Human hepatic microsomal metabolism of delta 1-tetrahydrocannabinol.

Hepatic microsomal metabolism of delta 1-tetrahydrocannabinol (THC) has been extensively studied in many rodent species, but there have been few reports describing such metabolism in humans. Because several THC metabolites are known to be pharmacologically active, identifying the P-450 subfamilies responsible for their formation is of clinical importance. We have found that, in addition to catalyzing the formation of significant amounts of 7-hydroxy-THC, hepatic microsomes from nine human livers also formed 6 beta-hydroxy-THC at approximately the same rate. In addition, 1 alpha,2 alpha-epoxyhexahydrocannabinol (EHHC) was formed at approximately one-third the rate of 7-hydroxy- and 6 beta-hydroxy-THC, and small amounts of 6 alpha-hydroxy- and 6-keto-THC were also found. Immunoinhibition studies with antibodies raised against human hepatic P-450 2C9, or a mouse hepatic P-450 isozyme belonging to the P-450 3A subfamily, revealed that P-450 2C9 catalyzed the formation of 7-hydroxy-THC, whereas P-450 3A catalyzed the formation of 6 beta-hydroxy-THC, EHHC, and the relatively minor metabolites. In contrast, antibodies raised against human P-450 2C8 had no affect on human microsomal THC hydroxylation. Excellent correlations were found between hepatic microsomal P-450 2C9 and 3A content and 7-hydroxy- and 6 beta-hydroxy-THC formation, respectively. In addition, purified P-450 2C9 catalyzed the formation of 7-hydroxy-THC at a 7-fold higher rate than that observed with microsomes. Microsomal 7-hydroxy-THC formation varied less than 5-fold between the livers, suggesting that this activity is normally expressed and probably not subject to environmental influences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inactivation of mouse liver cytochrome P-450IIIA by cannabidiol

Cannabidiol (CBD) inhibits hepatic drug metabolism in mice, particularly those activities known to be catalyzed by the cytochrome P-450IIIA (P-450IIIA) subfamily. CBD treatment (120 mg/kg) inhibited more than 75% of hepatic 6 beta-testosterone hydroxylase and erythromycin N-demethylase activities (functional markers of P-450IIIA) after 2 hr. An isozyme of the P-450IIIA subfamily (Mr 49,960) was purified to apparent homogeneity from hepatic microsomes of untreated mice and was found to catalyze testosterone hydroxylation at the 2 beta-, 6 beta-, and 15 beta-positions exclusively. Incubation of this isozyme with CBD in a reconstituted system resulted in a time- and concentration-dependent inactivation, with almost complete loss of P-450 chromophore and corresponding increase in P-420 content. NH2-terminal sequence analysis of the isozyme revealed an 86% similarity to the corresponding sequence of rat P-450IIIA2, a constitutive P-450 isozyme in the male rat liver. Pretreatment of mice with dexamethasone markedly (6-fold) increased the steroid-inducible P-450IIIA-dependent activities 6 beta-testosterone hydroxylation and erythromycin N-demethylation. CBD treatment of dexamethasone-pretreated animals failed to inhibit these activities, indicating that the steroid-inducible P-450IIIA was refractory to CBD-mediated inactivation. 3-Methylcholanthrene-inducible P-450IA and phenobarbital-inducible P-450IIB also appear to be refractory to CBD-mediated inactivation. On the other hand, erythromycin N-demethylase activity increased 4-fold after phenobarbital pretreatment and, as in untreated animals, was comparably inhibited by CBD, demonstrating its susceptibility to this drug. Thus, CBD appears to inactivate the P-450IIIA isozymes that are constitutively present in hepatic microsomes of untreated mice and/or inducible by phenobarbital pretreatment but not those that are steroid inducible.     

Tolbutamide hydroxylation by human, rabbit and rat liver microsomes and by purified forms of cytochrome P-450

