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.     

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

1. The metabolism of di-, tri- and tetrabromobiphenyls (PBBs) by hepatic microsomes isolated from control animals and animals treated with Arochlor 1254 was studied.2. Hepatic microsomes isolated fom control rats expressed higher rates of oxidations than avians3. Treatment of rats and pigeons with Arochlor 1254 induced cytochrome P450 dependent monooxygenases leading to an increased regioselective metabolism of PBB 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.     

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.     

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 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.     

Evidence for the induction of fatty acid desaturation in proliferating hepatic endoplasmic reticulum in response to treatment with polychlorinated biphenyls. Are fatty acid desaturases cytochrome P-450-dependent monooxygenases?

1. Polychlorinated biphenyls (PCBs) are abundant and persistent pollutants in the ecosystem which accumulate in biological systems. 2. We have shown previously (Borlakoglu et al., 1990; Eur. J. Biochem. 118, 327-332) that 120 hr after treating pigeons and rats with 1.5 mmol Aroclor 1254/kg body weight, hepatic microsomal membranes showed significant increases in the proportion of arachidonate (20:4,5, 8,11,14), in the concentration of cytochrome P-450 and in the activities of a wide range of cytochrome P-450-dependent enzymes involved in the metabolism of drugs and other xenobiotics. 3. After treating pigeons and rats in vivo with Aroclor 1254, linoleate desaturases activity increased significantly 3.35-, 4.35-, 5.83- and 8.61-fold 24, 48, 68 and 120 hr for pigeons and 2- and 7-fold for rats respectively 48 and 120 hr post treatment. The total activity of linoleate desaturases in the whole liver of pigeons and rats increased 40- and 10-fold respectively. 4. There were excellent correlations between the concentrations of cytochrome b5 and cytochrome P-450 and the activity of pigeon linoleate desaturases. Extrapolation of the concentration of cytochrome P-450 to zero is coincident with zero linoleate desaturase activity. 5. Evidence is presented to suggest the novel concept that linoleate desaturation is dependent upon the catalytic cycle of these monooxygenases.     

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.     

Dechlorination of Four Commercial Polychlorinated Biphenyl Mixtures (Aroclors) by Anaerobic Microorganisms from Sediments

The rate, extent, and pattern of dechlorination of four Aroclors by inocula prepared from two polychlorinated biphenyl (PCB)-contaminated sediments were compared. The four mixtures used, Aroclors 1242, 1248, 1254, and 1260, average approximately three, four, five, and six chlorines, respectively, per biphenyl molecule. All four Aroclors were dechlorinated with the loss of meta plus para chlorines ranging from 15 to 85%. Microorganisms from an Aroclor 1242-contaminated site in the upper Hudson River dechlorinated Aroclor 1242 to a greater extent than did microorganisms from Aroclor 1260-contaminated sediments from Silver Lake, Mass. The Silver Lake inoculum dechlorinated Aroclor 1260 more rapidly than the Hudson River inoculum did and showed a preferential removal of meta chlorines. For each inoculum the rate and extent of dechlorination tended to decrease as the degree of chlorination of the Aroclor increased, especially for Aroclor 1260. The maximal observed dechlorination rates were 0.3, 0.3, and 0.2 μg-atoms of Cl removed per g of sediment per week for Aroclors 1242, 1248, and 1254, respectively. The maximal observed dechlorination rates for Hudson River and Silver Lake organisms for Aroclor 1260 were 0.04 and 0.21 μg-atoms of Cl removed per g of sediment per week, respectively. The dechlorination patterns obtained suggested that the Hudson River microorganisms were more capable than the Silver Lake organisms of removing the last para chlorine. These results suggest that there are different PCB-dechlorinating microorganisms at different sites, with characteristic specificities for PCB dechlorination.     

Effect of inducers and inhibitors of monooxygenase on the hydroxylation of prostaglandins in the guinea pig. Evidence for several monooxygenases catalyzing omega- and omega-1-hydroxylation.

