Cytochrome P-450 isozymes in metabolic activation of delta 9-tetrahydrocannabinol by rat liver microsomes

delta 9-Tetrahydrocannabinol (THC) was incubated with a reconstituted system consisting of dilauroylphosphatidylcholine, NADPH-cytochrome c reductase, cytochrome b5, and cytochrome P-450 (P-450) isozyme UT-2, UT-4, or UT-5, which was purified from liver microsomes of adult male rats. It was biotransformed by UT-2 to 11-OH-delta 9-THC and 3'-OH-delta 9-THC, and by UT-4 to 8 beta-OH-delta 9-THC and 11-OH-delta 9-THC. UT-5, however, showed only a little activity for 11-OH-delta 9-THC formation. Activity of the isozyme UT-2 for 11-OH-delta 9-THC formation from delta 9-THC was calculated to be 4.07 nmol/min/nmol P-450 while those of UT-2 for the formations of 16 alpha-OH-testosterone (16 alpha-OH-T), 2 alpha-OH-T, and androstenedione from testosterone were 14.7, 6.6, and 2.2 nmol/min/nmol P-450, respectively. Anti-P-450 UT-2 IgG fraction obtained from rabbit serum dose-dependently suppressed formations of 16 alpha-OH-T, 2 alpha-OH-T, and androstenedione from testosterone with liver microsomes of adult male rats. The antibody, in the amount that inhibited above 90% of 16 alpha-OH-T and 2 alpha-OH-T formations from testosterone, also reduced 80% of the microsomal formations of 11-OH-delta 9-THC and 3'-OH-delta 9-THC from delta 9-THC, as compared with control experiments using preimmune IgG fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex difference in the oxidative metabolism of delta 9-tetrahydrocannabinol in the rat.

Oxidative metabolism of delta 9-tetrahydrocannabinol (THC), one of the major components of marihuana, was studied using liver microsomes of adult male and female rats. There was no significant difference in the rates of the cannabinoid oxidation in terms of nmol per min per nmol of liver microsomal cytochrome P450 or of nmol per min per mg of microsomal protein between male and female rats. delta 9-THC was biotransformed to various metabolites including 11-hydroxy-delta 9-THC (11-OH-delta 9-THC), 8 alpha-OH-delta 9-THC, 8 alpha,11-diOH-delta 9-THC, 3'-OH-delta 9-THC by liver microsomes of male rats, while it was oxidized selectively to 11-OH-delta 9-THC by liver microsomes of female rats. After intraperitoneal administration of delta 9-THC, various metabolites were again found in the liver of the male rat, while in the female rat oxidation of the methyl group at the 9-position was a major metabolic pathway. These results demonstrate that an apparent sex-related difference exists in the oxidative metabolism of delta 9-THC in the rat.     

Catalytic activity of cytochrome P450 isozymes purified from rat liver in converting 11-oxo-delta 8-tetrahydrocannabinol to delta 8-tetrahydrocannabinol-11-oic acid.

Cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyse the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid in the presence of NADPH, cytochrome P450 reductase and dilauroylphosphatidylcholine. The catalytic activities (nmol/min/nmol P450) of cytochrome P450s, UT-2 (IIC11), UT-4 (IIA2), UT-5 (IIC13), PB-1, PB-2 (IIC6), PB-4 (IIB1), MC-1 (IA2), MC-5 (IA1) and IF-3 (IIA1), were 0.69, 0.08, 0.07, 0.23, 0.46, 0.02, 0.06, 0.07 and 0.34, respectively, whereas the activities of cytochrome P450s, PB-5 (IIB2) and DM (IIE1), were less than 0.02 nmol/min/nmol P450. Cytochrome P450 IIC11 showed the highest catalytic activity of the cytochromes examined. The mechanism for the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid by cytochrome P450 IIC11 was established as being an oxygenation since one atom of oxygen-18 was exclusively incorporated into the carboxylic acid formed under 18O2. The antibody raised to cytochrome P450 IIC11 inhibited by 60% the hepatic microsomal oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in male rats. These results indicate that cytochrome P450 IIC11 is a major form of the cytochrome to catalyse the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in the hepatic microsomes of male rats and that the oxidation of aldehyde to carboxylic acid is a catalytic activity common to most isozymes of P450.     

