Metabolism of di-, tri-, tetra-, penta- and hexachlorobiphenyls by hepatic microsomes isolated from control animals and animals treated with aroclor 1254, a commercial mixture of polychlorinated biphenyls (pcbs)

1. The metabolism of a wide range of di-, tri-, tetra-, penta- and hexachlorobiphenyls by hepatic microsomes isolated from control animals and animals treated with Aroclor 1254 was studied.2. Hepatic microsomes isolated from control rats expressed higher rates of oxidations than avians.3. Treatment of rats and pigeons with Aroclor 1254 induced cytochrome P450 dependent mono-oxygenases leading to an increased regioselective metabolism of PCB isomer and congeneres.4. There was an inverse relationship between the degree of halosubstitution and microsomal oxidation. Meta-para carbon atoms free of halosubstitution were the preferred side for oxidation.5. A good correlation was found between the in vitro metabolism of PCBs and their relative abundance in tissue extracts, thus suggesting oxidative metabolism to be the major route of metabolic disposal.     

Microsomal oxidation of bromo-, chloro- and fluorobiphenyls

1. The metabolism of 2-, 3-, 4-bromo-, 2-, 4-chloro-, and 2-fluorobiphenyl by hepatic microsomes isolated from control and Aroclor 1254-treated rats and pigeons was studied.2. Meta and para as well as dihydroxylated metabolites were detected, but para hydroxylation was the preferred route of metabolism with all of the substrates used.3. The overall rates of hydroxylation were greater with hepatic microsomes from rats than from pigeons.4. Treatment with Aroclor 1254, a potent inducer of hepatic monooygenases, resulted in increased rates of metabolism and in the enhanced formation of diol metabolites. Metabolism of halobiphenyls by induced P450 isoenzymes altered the regioselective hydroxylation pathways.5. Ortho- and meta halosubstituted biphenyls were less rapidly metabolised when compared with paru substituted isomers.     

Neonatal phenobarbital imprinting of rat hepatic microsomal testosterone hydroxylations

The effects of neonatally administered phenobarbital (PB) on adult rat hepatic microsomal metabolism of testosterone were examined in 60-, 90-, and 120-day-old animals. Phenobarbital-induced imprinting was evident at all ages; however, female rats appeared to be more susceptible to the neonatal effects of phenobarbital than did male rats. In 60-day-old female rats, increased testosterone 2α-hydroxylase activity was observed in microsomes from noninduced rats, whereas decreased testosterone oxidation at all positions except 2α and 15β was observed in microsomes from Aroclor 1254-induced rats. The decreased oxidation of testosterone at specific sites in response to Aroclor 1254 induction was quite dramatic, decreasing the activities to near or below control levels. By contrast, phenobarbital-treated 60-day-old males exhibited approximately a twofold increase in Aroclor 1254-induced 16α and 2α-hydroxylase activities. The pattern of changes in testosterone metabolism observed in phenobarbital-treated animals was different at both 90 and 120 days from that at 60 days. Only minor alterations in the oxidation of testosterone were observed in 90-day-old animals of either sex. In 120-day-old animals the greatest effects of neonatal phenobarbital exposure were on Aroclor 1254–induced 16β-hydroxylase activities. In induced female rats 16β-hydroxylase activity was again decreased to noninduced levels, while in induced male rats a fourfold increase in this activity was observed. These results demonstrate that neonatal exposure to phenobarbital can alter both constitutive and Aroclor 1254–induced testosterone metabolism in adult rats and that the effects of neonatal phenobarbital exposure are age and sex differentiated.     

The metabolism of drugs and carcinogens in isolated subcellular fractions of Drosophila melanogaster. II. Enzyme induction and metabolism of benzo[a]pyrene