The incubation of prostaglandins (PG's) with liver microsomes from guinea pigs treated with inducers of monooxygenase (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzo[alpha]pyrene (benzpyrene), or a mixture of chlorinated biphenyls (Aroclor 1254)) exhibited marked elevation of 19-hydroxylation of PGE1, PGE2, PGA1, and PGA2 without affecting significantly 20-hydroxylation. However, with respect to effects on hydroxylation of a variety of xenobiotics, benzpyrene and Aroclor treatments differed markedly; whereas Aroclor treatment elevated the demethylation of ethylmorphine, benzphetamine, and p-chloro-N-methylaniline (PCMA), benzpyrene treatment had no effect on demethylation of ethylmorphine and only a marginal effect on that of PCMA. Both inducers elevated benzpyrene hydroxylation. By contrast, treatment with phenobarbital did not affect the hepatic microsomal PG's hydroxylation, although the hydroxylation of benzpyrene and the demethylation of ethylmorphine, benzphetamine, and PCMA were enhanced. Also, the hydroxylation of PG's by kidney cortex microsomes was not affected by either benzpyrene or Aroclor treatment. Inhibitors of monooxygenase were used to help delineate the type of monooxygenases induced. At low levels of alpha-naphthoflavone (ANF), benzpyrene hydroxylation in control- and Aroclor-treated guinea pigs was only little affected; by contrast, the same concentration of ANF markedly inhibited benzpyrene hydroxylation in benzpyrene-treated guinea pigs. On the other hand, metyrapone was most inhibitory in control guinea pigs. Support for the conclusion that benzpyrene induces in the guinea pig a hepatic monooxygenase with different characteristics than that found in control animals was provided by the observation that ANF (10 MICROM) inhibited PGE1 hydroxylation more pronouncedly in liver microsomes from benzpyrene-treated than from Aroclor-treated guinea pigs or controls. In addition, in benzpyrene and Aroclor-treated guinea pigs, ANF inhibited the (omega-1)-hydroxylation more pronouncedly than that of omega-hydroxylation. By contrast, metyrapone appeared to inhibit omega-hydroxylation more effectively than (omega-1)-hydroxylation. These results indicate that in the guinea pig, hydroxylation of PG's at the omega (20-) and omega-1 (19-) positions is catalyzed by different monooxygenases and that the inducers tested affect several hepatic monooxygenases with different specificities toward xenobiotics; however, with respect to PG's only the enzyme(s) involved in the 19-hydroxylation is affected.     

In Vitro Metabolism of 20(R)-25-Methoxyl-Dammarane-3, 12, 20-Triol from Panax notoginseng in Human,Monkey, Dog,Rat, and Mouse Liver Microsomes

The present study characterized in vitro metabolites of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol (20(R)-25-OCH₃-PPD) in mouse, rat, dog, monkey and human liver microsomes. 20(R)-25-OCH₃-PPD was incubated with liver microsomes in the presence of NADPH. The reaction mixtures and the metabolites were identified on the basis of their mass profiles using LC-Q/TOF and were quantified using triple quadrupole instrument by multiple reaction monitoring. A total of 7 metabolites (M1–M7) of the phase I metabolites were detected in all species. 25(R)-OCH₃-PPD was metabolized by hydroxylation, dehydrogenation, and O-demethylation. Enzyme kinetic of 20(R)-25-OCH₃-PPD metabolism was evaluated in rat and human hepatic microsomes. Incubations studies with selective chemical inhibitors demonstrated that the metabolism of 20(R)-25-OCH₃-PPD was primarily mediated by CYP3A4. We conclude that 20(R)-25-OCH₃-PPD was metabolized extensively in mammalian species of mouse, rat, dog, monkey, and human. CYP3A4-catalyzed oxygenation metabolism played an important role in the disposition of 25(R)-OCH₃-PPD, especially at the C-20 hydroxyl group.     

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.     

Dealkylation of pentoxyresorufin: A rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat

The O-dealkylation of pentoxyresorufin (7-pentoxyphenoxazone) by rat liver microsomes was examined. The reaction appeared highly specific for certain phenobarbital inducible forms of cytochrome P-450 and was increased 95- to 140-fold by animal pretreatment with phenobarbital (75 mg/kg/day, four ip injections) and ~50-fold by Aroclor 1254 (500 mg/kg, one ip injection) while animal pretreatment with 3-methylcholanthrene (50 mg/kg/day, three ip injections) resulted in less than a 2-fold increase over the rate detected in control microsomes. It was observed that this activity, in microsomes for Aroclor-pretreated rats, was dependent on O₂ and was inhibited by metyrapone and SKF 525-A, indicative of cytochrome(s) P-450 mediation in the reaction. When antibodies directed against purified cytochrome(s) P-450S were employed to inhibit the pentoxyresorufin O-dealkylation reaction, antibodies to P-450_PB-B greatly inhibited the reaction (>90%), while antibodies to P-450_PB-C or P-450_PB/PCN-E had minimal effects. Assay of hepatic microsomes from rats which were pretreated with varying doses of phenobarbital (0.9–75 mg/kg/day, four ip injections) indicated that while aminopyrine-N-demethylase activity was induced only 2-fold at the maximum dose (75 mg/kg/day), pentoxyresorufin O-dealkylase activity was induced ~140-fold at this dose and ~4-fold by a dose of phenobarbital as low as 0.9 mg/kg.     

