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K L Platt

Publications and source records attributed to K L Platt.

At least 55 records · Page 3Linked to original sources
PubMed1991

Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17 beta-estradiol and 2-aminofluorene in V79 Chinese hamster cells.

In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P450IA2. Full-length cDNA encoding rat P450IA2 was obtained by searching a cDNA library made from Aroclor 1254-induced rat liver mRNA and by joining a small 5'-end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full-length cDNA was inserted into a simian virus 40 early promoter-containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate-coprecipitation technique. G418-resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.

Amino Acid Sequence↗
PubMed1991

Differential stabilization of cytochrome P-450 isoenzymes in primary cultures of adult rat liver parenchymal cells.

Cytochrome P-450 dependent hydroxylation of testosterone was measured in 7-day-old cultures of primary rat liver parenchymal cells. Determinations were carried out in monocultures of parenchymal cells and co-cultures of parenchymal cells with rat liver nonparenchymal epithelial cells, or mouse embryo fibroblasts. In the monoculture system, testosterone metabolism was drastically reduced and hardly measurable after 7 days in culture. In the co-culture systems, individual P-450 isoenzymes were stabilized on different levels. P-450s p and presumably c were well preserved, P-450 a was reduced but clearly measurable, P-450 h was totally lost whereas P-450s b and e were not measurable after 7 days (the activities of these isoenzymes however were already low in freshly isolated parenchymal cells). The results were independent of the cell line used for co-cultivation and of the method of parenchymal cell isolation, that is whether collagenase or EDTA was used as the agent for dissociating the cells from the liver. The results showed that the co-cultivation of liver parenchymal cells with other nonparenchymal cells significantly improved the differentiated status of the former. In this cell culture system however, not every parameter was equally well stabilized.

Animals↗
PubMed1991

Regiospecific oxidation of polycyclic aromatic dihydrodiols by rat liver dihydrodiol dehydrogenase.

Rat liver dihydrodiol dehydrogenase (DDH, E.C. 1.3.1.20) has recently been shown to oxidize the highly carcinogenic benz[a]anthracene-3,4- dihydrodiol in an NADP(+)-dependent reaction to its corresponding catechol. The present study is a systematic investigation of the substrate specificity of the purified enzyme towards synthetic trans-dihydrodiol metabolites of phenanthrene, benz[a]anthracene, chrysene, dibenz[a, h]anthracene and benzo[a]pyrene. DDH exhibited a remarkable regiospecificity of enzymatic catalysis with regard to the site of the dihydrodiol moiety of the parent hydrocarbon. M-region- and, with lower efficiency, bay-region dihydrodiols were found to be good substrates of the enzyme with maximal velocities between 20-80 nmol/min per mg enzyme and Km values in the micromolar range. K-region dihydrodiols were not accepted as substrates. Dihydrodiols situated at the terminal ring of an anthracene-type structure such as benz[a]anthracene-8,9-dihydrodiol as well as the corresponding dihydrodiol epoxides were also not oxidized by DDH at measurable rates. The results provide evidence for a detoxifying role of DDH in the metabolism of the chemical carcinogens benz[a]anthracene, chrysene and dibenz[a, h]anthracene.

Alcohol Oxidoreductases↗
PubMed1991

The metabolic activation of dibenz[a,h]anthracene in mouse skin examined by 32P-postlabelling: minor contribution of the 3,4-diol 1,2-oxides to DNA binding.