The effect of various pretreatments on the activities of several drug metabolizing enzymes was investigated in microsomes and postmicrosomal supernatant fractions isolated from whole body homogenates of Drosophila melanogaster larvae of different strains. Pretreatments of larvae with either phenobarbital (PB), β-naphthoflavone (BNF) or a mixture of polychlorinated biphenyls (Aroclor 1254, PCB) for 24 h increased microsomal benzo[a]pyrene (BP) monooxygenase activity 2- to 6-fold in all strains as compared to untreated larvae. A simultaneous increase in the contents of cytochrome P-450 occurred after pretreatment with PB and PCB. Comparison of the turnover rates of BP per molecule of cytochrome P-450 indicated that BP was a poor substrate for control cytochrome P-450 whereas BNF induced a most active hemoprotein for this metabolism. Marked differences in the qualitative pattern of BP metabolites were obtained between microsomes isolated from BNF-treated larvae or rat liver microsomes. 3-Hydroxy-BP (3-OH-BP) was the dominating metabolite with both preparations, while the BP dihydrodiols were formed in minor quantities in Drosophila as compared to rat liver. Metyrapone and SKF 525-A inhibited BP metabolism in microsomes isolated from untreated and BNF treated larvae of all strains. In contrast, α-naphthoflavone (ANF) stimulated the BP monooxygenase activity of microsomes isolated from untreated larvae approx. 3-fold but only slightly influenced the activity of microsomes from BNF treated larvae indicating that the latter species of cytochrome P-450 was less sensitive to ANF.In all strains, PCB and PB treatments approximately doubled microsomal epoxide hydrolase activity and increased cytosolic glutathione-S-transferase activity 25–60%, significant only in strain Berlin K after PB treatment. The activities of epoxide hydrolase and glutathione-S-transferase in control larvae were comparable in the different strains, whereas the content of cytochrome P-450 and BP monooxygenase activity was higher in the Hikone R strain. Variability in the induction response to the various pretreatment was observed among the three strains.     

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.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

The mutagenicity testing of tertiary-butyl alcohol, tertiary-butyl acetate and methyl tertiary-butyl ether in Salmonella typhimurium

Tertiary-Butyl alcohol (TBA), tertiary-butyl acetate (TBAc) and methyl tertiary-butyl ether (MTBE) are chemicals to which the general public may be exposed either directly or as a result of their metabolism. There is little evidence that they are genotoxic; however, an earlier publication reported that significant results were obtained in Salmonella typhimurium TA102 mutagenicity tests with both TBA and MTBE. We now present results of testing these chemicals and TBAc against S. typhimurium strains in two laboratories. The emphasis was placed on testing with S. typhimurium TA102 and the use of both dimethyl sulphoxide and water as vehicles. Dose levels up to 5000 microg/plate were used and incubations were conducted in both the presence and absence of liver S9 prepared from male rats treated with either Arochlor 1254 or phenobarbital-beta-naphthoflavone. The experiments were replicated, but in none of them was a significant mutagenic response observed, thus the current evidence indicates the TBA, TBAc and MTBE are not mutagenic in bacteria.     

Cytosolic activation of 2-aminoanthracene: implications in its use as diagnostic mutagen in the Ames test.

The metabolic activation of 2-aminoanthracene to mutagens in the Ames test was investigated using hepatic S9, microsomal and cytosolic fractions from control and Aroclor 1254-treated rats as activation systems. Microsomal and S9 preparations from control animals could activate 2-aminoanthracene, but the efficiency of activation was suppressed by pretreatment of animals with Aroclor 1254. Cytosolic fractions from Aroclor 1254-treated rats could readily activate the promutagen more readily than microsomes. The cytosolic activation of 2-aminoanthracene required NADPH and could not be accounted for by possible microsomal contamination. The molybdenum oxygenases appear not to contribute to the cytosolic activation of this promutagen. It is concluded that (a) the microsomal activation of 2-aminoanthracene is catalysed more effectively by enzyme systems other than the P450 I family and (b) an enzyme system capable of activating this carcinogen in vitro is present in the hepatic cytosol. The implications of these findings in the use of 2-aminoanthracene as a positive control in the Ames test are discussed.     