Neonatal modulation of adult rat hepatic microsomal benzo[a]pyrene hydroxylase activities by Aroclor 1254 or phenobarbital

The constitutive and Aroclor 1254-induced activities of hepatic microsomal benzo[a]pyrene hydroxylases in male and female rats were determined in animals from ages 11 to 120 days. In 11-day-old noninduced male rats, benzo[a]pyrenediones and 9-hydroxybenzo[a]pyrene were the major microsomal metabolites; in 21-day-old males benzo[a]pyrene-diones and benzo[a]pyrene-9,10-dihydrodiol were predominant. In 60- and 120-day-old animals 3-hydroxybenzo[a]pyrene was the major microsomal metabolite. A similar trend was observed for the development of benzo[a]pyrene hydroxylase activities in female rats. With the exception of 4,5-dihydrodiol formation, the highest induction of individual and total benzo[a]pyrene hydroxylase activities by Aroclor 1254 was observed in the 21-day-old immature male rats, in which there was a 330- and 4.5-fold increase in the formation of 3-hydroxybenzo[a]pyrene and quinone metabolites, respectively. The induction of benzo[a]pyrene total metabolite formation by Aroclor 1254 in female rats from 11 to 120 days of age was relatively constant (i.e., 13.3- to 10.1-fold induction); however, the relative induction of the individual benzo[a]pyrene hydroxylases was highly variable. In a second set of experiments, male and female rats were neonatally exposed to phenobarbital (600 mumol/kg) or Aroclor 1254 (100 mumol/kg), and the effects of these xenobiotics on neonatal imprinting of hepatic microsomal benzo[a]pyrene hydroxylase activities were determined in the 120-day-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circulating thyroid hormone levels and iodothyronine deiodinase activities in Nile tilapia (Oreochromis niloticus) following dietary exposure to Endosulfan and Aroclor 1254

We evaluated the effects of two organochlorinated environmental contaminants, Endosulfan and Aroclor 1254 on peripheral thyroid hormone metabolism and thyroid hormone plasma levels in Nile tilapia (Oreochromis niloticus). Tilapia were exposed through diet to 0.1 and 0.5 microg g(-1) of Endosulfan and 0.5 microg g(-1) of Aroclor 1254 for 21 and 35 days. Decreased plasma T4 and rT3 levels were observed in tilapia exposed to the lower dose of Endosulfan, while treatment with a higher dose and Aroclor 1254 produced no changes. Plasma T3 levels were not affected by these compounds. Hepatic type I deiodinase (D1) activity was depressed by a lower dose of Endosulfan and hepatic type III (D3) activity was increased following 35 days of exposure to the lower dose of Endosulfan and following 21 and 35 days of exposure to Aroclor 1254; while type II (D2) remained unchanged in liver as well as in all other organs analysed. Apart from hepatic D3 activity, Endosulfan and Aroclor 1254 also increased D3 activity in gill, but not in other tested organs. It is concluded that dietary exposure of tilapia to Endosulfan or Aroclor 1254 can lead to changes in circulating thyroid hormone levels and/or in peripheral thyroid hormone metabolism. The changes in hormone metabolism differ between tissues, eventually reflecting tissue-specific differences in adaptation.     

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.     

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.     

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.     

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.     

Mutagenic activation of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline (MeIQ) by subcellular fractions and cells isolated from small intestine,kidney and liver of the rat

The mutagenic activity of the pyrolysis products 2-amino-3-methylimidazo[4,5-f]-quinoline and 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline in Salmonella typhimurium TA98 using rat intestinal and renal subcellular fractions as activation systems was approximately 1 and 5 revertants per nmol, respectively. This was 1,000 times less than the activity with a subcellular fraction from rat liver. The mutagenic activity of both compounds was considerably increased using intestinal, renal and hepatic preparations isolated from PCB (Aroclor 1254)-pretreated rats, compared to preparations from control animals. In addition, both compounds displayed a moderate direct-acting mutagenic activity at concentrations above 10^-5 M. Isolated cells from small intestine, kidney and liver incubated in nucleopore chambers were able to convert both compounds into products which mutated bacteria outside the chambers. The concentrations of chemicals required to yield responses of a similar magnitude were approximately 3 orders of magnitude higher in the intestinal and renal systems compared to the hepatic system. The formation of metabolites mutagenic for Salmonella typhimurium by hepatic subcellular and cellular systems was shown to be superior to the respective intestinal and renal systems.Abbreviations AHH arylhydrocarbon hydroxylase - IQ 2-amino-3-methylimidazo[4,5-f]-quinoline - MC 3-methylcholanthrene - MeIQ 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline - PCB polychlorinated biphenyls (Aroclor 1254) - S9 the 9,000 g supernatant tissue fraction - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin