Benzo(a)pyrene activation to 7,8-dihydrodiol 9,10-oxide by rat liver microsomes. Control by selective product inhibition

Metabolism of benzo(a)pyrene (BP) and 7,8-dihydrodiol by 3-methylcholanthrene (MC)-induced rat liver microsomes are both subject to severe inhibition by primary metabolites of BP, which was analyzed by determining individual inhibition constants for all primary BP metabolites for both BP and 7,8-dihydrodiol metabolism. Monooxygenation of 7,8-dihydrodiol was, surprisingly, 5 to 10 times more sensitive than monooxygenation of BP to inhibition by all primary metabolites, even though both reactions require the same enzyme, cytochrome P-450c. Two representative products, 1,6-quinone and 9-phenol, were both strong, competitive inhibitors of BP metabolism with Ki values of 0.12 and 0.74 microM, respectively. The total effect of product inhibition on the overall reactions was determined by fitting progress curves of BP, 7,8-dihydrodiol, and anti-7,8-dihydrodiol 9,10-oxide (determined as 7,10/8,9-tetrol) over a range of BP concentrations to integrated steady-state equations using experimental Vmax and Km values. The effective product inhibition factors for BP and 7,8-dihydrodiol metabolism, determined from progress curve fits, were only 2-fold higher than the corresponding calculated theoretical values. The effective product inhibition factors, obtained from progress curve analysis, confirmed that 7,8-dihydrodiol metabolism was substantially more sensitive to inhibition by primary BP metabolites than BP metabolism itself. This difference probably reflects the much higher affinity of cytochrome P-450c for BP (Kd = 6 nM), as compared to 7,8-dihydrodiol (Kd = 175 nM) that was established spectrophotometrically both for the purified cytochrome and for MC microsomes. The Km for BP metabolism is 50 to 100 times higher than the Kd, while the Km is similar to the Kd for 7,8-dihydrodiol metabolism. The discrepancy for BP between Km and Kd suggests that standard Michaelis-Menten kinetics may be perturbed by either slow substrate or product dissociation.     

Effect of inducers on metabolism of benzo(a)pyrene in vivo and in vitro: analysis by high pressure liquid chromatography

The characterisation of metabolites formed from benzo(a)pyrene (BP) by Aspergillus ochraceus TS and effect of inducers on BP metabolism are reported. The high pressure liquid chromatographic profile of BP metabolites was similar to that of mammalian microsomes furnishing diols, quinones and phenols. The production of BP-4,5-dihydrodiol (K-region diol) by Aspergillus ochraceus TS seems to be novel and provides first report on BP metabolism by eukaryotic fungi. In control, phenols and quinones were produced in excess over dihydrodiols while the induced preparation showed the reverse order. Presumably the induction effecting production of excess dihydrodiols influenced the synthesis of epoxide hydrolase. In addition, a differential increase in BP metabolism was observed with inducers of narrow and broad specificity.     

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.     

Product inhibition of benzo[a]pyrene metabolism in uninduced rat liver microsomes: Effect of diol epoxide formation

Conversion of benzo[a]pyrene (BP) to BP 7,8-dihydrodiol 9,10-oxides (DE) (measured as 7,10/8,9-tetrols) by untreated (UT) rat liver microsomes is over 10 times slower than following 3-methylcholanthrene (MC) induction. Time courses have been subjected to a kinetic analysis analogous to that previously reported for metabolism by MC-induced microsomes (J. Biol. Chem., 259 (1984) 13770–13776). Competition between BP and 7,8-dihydrodiol for P-450 is the major determinant of the rate of DE formation. Glucuronidation of quinones and phenols only increases the isolated BP metabolites including DE by 40%. This indicates far less inhibition by these products than for metabolism in MC-microsomes (4–6-fold). Thus stimulation may result from a decreased quinone-mediated oxidation of metabolites. In the presence of DNA, UT-microsomes metabolize BP to approximately equal amounts of 9-phenol-4,5-oxide (9-PO) and DE/DNA adducts. Addition of uridine diphosphoglucuronic acid (UDPGA) fails to enhance modification of DNA by DE, but formation of the 9-PO adduct is reduced as a result of lower free 9-phenol levels. The kinetic characteristics of BP metabolism by UT-microsomes are highly sensitive to the presence of very small but variable amounts (2–25 pmol/mg) of the very active cytochrome P-450_c, which is the predominant form in MC-microsomes. The major effect of elevated levels of P-450_c is an 8-fold increase in DE formation at low concentrations of BP due to a lowering of K_m (7.9–2.6 μM) and an increase in the regioselectivity for DE formation from 7,8-dihydrodiol (5–15% of total BP metabolites). The formation of DE was directly correlated with the content of P-450_c (r = 0.94). The presence of increased levels of P-450_c in UT-microsomes is probably due to previous exposure of the animals to environmental inducers and is minimized by controlled housing and feeding.     