Dibenz[a,h]anthracene (DB[a,h]A) and the related 3,4-diol and anti- and syn-3,4-diol 1,2-oxides were applied to the shaved dorsal skin of groups of four C57Bl/CB1 mice. Twenty-four hours later the mice were killed, DNA isolated from the treated skin, hydrolysed and examined for the presence of aromatic adducts using the nuclease P1 modification of the 32P-postlabelling technique. Autoradiography of the maps obtained by chromatography on polyethyleneimine-cellulose plates showed that six DNA adduct spots that were derived from DB[a,h]A were also present in the DNA of skin treated with the DBA 3,4-diol and that, whilst four of these adduct spots were also seen in maps prepared from the DNA of skin treated with the anti-3,4-diol-1,2-oxide, they were not present in DNA from skin to which the syn-isomer had been applied. The identity of these adduct spots was confirmed by their coincidence when mixtures of different DNA hydrolysates were chromatographed together. Quantitatively, the highest levels of mouse skin modification were obtained with the diol-epoxides and the lowest with DB[a,h]A. The results suggest that most of the DNA adducts formed in DB[a,h]A-treated mouse skin arise through metabolism of the hydrocarbon to the related 3,4-diol and that some may be formed following the conversion of this diol to the bay-region anti-3,4-diol-1,2-oxide.

Animals↗
PubMed1990

The oxidation of the highly tumorigenic benz[a]anthracene 3,4-dihydrodiol by rat liver dihydrodiol dehydrogenase.

Rat liver dihydrodiol dehydrogenase (DDH, EC 1.3.1.20) has been shown to reduce the mutagenicity of benz[a]anthracene (BA) in the bacterial Ames test. BA-3,4-dihydrodiol is a highly mutagenic and tumorigenic metabolite of BA. In order to test the hypothesis that this dihydrodiol may be a substrate of DDH, we established two novel assay systems for the NADP(+)-dependent oxidation of BA-3,4-dihydrodiol by rat liver DDH, an HPLC-based assay procedure and a radiometric assay with specifically labelled [3,4-3H]-BA-3,4-dihydrodiol as substrate. With the HPLC-based assay, the kinetic constants of the enzymatic catalysis were as follows: Km(app) = 21 microM for BA-3,4-dihydrodiol and Vmax = 20.0 nmol/min.mg enzyme. The reaction product was identified by cochromatography, fluorimetry and mass spectroscopy as BA-3,4-catechol, but interconversions between the catechol and the corresponding o-quinone during the analytical procedures were detected. With the radiolabelled substrate, a linear relationship between substrate concentration and reaction velocity was found. The V/K value for labelled substrate was 0.155 ml/min.mg enzyme and a (V/K)H/(V/K)T kinetic isotope effect of 6.7 was observed. The non-labelled substrate acted as a competitive inhibitor of the enzymatic oxidation of tritiated BA-3,4-dihydrodiol with a Ki value of 56.4 microM. The reaction rates determined in this study suggest an important role of DDH activity in the metabolism of BA.

Alcohol Oxidoreductases↗
PubMed1990

Comparative tumorigenicity of picene and dibenz[a,h]anthracene in the mouse.

The carcinogenic activity of the two polycyclic aromatic hydrocarbons (PAHs), picene (benzo[a]chrysene) and dibenz[a,h]anthracene (DBA), was determined in NMRI mice by five different experimental protocols in order to find out if picene is a carcinogen as predicted by recent quantum mechanical calculations in contrast to earlier observations which could not confirm any carcinogenic activity of picene. Single s.c. treatment of adult mice with picene or DBA (308 nmol/animal, each) led to the formation of fibrosarcomas in 63.3% of treated animals regardless of the PAH used. Chronic epicutaneous application of both PAHs (total dose 1.36 mumol) to the back of mice resulted in the development of papillomas with a tumor rate of 22% in the case of picene and of 32% in the case of DBA. When newborn mice were s.c. treated once on day 2 of their life with each of the two PAHs (400 nmol/animal), 27.8% of treated animals developed lung adenomas after 40 weeks in the case of picene compared to 92.1% in the case of DBA. Histopathological examination of the tumors in the three experimental models revealed no difference in the type of tumor between picene and DBA. Epicutaneous application of both PAHs (600 nmol/animal) followed by chronic treatment with 12-O-tetradecanoyl-phorbol-13-acetate for 24 weeks led to the formation of papillomas in 93% of animals treated with DBA while picene showed no tumorigenic activity at all. Initiation of tumorigenesis in the two-stage tumor model with 7,12-dimethylbenz[a]anthracene (1 mumol/animal) and chronic treatment with picene (total dose 4.8 mumol) for 24 weeks was equally ineffective in producing tumors in NMRI mice. This rare biological property of picene, which is a complete carcinogen, yet at most a very weak tumor initiator, is explained in terms of its inefficient biotransformation to mutagenic and carcinogenic metabolites as compared to the strong tumor initiator DBA.