Tolbutamide hydroxylation has been investigated in human, rabbit and rat liver microsomes and by six purified forms of hepatic rabbit cytochromes P-450. These studies were carried out to investigate whether an appropriate animal model could be developed for the human cytochrome(s) P-450 metabolizing tolbutamide. Selective induction was used in rats and rabbits to indicate the isozymes primarily responsible for tolbutamide hydroxylation in these species. Microsomal tolbutamide hydroxylase activity was significantly induced only by phenobarbital pretreatment in the rat which induces P-450 forms b (P-450IIB1) and/or e (P-450IIB2). Only pretreatment of rabbits with rifampicin, which induces cytochrome P-450 form 3c (P-450IIIA6), significantly increased the microsomal hydroxylation of tolbutamide. However, the increase in tolbutamide hydroxylase activity in rifampicin-induced microsomes (congruent to 50%) appears low compared to known levels of induction of P-450IIIA6 following rifampicin pretreatment (5-10-fold). These data suggest that P-450IIIA6 is at least partially involved in tolbutamide hydroxylation in rabbit liver but that other form(s) may be relatively more important. Reconstitution experiments with six purified forms of rabbit cytochrome P-450 indicated that the highest activity occurred with P-450IIIA6 (form 3c). As isozymes from different gene families or subfamilies appeared to metabolize tolbutamide in the three species studied, catalytic similarities between the P-450s with respect to inhibition was further investigated in microsomes using sulfaphenazole, alpha-naphthoflavone and mephenytoin. These studies showed that the catalytic characteristics in relation to inhibition differ markedly between species. Hence, it appears that the animal model approach is not likely to be successful in the identification and characterization of the cytochrome P-450 form(s) metabolizing tolbutamide in humans.     

Purification and characterization of a mouse liver cytochrome P-450 induced by cannabidiol

A cytochrome P-450 isozyme (Mr = 51,600) was purified to apparent homogeneity from hepatic microsomes of mice pretreated with cannabidiol (CBD), a major constituent of marijuana. The isozyme exhibited high pentoxyresorufin O-dealkylase, hexobarbital hydroxylase, and 16 alpha- and 16 beta-testosterone hydroxylase activities and formed a Fe+2-metyrapone complex, properties characteristic of the major hepatic cytochrome P-450s previously purified from phenobarbital (PB)-pretreated animals. In addition, the CBD-induced cytochrome P-450 was immunoreactive with an antibody raised against the major rat hepatic PB-inducible cytochrome P-450 and exhibited an NH2-terminal amino acid sequence greater than 90% homologous with that of the PB-inducible rat liver isozyme. Because of the many similarities between the CBD-induced isozyme and certain other isozymes previously purified from PB-pretreated animals, a cytochrome P-450 isozyme was purified from PB-pretreated mice by a chromatographic procedure similar to that employed for purification of the CBD-induced isozyme. The PB-inducible isozyme was indistinguishable from the CBD-inducible cytochrome P-450 on the bases of apparent molecular weight, absorption spectra, NH2-terminal amino acid sequence, peptide mapping, immunoreactivity, and catalytic activity. Although the CBD- and PB-inducible P-450 isozymes appear to be qualitatively very similar, PB appears to be a quantitatively better inducer of the isozyme. Thus, CBD exposure results in the induction of an isozyme that is refractory to CBD-mediated inactivation, thereby apparently altering the cytochrome P-450 isozymal composition of mouse hepatic microsomes.     

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)     

Self-catalyzed inactivation of cytochrome P-450 during microsomal metabolism of cannabidiol

When cannabidiol (CBD) was incubated with hepatic microsomes of mice in the presence of an NADPH-generating system, a significant decrease of cytochrome P-450 content was observed by measuring its carbon monoxide difference spectra. The decrease of cytochrome P-450 by CBD required NADPH and molecular oxygen. The effect was partially inhibited by SKF 525-A but not by various scavengers of active oxygen species, superoxide anion, hydroxyl radical and singlet oxygen. The incubation of CBD with hepatic microsomes did not affect total heme but decreased significantly free sulfhydryl contents in the microsomes. The derivatives of CBD modified in the resorcinol moiety, CBD-monomethyl- and dimethylethers, almost lost the effect on cytochrome P-450, whereas those modified in the terpene moiety, 8,9-dihydro- and 1,2,8,9-tetrahydro-CBDs exhibited some potency to inactivate cytochrome P-450. The inactivation of cytochrome P-450 by CBD and related compounds led to the inhibition of hepatic microsomal p-nitroanisole O-demethylase and aniline hydroxylase activities. These results suggest that the resorcinol moiety of CBD plays some role in the inactivation of cytochrome P-450 by the cannabinoid.     

