Metabolic study of 7-methylbenzo[a]pyrene with rat liver microsomes: Separation by reversed-phase and normal-phase high performance liquid chromatography and characterization of metabolites

The 7-methylbenzo[a]pyrene (7-MBaP) was incubated with liver microsomes of rats pretreated with polychlorinated biphenyls (Aroclor 1254) (PCBs). Metabolites of 7-MBaP were isolated by both reversed-phase and normal-phase high performance liquid chromatography (HPLC) and were characterized by nuclear magnetic resonance, UV-visible and mass spectral analyses. The predominant metabolite of 7-MBaP was found to be 3-hydroxy-7-methylbenzo[a]pyrene (3-hydroxy-7-MBaP). Other identified metabolites include 7-MBaP 4,5-, 7,8-, and 9,10-trans-dihydrodiols, 7-hydroxymethyl-BaP, 7-hydroxymethyl-BaP trans-9,10-dihydrodiol, 9-hydroxy-7-MBaP, 3-hydroxy-7-hydroxymethyl-BaP, 7-MBaP 1,6- and 3,6- quinones, and a hydroquinone which is also formed by further metabolism of the 3-hydroxy-7-MBaP. Comparative metabolic studies of 7-MBaP and BaP indicated that, relative to that of BaP, the methyl substituent of 7-MBaP slightly increases the formation of 3-hydroxy-7-MBaP and decreases the metabolism at other regions of the 7-MBaP molecule. The finding that a 7,8-dihydrodiol is a metabolite indicates that, like BaP, 7-MBaP may also be activated to the potentially reactive 7,8-dihydrodiol 9,10-epoxides although their formations are significantly reduced.     

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.     

Comparative metabolism of benzo[f]quinoline by liver microsomes from brown bullheads and rats

1. Liver microsomes from rats were considerably more active in metabolizing benzo[f]quinoline (B f Q) than those from brown bullheads (Ictalurus nebulosus). 2. The main B f Q metabolites formed by both rat and brown bullhead liver microsomes were qualitatively similar and included B f Q-7,8-dihydrodiol, B f Q-9,10-dihydrodiol, B f Q-N-oxide, 7-hydroxy B f Q, and 9-hydroxy B f Q. 3. The liver microsomes from control brown bullheads and rats metabolized B f Q primarily at the 7,8-and 9,10-positions, respectively, whereas in the case of microsomes from 3-methylcholanthrene (3-MC)-treated rats or brown bullheads, the major site of metabolic attack was the 7,8-position. 4. A 3-MC-type of cytochrome P-450 appears to be primarily responsible for the oxidation of B f Q by control brown bullhead liver microsomes, whereas a phenobarbital-inducible type of cytochrome P-450 seems to be involved in the metabolism of B f Q by control rat liver microsomes.     

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.     

Comparative metabolism of 7H-dibenzo[c,g]carbazole and dibenz[a,j]acridine by mouse and rat liver microsomes.

The comparative metabolism of the carcinogenic pollutants 7H-dibenzo[c,g]-carbazole (DBC) and dibenz[a,j]acridine (DBA) was investigated in vitro using 3-methylcholanthrene (3MC) induced Sprague-Dawley rat and Hsd:ICR(Br) mouse liver microsomal preparations with benzo[a]pyrene (BaP) as the positive control. Metabolites were isolated and separated by HPLC and identified by spectroscopic and co-chromatographic techniques using synthetic standards. The major metabolites of DBC were the phenols: the 5-OH-DBC, 3-OH-DBC, and 2-OH-DBC. Traces of 1-OH-DBC were also found yet no dihydrodiols were identified. The major metabolites of DBA were the 3,4-diol-DBA and 5,6-diol-DBA, 1,2-diol-DBA, DBA-5,6-oxide and 4-OH-DBA. Treatment of both mice and rats with 3MC resulted in significant (P less than or equal to 0.05) increases relative to control in the microsomal metabolism of DBA to dihydrodiol and phenol metabolites, similar to that observed for BaP. 3MC-induced rat liver microsomes significantly (P less than or equal to 0.05) increased DBC metabolism relative to control microsomes whereas DBC metabolism was not increased with 3MC-induced mouse liver microsomes. These data indicate that different enzymatic pathways are involved in the metabolic activation of DBC in the Hsd:ICR(Br) mouse and Sprague-Dawley rat.     

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.     

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.     

