Biosynthesis in the rat liver of a common form of monooxygenase induced by xenobiotics of a methylcholanthrene series

Injection of Wistar rats with five structurally different inducers of methylcholanthrene-type (polycyclic aromatic and heterocyclic hydrocarbons, chloro-derivatives of biphenyl and dibenzo-p-dioxin) results in the de novo synthesis of two P-448 hemoproteins (molecular weight 56 000 and 53 000 Da) differing in their functional and immunochemical properties in liver microsomes. A comparison of catalytic and immunochemical characteristics of five cytochrome P-448 forms (Mr = 56 000 Da) as well as the data from electrophoretic, proteolytic and inhibitory analyses revealed no differences in the preparations used, with the exception of 2,3,7,8-tetrachloro-dibenzo-p-dioxin-induced microsomes characterized by a low level of this cytochrome P-448 form and a higher molecular activity as compared with 3,4-benzpyrene and 7-ethoxyresorufin-induced microsomes. The experimental results do not confirm the hypothesis on the feasibility of induced synthesis of a variety of individual forms of monooxygenase that would correlate with the number of structural variants of inducers.     

The effects of pretreatment with cytochrome P-450 inducers and preincubation with a cytochrome P-450 effector on the mutagenicity of genotoxic carcinogens mediated by hepatic and renal S9 from two species of marine fish

A series of experiments was designed to characterize the cytochrome P-450-dependent activation of 7 genotoxic carcinogens in the Salmonella preincubation assay by hepatic postmitochondrial fractions (S9) from the oyster toadfish and the Americal eel and by renal S9 from the toadfish. Significant S9-dependent mutagenicity was observed for benzo[a]pyrene (BAP), 2-aminoanthracene (2AA), aflatoxin B1 (AFB1), 7,12-dimethylbenz[a]anthracene (DMBA) and cyclophosphamide (CP) with hepatic S9 from untreated fish (UI S9) of both species and with renal S9 from untreated toadfish, although renal UI S9 was only marginally effective for activating AFB1. Neither UI S9 from toadfish liver or kidney nor that from eel liver consistently affected the direct mutagenicity of ethylene dibromide (EDB) or substantially activated dimethylnitrosamine (DMN). Pretreatment of toadfish with 3-methylcholanthrene (MC) decreased the mutagenicity of 2AA and increased the mutagenicities of BAP, AFB1 and DMBA, whereas, pretreatment of eels with MC increased the mutagenicities of BAP, 2AA and AFB1. Pretreatment of toadfish with Aroclor 1254 (AC) decreased the mutagenicity of AFB1 and increased the mutagenicity of 2AA, whereas, pretreatment of eels with AC increased the mutagenicities of BAP and DMBA. Pretreatment of toadfish with beta-napthoflavone (BNF) effected changes similar to those by pretreatment with MC except that the mutagenicity of AFB1 was not increased. Coincubation with 10(-4) M alpha-napthoflavone (ANF) decreased the mutagenicity of BAP mediated by toadfish MC and BNF S9 and eel AC S9 and decreased the mutagenicity of AFB1 mediated by toadfish MC and BNF S9 and by eel MC S9. Coincubation with ANF increased the mutagenicity of AFB1 mediated by toadfish and eel AC S9 and increased the mutagenicity of 2AA mediated by eel AC S9. Pretreatment of toadfish with MC, BNF and AC decreased the mutagenicity of 2AA mediated by renal S9 and ANF decreased the mutagenicity of 2AA mediated by renal UI and BNF S9. MC pretreatment of toadfish and eels and BNF pretreatment of toadfish induced BAP monooxygenase activity in hepatic microsomes. ANF (10(-4) M) inhibited the BAP monooxygenase activity of MC microsomes from toadfish and eels and of BNF microsomes from toadfish. The conjugation effectors diethyl maleate and salicylamide alone or combined had little or no effect on the mutagenicities of BAP and 2AA mediated by toadfish and eel UI and MC S9.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential control of rat microsomal "aryl hydrocarbon" monooxygenase and epoxide hydratase.