Mechanism for inhibitory effect of cannabidiol on microsomal testosterone oxidation in male rat liver

Effects of four cannabinoids [cannabidiol (CBD), delta 8-tetrahydrocannabinol, delta 9-tetrahydrocannabinol, and cannabinol] on hepatic microsomal oxidation of testosterone (17 beta-hydroxy-androst-4-ene-3-one) were examined in adult male rats. Only CBD (30 microM) competitively inhibited 2 alpha-hydroxy-testosterone (2 alpha-OH-T) and 16 alpha-OH-T formation by hepatic microsomes but did not affect androstenedione (androst-4-ene-3,17-dione) and 7 alpha-OH-T formation. Kinetic analyses demonstrated that the inhibitory profile of CBD for testosterone oxidation was different from those of SKF 525-A, which caused competitive inhibition for 2 alpha- and 16 alpha-hydroxylations and noncompetitive inhibition for 6 alpha-hydroxylation, and of metyrapone, which inhibited only 6 beta-hydroxylation competitively. CBD also suppressed formation of 2 alpha-OH-T, 16 alpha-OH-T, and androstenedione from testosterone, catalyzed by a reconstituted system containing hepatic cytochrome P-450 purified from phenobarbital-treated rats. Pretreatment of the rat with CBD (10 mg/kg, ip, once a day for 3 days) decreased testosterone oxidation at the 2 alpha-, 16 alpha-, and 17-positions and increased 7 alpha-OH-T formation, while total cytochrome P-450 content was decreased. These results suggest that CBD suppresses hepatic testosterone oxidation at the 2 alpha-, 16 alpha-, and 17-positions through selective inhibition of the male-specific cytochrome P-450 in the adult male rat.     

Effect of the suicide substrate 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine on the metabolism of xenobiotics and on cytochrome P-450 apoproteins

Treatment of rats with the cytochrome P-450 suicide substrate, 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), produced a 95% inhibition of the in vivo demethylation of either aminopyrine or morphine within 2 hr. One-carbon metabolism of formaldehyde or formate to carbon dioxide was not altered. DDEP also produced a time-dependent decrease in total hepatic microsomal cytochrome P-450 but had no effect on either NADPH-cytochrome c reductase or p-nitrophenol glucuronyl-transferase activities up to 24 hr after administration. A rapid decrease in rat liver microsomal aniline hydroxylation and ethoxyresorufin deethylation was observed in vitro following DDEP administration. Although in vitro testosterone metabolism to 16 alpha-, 16 beta-, and 2 alpha-hydroxy metabolites was depressed profoundly by DDEP in microsomes from untreated and 3-methylcholanthrene-treated animals, 7 alpha-hydroxylation of testosterone was much less affected. Immunochemical quantification of various microsomal cytochrome P-450 protein moieties showed that cytochromes P-450 beta NF-B, P-450UT-A, P-450PCN-E, and P-450PB-C were decreased in hepatic microsomes from DDEP-treated rats. However, the protein moiety of cytochrome P-450UT-H was not diminished and the immunoreactive protein for cytochromes P-450UT-F, P-450PB-B, and P-450ISF-G was only slightly decreased. These results show that DDEP treatment leads to marked decreases in holoprotein and apoproteins of many but not all hepatic microsomal cytochrome P-450 isozymes.     

Hypophysectomy differentially alters P-450 protein levels and enzyme activities in rat liver: pituitary control of hepatic NADPH cytochrome P-450 reductase

Pituitary-determined hormones regulate the expression of hepatic cytochromes P-450 through processes involving both negative and positive controls. Accordingly, protein levels of several P-450 forms are elevated in rat liver following hypophysectomy [P-450 forms designated 2a (gene IIIA2), RLM2 (gene IIA2), and PB-4 (gene IIB1)], whereas protein levels of others are suppressed [e.g., P-450 2c (gene IIC11)]. In the present study, microsomal steroid hydroxylase activities associated with these same P-450 forms were found to be decreased by hypophysectomy, despite elevations in protein levels for several of them. Studies were, therefore, undertaken to determine the biochemical basis for this decrease in microsomal P-450 enzyme specific activity. In vivo treatment of hypophysectomized rats with gonadotropin, under conditions that restore heme to testis P-450, and heme reconstitution experiments carried out with liver homogenates indicated that a deficiency in P-450-associated heme is unlikely to account for the observed decreases in liver P-450 enzyme specific activity. Analysis of the flavoprotein P-450 reductase, however, revealed that the reductase protein and its associated cytochrome c reductase activity are decreased by 50 to 75% in liver microsomes isolated from hypophysectomized rats. Moreover, supplementation of isolated liver microsomes with exogenous purified P-450 reductase stimulated microsomal steroid hydroxylase activity preferentially in the hypophysectomized rats, to levels consistent with the observed changes in P-450 protein levels. Thus, a deficiency in P-450 reductase, which is a rate-limiting component for many P-450-dependent hydroxylation reactions, appears to be responsible for the decrease in steroid hydroxylase specific activity in the hypophysectomized rats. Although growth hormone, adrenocorticotropic hormone, and chorionic gonadotropin were each ineffective at restoring hepatic P-450 reductase when administered to hypophysectomized rats, substantial restoration of P-450 reductase levels could be achieved by treatment of the hypophysectomized rats with thyroxine. Thyroxine treatment of these rats also elevated the microsomal steroid hydroxylase activities associated with the individual hepatic P-450 forms to levels commensurate with their respective P-450 protein levels. These results establish that hepatic P-450 reductase is subject to hormonal controls that are distinct from those governing cytochrome P-450 expression and further demonstrate the complexity of endocrine control of hepatic steroid hormone metabolism.     