Effect of repeated administration of 11-hydroxy-delta 8-tetrahydrocannabinol, an active metabolite of delta 8-tetrahydrocannabinol, on the hepatic microsomal drug-metabolizing enzyme system of mice

The effects of delta 8-tetrahydrocannabinol (delta 8-THC) and its major and active metabolite, 11-hydroxy-delta 8-tetrahydrocannabinol (11-OH-delta 8-THC), on the hepatic microsomal drug-metabolizing enzyme system were studied in mice. The repeated administration of 11-OH-delta 8-THC (5 mg/kg/day, i.v.) for 3 or 7 days increased significantly the activities of aniline hydroxylase and p-nitroanisole O-demethylase. By the same treatment, cytochrome P-450 content (3 days) or NADPH-cytochrome c reductase activity (7 days) was also increased significantly. The treatment with delta 8-THC for 7 days (5 mg/kg/day, i.v.) significantly increased aniline hydroxylase only. 11-OH-delta 8-THC increased the Vmax, but not the Km, values for both drug-metabolizing enzymes, whereas delta 8-THC decreases significantly the Km value (270 microM) for p-nitroanisole O-demethylase as compared with the control (398 microM). Repeated administration of these cannabinoids for 7 days also increased the metabolism of delta 8-THC by hepatic microsomes; this was attributed to an enhanced formation of 11-OH-delta 8-THC. In contrast, microsomal formation of 7 alpha-OH-delta 8-THC was decreased significantly by treatment with delta 8-THC. 11-OH-delta 8-THC, but not delta 8-THC, treatment increased the metabolism of 11-OH-delta 8-THC by hepatic microsomes. These findings indicate that delta 8-THC and 11-OH-delta 8-THC treatment can induce hepatic microsomal drug-metabolizing enzymes and affect differently the catalytic properties of the enzymes.     

Immunochemical characterization of a cytochrome P450 isozyme and a protein purified from liver microsomes of male guinea pigs and their roles in the oxidative metabolism of delta 9-tetrahydrocannabinol by guinea pig liver microsomes.

A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.     

Identification and determination of 8 alpha, 9 alpha-epoxyhexahydrocannabinol as a metabolite of delta 8-tetrahydrocannabinol of mice in vitro

1. 8 alpha, 9 alpha-Epoxyhexahydrocannabinol (8 alpha, 9 alpha-EHHC) was formed from delta 8-tetrahydrocannabinol (delta 8-THC) by mouse liver microsomal preparation in the presence of an NADPH-generating system. 2. The epoxide was identified by t.l.c., g.l.c. and g.l.c.-mass spectrometry and, together with 11-hydroxy-delta 8-THC (11-OH-delta 8-THC), was determined by g.l.c. 3. When delta 8-THC was incubated with mouse liver microsomal preparation, 8 alpha, 9 alpha-EHHC and 11-OH-delta 8-THC was formed to the extents of 14% and 23%, respectively, of the added delta 8-THC.     

Enzymatic oxidation of 7-hydroxylated delta 8-tetrahydrocannabinol to 7-oxo-delta 8-tetrahydrocannabinol by hepatic microsomes of the guinea pig

