytes of 15 workers occupationally exposed to BD and 11 controls by (32)P-post-labelling using HPLC with radioactivity detection. The difference in the adduct levels between the BD-exposed workers (4.5 +/- 7.7 adducts/10(9) nucleotides) and the controls (0.8 +/- 1.2 adducts/10(9) nucleotides) was statistically significant (Wilcoxon rank sum test, P = 0.038). This study shows for the first time BD-induced DNA adducts in humans and suggests that N-1-THB-Ade adducts may be used to biomonitor human exposure to BD.     

New studies on trans-anethole oxide and trans-asarone oxide.

The widespread use of naturally occurring alkenylbenzenes as flavoring and fragrance agents has led to a long-standing interest in their toxicity and carcinogenicity. Among them several allyl- and propenylbenzenes have been found to be mutagenic and carcinogenic. It has been shown that the carcinogenicity of several allylbenzenes can be related to the formation of electrophilic sulfuric acid esters following 1'-hydroxylation. Unlike the allylbenzenes, the mechanisms of carcinogenesis of propenylbenzenes such as anethole and asarone are not clear. It has been reported that one of the main metabolic pathways of trans-anethole is the epoxidation of the side chain 1,2-double bond, which was responsible for cytotoxicity but not for genotoxicity. However, we report here that synthetic trans-anethole oxide prepared from trans-anethole and dimethyldioxirane is not only mutagenic for Salmonella tester strains but is also carcinogenic in the induction of hepatomas in B6C3F1 mice and skin papillomas in CD-1 mice. Synthetic trans-asarone oxide was also carcinogenic in the induction of hepatomas as well as mutagenic for Salmonella strains. Further studies are needed on these side chain oxides of trans-anethole and trans-asarone as possible metabolites in the toxicity, mutagenicity and carcinogenicity of these and other propenylbenzenes.     

Metabolism of the carcinogen (3H)6-nitrochrysene in the preweanling mouse: identification of 6-aminochrysene-1, 2-dihydrodiol as the probable proximate carcinogenic metabolite

6-Nitrochrysene (NC) is a potent lung and liver carcinogen whenadministered in multiple doses to preweanling mice. We haveinvestigated both the in vitro metabolism of (³H)NC by 9000g supernatants (S9) prepared from the livers of preweanlingmice and the in vivo metabolism of (³H)NC in these animals.The in vitro covalent binding of primary metabolites of NC toDNA after further reductive and/or oxidative metabolism wasthen examined in an attempt to define the metabolic activationpathway responsible for the formation of carcinogen-DNA adductsin NC-treated preweanling mice. NC-1,2-dihydrodiol, NC-9, 10-dihydrodiol,6-aminochrysene (AC), and several unidentified compounds werefound in ethyl acetate extracts of incubations containing (³H)NCand liver S9 from 1-or 8-day-old BLU: Ha mice. Comparison ofthe in vivo metabolism of NC in 1-day-old animals and 8-day-oldanimals which had been treated with NC on day 1 indicated thatthe formation of AC and the two NC dihydrodiols was greaterin the younger animals. Further metabolism of NC-1, 2-dihydridiolby S9 from 8-day-old mice yielded AC-1,2-dihydrodiol as a majorproduct. Incubation of AC-1,2-dihydrodiol, calf thymus DNA andliver microsomes from 3-methylcholanthrene-induced rats yieldeda single major adduct that was chromatographically and chemicallyidentical to the major adduct formed in (³HJNC-and (³H)-AC-treatedpreweanling mice. The results indicated that the major DNA adductfound in vivo is derived from the further metabolism of theproximate carcinogen AC-1, 2-dihydrodiol.     

Species differences in urinary butadiene metabolites; identification of 1,2-dihydroxy-4-(N-acetylcysteinyl)butane, a novel metabolite of butadiene.