A growing body of evidence implicates epoxide metabolites of mutagenic and carcinogenic polycyclic hydrocarbons as either the only species, or one of the contributing species responsible for these adverse effects. Selective induction of epoxide hydratase(s) catalyzing the transformation of epoxides to electrophilically unreactive dihydrodiols, under conditions not leading to increases in monooxygenase(s) responsible for epoxide formation would, therefore, be of interest. All inducers of rat hepatic epoxide hydratase (determined with [7-3H]styrene oxide as substrate) which have been discovered also induced monooxygenase (determined with benzo(a)pyrene as substrate) suggesting a possible common biosynthetic control of these enzymes. The enzyme levels observed in different sexes and at different stages of the ontogenetic development, possibly dependent on endogenous inducers, strengthened this view. No sex difference is epoxide hydratase activity was observed in young rats (1 to 5 days old) while epoxide hydratase levels were about 3-fold higher in adult males than in females, which was remarkably similar to the behavior of monooxygenase. Moreover, the prenatal development of epoxide hydratase and monooxygenase appeared to be similar--although the low enzyme levels precluded accurate determinations of the latter. Although different types of known monooxygenase inducers all led to epoxide hydratase induction in adult rat liver, their effect of epoxide hydratase and monooxygenase could be dissociated by transplacental treatment. Dissociation was clearest with inducers of the polycyclic hydrocarbon type which led to great induction of monooxygenase while epoxide hydratase remained unchanged. The increases in monooxygenase activity were very different when determined by two methods based on different principles, demonstrating that at least two monooxygenases are involved in oxidative metabolism of benzo(a)pyrene, and that the control of epoxide hydratase is not under common control with either of them.     

Mutagenicity and irreversible binding of the hepatocarcinogen, 2,4-diaminotoluene.

Mutagenicity of 2,4-diaminotoluene (DAT) in the Salmonella mutagenicity assay was increased with liver fractions from phenobarbital (PB) or beta-naphthoflavone (BNF) treated rats. Substitutions of the hydrogens in the methyl group of 2,4-DAT with deuterium resulted in a decrease in mutagenicity. Incubation of rat liver microsomes with tritiated 2,4-DAT in the presence of NADPH led to the formation of irreversibly bound products to microsomal protein. The rates of binding were not increased using microsomes from PB or BNF-treated rats and was not altered by deuterium substitution in the methyl group. Addition of superoxide dismutase, glutathione (GSH) or rat liver supernatant reduced 2,4-DAT irreversible binding, whereas 2,4-DAT mutagenicity was unaffected by superoxide dismutase addition. Injection of tritiated 2,4-DAT 100 mg/kg to rats lead to its irreversible binding to liver protein and ribosomal RNA and to kidney protein in vivo, again protein binding was not increased after prior treatment with PB or BNF. No irreversible interaction of tritiated 2,4-DAT with DNA either in vitro or in vivo could be demonstrated.     

Metabolism of benzo[c]phenanthrene by rat liver microsomes and by a purified monooxygenase system reconstituted with different isozymes of cytochrome P-450

Metabolism of the environmental pollutant and weak carcinogen benzo[c]-phenanthrene (B[c]Ph) by rat liver microsomes and by a purified and reconstituted cytochrome P-450 system is examined. B[c]Ph proved to be one of the best polycyclic aromatic hydrocarbon substrates for rat liver microsomes. It is metabolized by microsomes from control rats and by rats treated with phenobarbital or 3-methylcholanthrene at 3.9, 4.2 and 7.8 nmol/nmol cytochrome P-450/min, respectively. Principal metabolites are dihydrodiols along with small amounts (less than 10%) of phenols. The K-region 5,6-dihydrodiol is the major metabolite and accounts for 77-89% of the total metabolites. The 3,4-dihydrodiol with a bay-region 1,2-double bond is formed in much smaller amounts and accounts for only 6-17% of the total metabolites, the highest percentage being formed by microsomes from control rats. Highly purified monooxygenase systems reconstituted with cytochrome P-450a, P-450b and P-450c and epoxide hydrolase form predominantly the 5,6-dihydrodiol (95-97% of total metabolites) and only a small percentage of the 3,4-dihydrodiol (3-5% of total metabolites). The 3,4-dihydrodiol is formed with higher enantiomeric purity by microsomes from 3-methylcholanthrene-treated rats (88%) than by microsomes from control rats (78%) or phenobarbital-treated rats (60%). In each case the (3R,4R)-enantiomer predominates. B[c]Ph 5,6-dihydrodiol formed by all three microsomal preparations is nearly racemic.     

Rat pulmonary microsomal cytochrome P-450 enzymes involved in the activation of procarcinogens.