Selenium modified mutagenicity and metabolism of benzo[a]pyrene in an S9-dependent system

Selenium added to the incubation mix containing rat-liver S9 modified both the metabolism and mutagenicity of benzo[a]pyrene (BaP) and several of its metabolites. Selenium (Na2SeO3) inhibited the S9-dependent mutagenic effects of BaP on Salmonella typhimurium strain TA100 as indicated by the number of histidine-dependent revertants counted. This inhibition was concentration-dependent over a range of 12.5 to 100 ppm. When used as the substrate the BaP metabolites 7,8-dihydrodiol, 9,10-dihydrodiol and 3-hydroxy also produced significantly fewer revertants in TA100 when selenium was included in the incubation mix. High-performance liquid chromatographic analysis of metabolites from S9-dependent metabolism of BaP indicated that selenium inhibited the formation of 3-hydroxy-BaP, 9,10-dihydrodiol, 7,8-dihydrodiol, 1,3- and 3,6-quinone. Eluting samples on an alumina column to isolate the conjugated metabolites showed that selenium caused 12% less binding to glucuronides, no significant differences in binding to sulfate esters or glutathione but the amount of unmetabolized BaP and unconjugated metabolites was increased by 48%. These results suggest that selenium inhibits S9-dependent BaP metabolism therefore reducing the mutagenic effects of this compound.     

Inhibition of microsome-mediated binding of benzo[a]pyrene to DNA by flavonoids either in vitro or after dietary administration to rats.

The in vitro and in vivo effects of selected natural flavonoids (flavone, flavanone, tangeretin, quercetin, chrysin) on the microsome-catalysed binding of [3H]benzo[a]pyrene to calf thymus DNA were investigated and compared with those of two synthetic flavonoids, 7,8-benzoflavone and 5,6-benzoflavone. In vitro addition of these flavonoids (0.1 mM) to an incubation system containing hepatic microsomes prepared from Aroclor 1254-pretreated rats strongly inhibited BaP-DNA adduct formation (72-89%). The incubation of BaP with hepatic microsomes prepared from animals fed 0.3% quercetin, tangeretin and 7,8-benzoflavone for 2 weeks also resulted in less effective binding of BaP metabolites to added DNA, than with microsomes from untreated rats. Other tested compounds, chrysin, flavone, flavanone and 5,6-benzoflavone showed no or little effect. The influence of flavonoid pretreatment on hepatic microsomal enzymes involved in BaP metabolism has also been examined. Aryl hydrocarbon hydroxylase activity was moderately increased (1.5-1.8-fold) in microsomes prepared from rats fed flavone, tangeretin, 7,8-benzoflavone and 5,6-benzo-flavone. Epoxide hydrolase activity was enhanced by 7,8-benzoflavone (1,6-fold), and by flavone and flavanone (5-fold). These results confirm that flavonoids, in vitro, are potent inhibitors of carcinogen-DNA binding. Oral administration of 0.3% flavonoids alters the properties of liver microsomes, resulting in the decreased ability of BaP metabolites to bind DNA.     

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.     

Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]-phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system

Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.     

Metabolism of 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene by Cunninghamella elegans.

The fungal metabolism of 7-methylbenz[a]anthracene (7-MBA) and 7-hydroxymethylbenz[a]anthracene (7-OHMBA) was studied. 7-MBA was metabolized by Cunninghamella elegans to form 7-OHMBA-trans-8,9-dihydrodiol and 7-OHMBA-trans-3,4-dihydrodiol as the predominant metabolites. Other metabolites were identified as 7-OHMBA, 7-MBA-trans-8,9-dihydrodiol and 7-MBA-trans-3,4-dihydrodiol, and 7-MBA-8,9,10,11-tetraol. Incubation of 7-OHMBA with C. elegans cells indicated that 7-OHMBA-trans-8,9-dihydrodiol and 7-OHMBA-trans-3,4-dihydrodiol were major metabolites. The metabolism of 7-MBA by rat liver microsomes from 3-methylcholanthrene-treated rats showed that the metabolites were qualitatively similar to those formed by C. elegans, except additional dihydrodiol metabolites were formed at the 5,6 and 10,11 positions. The metabolites formed were isolated by high-performance liquid chromatography and identified by comparing their chromatographic, UV-visible absorption and mass spectral properties with those of reference compounds.     