Cytochrome P-450 isozymes involved in the oxidative metabolism of delta 9-tetrahydrocannabinol by liver microsomes of adult female rats.

Oxidative metabolism of delta 9-tetrahydrocannabinol (THC) by liver microsomes was studied in female rats. Delta 9-THC was mainly biotransformed to 11-hydroxy-delta 9-THC (11-OH-delta 9-THC) and 9 alpha,10 alpha-epoxy-hexahydrocannabinol (EHHC) by liver microsomal fraction of adult female rat. Two isozymes of cytochrome P-450 (P-450) [F-1 (IIC6) and F-2 (IIC12)] were purified from liver microsomes of female rats and oxidation activities toward delta 9-THC were assessed in the reconstituted system containing NADH-P-450 reductase and cytochrome b5. P-450 F-1 showed considerable activity toward 11-OH-delta 9-THC formation (10.62 nmol/min/nmol of P-450), whereas P-450 F-2 did not show any activity toward delta 9-THC oxidation under the conditions used. Preincubation of microsomes with antiserum against P-450 F-1 obtained from rabbits caused a marked decrease in 11-OH-delta 9-THC formation, whereas antiserum against P-450 F-2 did not exhibit any inhibitory effect on the oxidation of delta 9-THC by liver microsomes of adult female rats. Further, antiserum against P-450 F-1 or F-2 did not affect the microsomal formation of 9 alpha,10 alpha-EHHC from delta 9-THC. These results indicate that P-450 F-1 and its immunochemically related P-450 isozyme(s) play important roles in the formation of an active metabolite, 11-OH-delta 9-THC, from delta 9-THC by liver microsomes of adult female rats.     

The cytochrome P-450-dependent hydroxylation of T-2 toxin in various animal species

Gas-liquid chromatography was used to investigate the hepatic and intestinal metabolism of T-2 toxin, a cytotoxic and immunodepressive trichothecene produced by species of Fusarium. The hepatic S-9 and microsomal fractions of various species hydroxylated T-2 toxin to form 3'-hydroxy-T-2. HT-2 toxin, a deacetylated metabolite of T-2 toxin formed by reactions involving microsomal esterases, was also hydroxylated, to 3'-hydroxy HT-2 toxin. Experiments with inhibitors and inducers of the cytochrome P-450-dependent system revealed that these two hydroxylation reactions were catalysed by the cytochrome P-450-dependent monooxygenase system. Species comparisons using rats, mice, guinea-pigs, rabbits, pigs, cows and chickens showed that the rate of the hydroxylation reaction was highest in the hepatic microsomes of guinea-pigs, followed by mice. Chickens possessed a low activity both in the hydrolysis and hydroxylation reactions. No hydroxylated metabolites were produced by the intestinal microsomes of rabbits. These two hydroxylated metabolites were far less cytotoxic to Reuber hepatoma cells than the parent compound, T-2 toxin.     

Monoclonal antibodies to rat liver cytochrome P-450 2c/RLM5 that regiospecifically inhibit steroid metabolism

