Studies on the developmental and adrenal regulation of cytosolic and microsomal epoxide hydrolase activities in rat liver.

The present study was undertaken to ascertain developmental profiles of microsomal epoxide hydrolase (mEH) and cytosolic epoxide hydrolase (cEH) enzyme activities in rat liver. During development, mEH activity reached an optimum by 6 weeks of age (63 nmol/min/mg protein). Activities decreased thereafter in both sexes although in adult male liver the activity was twice that measured in adult female liver. Thus, the importance of pituitary maturation was suggested from these findings. Since glucocorticoids have been implicated in the regulation of mEH gene expression the effect of adrenalectomy on mEH activity was investigated in adult male rat liver. The procedure increased mEH activity almost two-fold and the increase was reversed by dexamethasone, but not deoxycorticosterone, administration. With respect to hepatic cEH activity, the developmental profiles indicated that enzyme activity was greatest in rats at 1 week of age (12-15 nmol/min/mg protein) and very little activity was detected beyond 4 weeks of age (approximately 5 nmol/min/mg protein); sex differences in cEH activity were not apparent at any age. Thus, the pituitary appears to be important in the developmental induction of mEH but not cEH. Glucocorticoids appear to provide the major hormonal influence on mEH expression. Thus, the hypothalamus-pituitary-adrenal axis is involved in the regulation of mEH but the regulation of the cEH enzyme remains unclear.

Evidence for complexation of P-450 IIC6 by an orphenadrine metabolite.

Removal of the orphenadrine metabolite from its complex with rat liver P-450 IIB1 is associated with a discrepancy in the reactivation of IIB1 activity. Two possible explanations are that either (1) NADPH-P-450-reductase is inaccessible to the restored IIB1, or (2) complexation of other P-450s may occur. Exogenous P-450 reductase increased all pathways of steroid hydroxylation (1.9 to 3.6-fold) but did not enhance reactivation of IIB1-dependent steroid 16 beta-hydroxylation. Instead, P-450 IIC6-dependent progesterone 21-hydroxylase activity was increased after dissociation to 122% of control. IIC6 activity was also inhibited in vitro in microsomes from phenobarbital-induced rats (ki = 151 microM). Thus, orphenadrine appears to complex P-450 IIC6 as well as IIB1 in rat liver.

In vitro inhibition of hepatic drug oxidation by thioridazine. Kinetic analysis of the inhibition of cytochrome P-450 isoform-specific reactions.

The phenothiazine tranquilizer thioridazine has been associated with drug interactions in man. This study investigated the capacity of the drug to inhibit hepatic drug oxidations mediated by cytochromes P-450 (P-450) in microsomes in vitro. Thioridazine was a potent linear mixed-type inhibitor of P-450b-dependent 7-pentoxyresorufin O-depentylase activity in phenobarbital-induced rat liver. The kinetic analysis revealed the enzyme-substrate dissociation constant (Ks) to be 1.6 microM whereas the dissociation constant of the enzyme-inhibitor complex (Ki) was 0.11 microM. In contrast, 7-ethoxyresorufin O-deethylase activity (mediated by P-450c) in beta-naphthoflavone-induced rat hepatic microsomes was inhibited to a lesser extent (Ki = 2.4 microM) in relation to the Ks value (0.5 microM). Spectral studies indicated that the efficiency of thioridazine binding in phenobarbital-induced microsomes was about 25-fold greater than in microsomes from beta-naphthoflavone-induced rat liver. This finding is consistent with the relative capacity of thioridazine to inhibit oxidase activities catalyzed by P-450b and P-450c. Mixed-function oxidase activities catalysed by other P-450s were also inhibited by thioridazine, although to a lesser extent than those catalysed by forms b and c. Thus, the 6 beta- and 16 beta-hydroxylations of androst-4-ene-3,17-dione in hepatic microsomes from untreated rats were inhibited to a similar extent (I50S = 52 and 43 microM, respectively). The 7 alpha- and 16 alpha-hydroxylase pathways were approximately only half as susceptible to inhibition by thioridazine. These findings demonstrate the capacity of thioridazine to inhibit a range of P-450-dependent drug oxidations, with those catalysed by forms b and c most susceptible. The present study strongly suggests that drug interactions elicited by thioridazine are most likely a consequence of inhibitory interactions with P-450 enzymes.

Effect of ofurace, oxadixyl, and alachlor on xenobiotic biotransformation in the rat liver.