Differential stereoselectivity on metabolism of triphenylene by cytochromes P-450 in liver microsomes from 3-methylcholanthrene- and phenobarbital-treated rats

Metabolism of triphenylene by liver microsomes from control, phenobarbital(PB)-treated rats and 3-methylcholanthrene(MC)-treated rats as well as by a purified system reconstituted with cytochrome P-450c in the absence or presence of purified microsomal epoxide hydrolase was examined. Control microsomes metabolized triphenylene at a rate of 1.2 nmol/nmol of cytochrome P-450/min. Treatment of rats with PB or MC resulted in a 40% reduction and a 3-fold enhancement in the rate of metabolism, respectively. Metabolites consisted of the trans-1,2-dihydrodiol as well as 1-hydroxytriphenylene, and to a lesser extent 2-hydroxytriphenylene. The (-)-1R,2R-enantiomer of the dihydrodiol predominated (70 to 92%) under all incubation conditions. Incubation of racemic triphenylene 1,2-oxide with microsomal epoxide hydrolase produced dihydrodiol which was highly enriched (80%) in the (-)-1R,2R-enantiomer. Experiments with 18O-enriched water showed that attack of water was exclusively at the allylic 2-position of the arene oxide, indicating that the 1R,2S-enantiomer of the oxide was preferentially hydrated by epoxide hydrolase. Thiol trapping experiments indicated that liver microsomes from MC-treated rats produced almost exclusively (greater than 90%) the 1R,2S-enantiomer of triphenylene 1,2-oxide whereas liver microsomes from PB-treated rats formed racemic oxide. The optically active oxide has a half-life for racemization of only approximately 20 s under the incubation conditions. This study may represent the first attempt to address stereochemical consequences of a rapidly racemizing intermediary metabolite.     

Specific labelling of microsomal proteins by reactive intermediates generated from 2-acetylaminofluorene in vitro

Incubation of 2-[9-¹⁴C] acetylaminofluorene (2-[9-¹⁴C]AAF) in vitro with rat liver microsomes, leads to covalent binding of label to microsomal proteins. The binding is NADPH-dependent, increases linearly with time, and is inhibited by SKF-525A and 7,8-benzoflavone (7,8-BF). Binding is increased more than 8-fold in microsomes from 3-methylcholanthrene(MC)-pretreated rats, but only less than 2-fold in those from phenobarbital(PB)-pretreated rats. In the presence of cytosolic proteins, there is slight enhancement of the labelling of microsomes and some labelling of the cytosolic proteins. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis indicate that covalent labelling by 2-AAF derivatives is concentrated in specific proteins. The pattern of labelling varies between microsomes from animals pretreated with PB, MC and 2-AAF. Factors which may contribute to the specificity of labelling are discussed.     

Metabolism of benzo(a)pyrene by microsomes from tissues of pregnant and fetal hamsters

Pretreatment of hamsters with benzo (a) pyrene (BaP) greatly increased the $\text{in vitro}$ metabolism of BaP by lung microsomes from pregnant hamsters, and had less effect on the metabolism of BaP by liver microsomes. The production of various metabolites of BaP by lung microsomes was increased to different extents: 3-hydroxy-BaP (3-OH-BaP) was one of the major metabolites; the metabolic yields of 9, 10-dihydrodihydroxy-BaP (9, 10-diol) and 7,8-diol were increased more than that of the 4,5-diol. In the case of liver microsomes, only the yields of 9,10-diol and 7,8-diol were increased over the control levels. The presence of cyclohexene oxide in the incubation mixtures decreased the production of the diols. Basal-level enzyme activities in placental, fetal liver, and fetal skin microsomes in metabolizing BaP were very low. Pretreatment of pregnant hamsters with BaP induced BaP-metabolizing enzymes in fetal tissue 2–3 fold.     

In vitro effects of L-ascorbic acid (vitamin C) on aryl hydrocarbon hydroxylase activity in hepatic microsomes of mice.

When aromatic hydrocarbon (Ah)-responsive and -non-responsive strains of mice were pretreated with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), vitamin C reduced the microsomal aryl hydrocarbon hydroxylase (AHH) activity. The AHH inhibitors 7,8-benzoflavone (7,8-BF) and 3-methylsulfonyl-3',4,4',5-tetrachlorobiphenyl (3-MSF-3',4,4',5-tetraCB) showed various inhibitory effects depending upon the types of microsomes, whereas vitamin C exhibited inhibition irrespective of the types of microsomes. 7,8-BF and 3-MSF-3',4,4',5-tetraCB as well as vitamin C suppressed the reverse mutation of the Salmonella typhimurium tester strains TA98 and TA100 induced by benzo[a]pyrene.     