Absolute stereochemistry of the trans-dihydrodiols formed from benzo[a]anthracene by liver microsomes.

Through application of the exciton chirality method, absolute stereochemistry has been assigned to the (+)-and (-)-enantiomers of four of the five metabolically possible trans-dihydrodiols of the polycyclic hydrocarbon benzo[a]anthracene (BA). The (+)- and (-)-enantiomers of each of these dihydrodiols can be separated as their diastereomeric bis-esters with (-)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid by high pressure liquid chromatography (HPLC). BA 3,4-, 5,6-, 8,9- and 10,11-dihydrodiol are formed in 38%, 36%, 78% and 66% enantiometric purity, respectively, by liver microsomes from phenobarbital-treated rats, whereas the liver microsomes from 3-methylcholanthrene(MC)-treated rats form BA 5,6-, 8,9- and 10,11-dihydrodiols with higher optical purity (62%, 96% and 96%, respectively). BA 3,4-dihydrodiol is formed from (+/-)-BA 3,4-oxide by microsomal epoxide hydrase in very high enantiometric purity (78%). The major enantiomer of the BA dihydrodiols formed by liver enzymes has R,R absolute stereochemistry in each case. In parallel with previous studies on the metabolism of benzo[a]pyrene, the more tumorigenic (-)-enantiomer is the predominant isomer of BA 3,4-dihydrodiol formed by liver microsomes from BA.     

Oxidative metabolism of the carcinogen 6-fluorobenzo[c]phenanthrene. Effect of a K-region fluoro substituent on the regioselectivity of cytochromes P-450 in liver microsomes from control and induced rats

Oxidative metabolism of the carcinogen 6-fluorobenzo[c]phenanthrene (6-FB[c]Ph) was compared with that of benzo[c]phenanthrene (B[c]Ph) to elucidate the enhancement of carcinogenicity of B[c]Ph by the 6-fluoro substituent. Liver microsomes from untreated (control), phenobarbital-treated, and 3-methylcholanthrene-treated rats metabolized 6-FB[c]Ph at rates of 3.5, 1.5, and 7.7 nmol of products/nmol of cytochrome P-450/min, respectively. The rates of metabolism of B[c]Ph by the same microsomes were 2.9, 1.6, and 5.5 nmol of products/nmol of cytochrome P-450/min, respectively. Whereas the K-region 5,6-dihydrodiol was the major metabolite of B[c]Ph, the major metabolite of 6-FB[c]Ph was the K-region 7,8-oxide, which underwent slow rearrangement to an oxepin. Thus, the 6-fluoro substituent blocks oxidation at the 5,6-double bond and inhibits hydration of the K-region 7,8-oxide by epoxide hydrolase. Substitution with fluorine at C-6 caused an almost 2.5-fold increase in the percentages of the putative proximate carcinogens, i.e. benzo-ring dihydrodiols with bay-region double bonds, when liver microsomes from 3-methylcholanthrene-treated rats were used. Little or no increase was observed in their formation by liver microsomes from control or phenobarbital-treated rats. Interestingly, liver microsomes from control rats formed almost 3-fold as much 3,4-dihydrodiol as isosteric 9,10-dihydrodiol. The R,R-enantiomers of the 3,4- and 9,10-dihydrodiols and the S,S-enantiomer of the 7,8-dihydrodiol were predominantly formed by all three microsomal preparations.     