Ofurace, oxadixyl, and alachlor were studied for their effect on hepatic xenobiotic biotransformation in male rats by dosing i.p. with 1 or 100 mg/kg of chemical for 7 days. Ofurace decreased ethoxyresorufin-O-deethylase and aniline p-hydroxylase activities but induced ethoxycoumarin-O-deethylase activity. Glutathione S-transferase and aminopyrine N-demethylase activities responded in a non dose-dependent manner, while cytochrome P-450 content and aldrin epoxidase activities were unchanged. Oxadixyl induced P-450 content, and ethoxycoumarin-O-deethylase and aminopyrine N-demethylase activities. It left ethoxyresorufin-O-deethylase, aldrin epoxidase and epoxide hydrolase activities unchanged and decreased aniline p-hydroxylase activities. Alachlor induced all activities excepting aldrin epoxidase (no change) and aniline p-hydroxylase (decreased). The data indicate that each of these acylanilide pesticides interacts with the monooxygenase system but with differing patterns.

In vitro formation of an inhibitory complex between an isosafrole metabolite and rat hepatic cytochrome P-450 PB-B.

A series of in vitro studies was performed, in rat liver microsomes, in which metabolite intermediate (MI) complexation of cytochrome P-450 (P-450) by the methylenedioxyphenyl compound isosafrole was related to P-450 isozyme-specific inhibition of drug oxidation. The C19-steroid androst-4-ene-3,17-dione was selected for initial study because the stereoselective hydroxylation of this substrate is specific for certain P-450s. In control microsomes only the 6 beta- and 16 beta-hydroxylations of the steroid (catalyzed, respectively, by the P-450s PCN-E and PB-B) were inhibited by isosafrole (I50 = 100 and 110 microM). In contrast, the 7 alpha- and 16 alpha-hydroxylases (P-450 UT-F- and UT-A-mediated, respectively) were refractory to inhibition. After phenobarbital (PB) induction, steroid 6 beta- and 16 beta-hydroxylase activities were again inhibited (I50 = 170 and 190 microM) but, in addition, the 16 alpha-hydroxylase pathway was also inhibited (I50 = 200 microM). Spectral studies revealed that MI complexation of P-450 in untreated microsomes was minimal but was enhanced markedly after PB induction (up to 50% of the total P-450 content complexed). Thus, it is apparent that a PB-inducible P-450 is involved in MI complex formation under these conditions. Indeed the I50 of isosafrole toward steroid 16 beta-hydroxylase activity was decreased if the inhibitor was preincubated with NADPH-fortified PB-induced microsomes prior to substrate addition; the preincubation step did not enhance the inhibition of any other steroid hydroxylase pathway by isosafrole.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics.

The anti-parkinsonian agent orphenadrine has been shown to form an in vitro metabolic intermediate (MI) complex in hepatic microsomes isolated from phenobarbital (PB)-treated rats. The present study was undertaken to assess the cytochrome P-450 isozyme specificity of inhibition and MI complexation. Spectral studies with untreated and PB-induced rat hepatic microsomes confirmed earlier reports on the selectivity of P-450 complexation by orphenadrine; MI complex formation was only observed with PB-induced microsomes. Inhibition studies with the P-450 substrates androst-4-ene-3,17-dione (androstenedione) and 7-pentoxyresorufin revealed selective inhibition of P-450 PB-B/D-associated monooxygenase activity. Thus, in microsomes from untreated male rats, orphenadrine failed to significantly inhibit (less than 50% inhibition up to a concentration of 300 microM) any of the major pathways of P-450-associated androstenedione metabolism. Preincubation of these microsomal fractions with orphenadrine and NADPH was not associated with increased inhibition of androstenedione metabolism. However, in PB-induced microsomes, P-450 PB-B/D-specific androstenedione 16 beta-hydroxylase activity was significantly and selectively inhibited (IC50 = 90 microM). Preincubation of orphenadrine with NADPH-supplemented PB-induced microsomes for 2, 4, or 8 min before androstenedione addition resulted in increased inhibition toward 16 beta-hydroxylase activity, lowering the observed IC50 to 6.6, 0.47, and 0.06 microM), respectively. Preincubation did not affect the selectivity of inhibition. In the absence of preincubation, orphenadrine appeared to be a potent mixed (competitive/noncompetitive)-type inhibitor of P-450 PB-B/D-associated pentoxyresorufin O-depentylation (Ki = 3.8 microM). Preincubation of orphenadrine with NADPH-supplemented microsomal fractions for 4 min resulted in a 30-fold lowering of the apparent inhibitor constant (Ki = 0.13 microM) and a change in the apparent inhibition kinetics to noncompetitive. Treatment of rats with orphenadrine (75 mg/kg/day intraperitoneally for 3 days) was associated with a 2-fold induction of total hepatic P-450, a 5- and 2.4-fold induction of androstenedione 16 beta- and 6 beta-hydroxylase activity, respectively, and formation of an orphenadrine-P-450 MI complex. Western blots of orphenadrine-induced microsomes revealed a 20-fold increase in P-450 PB-B/D-immunoreactive protein.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro inhibition of hepatic steroid hydroxylation by tamoxifen, a series of tamoxifen analogues and related compounds.