Stereoselective metabolism of the optical isomers of trans-1,2-dihydroxy-1,2-dihydrophenanthrene to bay-region diol epoxides by rat liver microsomes

Enantiomerically pure isomers of trans-1,2-dihydroxy-1,2-dihydrophenanthrene have been obtained by chromatographic separation of their diastereomeric bis esters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. Liver microsomes from control rats, as well as rats treated with phenobarbital or 3-methylcholanthrene, metabolize these dihydrodiols to a pair of diastereomerically related bay-region 1,2-diol-3,4-epoxides in which the benzylic hydroxyl group and the epoxide oxygen are either cis (isomer-1) or trans (isomer-2) to each other. In general, diol epoxide-1 was the major metabolite of the (+)-(1S,2S)-dihydrodiol, whereas diol epoxide-2 was the major metabolite of the (?)-(1R-2R)-dihydrodiol. The extent of this stereoselectivity is dependent on the source of the microsomes and is greatest for liver microsomes from 3-methylcholanthrene-treated rats; the ratio of diol epoxide-1 relative to diol epoxide-2 was 5.6 : 1 with the (+)-enantiomer as substrate and 1 : 5.5 with the (?)-enantiomer as substrate. For a given microsomal preparation, rates of metabolism were independent of the enantiomer composition of the substrate. Relative to microsomes from control animals, treatment of rats with 3-methylcholanthrene enhanced rates of metabolism by about 40%, whereas treatment with phenobarbital decreased rates to a similar extent when the amounts of metabolites formed per nanomole of cytochrome P?450 were compared. The failure of treatment by 3-methylcholanthrene to enhance markedly the rate of metabolism of a polycyclic aromatic hydrocarbon substrate is unusual.     

Stimulation of microsomal dimethylnitrosamine-N-demethylase by pretreatment of mice with acetone

To further investigate the relationship between in vivo microsomal enzyme modifiers and in vitro dimethylnitrosamine (DMN) metabolism, male C57BL/6J mice were pretreated with acetone or Aroclor 1254, two compounds known to influence DMN-N-demethylase activity. Pretreatment with acetone enhanced the in vitro microsomal activity of DMN-N-demethylase, as measured by formaldehyde production from DMN. Accompanying this acetone-enhanced demethylase activity was an increase in the covalent binding of [14C]DMN to RNA, protein and DNA. Four distinct Km values dependent on the substrate concentration were observed for the N-demethylase present in control microsomes. Only one Km value was observed for the demethylase in microsomes from acetone-treated animals, but it was significantly lower than the lowest Km observed in the control microsomes. At DMN concentrations of 1 and 10 mM, acetone significantly increased N-demethylation of DMN as compared to control, but not at 100 mM DMN. Aroclor 1254 pretreatment repressed DMN-N-demethylase at 1 mM DMN but enhanced it at 100 mM. These results suggest that there may be multiple forms of DMN-N-demethylase which are dependent on DMN concentration and respond differently to modifiers of the microsomal drug-metabolizing enzymes.     

Hepatic cytochrome P-448 and epoxide hydrase: enzymes of nuclear origin are immunochemically identical with those of microsomal origin.

Nuclear and microsomal sources of hepatic cytochrome P-448 and epoxide hydrase were compared using antibodies made against the pure antigens isolated from rat liver microsomes. Both antigens were easily detected in detergent-solubilized nuclei and microsomes from rats using the Ouchterlony double-diffusion technique. Epoxide hydrase from either whole nuclei or nuclear envelope was immunochemically identical with the enzyme isolated from microsomes. Similarly, in rats pretreated with 3-methylcholanthrene, the cytochrome P-448 of nuclear origin was immunochemically indistinguishable from the enzyme derived from microsomes. These results establish the immunochemical identity of these hepatic nuclear and microsomal enzymes and provide a firm basis for applying the knowledge gained with the microsomal system of metabolism to the nuclear system.     

Inhibition of CCl4 metabolism by oxygen varies between isoenzymes of cytochrome P-450

Oxygen inhibition of CCl4 metabolism by different isoenzymes of cytochrome P-450 was assessed by studying liver microsomes isolated from control rats and rats treated with phenobarbital or isoniazid. Rates of CCl4 metabolism were similar for all microsomes under a nitrogen atmosphere. An air atmosphere inhibited metabolism by microsomes from control rats to 12% of the value under nitrogen and metabolism by microsomes from rats treated with phenobarbital to 5%. It inhibited metabolism by microsomes from rats treated with isoniazid only to 32%. Rats treated with phenobarbital, which increases hepatic cytochrome P-450 content, or isoniazid, which does not increase hepatic cytochrome P-450 content, both metabolized more CCl4 than control rats as indicated by exhalation of greater quantities of CCl4 metabolites and by an increase in CCl4 toxicity. These results indicate that some isoenzymes of cytochrome P-450 are more effective than others in metabolizing CCl4 when oxygen is present.     