Induction and repair of DNA strand breaks in cultured human fibroblasts exposed to various phenols and dihydrodiols of benzo[a]pyrene

Cultured human fibroblasts from healthy donors were incubated for 30 min with nine different benzo[a]pyrene (BP) derivatives in the presence or absence of liver microsomes from 3-methylcholanthrene treated rats. The induction and repair of DNA strand breaks were analysed by alkaline unwinding and separation of double and single stranded DNA (SS-DNA) by hydroxylapatite chromatography immediately after the incubation or at various times after the treatment. In the absence of microsomes DNA stand breaks were detected in fibroblasts exposed to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP) and the three BP dihydrodiols (BP-4,5-, BP-7,8- or BP-9,10-dihydrodiol). After removal of the BP derivatives from the medium the DNA strand breaks disappeared within 24 h. alpha-Naphthoflavone (alpha-NF) caused a decrease in the induction of strand breaks by 1-, 3- and 9-OH-BP but did not affect the induction of strand breaks in cells exposed to BP-7,8-dihydrodiol. In the presence of microsomes DNA strand breaks were found after exposure to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP), as well as BP-7,8- and 9,10-dihydrodiol. In contrast BP-4,5-dihydrodiol did not induce strand breaks under these conditions. The induction of strand breaks by BP-7,8-dihydrodiol was enhanced in the presence of cytosine-1-beta-D-arabinofuranoside (AraC). In all cases the DNA strand breaks had disappeared 24 h after removal of the BP derivatives and microsomes except after treatment with BP-7,8-dihydrodiol.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Direct evidence that an arene oxide is a metabolic intermediate of 2,2',5,5'-tetrachlorobiphenyl.

Radiolabeled arene oxide was recovered from incubations containing [³H]-2,2′,5,5′-tetrachlorobiphenyl (³H-TCB), unlabeled 2,2′,5,5′-tetrachlorobiphenyl-3,4-oxide (TCBAO), 3,3,3-trichloropropene-1,2-oxide (TCPO), NADPH, and liver microsomes from phenobarbital-induced rats. No labeled arene oxide was generated in the absence of NADPH, nor during the metabolism of unlabeled TCB in the presence of [³H]-H₂O. The recovered oxide (radiolabeled and carrier) was characterized by mobility on silica gel and by conversion to 3- and 4-hydroxy-TCB. Formation of a dihydrodiol metabolite was apparently blocked by inhibition of epoxide hydrase. These data provide the first direct evidence that arene oxides are intermediates of halogenated biphenyl metabolism.     

An indirect thyroxine stimulation of microsomal fatt acid desaturation in vitro.

Metabolically active intestinal epithelial cells were isolated using collagenase plus hyaluronidase. Oxygen consumption was measured and was found to be inhibited by KCN, antimycin A, and rotenone. Cells from 3-methylcholanthrene(MC)-treated rats metabolized benzo(α)pyrene (BP) at a rate that was 30-fold greater than control cells. The addition of salicylamide to the incubation medium inhibited conjugation of BP metabolites and facilitated the accumulation of fluorescent and ethylacetate extractable metabolites. Metyrapone, SKF 525-A, α-naphthoflavone (α-NF), and rotenone inhibited BP-metabolism in intestinal cells from MC-treated rats, with α-NF being the most inhibitory. In intestinal cells from control animals, metyrapone and SKF 525-A both inhibited BP metabolism, while α-NF and rotenone both produced an increase in the formation of fluorescent BP products. The distribution of metabolites from MC-treated rats was determined by high-pressure liquid chromatography and compared with authentic BP derivatives. Incubations were conducted for 5 and 30 min in the presence and absence of salicylamide, and 30-min samples incubated in the absence of salicylamide were hydrolyzed with β-glucuronidase or aryl sulfatase. In the absence of salicylamide, large amounts of conjugates were formed, the formation of which were inhibited by salicylamide addition. A product corresponding to the 4,5-oxide constituted the major metabolite after a 5-min incubation, while little dihydrodiol formation occurred. Large amounts of phenolic BP derivatives were also formed. After 30-min incubations, the percentage of products corresponding to the 4,5-oxide decreased, and an increase in dihydrodiol formation was observed. The slow metabolism of the 4,5-oxide and the slow accumulation of dihydrodiols is due to the presence of low epoxide hydrase activity in the intestine. Intestinal cells are capable of xenobiotic metabolism, and offer a convenient method of studying intestinal drug metabolizing processes which may significantly contribute to the overall xenobiotic metabolis in the body.     