Animals↗
PubMed1990

The initiator tRNA acceptance assay as a short-term test for carcinogens. 6. Results with 78 polycyclic aromatic compounds.

A total of 78 polycyclic aromatic compounds (PAH), including pure hydrocarbons, PAH metabolites, aromatic amines and nitroarenes, were tested in the initiator tRNA acceptance assay (tR assay) for carcinogens. Among the pure hydrocarbons, all strong carcinogens were highly active in the tR assay. Some weak carcinogens showed moderate positive responses, others as well as all possible non-carcinogens were inactive. Various PAH metabolites, including phenols, dihydrodiols, arene oxides, dihydrodiol epoxides, quinones and benzylic sulfate esters, were positive as well. Strikingly, however, their effects rarely reached the levels observed with the strong carcinogens among the pure hydrocarbons. Moreover, the correlation with carcinogenicity was less clear, partially due to limitations in the available carcinogenicity data. The activities in the tR assay were also compared with the mutagenicity in Salmonella typhimurium. No appreciable correlation was observed. For example, trans-9,10-dihydroxy-9,10-dihydrobenzo[c]chrysene, in the absence of a mammalian metabolic system, was highly active in the tR assay, but non-mutagenic. Upon addition of rat liver enzymes, the reverse result was obtained. syn-Benzo[c]chrysene-9,10-dihydrodiol-11,12-oxide, on the other hand, was a potent direct mutagen, but required the presence of liver microsomes for a positive response in the tR assay. Thus, the metabolic basis for these two activities is different, and not yet understood for the tR assay. The partial correlations in the tR assay and in the Salmonella mutagenicity assay with carcinogenicity, and the pronounced discrepancies between these in vitro tests, may suggest that they detect different mechanisms involved in carcinogenicity. However, the tR assay was less predictive for the carcinogenicity of PAHs as compared to the previously investigated N-nitroso compounds and mycotoxins.

Carcinogenicity Tests↗
PubMed1990

Expression of xenobiotic-metabolizing enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds.

Activities of various xenobiotic-metabolizing enzymes were determined in 18 cell lines. Activities of cytochrome P450 reductase, microsomal epoxide hydrolase and glutathione transferase were detectable in all lines. The highest values were similar to the activities found in freshly isolated rat hepatocytes. Catalase activity was also present in all 12 investigated cell lines. Activity of UDP-glucuronosyl transferase was high in some lines, but low or undetectable in others. Activity of cytosolic epoxide hydrolase was not measurable in most lines, and was low in the others. Metabolism of benzo[a]pyrene was observed in eight out of nine examined lines, no activity being found in V79 cells. V79 and three epithelial cell lines were then used as target cells in a genotoxicity assay in which the frequency of micronucleated cells was determined. In V79 cells, 7,12-dimethyl- benz[a]anthracene, benzo[a]pyrene, benzo[a]pyrene-trans-7,8-dihydrodiol, aflatoxin B1, N-nitrosomorpholine and 2-acetylaminofluorene showed negative responses, whereas N-methyl-N'-nitro-N-nitrosoguanidine, 9-hydroxybenzo[a]pyrene, 2-nitrofluorene, dibenz[a,h]anthracene 1,2-catechol, dibenz[a,h]anthracene, 1,2-quinone hydroquinone and p-benzoquinone proved positive in the test. All 13 compounds, however, induced micronuclei in rat intestinal cells (IEC-17 and IEC-18) and in embryonal human liver cells (HuFoe-15). Thus, these epithelial cell lines are capable of activating and detecting a broad spectrum of chemically diverse genotoxic compounds. They may also be useful for the detection of hazardous compounds whose active metabolites are not able to penetrate from the extracellular space into the indicator cell.