Hybridomas were formed from myeloma cells and spleen cells derived from BALB/c female mice immunized with purified liver microsomal cytochrome P-450 2c/RLM5 (P-450 gene IIC11) isolated from untreated adult male rats. Six hybridoma clones produced monoclonal antibodies (MAbs) of the IgM(kappa) type. All the MAbs bound strongly to P-450 2c/RLM5 when measured by radioimmunoassay, and four of the six specifically immunoprecipitated P-450 2c/RLM5 in an Ouchterlony double-immunodiffusion test. These four MAbs also bound but did not immunoprecipitate P-450 RLM3. The MAbs that precipitated P-450 2c/RLM5 neither bound nor precipitated P-450 PB-B (gene IIB1) and P-450 BNF-B (gene IA1) of rats or P-450 LM2 and P-450 LM4 of rabbits. In contrast, mouse polyclonal anti-P-450 2c/RLM5 antibody strongly immunoprecipitated P-450 RLM3 as well as P-450 2c/RLM5 and to a lesser extent P-450 PB-B and P-450 LM2. The MAbs that precipitated P-450 2c/RLM5 also inhibited by more than 90% androstenedione 16 alpha-hydroxylase activity of untreated rat microsomes, but did not inhibit microsomal 6 beta- or 7 alpha-hydroxylation. In addition, complete inhibition of both androstenedione 16 alpha-hydroxylation and testosterone 16 alpha-hydroxylation was observed in a reconstituted system with P-450 2c/RLM5. Androstenedione 6 beta-hydroxylation catalyzed by P-450 2c/RLM5 was also inhibited, whereas P-450 3-catalyzed 7 alpha-hydroxylation was not inhibited by the MAbs. P-450 2c/RLM5 catalyzed 2 alpha-, 16 alpha- and 6 beta-hydroxylation of progesterone in a reconstituted system were also inhibited by the MAb by 60-80%. These MAbs should prove useful for "reaction phenotyping," i.e. for defining the contribution of microsomal P-450 2c/RLM5 to the oxidative metabolism of endogenous steroids and other P-450 substrates in animal and human tissues.     

Stimulation of microsomal drug oxidation activities by incorporation into microsomes of purified NADPH-cytochrome c (P-450) reductase.

The effects of addition of purified NADPH-cytochrome c (P-450) reductase on microsomal activities of aniline hydroxylation, p-phenetidine O-deethylation and ethylmorphine and aminopyrine N-demethylations were investigated utilizing microsomes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated rats. The purified reductase was incorporated into microsomes. The drug oxidation activities were increased by the fortification of microsomes with the reductase while the extent of increase in the activities varied with the substrate and microsomes employed. The most pronounced enhancement was seen in p-phenetidine O-deethylation, followed by aniline hydroxylation and aminopyrine and ethylmorphine N-demethylations. The enhancement was more remarkable in microsomes from rats treated with 3-methylcholanthrene or phenobarbital. alpha-Naphthoflavone inhibited p-phenetidine O-deethylation activity markedly when the reductase was incorporated into microsomes, indicating that a larger amount of a species of cytochrome P-450 sensitive to the inhibitor was capable of participating in the oxidation of this substrate in the presence of the added reductase. One of the two Km values seen with higher concentrations of aniline or aminopyrine was altered by the fortification of microsomes with the purified NADPH cytochrome c (P-450) reductase. From these results, we propose that NADPH-cytochrome c (P-450) reductase transfers electrons to the selected one or two of multiple species of cytochrome P-450 more preferentially depending upon the substrate and the concentration of the substrate in microsomal membranes.     

Metabolite intermediate complexation of microsomal cytochrome P450 2C11 in male rat liver by nortriptyline.