Effects of a 6-fluoro substituent on the metabolism of benzo(a)pyrene 7,8-dihydrodiol to bay-region diol epoxides by rat liver enzymes

Metabolism of trans-7,8-dihydroxy-7,8-dihydro-6-fluorobenzo(a)pyrene by liver microsomes from 3-methylcholanthrene-treated rats and by a highly purified monooxygenase system, reconstituted with cytochrome P-450c, has been examined. Although both the fluorinated and unfluorinated 7,8-dihydrodiol formed from benzo(a)pyrene by liver microsomes share (R,R)-absolute configuration, the fluorinated dihydrodiol prefers the conformation in which the hydroxyl groups are pseudodiaxial due to the proximate fluorine. The fluorinated 4,5- and 9,10-dihydrodiols are also greater than 97% the (R,R)-enantiomers. For benzo(a)pyrene, metabolism of the (7R,8R)-dihydrodiol to a bay-region 7,8-diol-9,10-epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans constitutes the only known pathway to an ultimate carcinogen. With the microsomal and the purified monooxygenase system, this pathway accounts for 76-82% of the total metabolites from the 7,8-dihydrodiol. In contrast, only 32-49% of the corresponding diol epoxide is obtained from the fluorinated dihydrodiol and this fluorinated diol epoxide has altered conformation in that its hydroxyl groups prefer to be pseudodiaxial. Much smaller amounts of the diastereomeric 7,8-diol-9,10-epoxides in which the benzylic hydroxyl groups and the epoxide oxygen are cis are formed from both dihydrodiols. As the fluorinated diol epoxides are weaker mutagens toward bacteria and mammalian cells relative to the unfluorinated diol epoxides, conformation appears to be an important determinant in modulating the biological activity of diol epoxides. One of the more interesting metabolites of 6-fluorinated 7,8-dihydrodiol was a relatively stable arene oxide, probably the 4,5-oxide, which is resistant to the action of epoxide hydrolase.     

Effects of the epoxide hydrase inhibitor, 1,1,1-trichloropropane-2,3-oxide on the genetic activity of aflatoxin B1 metabolites in in vitro activation test systems.

The epoxide hydrase inhibitor 1,1,1-trichloroprophane-2,3-oxide (TCPO) was genetically active to cells of S. cerevisiae and conidia of N. crassa. This genetic activity could be eliminated or reduced to near spontaneous levels in the presence of the S-9 fraction of hamster liver homogenate. The addition of TCPO to an in vitro activation system containing aflatoxin B1 resulted in an increase in the genetic activity of aflatoxin B1, and this increase was dependent on the dose of TCPO. These results are discussed in relation to the possible metabolism of the promutagen aflatoxin B1.     

The formation of dihydrodiols from benzo[alpha]pyrene by oxidation with an ascorbic acid/ferrous sulphate/EDTA system.

In the oxidation of benzo[alpha]pyrene in an abscorbic acid-ferrous sulphate-EDTA system, four dihydrodiols were detected. Three, trans-4,5-dihydro-4,5-dihydroxybenzo[alpha]pyrene, trans-7,8-dihydro-7,8-dihydroxybenzo[alpha]pyrene and trans-9,10-dihydro-9,10-dihydroxybenzo[alpha]pyrene were identified by their UV spectra and by direct comparisons of their chromatographic properties, using HPLC, with those of the authentic compounds. The fourth compound appeared to be trans-11,12-dihydro-11,12-dihydroxybenzo[alpha]pyrene since its ultraviolet spectrum was identical to that of the cis-dihydrodiol. Time-course experiments showed that the maximum amounts of products were obtained after 8 h of oxidation. A re-examination of the dihydrodiols formed from benzo[alpha]pyrene by rat-liver microsomal fractions failed to show the formation of the trans-11,12-dihydrodiol.     