Benzo[a]pyrene metabolism and induction of enzyme-altered foci in regenerating rat liver

The metabolism of benzo[a]pyrene (BP) in regenerating rat liver and the induction of enzyme-altered foci (EAF) in the liver of partially hepatectomized rats, treated with BP and promoted with 2-acetylaminofluorene (2-AAF)/CCl4 was investigated. The aim was to examine factors that might be of importance for the tumorigenicity of BP in the regenerating rat liver, such as cytochrome P-450 activity and glutathione levels. In regenerating rat liver, obtained 18 h after partial hepatectomy (PH), the amount of microsomal cytochrome P-450 was reduced by 20% whereas the level of glutathione was elevated by 15% and the cytosolic glutathione transferase activity towards chlorodinitrobenzene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE) was unaffected. Microsomes from these animals had a reduced capacity to activate (-)-trans-7,8-dihydroxy-7,8-dihydro-BP (BPD) to DNA-binding products but the pattern of BP metabolites was similar to that observed with control rat liver microsomes. Treatment of rats with 3-methylcholanthrene (MC, 50 mg/kg body wt.) increased cytochrome P-450 levels and glutathione transferase activity towards both substrates. Regenerating livers from these animals retained their cytochrome P-450 level and enzymatic activity towards BP and BPD. Regenerating rat liver microsomes from MC-treated animals were about 35 times more efficient in activating BPD than microsomes from uninduced, partially hepatectomized animals. Intraperitoneal administration of BP (50 mg/kg body wt.) 18 h after PH induced EAF in rats subsequently promoted with 2-AAF/CCl4. Pretreatment of rats with MC 66 h before PH and 84 h before BP administration, increased the number of EAF. In accordance with results by Tsuda et al. (Cancer Res., 40 (1980) 1157-1164), these studies demonstrate that BP is tumorigenic in regenerating rat liver, despite a reduced ability of the liver to activate this compound. Furthermore, MC, an inducer of certain cytochrome P-450 species ("aryl hydrocarbon hydroxylase"), potentiates the effect of BP.     

The in vitro metabolism of benzo[a]pyrene by polychlorinated and polybrominated biphenyl induced rat hepatic microsomal monooxygenases

The metabolism of benzo[a]pyrene by halogenated biphenyl-induced rat hepatic microsomal monooxygenases was determined using a high pressure liquid chromatographic assay system. Incubation of benzo[a]pyrene with microsomes from rats pretreated with phenobarbitone or phenobarbitone-type inducers (2,2',4,4',5,5'-hexachlorobiphenyl, 2,2',4,4',6,6'-hexachlorobiphenyl, 2,2',5,5'-tetrachlorobiphenyl, 2,2',4,4',5,5'-hexabromobiphenyl, and 2,2',5,5'-tetrabromobiphenyl) resulted in increased overall metabolism of the hydrocarbon (less than fourfold) into phenolic, quinone, and diol metabolites, with the most striking increase observed in the formation of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene. In contrast, the metabolism of benzo[a]pyrene by microsomes from rats induced with 3-methylcholanthrene or 3,3',4,4'-tetrachlorobiphenyl resulted in a greater than 10-fold increase in overall benzo[a]pyrene metabolism, with the largest increases observed in the formation of the trans-7,8- and -9,10-dihydrodiol metabolites of benzo[a]pyrene. However, in comparison to control and phenobarbitone-induced microsomes, the oxidative conversion of benzo[a]pyrene by microsomes induced with 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl into the 6,12-quinone was substantially inhibited. Previous reports have shown that the commercial halogenated biphenyl mixtures, fireMaster BP-6, and Aroclor 1254 are mixed-type inducers and that microsomes from rats pretreated with these mixtures markedly enhance the overall metabolism of benzo[a]pyrene. Not surprisingly, the metabolism of benzo[a]pyrene by microsomes from rats pretreated with the mixed-type inducers, 2,3,3',4,4'-penta-,2,3,3',4,4',5-hexa-, and 2',3,3',4,4',5-hexa- chlorobiphenyl was also increased and the metabolic profile was similar to that observed with fireMaster BP-6 and Aroclor 1254 induced microsomes.     

Ethoxyquin feeding to rats increases liver microsome-catalyzed formation of benzo(a)pyrene diol epoxide — DNA adduct

The ability of rat liver microsomes to catalyze the formation of benzo(a)pyrene 7,8-diol-9,10-epoxide — DNA nucleoside adduct was increased threefold by feeding 0.5% ethoxyquin to the animals. Microsomal epoxide hydratase activity was enhanced i parallel by a factor of 3 while aryl hydrocarbon hydroxylase activity was not induced. Liver microsomes from rat pretreated with 3-methylcholanthrene produced an increased proportion of diol epoxide — DNA adduct when ethoxyquin had been fed to the animals. The main chromatographic peak formed by microsomes from 3-methylcholanthrene treated rats which contains DNA adducts of secondary benzo(a)pyrene phenol metabolites is reduced when the animals had received ethoxyquin.     

Cutaneous metabolism of benzo[a]pyrene: comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague--Dawley rats

The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.