The homogeneity of rat liver microsomal cytochrome P-448 activity and its role in the activation of benzo[a]pyrene to mutagens

The O-deethylation of ethoxyresorufin and the metabolic activation of benzo[a]pyrene to mutagens were determined in hepatic microsomal preparations from control and induced animals. An excellent direct correlation (r = 0.95) has been observed between ethoxyresorufin O-deethylase and the metabolic activation of benzo[a]pyrene to mutagens when the fraction of cytochromes P-450 present as cytochrome P-448 was altered by the administration of phenobarbitone and 3-methylcholanthrene alone or in combination with 9-hydroxyellipticine. The correlation between these activities was maintained following treatment of animals with Arochlor 1254, benzo[a]pyrene, benzo[e]pyrene, 7,12-dimethylbenzo[a]anthracene,2-anthramine and 2-naphthylamine.     

Diazepam metabolism by multiple forms of cytochrome P-450 in rat liver microsomes

The synthesis of pharmacologically active diazepam metabolites (oxazepam, 4-hydroxydiazepam, N-demethyldiazepam) in liver microsomes of intact and phenobarbital-, 3-methylcholanthrene- and dexamethasone-induced male and female Wistar rats as well as in a reconstituted system with isolated forms of cytochrome P-450 (P-450a, P-450b, P-450c, P-450d and P-450k according to the Ryan nomenclature) was studied. Marked sex-dependent differences in the rates of diazepam metabolism in liver microsomes of intact and induced animals were revealed. The changes in the spectrum of diazepam metabolites in liver microsomes of induced rats (as compared to control animals) were revealed. In a reconstituted system only phenobarbital-induced cytochromes P-450b and P-450k were found to be active participants of diazepam N-demethylation; none of the isoenzymes tested were shown to be involved in diazepam hydroxylation.     

In vitro metabolism of [14C]4-chlorobiphenyl and [14C]2,2',5,5'-tetrachlorobiphenyl by hepatic microsomes from rats and pigeons. Evidence against an obligatory arene oxide in aromatic hydroxylation reactions.

1. The catalytic activities of cytochromes P-450IA1 and P-450IIB1 in control and Aroclor 1254 treated rats and pigeons (1 mmol/kg) were assessed using [14C]4-chloro- and [14C]2,2',5,5'-tetrachlorobiphenyl as substrates. Treatment of rats resulted in increases of the total amount of chloroform-extractable metabolites of [14C]4-chlorobiphenyl from 37.2 (control) to 199.4 and 221.6 nmol/hr per mg microsomal protein at 48 and 120 hr post treatment. The portion of [14C]4-chloro-3',4'-dihydroxybiphenyl (M4) and of a second unidentified dihydroxylated metabolite (M3) increased during these incubations from 13.7% for controls to 53.5% at 48 hr and 69.12% at 120 hr post treatment. 2. [14C]4-chloro-3'-hydroxybiphenyl (M1) and [14C]4-chloro-4'-hydroxybiphenyl (M2) were the major metabolites formed by pigeon hepatic microsomes; however, the amounts formed were 38.7- and 29.3-fold less, respectively, than in untreated rats. Treatment of pigeons with Aroclor 1254 increased the metabolite formation from 1.0 (control) to 13.6 and 22.4 nmol/hr per mg microsomal protein at 48 hr and 120 hr post treatment respectively; however, only small amounts of metabolites M3 (0.5 nmol/hr per mg protein) and M4 (2.0 nmol/hr per mg protein) were detected. 3. Treatment of rats with Aroclor 1254 resulted in an approximately two-fold increase in the rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl, and the ratio of 3- to 4-hydroxylation increased from 0.45 (control) to 0.6 and 0.8 at 48 hr and 120 hr post treatment respectively. The rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl by control and Aroclor 1254 treated pigeons was up to 23-fold lower than in rats and there was no evidence for the formation of the diol metabolite M3. However, as with rats, the ratio of meta- to para-carbon atom hydroxylation increased from 0.58 (controls) to 0.72 at 120 hr post treatment. 4. From the evidence presented, it is suggested that cytochromes P-450IA1 and P-450IIB1 may not metabolize PCB-congeneric substrates via an obligatory arene oxide intermediate.     