Antidepressant drugs that contain alkylaminoalkyl substituents have been associated with serious pharmacokinetic interactions in humans that may be related to the inhibition of cytochrome P450 (P450) enzymes. In this study, the propensity of the tricyclic antidepressant nortriptyline (NOR) to inhibit individual microsomal P450 enzymes in rat liver was investigated to provide a mechanistic explanation for these pharmacokinetic interactions. Enzyme kinetic studies revealed that NOR inhibited steroid 2 alpha-, 6 beta, 7 alpha-, and 16 alpha-hydroxylation in untreated rat liver with Km/Ki ratios of 0.53, 0.59, 0.25, and 0.29, respectively. When the drug was preincubated with microsomes and NADPH before testosterone hydroxylation was conducted, marked increases in the Km/Ki ratios were observed (to 8.8, 3.9, 0.62, and 13, respectively). Thus, enzymic oxidation of NOR enhanced its inhibition capacity against P450 activities. Indeed, the altered Km/Ki ratios indicate 17-, 6.6-, 2.5-, and 47-fold increases in inhibition of the four pathways of testosterone hydroxylation after the biotransformation of NOR to its metabolites. From these experiments it was apparent that testosterone 2 alpha- and 16 alpha-hydroxylations, catalyzed predominantly by P450 2C11, were subject to the most pronounced increase in inhibition. Under these conditions, the apparent content of microsomal P450 was decreased, thus suggesting the formation of a NOR metabolite intermediate (MI) complex with the cytochrome. Further, optical difference spectroscopy of NADPH-supported metabolism of NOR in microsomes and in a reconstituted system incorporating purified P450 2C11 indicated the appearance of an absorbance peak near 454 nm, similar to those produced by triacetyloleandomycin, SKF 525-A, and orphenadrine. Formation of this absorbance peak in microsomes was inhibited by an antibody raised against the male-specific P450 2C11. Because oxidative metabolism of NOR to inhibitory products would not necessarily involve MI complexation, additional experiments were undertaken in which NOR-related free metabolites produced in microsomal incubations were removed on Sep-Pak mini-C18 columns before estimation of testosterone hydroxylation. The principal finding from this experiment was that P450 3A2-dependent steroid 6 beta-hydroxylase activity was inhibited to a much lesser extent after removal of unbound NOR metabolites on Sep-Pak columns (25% inhibition after Sep-Pak extraction, compared with 82% inhibition observed when all NOR metabolites were present during subsequent testosterone hydroxylation); inhibition of P450 2C11-mediated 2 alpha- and 16 alpha-hydroxylation was not noticeably different after Sep-Pak treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of cytochrome P-450IIB1 as a cocaine-bioactivating isoform in rat hepatic microsomes and in cultured rat hepatocytes.

Induction of cytochrome P-450-dependent monooxygenases with phenobarbital (PB) or other hepatic drug-metabolizing enzyme inducers in the rat is associated with enhanced cocaine hepatotoxicity both in vivo and in cultured rat hepatocytes. To demonstrate whether the major PB-inducible P-450 subfamily (P-450IIB) could be involved in the metabolic activation of cocaine, rates of cocaine N-demethylation (the first step of cocaine bioactivation) and the rate of irreversible (covalent) binding of tritiated cocaine to hepatic microsomal proteins (a measure for the overall bioactivation) were determined in microsomes from saline or PB-pretreated rats. PB pretreatment augmented Vmax (6-fold), but not KM, of cocaine N-demethylation. Similarly, the rate of irreversible protein binding was 3-fold increased in microsomes from PB-pretreated rats as compared with those from saline controls. Addition of benzphetamine, a substrate of P-450IIB, markedly inhibited cocaine irreversible binding. In addition, various concentrations of cocaine inhibited microsomal pentoxyresorufin O-depentylase activity in a competitive-type pattern. A polyclonal antibody raised against purified rat P-450IIB1 markedly inhibited cocaine N-demethylation as compared with control incubations with preimmune IgG. Finally, pretreatment of rats with PB potentiated cocaine-induced cytotoxicity in primary, short-term cultured hepatocytes, assessed as lactate dehydrogenase release into the culture medium. This enhancing effect of PB became even more evident in glutathione-depleted cells. These results suggest that cocaine is metabolized and bioactivated by P-450IIB1 in the rat liver, and that induction of this isoform with various agents may be associated with enhanced lethal hepatocyte injury in the rat.     

Monoclonal antibodies inhibitory to rat hepatic cytochromes P-450: P-450 form specificities and use as probes for cytochrome P-450-dependent steroid hydroxylations