Rat lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic specificities towards activation of several procarcinogens to genotoxic metabolites in Salmonella typhimurium TA1535/pSK1002. We first examined the roles of rat liver microsomal P-450 enzymes in the activation of benzo[a]pyrene and its 7,8-diol enantiomers to genotoxic products, and found that P-450 1A1 is a major catalyst for the activation of these potential procarcinogens in rat livers. Using lung microsomes isolated from rats treated with various P-450 inducers we obtained evidence that at least three P-450 enzymes are involved in the activation of several procarcinogens. Immunoinhibition studies support the view that benzo[a]pyrene and its 7,8-diol derivatives, other dihydrodiol derivatives of polycyclic aromatic hydrocarbons, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole are activated to genotoxins mainly by rat P-450 1A1, which is inducible in rat lungs by 5,6-benzoflavone and the polychlorinated biphenyl mixture Aroclor 1254. Activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline and 2-amino-3-methylimidazo[4,5-f]quinoline may be catalyzed by another P-450 enzyme because the activities were not induced by treatment with 5,6-benzoflavone or Aroclor 1254. The observation that both activities were inhibited by antibodies raised against P-450 1A2 and by 7,8-benzoflavone suggests a role for an enzyme of P-450 1A family, probably P-450 1A2, in rat lung microsomes. The activation of aflatoxin B1 and sterigmatocystin appears to be catalyzed by other P-450 enzyme(s) rather than the P-450 1A family as judged by the different responses of activities to the P-450 inducers and the specific antibodies in rat lung microsomes. Interestingly, lung microsomal activation of several procarcinogens was found to be suppressed in rats treated with isosafrole and pregnenolone 16 alpha-carbonitrile. Thus, the results support the roles of different P-450 enzymes in the activation of procarcinogens in rat lung microsomes.     

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.     

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.     

B-naphthoflavone induction and its effect on hepatic phospholipid metabolism in rainbow trout (Salmo gairdneri)

1. B-naphthoflavone (BNF) induction of microsomal cytochrome P-488 and its effect on liver phospholipid metabolism were examined in rainbow trout (Salmo gairdneri) 24 and 96 hr after intraperitoneal injection. 2. Cytochrome P-448 content increased at 96 hr to approximately double the cytochrome content of control fish. 3. Computer-analyzed laser densitometry scans of LDS-polyacrylamide gels of 96 hr BNF-treated liver microsomes showed a 90% increase in cytochrome P-448 levels. 4. A 34% increase in microsomal phospholipid (mumol/mg protein) was observed 24 hr after BNF injection, with a marked increase in choline, ethanolamine and inositol phospholipids. 5. Following 96 hr of exposure to BNF some differences in enzyme activity were noted; choline kinase and cytidylyltransferase activities were reduced, while a marked increase was observed in choline phosphotransferase activity. In light of current information on induction of liver microsomal phospholipid metabolism in mammals, the results of the this study suggest that trout do not respond like mammals to inducers of monooxygenase activity.     

The effect of monooxygenase inducing agents on the incorporation of [35S]methionine into hepatic microsomal protein of rainbow trout (Salmo gairdneri)

Treatment of rainbow trout (Salmo gairdneri) with 150 mg/kg BNF resulted in an increase in hepatic microsomal monooxygenase activity as assessed by ECOD and EROD when compared to those activities in corn oil-pretreated animals. Administration of 100 mg/kg 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) to trout had no significant effect on these catalytic activities or on BeND. The amount of radioactivity in hepatic microsomes at 24, 48 or 72 hr following the administration of 75 muCi of [35S]methionine was consistently higher in animals pretreated with BNF than in those treated with corn oil or 6-CB. Autoradiography/fluorography of electrophoretograms demonstrated the appearance of at least three radiolabeled bands in the 50,000-60,000 mol. wt range in solubilized microsomes from BNF-treated fish which were not present in microsomes from control animals or fish treated with 6-CB. These data indicate that the stimulation of hepatic microsomal catalytic activities observed following the administration of 3-MC-type agents to rainbow trout is due, at least in part, to induction of enzyme(s) rather than activation of existing enzyme(s). These results further support the observation that fish appear to be non-responsive to phenobarbital-type inducing agents.     