Animals↗
PubMed1990

Toxicological implications of enzymatic control of reactive metabolites.

Many foreign compounds are transformed into reactive metabolites, which may produce genotoxic effects by chemically altering critical biomolecules. Reactive metabolites are under the control of activating, inactivating and precursor sequestering enzymes. Such enzymes are under the long-term control of induction and repression, as well as the short-term control of post-translational modification and low molecular weight activators or inhibitors. In addition, the efficiency of these enzyme systems in preventing reactive metabolite-mediated toxicity is directed by their subcellular compartmentalization and isoenzymic multiplicity. Extrapolation from toxicological test systems to the human requires information of these variables in the system in question and in man. Differences in susceptibility to toxic challenges between species and individuals are often causally linked to differences in these control factors.

Base Sequence↗
PubMed1990

Stereoselective metabolism of dibenz(a,h)anthracene to trans-dihydrodiols and their activation to bacterial mutagens.

Dibenz(a,h)anthracene (DBA), a carcinogenic, polycyclic aromatic hydrocarbon ubiquitous in the environment, is metabolized by the hepatic microsomal fraction of immature Sprague-Dawley rats pretreated with Aroclor 1254 to 27 ethyl acetate-extractable metabolites. More than half of these metabolites (51%) consisted of trans-1,2-; -3,4-; and -5,6-dihydrodiols including their identified secondary metabolites. The three trans-dihydrodiols (4.9, 15.8, and 0.6% of total metabolic conversion) were highly enriched in their R,R enantiomers (85, 71, and 98%) as determined by high performance liquid chromatography on suitable chiral stationary phases. This is explained on the basis of the stereoselective epoxidation of DBA by cytochrome P-450c (induced by Aroclor 1254) followed by regioselective hydration catalyzed by microsomal epoxide hydrolase. Determination of the bacterial mutagenicity by measuring the reversion rate of histidine-dependent Salmonella typhimurium TA100 to histidine prototrophy revealed marked differences in the mutagenicity of the enantiomers of the trans-dihydrodiols of DBA when activated by the same metabolizing system as used in the metabolism studies. In the case of trans-1,2- and -5,6-dihydrodiol, the S,S enantiomers were converted to more mutagenic metabolites than their corresponding optical antipodes, whereas in the case of trans-3,4-dihydrodiol it was the R,R enantiomer that produced the stronger mutagens. Therefore, both regio- and stereoselectivity of the metabolizing enzymes attribute to the dominant role of trans-3,4-dihydrodiol in the mutagenicity of DBA.

Animals↗
PubMed1990

Rat liver endothelial and Kupffer cell-mediated mutagenicity of polycyclic aromatic hydrocarbons and aflatoxin B1.

The ability of isolated rat liver endothelial and Kupffer cells to activate benzo(a)pyrene (BP), trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (DDBP), trans-1,2-dihydroxy-1,2-dihydrochrysene (DDCH), and aflatoxin B1 (AFB1) to mutagenic metabolites was assessed by means of a cell-mediated bacterial mutagenicity assay and compared with the ability of parenchymal cells to activate these compounds. Endothelial and Kupffer cells from untreated rats were able to activate AFB1 and DDBP; DDBP was activated even in the absence of an NADPH-generating system. Pretreating the animals with Aroclor 1254 strongly enhanced the mutagenicity of the dihydrodiol, whereas the mutagenicity of AFB1 showed a slight increase. BP and DDCH were only activated by endothelial and Kupffer cells isolated from Aroclor 1254-pretreated rats. Parenchymal cells from untreated animals activated all four carcinogens tested; Aroclor 1254 enhanced the parenchymal cell-mediated mutagenicity of BP and DDCH but did not affect that of DDBP and clearly reduced that of AFB1. The reduced mutagenicity of AFB1 correlates with the decrease in the amount of 2 alpha-hydroxytestosterone formed when testosterone was incubated with parenchymal cell microsomes from Aroclor 1254-pretreated rats (compared with microsomes from untreated animals): the formation of 2 alpha-hydroxytestosterone is specifically catalyzed by cytochrome P-450h, a hemoprotein thought to be involved in the activation of AFB1. These results show that not only rat liver parenchymal cells, but also endothelial and Kupffer cells, activate several carcinogens to mutagenic metabolites.

Aflatoxin B1↗
PubMed1989

Genetically engineered V79 Chinese hamster cell expression of purified cytochrome P-450IIB1 monooxygenase activity.

Chinese hamster V79 fibroblasts, frequently used as target cells in short-term tests for mutagenicity, do not possess measurable monooxygenase activity; in particular, enzymatic oxidation of testosterone (T) cannot be demonstrated. If these V79 cells, however, had been transfected with the cDNA-encoding rat liver cytochrome P-450IIB1 under control of the SV40 early promoter, they stably expressed monooxygenase activity. These so-called SD1 cells then oxidatively metabolized T at a rate of 27 pmol/mg protein/min, converting it to 16 alpha- and 16 beta-hydroxy-T as well as 4-androsten-3,17-dione as sole metabolites in a ratio of 1.1:1.0:1.6. The regio- and stereoselective conversion of T by SD1 cells, as well as the quantitative distribution of the metabolites, corresponds well with the results reported for pure cytochrome P-450IIB1 in a reconstituted system.

Animals↗
PubMed1989

Multiple activation pathways of benzene leading to products with varying genotoxic characteristics.

Benzene and 13 potential metabolites were investigated for genotoxicity in Salmonella typhimurium and V79 Chinese hamster cells. In the presence of NADPH-fortified hepatic postmitochondrial fraction (S9 mix), benzene reverted his- S. typhimurium strains. The effect was strongest in strain TA1535. Among the potential metabolites, only the trans-1,2-dihydrodiol, in the presence of S9 mix, and the diol epoxides, in the presence and absence of S9 mix, proved mutagenic in this strain. The anti-diol epoxide was more potent than the syn-diastereomer. Both enantiomers of the anti-diastereomer showed similar activities. S9 mix did not appreciably affect the mutagenicity of the anti-diol epoxide. However, detoxification was observed when purified rat liver dihydrodiol dehydrogenase (EC 1.3.1.20) was used at concentrations comparable to that present in the liver. The (1S)-anti-diol epoxide was a much better substrate than the (1R)-enantiomer, as was true also for (1S)-versus (1R)-trans-1,2-dihydrodiol. The anti-diol epoxide reverted all six strains of S. typhimurium used and induced all four genotoxic effects studied in V79 cells (sister chromatid exchange greater than acquisition of 6-thioguanine resistance, acquisition of ouabain resistance, micronuclei). However, other potential benzene metabolites showed genotoxic effects in V79 cells, as well: sister chromatid exchange was induced by the syn-diol epoxide, 1,2,4-trihydroxybenzene, hydroquinone, catechol, and 1,2,3-trihydroxybenzene. Elevated frequencies of micronucleated cells were observed after treatment with hydroquinone, 1,2,4-trihydroxybenzene, catechol, phenol, 1,2,3-trihydroxybenzene, and quinone. Mutations to 6-thioguanine resistance were induced by quinone, hydroquinone, 1,2,4-trihydroxybenzene, catechol, and the trans-1,2-dihydrodiol.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗
PubMed1988

Microsomal metabolism of picene.

Picene, a polycyclic aromatic hydrocarbon (PAH) of environmental relevance has recently been predicted to be carcinogenic, based on quantum mechanical calculation, although in several animal studies no carcinogenicity could be detected. In order to find out if the metabolism of this PAH can provide an explanation for its lack of carcinogenicity, picene was incubated with the hepatic microsomal fraction of Sprague-Dawley rats, which had been pretreated with Aroclor 1254. Sixteen ethyl acetate-extractable metabolites could be separated by reversed-phase high-performance liquid chromatography. Comparison of the chromatographic behavior and the UV and mass spectral properties of the metabolites with those of synthetic derivatives of picene allowed the identification of trans-1,2-, -3,4-, -5,6-dihydrodiol as well as 2- and 4-phenol as microsomal metabolites of picene. At a substrate concentration of 2.7 microM and an amount of 68 micrograms microsomal protein per ml incubation volume, 4-picenol was the main microsomal metabolite with 32.2% of total metabolic conversion, followed by the 1,2-(bay-region)dihydrodiol with 16.7%, the 3,4-(M-region)dihydrodiol with 15.9%, 2-picenol with 9.1% and the 5,6-(K-region)dihydrodiol with 1.6%. In this respect the metabolism of picene is not significantly different from that of the carcinogenic PAH benzo[a]pyrene and dibenz[a,h]anthracene. The M-region dihydrodiols, potential precursors of electrophilically reactive dihydrodiol bay-region epoxides, are formed from all three PAHs at 11-16% of total metabolic conversion. From the 2.8- to 4.4-fold lower amounts of polar and water-soluble metabolites of picene as compared to dibenz[a,h]anthracene and benzo[a]pyrene it is deduced that dihydrodiol epoxides are generated from picene to a much smaller extent than from the two carcinogenic PAHs. The lacking carcinogenicity of picene could therefore result from the inability of microsomal enzymes to transform its M-region dihydrodiol to dihydrodiol bay-region epoxides in amounts necessary to initiate carcinogenesis.

Animals↗
PubMed1987

Individual differences in DNA repair capacities in man.

After metabolic activation of benzo[a]pyrene to the 7,8-dihydrodiol-9,10-epoxide, this ultimate carcinogen preferentially binds to the exocyclic amino group of guanine. In order to investigate possible interindividual differences in the capacity of repair of the DNA adducts formed from benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide, their persistence in freshly isolated lymphocytes of several donors was studied. The results show a surprisingly large interindividual variation in DNA adduct formation and their persistence in freshly isolated lymphocytes. This range included several individuals with an apparent complete lack of repair capability for these adducts. Compared with controls, smokers showed on average a lower initial extent of the DNA adducts formed from benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide, suggesting induction of inactivating enzymes. However, one of the smokers was an individual with apparent complete lack of repair for the DNA adducts formed from benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide, combining exposure to benzo[a]pyrene with a long persistence of the DNA adducts formed from benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide. The investigation of the DNA repair of methylnitrosourea-induced lesions showed significant interindividual differences in the adaptive response triggered by repeated exposure to the carcinogen, whereas the interindividual variations after single doses were low.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1987

Regioselectivity of rat liver microsomal UDP-glucuronosyltransferase activities toward phenols of benzo(a)pyrene and dibenz(a,h)anthracene.