Cytochrome P-450 (P-450) form specificities were established for a total of nine monoclonal antibodies (MAbs) raised to four distinct rat hepatic P-450 enzymes (P-450s 2c, PB-2a, PB-4, and BNF-B), using a combination of enzyme-linked immunosorbent analysis, dot immunoblotting, Western blotting, Ouchterlony immunodiffusion, and immunoinhibition analyses. Four of the MAbs were fully (greater than or equal to 85%) inhibitory toward the corresponding immunoreactive P-450s when assayed in purified, reconstituted enzyme systems, while two of the MAbs were partially inhibitory, with a maximum of 50 or 80% inhibition achieved in the presence of saturating MAb. Inhibitory MAbs reactive with P-450s 2c, 3, and PB-4, respectively, were used to demonstrate that the formation of multiple hydroxytestosterone metabolites by each of the respective purified P-450 enzymes is reflective of their inherent catalytic specificities and not due to the presence of immunochemical distinguishable P-450 enzyme contaminants. P-450 form-specific contributions to rat hepatic microsomal steroid hormone hydroxylase activities were then assessed using the inhibitory MAbs as probes. MAb-reactive P-450 2c was shown to be the major (greater than or equal to 85%) catalyst of microsomal testosterone and androstenedione 16 alpha-hydroxylation in both untreated and beta-naphthoflavone-induced rats. However, this P-450 form catalyzed only approximately 30% of hepatic microsomal steroid 16 alpha-hydroxylase activity in phenobarbital-induced adult males, and less than or equal to 10% of steroid 16 alpha-hydroxylase activity in (phenobarbital-induced immature males or adult females, where the balance of 16 alpha-hydroxylase activity is catalyzed by MAb-reactive P-450 PB-4. Although MAb-reactive P-450 PB-4 catalyzed the majority (greater than or equal to 90%) of microsomal androstenedione 16 beta-hydroxylation in phenobarbital-induced rats, this P-450 enzyme did not contribute to the low level 16 beta-hydroxylase activity of uninduced liver samples. Finally, MAb-reactive P-450 3 catalyzed at least 85% of microsomal androstenedione 7 alpha-hydroxylation, independent of the age, sex, or induction status of the animals used as source of liver microsomes. These findings demonstrate the usefulness of MAbs as probes for the contributions of individual P-450 enzymes to the metabolism of steroid hormones susceptible to hydroxylation at multiple sites.     

Role of P-450c in the formation of a reactive intermediate of chlorotrianisene (TACE) by hepatic microsomes from methylcholanthrene-treated rats

A previous study has shown that chlorotrianisene is metabolized by hepatic microsomal cytochrome P-450 monooxygenase(s) to a reactive intermediate that binds covalently to microsomal proteins [Juedes, Bulger, and Kupfer: Drug Metab. Dispos. 15, 786 (1987)]. Covalent binding of chlorotrianisene in hepatic microsomes is dramatically stimulated by treatment of rats with methylcholanthrene (MC), which is known to induce two major P-450 isozymes, P-450c (IA1) and P-450d (IA2). To determine whether P-450c and/or P-450d are involved in catalysis of covalent binding of chlorotrianisene, antibodies to P-450c and P-450d were used. Incubations of chlorotrianisene were conducted with liver microsomes from MC-treated rats (MC microsomes) and a monoclonal antibody (mAb) raised to the major MC-induced isozyme P450c, mAb 1-7-1, or a polyclonal monospecific antibody (pAb) to P-450d, pAb anti-d (-c). At a 5:1 ratio of antibody to microsomal protein, mAb 1-7-1 inhibited covalent binding by 67%, whereas pAb anti d (-c) showed a 10% inhibition. Maximal inhibition by mAb 1-7-1 was 89% at a 100:1 ratio of antibody to microsomal protein. From these findings it was concluded that P-450c is the major isozyme responsible for the metabolism of chlorotrianisene to the covalently binding reactive intermediate in MC microsomes. Additionally, it was observed that potentiation of covalent binding occurred with the noninhibitory mAbs used in these incubations. Substituting bovine serum albumin (BSA) for antibodies showed that this increase in binding is probably due to an increase in acceptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the rabbit and human cytochromes P-450IIIA as the major enzymes involved in the N-demethylation of diltiazem

Oxidative metabolism of diltiazem (DTZ), a calcium channel blocker, was investigated in rabbit and human liver microsomes as well as in primary cultures of human hepatocytes. DTZ N-demethylation, the major metabolic pathway in man, was strongly increased by treatment of animals, patients, and hepatocyte cultures with rifampicin and other inducers of the P-450IIIA subfamily. In a reconstituted system with purified forms of P-450 and NADPH cytochrome P-450 reductase, P-450IIIA7 exhibited the highest DTZ N-demethylase activity. In both rabbit and human liver microsomes, this activity was highly correlated with erythromycin demethylase, a characteristic substrate of P-450IIIA, or with an immunoquantitated level of P-450IIIA, and was specifically inhibited by anti-P-450IIIA7 polyclonal and monoclonal antibodies. Cyclosporin A, another specific substrate of P-450IIIA in rabbit and human, competitively inhibited DTZ N-demethylase in both species. In primary cultures of human hepatocytes treated with various inducers, including rifampicin, dexamethasone, phenobarbital, phenylbutazone or beta-naphthoflavone, the rate of release of N-demethyl-DTZ in the extracellular medium was highly correlated with the intracellular level of P-450IIIA, which appeared to be strongly induced by rifampicin and phenobarbital and to a lesser extent by dexamethasone and phenylbutazone. In aggregate, these results are consistent with the view that in both rabbit and human, cytochromes P-450 from the P-450IIIA subfamily are the major enzymes involved in the N-demethylation of DTZ. Accordingly, drugs which may be specific substrates or inducers of this P-450 are likely to influence both the side effects and the efficacy of this molecule.     