Hepatic microsomes of the guinea pig converted delta 8-tetrahydrocannabinol (delta 8-THC) to various oxidized metabolites, including 7 alpha-hydroxy-delta 8-THC (7 alpha-OH-delta 8-THC), 7 beta-OH-delta 8-THC, and 7-oxo-delta 8-THC. The enzyme which mediates biotransformation of 7-OH-delta 8-THCs to 7-oxo-delta 8-THC was characterized in the present study. The oxidative activity was mainly located in microsomes. The microsomal reaction required NADPH and oxygen and showed an optimal pH around 7.5. The reaction was inhibited by beta-diethylaminoethyl diphenylpropylacetate (SKF 525-A), an inhibitor of cytochrome P-450, but not by pyrazole, a specific inhibitor of alcohol dehydrogenase. However, 7-oxo-delta 8-THC formation was not affected by carbon monoxide or by pretreatment of animals with cobaltous chloride (40 mg/kg, ip, once a day for 3 days). Atmospheric oxygen was incorporated into 7-oxo-delta 8-THC formed from 7 alpha-OH-delta 8-THC, but not into that from 7 beta-OH-delta 8-THC. Further, 7-oxo-delta 8-THC formed from 7 alpha-18OH-delta 8-THC released about half of 18O at the 7-position, whereas the 7-oxo metabolite from 7 beta-18OH-delta 8-THC lost little of the isotope at the 7 beta-position during the oxidative reaction. From these results, it is likely that hepatic microsomal monooxygenase (probably cytochrome P-450) plays a main role in the oxidation. In addition, mechanisms for 7-oxo-delta 8-THC formation from 7 alpha-OH-delta 8-THC or 7 beta-OH-delta 8-THC are different.     

Mouse hepatic microsomal enzyme that catalyzes oxidation of 11-oxo-delta 8-tetrahydrocannabinol to delta 8-tetrahydrocannabinol-11-oic acid.

Mouse hepatic microsomes oxidized 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC, aldehyde) to delta 8-THC-11-oic-acid (carboxylic acid). The reaction required NADPH and molecular oxygen and showed an optimal pH around 7.5. The activity of NADPH-dependent carboxylic acid formation was mainly localized in microsomes. The reaction was inhibited by various inhibitors of cytochrome P-450-dependent oxidation such as SKF 525-A, alpha-naphthoflavone, and metyrapone. Disulfiram and menadione also inhibited the microsomal oxidation of the aldehyde to the carboxylic acid, but pyrazole did not inhibit the reaction. The pretreatment of mice with phenobarbital significantly increased the oxidation activity on the basis of microsomal protein, but did not affect it on the basis of cytochrome P-450 content. The mechanism for the oxidation of the aldehyde to the carboxylic acid was confirmed to be oxygenation, since oxygen-18 was incorporated into delta 8-THC-11-oic acid from molecular oxygen during the hepatic microsomal oxidation of 11-oxo-delta 8-THC.     

Oxidation of 1,1,1,2-tetrafluoroethane in rat liver microsomes is catalyzed primarily by cytochrome P-450IIE1.

1,1,1,2-Tetrafluoroethane (R-134a), a nonozone-depleting alternative air-conditioning refrigerant and propellant for pharmaceutical preparations, is oxidatively defluorinated by rat hepatic microsomes. In this report we show that induction of cytochrome P-450IIE1 in rats, by pyridine administration, resulted in an 8-fold increase in the rate of R-134a metabolism by hepatic microsomes (Vmax 47 vs. 6 nmol F-/mg microsomal protein/15 min). Furthermore, when data were normalized for P-450 content, a 4-fold increase in R-134a metabolism was noted for IIE1-enriched microsome preparations. In contrast, phenobarbital and Aroclor 1254 decreased the specific activity of hepatic microsomes for this function. The microsomal content of P-450IIE1, as evaluated by Western blot, was elevated significantly only in microsomes from pyridine-treated rats. p-Nitrophenol and aniline, which are metabolized at high rates by rat P-450IIE1, decreased the rate of R-134a defluorination by hepatic microsomes; Dixon plot analysis indicated competitive inhibition with a Ki of 36 microM p-nitrophenol or 115 microM aniline. Pyridine also potently induced defluorination of R-134a catalyzed by rabbit liver microsomes. Studies with individual P-450 isozymes purified from rabbit liver showed that the phenobarbital- and polycyclic hydrocarbon-induced isozymes (IIB1 and IA2) defluorinated R-134a at negligible rates (1.9 and 0.4 nmol F-/nmol P-450/60 min, respectively). In contrast, P-450IIE1 catalyzed defluorination of R-134a at a relatively high rate (16.2 nmol F-/nmol P-450/60 min); isozyme IA1, which also is induced by nitrogen-containing heterocycles such as pyridine, was somewhat active (5.3 nmol F-/nmol P-450/60 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Monkey hepatic microsomal alcohol oxygenase: purification and characterization of a cytochrome P450 enzyme catalyzing the stereoselective oxidation of 7alpha- and 7beta-hydroxy-delta8-tetrahydrocannabinol to 7-oxo-delta8-tetrahydrocannabinol

The formation of 7-oxo-delta8-tetrahydrocannabinol (7-Oxo-delta8-THC) from 7alpha- or 7beta-hydroxy-delta8-THC (7alpha- or 7beta-OH-delta8-THC) was found in hepatic microsomes of monkeys. The activity in 7beta-OH-delta8-THC was stereoselectively 2.5- to 4.6-fold higher than that from 7alpha-OH-delta8-THC. The oxidative activities of 7alpha- and 7beta-OH-delta8-THC to 7-Oxo-delta8-THC were inhibited to 35% and 10%, respectively, of the control value by the antibody against P450GPF-B (CYP3A), a major enzyme responsible for the formation of 7-Oxo-delta8-THC in guinea pigs. In the Lineweaver-Burk double-reciprocal plot analysis, testosterone 6beta-hydroxylase activity was competitively inhibited by 7beta-OH-delta8-THC. Two cytochrome P450 enzymes, called P450JM-D and P450JM-E, were purified from hepatic microsomes of Japanese monkeys. P450JM-E, assumed to be CYP3A8, immunologically reacted with the antibody against P450GPF-B and showed high forming activity of 7-Oxo-delta8-THC from 7-OH-delta8-THC. On the other hand, 7-Oxo-delta8-THC forming activity of P450JM-D, assumed to be CYP2C, was less than 10% of that of P450JM-E (CYP3A8). Oxygen-18 (18O) derived from atmospheric oxygen was incorporated into about 40% of the corresponding ketone formed from 7alpha-OH-delta8-THC or 8beta-OH-delta9-THC by P450JM-E (CYP3A8), although the incorporation of the stable isotope into the oxidized metabolite from 7beta-OH-delta8-THC or 8alpha-OH-delta9-THC was negligible. These results indicate that P450JM-E (CYP3A8) is a major enzyme of the oxidation of 7-OH-delta8-THC in monkey hepatic microsomes. The oxidation mechanism may proceed as follows: the alpha- and beta-epimers of 7-OH-delta8-THC or 8-OH-delta9-THC may be converted to ketone through dehydration of an enzyme-bound gem-diol by P450JM-E (CYP3A8), although this stereoselective dehydration differentiates between two epimers.     

Purification and characterization of the major hepatic cannabinoid hydroxylase in the mouse: a possible member of the cytochrome P-450IIC subfamily

Acute cannabidiol treatment of mice inactivated hepatic microsomal cytochrome P-450IIIA (P-450IIIA) and markedly inhibited in vitro cannabinoid metabolism. Antibodies raised against purified P-450IIIA inhibited the microsomal formation of quantitatively minor cannabinoid metabolites but had no effect on the major metabolites. Cannabinoid hydroxylation to the major metabolites was used as a functional probe to isolate and purify a P-450 (termed P-450THC) from hepatic microsomes of untreated mice. The purified protein had an apparent molecular weight of 47,000 and a specific content of 15.4 nmol/mg and exhibited an absorbance maximum at 452 nm for the reduced carbon monoxide complex. NH2-terminal sequence analysis of the first 16 residues of P-450THC suggests that it is a member of the P-450IIC subfamily, because its sequence is 85 and 69% identical to published sequences of rat hepatic P-450IIC7 and P-450IIC6, respectively. P-450THC exhibited high activity for cannabinoid hydroxylation and specifically produced 6 alpha- and 7-hydroxy-delta 1-tetrahydrocannabinol, as well as 6 alpha-, 7-, and 4"-hydroxycannabidiol. Unlike anti-P-450IIIA antibody, antibody raised against purified P-450THC markedly inhibited the microsomal formation of all major cannabinoid metabolites. Similar immunoinhibition studies also revealed the existence of orthologs of mouse P-450THC and P-450IIIA in human liver microsomes. Thus, cannabidiol treatment of mice resulted in the inactivation of at least two constitutive P-450 isozymes, which together account for the majority of the detected cannabinoid metabolites.     

Metabolic disposition of delta 8-tetrahydrocannabinol and its active metabolites, 11-hydroxy-delta 8-tetrahydrocannabinol and 11-oxo-delta 8-tetrahydrocannabinol, in mice

Metabolic disposition of delta 8-tetrahydrocannabinol (delta 8-THC), 11-hydroxy-delta 8-THC (11-OH-delta 8-THC), and 11-oxo-delta 8-THC was studied in mouse blood, liver, and brain. After administration of these cannabinoids at a dose of 10 mg/kg iv, the concentration in blood declined biphasically. The biological half-lives of the slower phases were 32, 12, and 6 min, respectively, for delta 8-THC, 11-OH-delta 8-THC, and 11-oxo-delta 8-THC. 11-OH- and 11-oxo-delta 8-THC were also eliminated faster from brain than is delta 8-THC. The peak levels of 11-OH- and 11-oxo-delta 8-THC in brain were, however, higher (10.64 and 4.25 microgram/g, respectively) than that of delta 8-THC (3.48 microgram/g) at 0.5 min after the iv injection (10 mg/kg). These results indicate that 11-OH- and 11-oxo-delta 8-THC are distributed more readily from blood to brain in mice than is delta 8-THC, and explain the greater pharmacological activity of these metabolites, as reported previously. It was also interesting to note that a much higher level of 11-OH-delta 8-THC (3.27 microgram/g) was found in brain than in liver(0.74 microgram/g) and blood (0.29 microgram/ml) at 15 min after the injection of 11-oxo-delta 8-THC (10 microgram/kg, iv). In this case the levels of 11-OH-delta 8-THC were always higher than those of 11-oxo-delta 8-THC. The results suggest that 11-OH-delta 8-THC may play an important role in the pharmacological effects of 11-oxo-delta 8-THC. In additional experiments, SKF 525-A (25 mg/kg, ip) inhibited the metabolism of 11-OH-delta 8-THC to 11-oxo-delta 8-THC, supporting the previous suggestion that this oxidation, as well as the 11-hydroxylation of delta 8-THC, is mediated by the microsomal mono-oxygenase system.     

Bioactivation and detoxication of the pyrrolizidine alkaloid senecionine by cytochrome P-450 enzymes in rat liver

Rats display a marked sex difference in the oxidation of the pyrrolizidine alkaloid senecionine, especially with respect to N-oxidation. This sex difference was largely eliminated following treatment with dexamethasone. These observations suggested the potential involvement of the male-specific cytochrome P-450 UT-A and the P-450 PCN-E in the metabolism of this pyrrolizidine alkaloid. Reconstituted rat P-450 UT-A exhibited a high rate of N-oxidation (15 nmol min-1 nmol P-450-1) which is almost 3-fold higher than the turnover number observed with male rat liver microsomes. In contrast, rat P-450 UT-A displayed a much lower activity toward necine pyrrole [+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, DHP) formation (1.0 nmol min-1 nmol P-450-1). The N-oxygenation and pyrrole formation activities displayed by rat cytochromes P-450 PB-B and P-450 BNF-B toward senecionine were low, with rates less than 1 nmol min-1 nmol P-450-1. Rabbit antibody to rat P-450 UT-A inhibited the senecionine-N-oxidation activity of untreated male rat liver microsomes by 60%, with lesser inhibition of DHP production. Rabbit antibody to human P-450NF (the human homologue to rat P-450 PCN-E) was a potent inhibitor of DHP production by untreated male rat liver microsomes. With microsomes from dexamethasone-pretreated rats, anti-P-450NF inhibited DHP and N-oxide production in parallel. We conclude that the large sex difference in senecionine N-oxidation probably is the result of the specificity of P-450 isozymes UT-A and PCN-E.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of microsomal alcohol oxygenase catalyzing the oxidation of 7-hydroxy-delta 8-tetrahydrocannabinol to 7-oxo-delta 8-tetrahydrocannabinol in rat liver

The formation of 7-oxo-delta8-tetrahydrocannabinol (7-oxo-delta8-THC) from 7beta-hydroxy-delta8-THC was found in hepatic microsomes of rats. The activity was stereoselective and about 3-fold higher than that from 7alpha-hydroxy-delta8-THC. The oxidative activity of 7alpha- and 7beta-hydroxy-delta8-THC to 7-oxo-delta8-THC was significantly higher in male than in female, and significantly enhanced by both dexamethasone and phenobarbital, and then inhibited up to about 20% of the control value by antibody against P450GPF-B, presumably a member of the 3A subfamily, a major enzyme responsible for the formation of 7-oxo-delta8-THC in guinea pigs. This antibody also inhibited the formation of 7alpha- and 7beta-hydroxy-delta8-THC, and 7-oxo-delta8-THC from delta8-THC by hepatic microsomes of rats. These results indicate that there is a sex-related difference in the oxidation of 7-hydroxy-delta8-THC to 7-oxo-delta8-THC and the reaction is mainly catalyzed by P450 enzyme(s) belonging to the 3A subfamily as major enzyme(s) of microsomal alcohol oxygenase in rats.     

On the sulphoxidation of cimetidine and etintidine by rat and human liver microsomes

1. Sulphoxidation of cimetidine and etintidine was investigated by in vitro assays with liver microsomes from untreated 5,6-benzoflavone- and phenobarbital-pretreated rats as well as with human liver microsomes. The formation rate of cimetidine sulphoxide and etintidine sulphoxide with liver microsomes of normal or pretreated rats reached to 1.1 and 0.9 nmol/min mg microsomal protein, respectively. 2. Inhibition experiments with carbon monoxide and n-octylamine indicated that this sulphoxidation is catalyzed by cytochrome(s) P-450, whereas flavin-containing monooxygenase and/or non-enzymatic reactions (via peroxides) seems not to be involved: no inhibition was observed by methimazole, N,N-dimethylaniline, preheating or glutathione and EDTA. 3. With human liver microsomes the cytochrome P-450-dependent sulphoxidation accounted for no more than 40% of the total oxidation.     

Oxidative defluorination of 1,1,1,2-tetrafluoroethane by rat liver microsomes

1,1,1,2-Tetrafluoroethane (R-134a) is a non-ozone-depleting alternative to dichlorodifluoromethane for use as an air-conditioning refrigerant and as a propellant in anti-asthmatic and other pharmaceutical preparations. Hepatic microsomes, supplemented with NADPH, catalyzed the release of F- from R-134a; metabolite production was positively correlated with both duration of incubation and gas phase [R-134a]. Defluorination of R-134a was inhibited by CO, lack of NADPH, or heat denaturation of microsomes. Release of F- from R- 134a biotransformation as shown by the near-total lack of dehalogenation during anaerobic incubations. R-134a did not produce a difference spectrum (360 to 500 nm) with either oxidized or dithionite-reduced microsomes. Microsomes from phenobarbital- or Aroclor 1254-treated rats produced greater amounts of F- per mg protein from high concentrations of R-134a than did microsomes from untreated rats, but when normalized for microsomal cytochrome P-450 content both phenobarbital and Aroclor treatment decreased the specific activity (nmol F-/nmol cytochrome P-450) of R-134a metabolism. Furthermore, while defluorination of R-134a by microsomes from livers of untreated rats was substrate-saturable (Vmax, 11 nmol of F-/nmol cytochrome P-450/15 min; KM, 8% R-134a), R-134a dehalogenation by microsomes from Aroclor-treated rats was nonsaturable with [R-134a] as high as 69%. Microsomes from phenobarbital-treated rats retained the saturable, low KM activity, but also exhibited the apparently nonsaturable kinetic component when [R-134a] was greater than 24%.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