A comparison of hepatic P450 induction in rat and trout (Oncorhynchus mykiss): delineation of the site of resistance of fish to phenobarbital-type inducers

1. A comprehensive approach was taken to delineate the site of refractivity of trout to phenobarbital-type (PB-type) hepatic monoxygenase (MO) inducers. 2. Model inducers beta-naphthoflavone (BNF; 3-MC-type), and PB as well as the polychlorinated biphenyl isomers, 3,4,5,3',4',5'-hexachlorobiphenyl (3,4,5-HCB; 3-MC-type) and 2,4,5,2',4',5'-hexachlorobiphenyl (2,4,5-HCB; PB-type) were used to assess MO activities, total cytochromes P450, and [35S]-methionine incorporation into de novo synthesized microsomal protein in both trout and rats. 3. In rainbow trout immunodetection of P450 isozymes and nucleic acid hybridization of rainbow trout P(1)450 mRNA using pfP(1)450-3' (trout 3-MC-inducible, P450IA1 gene) and genomic DNA using pfP(1)450-3' or pSP450-oligo (rat PB-inducible, P450IIB1 gene) cDNAs were carried out. 4. In rainbow trout, PB and 2,4,5-HCB do not increase hepatic MO activities, total cytochromes P450, de novo synthesis of microsomal protein, levels of P450 isozymes, or levels of P(1)450 mRNA. 5. Rainbow trout have, within their genome, DNA with sequence(s) similar to rat P450IIB1, but inducibility of this P450 in trout by PB-type inducers is lacking.     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

Evidence for the presence of distinct flavin-containing monooxygenases in human tissues

Catalytic activities and substrate specificity of flavin-containing monooxygenase were examined in human tissues. During incubation with imipramine, human hepatic microsomes efficiently carried out cytochrome P450-dependent reactions but not the formation of N-oxide, while in kidney imipramine N-oxide was the only metabolite formed during in vitro incubation. The production of imipramine N-oxide was essentially due to flavin-containing monooxygenase as shown by thermal inactivation. In contrast, thiobenzamide and dimethylaniline were actively transformed by both human liver and kidney flavin-containing monooxygenase. Neither the modification of pH nor the solubilization of microsomal membranes increased imipramine N-oxidation in human liver. Kinetic analysis indicated a poor affinity (about 7 mM) of human liver microsomes for imipramine versus 0.3 mM in kidney. Immunological studies were undertaken to support enzymatic data. Antibodies raised against rat liver flavin-monooxygenase reacted strongly with human kidney microsomes but extremely weakly with liver microsomes. The relative amount of immunochemically determined protein correlated well with imipramine N-oxidation activity. A dose-dependent inhibition of imipramine N-oxidation by anti-flavin-monooxygenase antibodies was observed in human kidney, as well as in rat kidney and liver. Taken together, the results can be interpreted by the possible existence in human tissues of distinct flavin-containing monooxygenases exhibiting a partial overlapping substrate specificity. The protein involved in imipramine N-oxidation is missing from human liver but actively carries out the reaction in kidney, while another protein catalyzes the oxidation of thiobenzamide and dimethylaniline in both tissues.     

The effects of tetrachlorobiphenyls on the electron transfer reaction of isolated rat liver mitochondria

A comparative study was made of the effects of several symmetrical tetrachlorobiphenyls (TCBs) on the electron transfer from succinate to oxygen of rat liver mitochondria, and some differences in effects caused by the different chlorine positions of the biphenyl ring were clarified. TCBs used in this study included 2,3,2',3'-, 2,4,2',4'-, 2,5,2',5'-, 2,6,2',6'-, and 3,4,3',4'-TCBs. The inhibitory actions of 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs on succinate oxidase were potent, while those caused by 2,6,2',6'- and 3,4,3',4'-TCBs were significantly weak. The inhibition sites of 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs in succinate oxidase were succinate dehydrogenase and cytochrome b-c segment of the electron transport chain. In the cytochrome b-c segment, these TCBs acted on myxothiazol-sensitive site rather than antimycin-sensitive site. Cytochrome c oxidase was hardly affected by TCBs. These results indicate that 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs severely depress the electron transfer with succinate as the substrate, which secondarily reduces the synthesis of ATP. The relationship between the activity and chemical structure of TCBs is also discussed.     