Stereoselective formation and hydration of benzo[c]phenanthrene 3,4- and 5,6-epoxide enantiomers by rat liver microsomal enzymes

The K-region 5,6-epoxides, formed in the metabolism of benzo[c]phenanthrene (BcPh) in the presence of an epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide (TCPO) by liver microsomes from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and polychlorinated biphenyls (Aroclor 1254)-treated rats of the Sprague-Dawley and the Long-Evans strains, were found by chiral stationary phase high-performance liquid chromatography analyses to be enriched (58-72%) in the 5S, 6R enantiomer. In the absence of TCPO, the metabolically formed BcPh trans-5,6-dihydrodiol was enriched (78-86%) in the 5S,6S enantiomer. The major enantiomer of the BcPh 3,4-epoxide metabolite was found to be enriched in the 3S,4R enantiomer which undergoes racemization under the experimental conditions. The major enantiomer of the 5,6-dihydrodiol metabolite was elucidated by the exciton chirality circular dichroism (CD) method to have a 5S,6S absolute stereochemistry. Absolute configurations of enantiomeric methoxylation products derived from each of the two BcPh 5,6-epoxide enantiomers. Optically pure BcPh 5S,6R-epoxide was enzymatically hydrated exclusively at the C6 position to form an optically pure BcPh 5S,6S-dihydrodiol. However, optically pure BcPh 5R,6S-epoxide was hydrated at both C5 and C6 positions to form a BcPh trans-5,6-dihydrodiol with a (5S,6S):(5R,6R) enantiomer ratio of 32:68.     

Two K-regions of 5-methylchrysene are sites of oxidative metabolism

Two K-region trans-dihydrodiols were identified as products formed in the metabolism of 5-methylchrysene by liver microsomes from phenobarbital-treated male Sprague-Dawley rats. These two dihydrodiols were isolated from a mixture of metabolites by reversed-phase and normal-phase high-performance liquid chromatographies. Both K-region dihydrodiols were characterized by ultra-violet, mass, and circular dichroism spectral analyses. Chiral stationary phase high-performance liquid chromatographic analyses indicated that 5-methylchrysene 5,6-dihydrodiol and 11,12-dihydrodiol contain (S,S): (R,R) enantiomer ratios of 2:98 and 12:88, respectively. Although it is a bay-region dihydrodiol, the hydroxyl groups of 5-methylchrysene trans-5,6-dihydrodiol adopt a quasidiequatorial conformation.     

Fluorobenzo[a]pyrenes as probes of the mechanism of cytochrome P450-catalyzed oxygen transfer in aromatic oxygenations

Fluoro substitution of benzo[a]pyrene (BP) has been very useful in determining the mechanism of cytochrome P450-catalyzed oxygen transfer in the formation of 6-hydroxyBP (6-OHBP) and its resulting BP 1,6-, 3,6-, and 6,12-diones. We report here the metabolism of 1-FBP and 3-FBP, and PM3 calculations of charge densities and bond orders in the neutral molecules and radical cations of BP, 1-FBP, 3-FBP, and 6-FBP, to determine the mechanism of oxygen transfer for the formation of BP metabolites. 1-FBP and 3-FBP were metabolized by rat liver microsomes. The products were analyzed by HPLC and identified by NMR. Formation of BP 1,6-dione and BP 3,6-dione from 1-FBP and 3-FBP, respectively, can only occur by removal of the fluoro ion from C-1 and C-3, respectively, via one-electron oxidation of the substrate. The combined metabolic and theoretical studies reveal the mechanism of oxygen transfer in the P450-catalyzed formation of BP metabolites. Initial abstraction of a pi electron from BP by the [Fe(4+)=O](+)(*) of cytochrome P450 affords BP(+)(*). This is followed by oxygen transfer to the most electropositive carbon atoms, C-6, C-1, and C-3, with formation of 6-OHBP (and its quinones), 1-OHBP, and 3-OHBP, respectively, or the most electropositive 4,5-, 7,8-, and 9,10- double bonds, with formation of BP 4,5-, 7,8-, or 9,10-oxide.     

The use of high pressure liquid chromatography to study chemically induced alterations in the pattern of benzo[a]pyrene metabolism.