The in vitro inhibition of the cytochrome P-450 (P-450) isozyme specific positional hydroxylation of androst-4-ene-3,17-dione (androstenedione) by the alkylamino containing compounds trans- and cis-tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, SKF 525-A and the non-alkylamino containing compounds tamoxifen metabolite E, and tamoxifen analogue U-23469 was assessed in pooled hepatic microsomes isolated from untreated male rats. P-450 IIA 1-mediated androstenedione 7 alpha-hydroxylation appeared refractory to inhibition, with the lowest I50s being approximately 200 microM (cis- and and trans-tamoxifen, 4-hydroxytamoxifen). (According to the recently recommended nomenclature for cytochromes P-450 (Nebert DW and Gonzalez FJ, Ann Rev Biochem 56: 945-993, 1987), rat hepatic cytochromes P-450 UT-A, PB-B, PCN-E and UT-F are encoded by genes IIC 11, IIB 1, IIIA 1/2 and IIA 1, respectively. I50s toward the P-450 IIC 11-, IIB 1-, and IIIA 1/2-catalysed reactions, androstenedione 16 alpha-, 16 beta- and 6 beta-hydroxylations, respectively, were generally in the range 70-190 microM. However, metabolite E exhibited a rather specific and potent capacity to inhibit androstenedione 16 alpha-hydroxylase activity (I50 = 18 microM). Since a number of alkylamine compounds have been shown to sequester microsomal P-450 as an inactive metabolite intermediate (MI), the tamoxifen analogues were investigated for their in vitro MI complexation capacity. However, spectral binding studies revealed that the incubation of these compounds with NADPH-fortified microsomal fractions did not result in MI complex formation. In binding experiments conducted with oxidised microsomal fractions it was apparent that most of the tamoxifen analogues are type I ligands of quite high affinity for ferric P-450 (Ks range 10-60 microM). It seems unlikely that MI formation is involved in the observed inhibition of androstenedione hydroxylation by tamoxifen and congeners. Instead, and in contrast to the situation observed with SKF 525-A, it would appear that the inhibitory capacity of the tamoxifen analogues is more closely related to type I binding capacity with ferric P-450. A finding of particular interest is that metabolite E, in which the alkylamino side-chain is absent, elicited a type I interaction of high capacity. The maximal absorbance change of the type I interaction of this compound with microsomal P-450 was about three-fold greater than the other compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of propiconazole on hepatic xenobiotic biotransformation in the rat.

Propiconazole, a foliar fungicide used for agricultural purposes was studied for its effects on the hepatic xenobiotic biotransformation in the rat. Rats were given an intraperitoneal injection of 0.1, 1, 10 or 100 mg/kg in corn oil for seven consecutive days. Induction was seen for cytochrome P-450, ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, aldrin epoxidase, aminopyrine N-demethylase and microsomal expoxide hydrolase activities. Aniline p-hydroxylase and cytosolic glutathione S-transferase activities were unchanged. All responses occurred at only 100 mg/kg, except for that of aminopyrine N-demethylase which also occurred at the 10 mg/kg dose. SDS polyacrylamide gel electrophoresis showed increased staining of a protein band of molecular weight 54,000 corresponding to cytochrome P-450b and/or P-450d. Collectively these results suggest that cytochromes P-450b and P-450d have been induced after exposure of rats to propiconazole.

Induction of xenobiotic biotransformation by the insecticide chlordimeform, a metabolite 4-chloro-o-toluidine and a structurally related chemical o-toluidine.