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.     

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.     

Arylamine activation following chronic ethanol ingestion by rats: studies on the liver S9, microsomal and cytosolic fractions and comparison with Aroclor 1254 pretreatment

That enzyme fractions derived from animals chronically fed alcohol can alter the metabolism of carcinogenic xenobiotic compounds has been documented. To further understand this relationship the mutagenicity of 3 aromatic amines was determined in the Ames test, employing activation systems derived from rats maintained on an alcohol-containing liquid diet, an isocaloric control liquid diet or Aroclor 1254-pretreated animals fed standard laboratory chow. Depending upon protein and substrate concentrations, S9 from ethanol-fed rats was 30-50% less efficient than S9 from pair-fed rats in activating arylamines (2-aminofluorene, 2-aminoanthracene and 2-acetylaminofluorene) to mutagens in Salmonella typhimurium TA98 and TA100. Cytosolic fractions from ethanol-fed animals always resulted in greater arylamine activation than that of controls whereas the opposite was true of the microsomal compartment in which the ethanol-treated group was consistently less active than the controls. The cytosolic N-acetyltransferase activities with respect to 2 different substrates, isoniazid and 2-aminofluorene, were unaffected by ethanol consumption, indicating that this activity probably does not account for the different activation profiles exhibited by the ethanol and pair-fed cytosolic systems. Both the cytosolic and microsomal compartments are required for maximal expression of the mutagenicity of each arylamine however, each compartment can activate arylamines independently of the other. Reconstituting cytosol with microsomes from ethanol- and pair-fed rats, but not Aroclor-pretreated rats, resulted in a synergistic activation of the aromatic amines and displayed an effect similar to that of S9. Compared to Aroclor pretreatment and pair-fed controls, microsomes from ethanol-fed rats displayed the least capacity for activating any of the arylamines to mutagens. Microsomes from Aroclor-pretreated rats accounted for at least 80% of the S9-mediated activation of each of the arylamines to mutagenic metabolites which was in marked contrast to the contribution of the microsomal fractions to the S9 activity in the ethanol- (5-20% of S9 activity) and pair-fed systems (22-30% of S9 activity). The data indicate that 2 opposing reactions occur in S9, a cytosolic activity that augments and a microsomal activity that attenuates the mutagenicity of arylamines. Both activities are modified by ethanol consumption and Aroclor pretreatment.     

Metabolic activation of promutagens, detectable in Ames' Salmonella assay, by 5000 X g supernatant of rat ventral prostate

Induction of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin-O-deethylase (7-EOD) activities as well as of benzo[a]pyrene (BP) metabolite formation in rat prostatic microsomes has been demonstrated after treatment with beta-naphthoflavone (BNF). The capacity to convert promutagenic compounds to ultimate mutagenic metabolites in the Ames' Salmonella assay by 5000 X g supernatant of rat ventral prostate was investigated. Male rats were treated with BNF, polychlorinated biphenyls (PCB; Arochlor 1254), phenobarbital (PB) and the vehicle, corn oil. PCB or BNF pretreatment increased the AHH- and 7-EOD activities 100-200-fold in the rat prostate 5000 X g supernatant (S-5 fraction). Epoxide hydrolase (EH) and glutathione-S-transferase (GST) activities were not affected while UDP-glucuronosyltransferase (UDP-GT) was increased 2.2- and 2.5-fold by PCB and BNF, respectively. PB did not significantly affect any of the enzyme activities measured. A dose-dependent increase in mutagenic response versus amount of 5000 X g supernatant and promutagen (aflatoxin B1 (AFB), 2-aminofluorene (2-AF), BP) was observed. The most pronounced activation was obtained with S-5 fraction from BNF- or PCB-treated rats. The great sensitivity of prostatic AHH to certain inducers and the capacity of the prostate to produce mutagenic metabolites might be of importance for initiation of prostatic cancer by environmental factors.