The metabolism of radiolabeled benzo[a]pyrene (BP) by control, 3-methyl-cholanthrene (3-MC) induced, and 1,1,1-trichloropropene-2,3-oxide (TCPO)-inhibited rat liver microsomes was measured using fluorescence, radiometric, and high-pressure liquid chromatographic (HPLC) assays. Significant differences in the total measurable metabolism of BP by the three microsomal enzyme incubations resulted from the use of the three assay procedures. Appreciable differences in the concentration of the metabolite fractions after 3-MC induction and TCPO inhibition are clearly demonstrated. NMR analysis revealed that while the 3-hydroxy-BP fraction is greater than 90% pure, the 9-hydroxy fraction contains a number of metabolites having essentially identical retention times.     

Effect of induction by DDT on metabolism and mutagenicity of benzo[a]pyrene

Liver microsomal enzymes are essential for the detection of benzo[a]pyrene (B[a]P)-mediated mutagenesis in the Salmonella/mammalian microsome mutagenicity test and, furthermore, this mutagenicity is considerably enhanced by induction of hepatic enzymes involved with drug metabolism. Although Aroclor 1254 is most commonly used for induction of S9 enzymes, DDT is also capable of this induction. This paper reports a comparison of liver S9 fraction induced by the two agents: there is a marked difference in their concentration optima for metabolism of B[a]P; greater numbers of revertant colonies are seen with Aroclor-induced S9, which is optimal at a concentration of 10% (v/v), whereas DDT-induced S9 is optimal at 2.5% (v/v); Aroclor induces aryl hydrocarbon hydroxylase (AHH), cytochrome P-450 and epoxide hydrase while DDT induces only AHH, to about half the level detected in the Aroclor-induced S9 fraction. A comparison of metabolite distribution for Aroclor- and DDT-induced hepatic microsomes reveals quantitative differences only. DDT-induced microsomes yield a greater proportion of B[a]P-4,5-oxide and its metabolic product B[a]P-4,5-dihydrodiol than do Aroclor-induced microsomes. Time course studies on the mutagen half-life measured on the agar plate provides good evidence that metabolites responsible for mutagenicity were different for each inducer.     

Identification of metabolites of benzo[j]fluoranthene formed in vitro in rat liver homogenate

The metabolites of benzo[j]fluoranthene (BjF) as formed in vitro using the 9000 X g supernatant from Aroclor-pretreated rats have been identified. Two dihydrodiols, trans-4,5-dihydro-4,5-dihydroxyBjF and trans-9,10-dihydro-9,10-dihydroxyBjF have been identified as major metabolites by comparison of their spectral and chromatographic properties with those of pure synthetic standards. There was no evidence that any of the isomeric 2,3-dihydrodiol was formed as a metabolite of BjF under these incubation conditions. Neither of the metabolic dihydrodiols of BjF were formed with a high degree of stereoselectivity. The enantiomeric purity of the 4,5-dihydrodiol was 20% while that of the 9,10-dihydrodiol was 46%. At least four phenols were detected among the metabolites of BjF. These were identified as 3-, 4-, 6- and 10-hydroxyBjF based upon comparison of their UV spectra and HPLC retention times with those of synthetic reference standards. BjF-4,5-dione was also identified as a metabolite under these incubation conditions.     

Subcellular distribution, catalytic properties and partial purification of epoxide hydrolase in the human adrenal gland

Epoxide hydrolase in human adrenal gland was characterized with respect to catalytic properties and subcellular distribution. With human adrenal microsomes and the substrates styrene-7,8-oxide, cis-stilbene oxide, estroxide and androstene oxide the specific activities were between 1.9 and 19.0 nmol/min/mg protein. With styrene-7,8-oxide as substrate the apparent Km-value was 0.98 mM and the pH optimum was 9.2. Subcellular fractionation revealed that the bulk of the activity was confined to the endoplasmic reticulum. Different compounds known to influence rodent microsomal epoxide hydrolase activity were also tested on the human adrenal enzyme. 1,1,1-Trichloropropene-2,3-oxide (TCPO) and cyclohexene oxide (CHO) inhibited the activity while benzil and clotrimazole stimulated the activity. Partial purification of human adrenal epoxide hydrolase indicates that its molecular weight is about 51 000 and that its concentration relative total protein in the human adrenal microsomes is about 10%.