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Glucuronidation of morphine in humans is predominantly catalyzed by UDP-glucuronosyltransferase 2B7 (UGT2B7). Since our recent research suggested that cytochrome P450s (P450s) interact with UGT2B7 to affect its function [Takeda S et al. (2005) Mol Pharmacol 67:665-672], P450 inhibitors are expected to modulate UGT2B7-catalyzed activity. To address this issue, we investigated the effects of P450 inhibitors (cimetidine, sulfaphenazole, erythromycin, nifedipine, and ketoconazole) on the UGT2B7-catalyzed formation of morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G). Among the inhibitors tested, ketoconazole was the most potent inhibitor of both M-3-G and M-6-G formation by human liver microsomes. The others were less effective except that nifedipine exhibited an inhibitory effect on M-6-G formation comparable to that by ketoconazole. Neither addition of NADPH nor solubilization of liver microsomes affected the ability of ketoconazole to inhibit morphine glucuronidation. In addition, ketoconazole had an ability to inhibit morphine UGT activity of recombinant UGT2B7 freed from P450. Kinetic analysis suggested that the ketoconazole-produced inhibition of morphine glucuronidation involves a mixed-type mechanism. Codeine potentiated inhibition of morphine glucuronidation by ketoconazole. In contrast, addition of another substrate, testosterone, showed no or a minor effect on ketoconazole-produced inhibition of morphine UGT. These results suggest that 1) metabolism of ketoconazole by P450 is not required for inhibition of UGT2B7-catalyzed morphine glucuronidation; and 2) this drug exerts its inhibitory effect on morphine UGT by novel mechanisms involving competitive and noncompetitive inhibition.

The effects of cytochrome P450 (P450, CYP) ligands and permeabilization of microsomes on 3-hydroxybenzo(a)pyrene [3-OH-B(a)P] glucuronidation mediated by rat hepatic microsomes were studied. While the UDP-glucuronosyltransferase (UGT) activity with non-permeabilized microsomes from 3-methylcholanthrene (MC)-treated rats was markedly reduced by alpha-naphthoflavone (NF), this inhibitor had hardly any effect when permeabilized microsomes were used in which the inhibitor was expected to have easy access to UGT. Kinetic analysis indicated that the inhibitory effect of alpha-NF is competitive. These results suggest that a UGT isoform(s) involved in 3-OH-B(a)P glucuronidation is interfered by a CYP1A inhibitor via a mechanism dependent on the intact nature of microsomal membranes in MC-treated rats. It is likely that P450 functions as a substrate transporter for some isoforms of UGT via possible interactions between UGT and P450.

The effect of 3,3',4,4',5-pentachlorobiphenyl (PenCB) on the synthesis of unsaturated fatty acids was studied in male Wistar rats. The arachidonic acid (20:4) content in the total lipids of liver homogenates was significantly reduced on day 5 of PenCB administration, while those of linoleic acid (18:2) and bishomo-gamma-linolenic acid (20:3) were increased. These changes in the total lipids of liver homogenates were observed following doses of PenCB ranging from 0.5 to 25 mg/kg of body weight. The same changes in these fatty acids were seen with four subtypes of microsomal glycerophospholipids in the liver. The marked reduction in the molar ratio of 20:4 to 18:2 in the lipids suggests alteration of the activity of the enzymes responsible for the synthesis of unsaturated fatty acids. The activity of Delta5 and Delta6 desaturases (arachidonic acid synthetase) in the liver microsomes was 17 and 13% of that of pair-fed control animals, whereas the activity of 1-acylglycerophosphorylcholine or 1-acylglycerophosphate acyltransferase, which transfers 20:4 or 18:2 to phospholipids, was not affected by the treatment. Thus, the reduction in the level of 20:4 that was observed can be explained by a reduction in desaturase activity. These results are evidence that the coplanar PenCB has a significant effect on the reduced synthesis of physiologically essential long-chain unsaturated fatty acids.