Polymorphism in stereoselective hydroxylations of mephenytoin and hexobarbital by Japanese liver samples in relation to cytochrome P-450 human-2 (IIC9).

1. Stereoselective 4'-hydroxylations of R-(--)-mephenytoin and S-(+)-mephenytoin were determined in liver microsomes of 19 Japanese subjects. 2. The content of P-450 human-2 assessed by Western-blots correlated with microsomal S-(+)-mephenytoin 4'-hydroxylation. Antibody raised against P-450 human-2 effectively inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation, but was less efficient for inhibition of R-(--)-mephenytoin 4'-hydroxylation in extensive metabolizers, and 4'-hydroxylation of both mephenytoin enantiomers in poor metabolizers. 3. Similar results were observed on the stereoselective hydroxylations of R-(--)- and S-(+)-hexobarbital. Clear correlations were observed for the content of P-450 human-2 and microsomal R-(--)-hexobarbital 3'alpha-hydroxylation and S-(+)-hexobarbital 3'beta-hydroxylation. 4. Moreover, yeast microsomes expressing P-450 human-2 cDNA showed high stereoselectivities for hydroxylations of mephenytoin and hexobarbital similar to those observed in human liver. 5. Two other cytochromes P-450(IIC 9/10) expressed in yeast, whose cDNA were synthesized by site-directed mutagenesis from human-2 cDNA, showed no stereoselectivity for the hydroxylations of mephenytoin and hexobarbital, in spite of the modification of only two amino acid substitutions or deletions in the whole sequence. 6. Only a cytochrome derived from P-450 human cDNA corresponding to P-450 human-2 was expressed in human livers, the two cytochromes of the three related IIC9/10 forms were not expressed. 7. These findings indicate that P-450 human-2 is the major cytochrome P-450 responsible for the polymorphisms in stereoselective hydroxylations of mephenytoin and hexobarbital.     

Immunochemical study on the contribution of hypolipidaemic-induced cytochrome P-452 to the metabolism of lauric acid and arachidonic acid

The influence of four hypolipidaemic drugs (clofibrate, WY-14,643, clobuzarit and bezafibrate) on hepatic cytochrome P-450 and fatty acid metabolism in male rat liver microsomes has been investigated. All of the hypolipidaemic drugs tested significantly induced the hydroxylation of lauric acid and, furthermore, this was accompanied by a concomitant 3-fold induction of a specific isoenzyme of cytochrome P-450 (termed cytochrome P-452) as determined by a single radial immunodiffusion technique. In addition, immunochemical quantitation of cytochrome P-452 in control, uninduced rat liver microsomes revealed that this particular isoenzyme constituted 22% of the total carbon monoxide-discernible cytochrome P-450 population. This has led us to the conclusion that cytochrome P-452 is a constitutive cytochrome P-450 isoenzyme and therefore that hypolipidaemic agents function as inducers of constitutive haemoprotein isoenzymes. Cytochrome P-452 plays a significant role in the hydroxylation of lauric acid as evidenced by inhibition of hydroxylase activity in the presence of an anti-P-452 IgG fraction. In addition, this antibody preferentially inhibits the 12-hydroxylation of lauric acid in rat liver microsomes by comparison to the 11-hydroxylase activity. Our studies have also shown that arachidonic acid serves as an excellent substrate for hypolipidaemic-induced cytochrome P-452, resulting in the formation of several metabolites that have been separated by reverse phase HPLC. Furthermore, a specific metabolite (or group of metabolites) of arachidonic acid is induced by clofibrate pretreatment and that the formation of this metabolite(s) is inhibited by an antibody to cytochrome P-452. By comparison, other metabolites of arachidonic acid remain refractory to induction by clofibrate and are not inhibited by the presence of anti-P-452 IgG. In addition, a reconstituted enzyme system containing highly purified cytochrome P-452 actively catalyses the above specific oxidation of arachidonic acid, a reaction that is significantly stimulated by the presence of cytochrome b5. Taken collectively, our data provide compelling evidence that hypolipidaemic agents induce a specific isoenzyme of hepatic microsomal P-450 that readily oxidizes fatty acids and that arachidonic acid may serve as an excellent endogenous substrate for this novel haemoprotein.