In vitro and in vivo studies of the effect of vitamin E on microsomal cytochrome P450 in rat liver

Administration of a diet supplemented with 0.06% vitamin E acetate to male rats over a 6-week period doubled hepatic microsomal stores of alpha-tocopherol over those in control (vitamin E adequate) rat liver. Total cytochrome P450 content and NADPH-cytochrome P450 reductase activity were significantly elevated in hepatic microsomes from vitamin E-supplemented rats to 111% and 123% of respective control values. Androstenedione 16 alpha-hydroxylase activity was increased in these fractions (2.57 +/- 0.31 nmol product/min/mg protein vs 1.81 +/- 0.38 in controls) whereas activities of the 6 beta-, 7 alpha- and 16 beta-hydroxylase pathways were unchanged. Immunoquantitation of the microsomal 16 alpha-hydroxylase, P450 IIC11, indicated a corresponding increase in the hepatic content of the enzyme. In view of the established antioxidant role of tocopherols, the effects of dietary vitamin E manipulation on the concentration of protein sulphydryl groups and the susceptibility of microsomes to ferric sulphate-ADP-NADPH-mediated lipid peroxidation were also assessed. Dietary supplementation did not influence microsomal protein sulphydryl content (68 +/- 10 nmol glutathione equivalents/mg protein) but decreased the extent of lipid peroxidation produced by the ferric sulphate-ADP-NADPH system in vitro. Further in vitro experiments demonstrated that vitamin E acetate (2 microM) protected protein sulphydryl groups and lipids against peroxidation in control microsomes and partially reduced the associated losses of P450-mediated steroid hydroxylase activities. Western immunoquantitation of P450 IIC11 revealed that exogenous vitamin E acetate protected completely against peroxidation-induced apoprotein loss. These studies establish that the in vitro protective effects of vitamin E acetate against sulphydryl and lipid peroxidation extend to protection of the P450 apoprotein but that enzyme activity is only partially protected. This finding suggests that peroxidation-dependent loss of P450 in vitro is mediated by haem degradation from the P450 holoenzyme and is not directly related to lipid/sulphydryl oxidation. In contrast, the in vivo effects of dietary vitamin E on drug metabolizing enzymes are regulatory in nature and are unrelated to effects on lipid peroxidation.     

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.     

On the sulphoxidation of cimetidine and etintidine by rat and human liver microsomes

1. Sulphoxidation of cimetidine and etintidine was investigated by in vitro assays with liver microsomes from untreated 5,6-benzoflavone- and phenobarbital-pretreated rats as well as with human liver microsomes. The formation rate of cimetidine sulphoxide and etintidine sulphoxide with liver microsomes of normal or pretreated rats reached to 1.1 and 0.9 nmol/min mg microsomal protein, respectively.

2. Inhibition experiments with carbon monoxide and n-octylamine indicated that this sulphoxidation is catalyzed by cytochrome(s) P-450, whereas flavin-containing monooxygenase and/or non-enzymatic reactions (via peroxides) seems not to be involved: no inhibition was observed by methimazole, N,N-dimethylaniline, preheating or glutathione and EDTA.

3. With human liver microsomes the cytochrome P-450-dependent sulphoxidation accounted for no more than 40% of the total oxidation.