1,3-Butadiene (BD) is used in the manufacture of styrene-BD and polybutadiene rubber. Differences seen in chronic toxicity studies in the susceptibility of B6C3F1 mice and Sprague-Dawley rats to BD raise the question of how to use the rodent toxicology data to predict the health risk of BD in humans. The purpose of this study was to determine if there are species differences in the metabolism of BD to urinary metabolites that might help to explain the differences in the toxicity of BD. The major urinary metabolites of BD in F344/N rats, Sprague-Dawley rats, B6C3F1 mice, Syrian hamsters, and cynomolgus monkeys were identified as 1,2-dihydroxy-4-(N-acetylcysteinyl)-butane (I) and the N-acetylcysteine conjugate of BD monoxide [1-hydroxy-2-(N-acetylcysteinyl)-3-butene] (II). These mercapturic acids are formed by addition of glutathione at either the double bond (I) or the epoxide (II) respectively. When exposed to approximately 8000 p.p.m. of BD for 2 h, the mice excreted 3-4 times as much metabolite II as I, the hamster and the rats produced approximately 1.5 times as much metabolite II as I, while the monkeys produced primarily metabolite I. The ratio of formation of metabolite I to the total formation of the two mercapturic acids correlated well with the known hepatic epoxide hydrolase activity in the different species. These data suggest that (i) the availability of the monoepoxide for conjugation with glutathione is highest in the mouse, followed by the hamster and the rat, and is lowest in the monkey; and (ii) the epoxide availability is inversely related to the hepatic activity of epoxide hydrolase, the enzyme that removes the epoxide by hydrolysis. The ratio of the two mercapturic acids in human urine following BD exposure may indicate the pathways of BD metabolism in humans and may aid in the determination of the most appropriate animal model for BD toxicity.     

Epidemiological and mechanistic data suggest that 1, 3-butadiene will not be carcinogenic to humans at exposures likely to be encountered in the environment or workplace

1, 3-Butadiene (BD) is a carcinogen in both rats and mice withmice being substantially more sensitive than rats. It is notknown if BD poses a carcinogenic risk for humans. Findings fromexposure assessment studies indicate that potential industrialexposure to BD in monomer, polymer, and end-user industriesis typically <2 p.p.m. Epidemiologic studies of persons occupationallyexposed to BD are inconclusive. In vitro metabolism of BD inrats, mice and human tissues indicate that there are significantquantitative species differences in the metabolic activationof BD to butadiene monoepoxide (BMO) and butadiene diepoxide(BDE) and the detoxication of BMO. Activation/detoxica-tionratios calculated using in vitro kinetic constants reveal thatratios in mice were 12-fold greater than rats and humans. Inrats and mice exposed to BD, concentrations of BMO in bloodand tissues of mice were up to 14-fold higher than in rats andBDE was only detected in mice thereby providing a strong argumentfor why mice are highly sensitive to BD carcinogenicity. Thefact that human tissues do not appear to metabolize BMO to BDEto any significant extent suggest that humans may not be sensitiveto BD carcinogenicity. In mice, BDE is a more potent carcinogenthan BMO. BDE is mutagenic in vitro at the hprt locus in humanTK6 lymphoblasts at concentrations that were 100-fold less thanthe concentration of BMO required to yield a similar mutationfrequency. Importantly, the concentrations of BDE that weregenotoxic in vitro are nearly identical to the concentrationsof BDE measured in blood and tissues of mice exposed to BD byinhalation. BD is genotoxic in mice, but not rats, followinginhalation exposure and this is paralleled by species differencesin observed tumor susceptibility. BD is not genotoxic in occupationally-exposedworkers. The genetic basis for BD carcinogenicity appears tobe primarily through induction of point mutations and deletionevents mediated via the potent genotoxic metabolite, BDE. Thegenotoxic endpoints induced by BDE (e.g., deletion and pointmutations) rather than BMO (e.g., point mutations) likely representthe underlying mechanism responsible for the striking speciesdifferences observed in the genotoxicity and carcinogenicityof BD in mice versus rats. In summary, the preponderance ofevidence which includes both epidemiological and mechanisticdata in mice, rats, and humans strongly suggests that BD willnot be carcinogenic to humans at occupational or environmentalexposures. Any cancer risk assessment for BD should use in vitrohuman tissue metabolic data and in vitro and in vivo rat datafor estimation of human cancer risks.     