Chlordimeform, 4-chloro-o-toluidine and o-toluidine have all been found to have carcinogenic properties. Due to an empirical link between such properties and alteration of some biotransformation enzymes, the abilities of these three chemicals to affect cytochrome P-450 mediated biotransformation, epoxide hydrolase and glutathione S-transferase have been examined. Chlordimeform had no effect on the cytochrome P-450 content, aniline p-hydroxylase or glutathione S-transferase activities, but induced ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase and epoxide hydrolase activities and decreased aldrin epoxidase and aminopyrine N-demethylase activities. The metabolite 4-chloro-o-toluidine increased cytochrome P-450, ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, glutathione S-transferase and epoxide hydrolase activities. o-Toluidine induced cytochrome P-450, ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, and aldrin epoxidase activities. Ethoxy-resorufin-O-deethylase activity was induced approximately eight times by chlordimeform and 18 times by 4-chloro-o-toluidine and o-toluidine. Induction was seen at 50 mg/kg with chlordimeform and at 10 mg/kg with the other treatments. Chlordimeform increased the 7 alpha and 16 alpha androstenedione hydroxylase pathways. 4-Chloro-o-toluidine increased the 7 alpha, 16 beta and 16 alpha hydroxylase pathways, while o-toluidine increased the 7 alpha, 6 beta, 16 beta and 16 alpha hydroxylase pathways. All three chemicals marginally decreased the testosterone pathways. SDS-PAGE of rat microsomes revealed an increase in a protein band of MW c54,000 for the chlordimeform and 4-chloro-o-toluidine treated groups. Taken together with the increase in ethoxyresorufin-O-deethylase activity these observations are consistent with the induction of hepatic isozyme P-450d. Thus each chemical has been shown to induce various pathways of biotransformation with increases in the P-450c and P-450d specific substrate ethoxyresorufin-O-deethylase being a consistent finding.

Identification of the hepatic cytochrome P-450 isozymes induced and decreased by picloram.

Microsomes from male rats treated with picloram (100 mg/kg/day) for 7 days showed a 48% decrease in 16 alpha-hydroxylase activity when incubated with (4-14C) androstenedione. These data are consistent with the assertion that picloram decreases the titer of hepatic male specific cytochrome P-450h. Several lines of evidence suggested that picloram is an inducer of hepatic cytochrome P-450 in male rats. First, SDS polyacrylamide gel electrophoresis revealed an intensified hepatic microsomal polypeptide (MW 54,000) following picloram pretreatment. This polypeptide co-migrated with protein bands which were correspondingly intensified after pretreatment with known inducers of cytochrome P-450d (3-methylcholanthrene and isosafrole). Second, no increase in the binding of metyrapone to picloram treated microsomes was noted compared with controls, suggesting no increase in phenobarbital-inducible forms of cytochrome P-450. Third, hepatic microsomes from picloram treated rats activated 2-amino-3-methylimidazo [4,5-f] quinoline (a cytochrome P-450d mediated catalysis) causing a 5-fold increase in the number of induced Salmonella typhimurium TA98 revertant colonies formed compared with control microsomes. Fourth, the binding of n-octylamine to hepatic microsomes from picloram-treated rats showed, like microsomes from 3-methylcholanthrene-treated rats, an increase in the proportion of high-spin cytochrome P-450 present. Cytochrome P-450d is known to be a high spin haemoprotein.

Effects of picloram on xenobiotic biotransformation in rat liver.

1. The effect of picloram on model xenobiotic substrate biotransformation in vivo was studied in female and male rat liver. 2. Treatment with picloram had little effect on epoxide hydratase and glutathione S-transferase activity, but caused a dose-dependent increase in ethoxyresorufin-O-deethylase activity and a concomitant decrease in aldrin epoxidase activity in male rats. 3. Treatment of male rats with equivalent doses of 2-acetylaminofluorene, 2-amino-anthracene and picloram induced ethoxyresorufin-O-deethylase activity to the same degree. 4. Treatment of female rats with picloram resulted in dose-dependent increases in ethoxyresorufin and ethoxycoumarin-O-deethylation without decreasing aldrin epoxidase activity. 5. Picloram binds to liver microsomal preparations from rats pretreated with phenobarbitone and/or 3-methylcholanthrene, giving a type I spectrum. 6. The results indicate that picloram is a 3-methylcholanthrene-type inducer, and the implications are discussed.

Effect of length of exposure to malathion on xenobiotic biotransformation in male rat liver.

The effect of exposure to malathion on several parameters of hepatic xenobiotic biotransformation was studied in male Sprague-Dawley rats. Groups of rats dosed i.p. daily for 1 or 2 weeks with 40 or 200 mg/kg malathion showed an increase in epoxide hydrolase activity (1 week, 200 mg/kg) and glutathione S-transferase activity (1 week, 200 mg/kg; 2 weeks 40 and 200 mg/kg). Aldrin epoxidation was decreased after 1 week of exposure to 200 mg/kg and by both dosage regimens after 2 weeks. After 9 weeks exposure to 40 mg/kg malathion administered i.p. 3 times per week, however, no changes in hepatic xenobiotic biotransformation were noted. The results demonstrate that only continuous exposure to high doses of malathion results in an induction of epoxide hydrolase and glutathione S-transferase activities. Inductive effects on hepatic cytochrome P-450 monooxygenase activity were not observed irrespective of whether exposure was short- or medium-term.