Cell-specific metabolic activation of 7,12-dimethylbenz[a]anthracene in rat testis

The binding of metabolites of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) to protein in rat testis seminiferous tubules was studied. Treatment of cultured seminiferous tubule segments with DMBA resulted in very little binding to protein, suggesting that the seminiferous epithelium from rat testis lacks the cytochrome P-450-dependent monooxygenase(s) required for DMBA metabolism. In contrast, Leydig cells from rat testis contain monooxygenase systems which catalyze the metabolism of PAH, such as DMBA. This metabolic activation of DMBA was localized in both mitochondria and microsomes derived from Leydig cells and was decreased by inhibitors of the cytochrome P-450 system and by free radical scavengers, suggesting that the metabolism involved both cytochrome P-450 and free radical-dependent pathways. In the presence of whole Leydig cells or microsomes prepared from Leydig cells, the covalent binding of DMBA metabolites to protein of rat testis seminiferous tubules was increased 5- and 13-fold, respectively. These results suggest that DMBA is metabolized primarily in rat testis Leydig cells and that part of the produced metabolites find their way to the seminiferous epithelium, where they undergo further metabolism producing reactive metabolites, possibly cation radicals and diolepoxides, which interfere with the functions of spermatogonia and spermatocytes by modifying key proteins covalently.     

Induction of cytochrome P-450 forms in liver microsomes of rats in the early neonatal period after administration of phenobarbital and 3-methylcholanthrene

The activity of cytochrome P-450 dependent monooxygenase system from rat liver microsomes after induction by phenobarbital and 3-methylcholantrene in early neonatal period (3-16 days after birth) was studied. It was found that the total amount of cytochrome P-450 increases after injection of these inducers in neonatal rats of all age groups. In parallel, in the case of 3-methylcholantrene induction the benz(a)pyrene hydroxylase and 7-ethoxyresorufin deethylase activities increase; phenobarbital induction causes a rise in the benzphetamine-N-demethylase and benz(a)pyrene hydroxylase activities. Immunochemical analysis involving the use of antibodies specifically directed against cytochrome P-450 of adult rats revealed that the level of cytochrome P-450 in the case of 3-methylcholantrene induction increases from 5 to 50%, whereas that of cytochrome P-450 upon phenobarbital induction increases from 5 to 40% in liver microsomes of 3- and 16-day-old rats. The mode of inhibition of various substrates metabolism by antibodies in neonatal rat microsomes suggests that the 3-methylcholantrene-induced cytochrome P-448, like in adult rats, participates in the hydroxylation of benz(a)pyrene and O-deethylation of 7-etoxyresorufin. The participation of phenobarbital-induced cytochrome P-450 in the metabolism of benzphetamine and aldrin in neonatal rats is much lower than in the adult ones. The metabolism of benz(a)pyrene in phenobarbital-induced neonatal rat microsomes in all age groups is not inhibited by antibodies. The age-dependent differences in inhibition of metabolism and the increase in the benz(a)pyrene hydroxylase activity in phenobarbital-induced rats suggest that the spectrum of inducible forms of cytochrome P-450 in neonatal rats differ from that in adult animals.     

In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.     

Characterization of covalent binding of [14C]-2-chloro-4-acetotoluidide to microsomes of starling liver and kidney

In this study, we have characterized the covalent binding of [14C]-2-chloro-4-acetotoluidide (CAT) radioactivity to microsomes of starling liver and kidney. The maximal velocity (Vmax) of covalent binding and apparent Michaelis constant (Km) for both tissues were similar. The Vmax for liver and kidney were 52.8 and 68.9 pmol/min/mg protein, and the apparent Kms were 0.54 and 0.87 mM, respectively. The covalent binding of radioactivity to heat-denatured microsomes of liver and kidney was reduced by 62% and 15%, respectively. Incubation at 0 degrees C reduced the binding by 80% to liver and 70% to kidney microsomes. Absence of nicotinamide adenine dinucleotide phosphate (NADP) and molecular O2 reduced the binding to liver microsomes by 36 and 53%, as opposed to 28% increase and 26% decrease in binding to kidney microsomes, respectively. Inducers of cytochrome P450 monooxygenase (P450), phenobarbital, and 3-methylcholanthrene (3-MC), had opposite effects on the covalent binding of [14C]-CAT radioactivity to hepatic and renal microsomes. Phenobarbital increased the binding to hepatic microsomes by 100% and had no effect on binding to renal microsomes. 3-MC, on the other hand, increased the binding to kidney microsomes by threefold and had no effect on the binding to hepatic microsomes. SKF 525A, an inhibitor of P450, inhibited the binding to hepatic microsomes by 60% at 0.5 mM but failed to have any effect on binding to renal microsomes. alpha-Naphthoflavone, another inhibitor of P450, had no effect on the covalent binding of [14C]-CAT radioactivity to microsomes of either tissue.(ABSTRACT TRUNCATED AT 250 WORDS)