Synthesis and mutagenicity of 5-alkyl-substituted chrysene-1,2-diol-3,4-epoxides

In order to explore the relationship between structure and mutagenicityof bay region diol-epoxides of chrysene substituted with analkyl group in the bay region, we compared the mutagenicityin Salmonella typhimurium TA 100of anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysenewith its 5-methyl, 5-ethyl and 5-propyl derivatives. The resultsshowed that anti-l,2-dihydroxy-3,4-epoxy-l,2,3,4-tetrahydro-5-methylchrysene(7400 revertants/nmol) was the most mutagenic of these diol-epoxidesfollowed by anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochryseneand its 5-ethyl derivative (1100 revertants/nmol). The 5-propylsubstituted diol-epoxide was inactive at the doses tested. Theresults demonstrate that steric factors are dominant in theexpression of methylchrysene diol-epoxide mutagenicity in S.typhimuriumand suggest that the molecular shape of the 5-methyl substituteddiol-epoxide leads to a unique reaction with DNA associatedwith high mutagenicity and tumorigenicity.     

Antimutagenic effects of 2(3)-tert-butyl-4-hydroxyanisole and of antimicrobial agents.

Administration of the antioxidants 2(3)-tert-butyl-4-hydroxyanisole (BHA) and ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) with the diet resulted in a marked decrease in the levels of mutagens present in mice treated with benzo(a)pyrene. This was reflected in the results of the host-mediated assay and determinations of the mutagenic activities of the urine, with the use of the sensitive tester strains TA100 and TA98 of Salmonella typhimurium his- developed by Ames and coworkers. Treatment with BHA was effective also in reducing the mutagenic activities in vivo of hycanthone, three other antischistosomal compounds, metronidazole, diazepam, and mebendazole. These effects were accompanied by increases in the thiol levels of some tissues. The production of mutagenic metabolites of two other antischistosomal drugs, 4-isothiocyano-4'-nitrodiphenylamine and oxamniquine, was not reduced by BHA treatment. However, such reductions in mutagenicity could be achieved by the administration of enteric antibacterial agents, implicating the role of intestinal microorganisms in the mutagenic activation of certain chemical agents. Combined treatment of mice with BHA and enteric antimicrobial agents reduced the levels of mutagens derived from metronidazole by more than 90%, and the combined treatments were more effective than was either treatment alone.     

Comparative gastrointestinal enzyme activity and activation of the promutagen 2,6-dinitrotoluene in male CD-1 mice and male Fischer 344 rats

Comparative intestinal nitroreductase, azo reductase, beta-glucuronidase, dechlorinase and dehydrochlorinase activities in young male Fischer 344 rats and young male CD-1 mice were measured in vitro while the comparative biotransformation of 2,6-dinitrotoluene to mutagenic metabolites was determined in vivo. The mice, which exhibit a high spontaneous incidence of hepatomas, had markedly greater nitroreductase activity and metabolized significantly more 2,6-dinitrotoluene to mutagenic metabolites than did Fischer 344 rats, which show a low incidence of liver tumors. Results of this study indicate that species differences in the incidence of hepatomas may be influenced by microbial flora and/or the biotransformation of xenobiotics in the G.I. tract.     

Physiologically based pharmacokinetic modeling of 1,3-butadiene, 1,2- epoxy-3-butene, and 1,2:3,4-diepoxybutane toxicokinetics in mice and rats

1,3-Butadiene (BD) is a more potent tumor inducer in mice than in rats. BD also shows striking differences in metabolic activation, with substantially higher blood concentrations of 1,2:3,4-diepoxybutane (butadiene diepoxide; BDE) in BD-exposed mice than in similarly exposed rats. The objective of this study was to develop a single mechanistic model structure capable of describing BD disposition in both species. To achieve this objective, known pathways of 1,2-epoxy-3-butene (butadiene monoepoxide; BMO) and BDE metabolism were incorporated into a physiologically based pharmacokinetic model by scaling rates determined in vitro. With this model structure, epoxide clearance was underestimated for both rats and mice. Improved simulation of blood epoxide concentrations was achieved by addition of first-order metabolism in the slowly perfused tissues, verified by simulation of data on the time course for BMO elimination after i.v. injection of BMO. Blood concentrations of BD were accurately predicted for mice and rats exposed by inhalation to constant concentrations of BD. However, if all BD was assumed to be metabolized to BMO, blood concentrations of BMO were overpredicted. By assuming that only a fraction of BD metabolism produces BMO, blood concentrations of BMO could be predicted over a range of BD exposure concentrations for both species. In vitro and in vivo studies suggest an alternative cytochrome P-450-mediated pathway for BD metabolism that does not yield BMO. Including an alternative pathway for BD metabolism in the model also gave accurate predictions of blood BDE concentrations after inhalation of BD. Blood concentrations of BMO and BDE observed in both mice and rats are best explained by the existence of an alternative pathway for BD metabolism which does not produce BMO.      

Mutagenic and cell-transforming activities of triol-epoxides as compared to other chrysene metabolites

The syn- and anti-isomers of the bay-region diol-epoxides of chrysene and of 3-hydroxychrysene and their metabolic precursors have been investigated for mutagenicity in Salmonella typhimurium (reversion to histidine prototrophy) and V79 Chinese hamster cells (acquirement of resistance to 6-thioguanine) and for transforming activity in M2 mouse prostate cells. Other known and potential chrysene metabolites have been included in mutagenicity experiments. Direct mutagenic activity in S. typhimurium TA 100 exhibited, in order of potency, anti-triol-epoxide greater than syn-triol-epoxide greater than anti-diol-epoxide greater than syn-diol-epoxide greater than chrysene 5,6-oxide much greater than chrysene-1,2-quinone, chrysene-3,4-quinone, and chrysene 5,6-quinone. Chrysene, the six isomeric chrysenols, and the trans-dihydrodiols [trans-1,2-dihydroxy-1,2-dihydrochrysene (chrysene-1,2-diol), trans-3,4-dihydroxy-3,4-dihydrochrysene, trans-5,6-dihydroxy-5,6-dihydrochrysene, and 9-hydroxy-trans-1,2-dihydroxy-1,2-dihydrochrysene (9-hydroxychrysene-1,2-diol)] were inactive per se but were activated to mutagens in the presence of reduced nicotinamide adenine dinucleotide phosphate-fortified postmitochondrial fraction (S9 mix) of liver homogenate from Arochlor 1254-treated rats. Chrysene, 3-hydroxychrysene, chrysene-1,2-diol, and 9-hydroxychrysene-1,2-diol were activated efficiently; the other compounds were activated weakly. In S. typhimurium TA 98, the mutagenic activities of the chrysene derivatives were weak in comparison with those in the strain TA 100. trans-3,4-Dihydroxy-3,4-dihydrochrysene (in the presence of S9 mix) was the most efficacious mutagen in strain TA 98. The relative mutagenic potencies of the directly active compounds differed from the results obtained in strain TA 100, in that in strain TA 98 the anti-diol-epoxide was more mutagenic than the triol-epoxides and chrysene 5,6-oxide was more mutagenic than syn-diol-epoxide and syn-triol-epoxide. In V79 cells, the order of mutagenic potency was: anti-triol-epoxide greater than anti-diol-epoxide greater than syn-triol-epoxide greater than syn-diol-epoxide greater than chyrsene 5,6-oxide greater than chrysene-1,2-diol (in the presence of S9 mix) greater than 9-hydroxychrysene-1,2-diol (in the presence of S9 mix) greater trans-3,4-dihydroxy-3,4-dihydrochrysene in the presence of S9 mix). Chrysene, 3-hydroxychrysene, 5-hydroxychrysene, and 6-hydroxychrysene showed no mutagenic effects in V79 cells, either in the presence or absence of S9 mix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased mutagenicity of 1,2-dibromo-3-chloropropane and tris(2,3-dibromopropyl)Phosphate in Salmonella TA100 expressing human glutathione S-transferases

We have expressed human glutathione S-transferases GSTA1-1 andGSTP1-1 in Salmonella typhimurium TA100 in order to assess theability of these enzymes to modulate the mutagenicity of l,2-dibromo-3-chloropropane (DBCP) and tris(2, 3-dibromopropyl)phosphate(Tris-BP). Both compounds were mutagenic when activated by Aroclor-inducedrat liver microsomes. However, when Aroclor-induced rat livermicrosomes were used together with the GST-expressing strainsthe mutagenicity of both DBCP and Tris-BP was markedly potentiated.Neither of the GST-expressing strains potentiated the mutagenicityin the absence of microsomes, indicating that cytochrome P450-mediatedmetabolism was a prerequisite for GST-mediated potentiation.With DBCP both isozymes had comparable effects on mutagenicfrequency, although the highest dose of DBCP was toxic in strainsexpressing GSTP1-1. In the case of Tris-BP, GSTP1-1 was muchmore active in potentiating the mutagenicity. These resultsindicate that human GSTs can play an important role in the activationof compounds such as DBCP and Tris-BP to mutagenic metabolites.     

Disposition of butadiene monoepoxide and butadiene diepoxide in various tissues of rats and mice following a low-level inhalation exposure to 1,3-butadiene

1,3-Butadiene (BD), a chemical used extensively in the productionof styrene-butadiene rubber, is carcinogenic in Sprague-Dawleyrats and B6C3F₁ mice. Chronic inhalation studies revealed profoundspecies differences in the potency and organ-site specificityof BD carcinogenesis between rats and mice. BD is a potent carcinogenin mice and a weak carcinogen in rats. Previous studies fromour laboratory and others have shown marked differences betweenrats and mice in the metabolism of BD, which may account forspecies differences in carcinogenicity. The purpose of the presentstudy was to examine the production and disposition of two mutagenicBD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide(BDO₂), in blood and other tissues of rats and mice during andfollowing inhalation exposures to a target concentration of62.5 p.p.m. BD. BDO was increased above background in blood,bone marrow, heart, lung, fat, spleen and thymus tissues ofmice after 2 h and 4 h exposures to BD. In rats, levels of BDOwere increased in blood, fat, spleen and thymus tissues. Noincreases in BDO were observed in rat lungs. BDO₂, the moremutagenic of the two epoxides, was increased in the blood ofrats and mice at 2 and 4 h after initiation of exposure to BD.In mice, BDO₂ was detected in all tissues examined immediatelyfollowing the 4 h exposure. This metabolite was detected inheart, lung, fat, spleen and thymus of rats, but at levels 40-to 160-fold lower than those seen in mice. Immediately afterthe 4 h exposure, blood levels of BDO₂ were 204±15 pmol/gfor mice but were 41-fold lower for rats. In the sensitive mousetarget organs, heart and lungs, levels of BDO₂ exceeded BDOlevels immediately after the exposure. This study shows thatthe levels of BD epoxides are markedly greater in the mouseBD target organs. The high concentrations of BDO₂ in these organssuggest that this compound may be particularly important inBD-induced carcinogenesis. Thus, although BD is oxidativelymetabolized by similar metabolic pathways in rats and mice,the substantial quantitative differences in tissue levels ofmutagenic epoxides between species may be responsible for theincreased sensitivity of mice to BD-induced carcinogenicity.     

Metabolic activation of 2‐amino‐1‐methyl‐6‐phenylimidazo [4,5‐b]pyridine and DNA adduct formation depends on p53: Studies in Trp53(+/+),Trp53(+/−) and Trp53(−/−) mice

The expression of the tumor suppressor p53 can influence the bioactivation of, and DNA damage induced by, the environmental carcinogen benzo[a]pyrene, indicating a role for p53 in its cytochrome P450 (CYP)‐mediated biotransformation. The carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), which is formed during the cooking of food, is also metabolically activated by CYP enzymes, particularly CYP1A2. We investigated the potential role of p53 in PhIP metabolism in vivo by treating Trp53(+/+), Trp53(+/?) and Trp53(?/?) mice with a single oral dose of 50 mg/kg body weight PhIP. N‐(Deoxyguanosin‐8‐yl)‐2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP‐C8‐dG) levels in DNA, measured by liquid chromatography‐tandem mass spectrometry, were significantly lower in liver, colon, forestomach and glandular stomach of Trp53(?/?) mice compared to Trp53(+/+) mice. Lower PhIP‐DNA adduct levels in the livers of Trp53(?/?) mice correlated with lower Cyp1a2 enzyme activity (measured by methoxyresorufin‐O‐demethylase activity) in these animals. Interestingly, PhIP‐DNA adduct levels were significantly higher in kidney and bladder of Trp53(?/?) mice compared to Trp53(+/+) mice, which was accompanied by higher sulfotransferase (Sult) 1a1 protein levels and increased Sult1a1 enzyme activity (measured by 2‐naphthylsulfate formation from 2‐naphthol) in kidneys of these animals. Our study demonstrates a role for p53 in the metabolism of PhIP in vivo, extending previous results on a novel role for p53 in xenobiotic metabolism. Our results also indicate that the impact of p53 on PhIP biotransformation is tissue‐dependent and that in addition to Cyp1a enzymes, Sult1a1 can contribute to PhIP‐DNA adduct formation.     

Epithelial binding of 1,2-dibromoethane in the respiratory and upper alimentary tracts of mice and rats

The metabolism and binding of the volatile carcinogen 1,2-dibromo[14C]ethane (DBE) were studied in C57BL mice, Sprague-Dawley rats, and Fischer rats. As shown by the whole-body and light microscopic autoradiography with heated and/or extracted sections, a selective accumulation of metabolites occurred in a number of tissues, preferentially in the reported target tissues for DBE-induced lesions [i.e., in the nasal cavity, lung, forestomach, and liver (tumors) and the adrenal, testicle, liver, and kidney (nonneoplastic lesions)]. High levels of nonextractable metabolites were registered in the epithelia of the entire respiratory tract, the upper alimentary tract, the vagina, and the subepithelial glands of the olfactory mucosa. Lower levels of metabolites were observed in the liver, adrenal cortex, testicular interstitium, and kidney. Autoradiography of slices from various extrahepatic tissues incubated in vitro with DBE showed that most epithelia of the respiratory tract, upper alimentary tract, vagina, and the testicular interstitium have a marked ability to activate DBE to metabolites that become bound to the tissue. Further in vitro experiments, performed with S-1 fractions prepared from various tissues, indicated that the nasal mucosa was most active in transforming DBE to products which could not be extracted from the protein precipitate. It is proposed that tissue-selective metabolism and activation of DBE in the epithelia of the respiratory and upper alimentary tract are responsible for the observed DBE-induced lesions in these organ systems.     

Fluorine probes for investigating the mechanism of activation of indeno[1,2,3-cd]pyrene to a tumorigenic agent

Indeno[1,2,3-cd]pyrene (IP) is a non-alternant polycyclic aromatic hydrocarbon that has tumor-initiating activity on mouse skin and is carcinogenic in newborn mice and in rat lungs. Previous studies have shown that 8- and 9-hydroxyIP and IP-1,2-diol are major metabolites formed in vivo in mouse skin. 8-HydroxyIP-1,2-diol and 9-hydroxyIP-1,2-diol are also observed as in vivo metabolites of IP. Although 8-hydroxyIP had marginal tumor-initiating activity on mouse skin, IP-1,2-diol and its epoxide precursor, IP-1,2-oxide, had similar tumorigenic activity as IP. In the present study fluorine probes have been employed to investigate the contribution of metabolic activation at the 1,2 and 7-10 positions of IP. At a total initiating dose of 4.0 mumol, 2-fluoroIP induced skin tumors in 76% of the treated animals with an average of 3.9 tumors/mouse. At the same dose, IP induced a 72% incidence of tumor-bearing mice with 2.1 tumors/mouse. In contrast, 8,9-difluoroIP elicited a tumorigenic response in 40% of the treated animals with 0.6 tumors/animal. Five mice from each experimental group were killed at the conclusion of the initiation phase of the bioassay and DNA was isolated from the treated areas of skin. 32P-Postlabeling analysis of the hydrolyzed DNA indicated that IP forms one major detectable DNA adduct that migrates close to the origin. This adduct is absent in mice treated with 8,9-difluoroIP. In contrast, 2-fluoroIP forms one major adduct spot with different retention behavior as compared with the adduct formed from IP. DNA from mice treated topically with IP-1,2-diol and IP-1,2-oxide was subjected to 32P-postlabeling analysis. IP-1,2-diol forms one major DNA adduct spot with mobility similar to that observed for the IP-DNA adduct. IP-1,2-oxide displayed an intense pattern of DNA adducts centered around the location of the IP-DNA adduct. No adducts were detected which had mobility similar to that formed from 2-fluoroIP. These results are consistent with IP undergoing metabolic activation at positions 7-10 either alone or in conjunction with dihydrodiol formation at the 1,2 position.     