Inducibility of rat liver microsomal UDP-glucuronosyltransferase was investigated with regard to the substrate structure using 3-, 6-, 7-, 8-, and 9-hydroxybenzo(a)pyrene, all seven phenols of dibenz(a,h)anthracene, 3-hydroxybenz(a)anthracene, and 3-hydroxyfluoranthene as substrates. Glucuronide formation of the majority of the planar phenols was preferentially inducible by 3-methylcholanthrene (4- to 8-fold, group 1). However, glucuronidation of 8-hydroxybenzo(a)pyrene, 3-hydroxybenz(a)anthracene, and 3-hydroxydibenz(a,h)anthracene was markedly inducible by phenobarbital (3- to 8-fold, group 2). Glucuronidation of 9-hydroxybenzo(a)pyrene and 3-hydroxyfluoranthene was only moderately induced by the two inducers (less than 2-fold, group 3). Glucuronidation was also determined with purified phenol UDP-glucuronosyltransferase from 3-methylcholanthrene-treated rats. A close correlation (r = 0.95) was observed between purification factors (ratio of enzyme activities in purified enzyme and microsomes) and induction factors (ratio of microsomal enzyme activities from 3-methylcholanthrene-treated rats and untreated controls). Interestingly, differences in size and shape of group 1 and 2 substrates could be recognized. Group 1 substrates were shorter (less than 1.3 nm) and broader (greater than 1.1 nm) than group 2 substrates when viewed from the hydroxy group, along the axis of the C-O bond, to the plane of the polycyclic aromatic hydrocarbon, suggesting a distinct geometry of the binding site of the 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase.

Animals↗
PubMed1987

Regio- and stereoselective metabolism of dibenz[a,h]anthracene: identification of 12 new microsomal metabolites.

Incubation of the carcinogenic polycyclic aromatic hydrocarbon dibenz[a,h]anthracene (DBA) with liver microsomes of Sprague-Dawley rats, pretreated with Aroclor 1254, yielded more than 30 metabolites. Fifteen of these could be identified, and they account for 95% of the ethyl acetate-extractable metabolites of DBA. Twelve metabolites were identified for the first time, by chromatographic and spectroscopic methods: these were DBA-5,6-oxide, 1-, 2-, 3-, 4-, 5-, 6-phenols, 3,4:12,13-bis-dihydrodiol, 1,4/2,3-tetrol, 1,3/2,4-tetrol, 3,4-catechol, and a phenol dihydrodiol derived from the 2-phenol. Quantitative determination revealed that the attack of cytochrome P-450 dependent monooxygenases occurs at the 1,2-, 3,4- and 5,6-positions of the DBA molecule in the ratio 1.7:1.9:1.0. Evidence is presented which indicates that the phenols of DBA are formed by aromatization of the initially generated arene oxides, rather than by direct hydroxylation. The index Ni obtained by refined perturbational molecular orbital calculations was found to be superior to the reactivity number Nt in predicting the predominant phenols, i.e., 2-, 4-, and 5-phenols, formed by aromatization of the corresponding arene oxides. Their enzymatic hydrolysis leads to the formation of trans-dihydrodiols, of which the 3,4-isomer dominates the microsomal metabolites of DBA accounting for more than 22% of the total metabolic conversion, compared to the 1,2-dihydrodiol with 11-16% and the 5,6-dihydrodiol with 2%. These metabolites were obtained as enantiomeric-enriched mixtures in which the R,R enantiomer of the 1,2-dihydrodiol prevailed with 84%, of the 3,4-dihydrodiol with 79% and of the 5,6-dihydrodiol with 96%. The metabolic pathway via the 1,2-dihydrodiol proceeds to the vicinal diol epoxides, as indicated by the products of hydrolysis the 1,4/2,3- and 1,3/2,4-tetrols. No evidence for the formation of vicinal dihydrodiol epoxides from the 3,4-dihydrodiol, one of the most mutagenic and carcinogenic metabolite of DBA, could be found. In this case, tetrol epoxides have been proposed as ultimate reactive metabolites. Tetrol epoxides can also be formed from DBA-5,6-dihydrodiol via the identified 3,4:12,13-bis-dihydrodiol. This unprecedented metabolic behavior of a carcinogenic polycyclic aromatic hydrocarbon could have its cause in the high molecular symmetry of DBA which permits subsequent metabolic attacks at discrete, but structurally equivalent sites of the molecule.

Animals↗