Covalent binding of 1,2-dihaloalkanes to DNA and stability of the major DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione

The major DNA adduct formed from the carcinogen ethylene dibromide (1,2-dibromoethane, EDB) is S-[2-(N7-guanyl)ethyl]glutathione, resulting from the reaction of guanyl residues with the half-mustard S-(2-bromoethyl)glutathione, which is generated by glutathione S-transferase-catalyzed conjugation of EDB with glutathione. The half-life of the alkylating species [putative S-(2-bromoethyl)glutathione or the derived episulfonium ion] was estimated to be less than 10 s. However, the stability was enough for approximately half of the alkylating metabolites to leave isolated rat hepatocytes before reacting with nucleic acids. Treatment of isolated rat hepatocytes with diethylmaleate decreased covalent binding of EDB to DNA, but treatment with 1-phenylimidazole did not, consistent with the view that conjugative metabolism is of greater importance than oxidation with regard to DNA binding. When EDB was administered to rats in vivo, only one major adduct, S-[2-(N7-guanyl)ethyl]glutathione, was formed in liver or kidney. S-[2-(N7-Guanyl)ethyl]glutathione was found in liver and kidney DNA of rats treated with 1,2-dichloroethane, but other adducts were also present. The gamma-glutamyl transpeptidase inhibitor AT-125 [L-(alpha-(5S)-alpha-amino-S-chloro-4,5-dihydro-5-isoxazoleacetic acid] did not affect the level of EDB bound to DNA by glutathione-fortified rat kidney homogenates or bound to liver or kidney DNA in vivo. The in vitro half-life of S-[2-(N7-guanyl)ethyl]glutathione in calf thymus DNA was 150 h; the half-life of the adduct in rat liver, kidney, stomach, and lung was between 70 and 100 h. Isolated S-[2-(N7-guanyl)ethyl]glutathione did not react with DNA to form new adducts. These results provide a further basis for understanding the carcinogenic action of 1,2-dihaloethanes.     

Mutagenicity of 1-nitropyrene metabolites from lung S9

The mutagenicity of 1-nitropyrene metabolites from rabbit lung S9 incubates was evaluated using the Salmonella typhimurium plate incorporation assay with strain TA98, with and without Aroclor-induced rat liver S9. The following metabolites were isolated, identified and quantitated by HPLC: 1-nitropyrene -4,5- or -9,10-dihydrodiol (K-DHD), N-acetyl-1-aminopyrene ( NAAP ), 1-aminopyrene (1-AMP), 10-hydroxy-1-nitropyrene, 4-, 5-, 6-, 8- or 9-monohydroxy-1-nitropyrene (phenols) and 3-hydroxy-1-nitropyrene. The predominant metabolites formed by lung S9 incubates were K-DHD, 3-OH-1-nitropyrene and phenols. All of the metabolites were mutagenic in the absence of the exogenous rat liver S9 metabolic activation system, and several, including two unidentified metabolites were more potent than the parent 1-nitropyrene. The mutagenicity of 3 of the metabolites ( NAAP , 10-OH-1-nitropyrene and phenols) were enhanced by S9 while most of the other metabolites were less mutagenic in the presence of S9. These results indicate that lung tissue is capable of both oxidative and reductive metabolism which produced mutagenic metabolites, several of which were more potent than the parent compound, 1-NP.