The peripheral benzodiazepine receptor plays a role in the translocation of cholesterol into mitochondria where steroidogenesis occurs. Sterols have been suggested to be involved in the regulation of the cytochrome P-450 (CYP)2B subfamily as the endogenous suppressor of this CYP. To investigate the role of cholesterol metabolites on the expression of CYPs, the effect of PK11195, a specific ligand of the peripheral benzodiazepine receptor and a stimulator of cholesterol transportation, on CYP expression was examined in rats in vivo and in cultured hepatocytes. As judged by the change in testosterone metabolic activity catalyzed by liver microsomes, i.p. injection of PK11195 into rats increased the CYP2B subfamily significantly. A trend in the induction of the CYP2A1, 2C11, and 3A isozymes was also observed. When PK11195 was given to rats together with phenobarbital, an additive effect of these compounds on testosterone metabolic activity was observed. In cultured hepatocytes, PK11195 exhibited the same effect on CYP expression as seen in vivo, but the magnitude of the effect was much greater than that observed in vivo. The inductive effect of PK11195 toward the CYP2B and 3A subfamilies was 2.3- and 6.5-fold greater, respectively, than that with phenobarbital. The inductive effect of PK11195 was confirmed by immunoblotting with antibodies against CYP2A, 2B, 2C, and 3A proteins. These results indicate that PK11195 has an inductive effect on several subfamilies of CYPs by directly acting on liver cells and has no ability to suppress the expression of these CYPs. This observation suggests that, if certain sterols play a role in the suppressive control of the CYP2B subfamily, they are produced in organelles other than the mitochondria.

"Animal-Plant Warfare" is one of the hypotheses for the evolution of drug-metabolizing P450s. To address the validity of this hypothesis, we examined the induction of xenobiotic-metabolizing P450s by 12 plant toxins in rats, using hepatic activity for testosterone metabolism as the index. The compounds tested were aconitine, morphine, tubocurarine, physostigmine, pilocarpine, muscarine, cocaine, atropine, amygdalin, digitonin, nicotine and solanine. Drinking water containing a test compound was given to rats for 4 days, and the hepatic activity of testosterone metabolism was determined together with monitoring body weight gain and liver weight as the indices of toxicity. The results showed that while cocaine and nicotine have a minor ability to increase testosterone 16 beta-hydroxylase activity, a marker activity for the CYP2B1 and 2, all other compounds did not have any such effect. No correlation was observed between a change in 16 beta-hydroxylase and toxicity caused by toxins. Therefore, these results did not support the idea that the inducibility of the CYP2B subfamily in animals is acquired through "Animal-Plant Warfare". Several compounds examined here increased or decreased hepatic activities of testosterone 2 alpha-, 6 beta-, 7 alpha- and 16 alpha-hydroxylation and 17-oxidation, indicating a possible effect on the CYP2A, 2C and 3A subfamily. Of these effects, a moderate correlation (r < 0.49) was observed in the changes in the activities of 2 alpha-/16 alpha-hydroxylation and 17-oxidation vs. that in toxicity. It is therefore suggested that inhibition or suppression of the expression of CYP2C11 is one of the mechanisms in the toxicity of plant toxins for rats, although it comes from an examination using limited numbers of compounds.

To study the specific target to which phenobarbital (PB) binds, resulting in the induction of cytochrome P450, we prepared two azido-PBs (AZPBs) as photoaffinity ligands. The azido substituent was introduced at the para- or meta-position of the PB aromatic ring. In this study, we estimated the utility of these compounds by examining their inducing activities in vivo in rats. Induction was assessed by immunoblotting with anti-CYP2B1/2 antibody and measuring testosterone-metabolizing activity, using hepatic microsomes. Administration of p-AZPB to rats increased hepatic CYP2B1/2 protein and testosterone 16beta-hydroxylase activity, although the effects were less than those of unmodified PB. m-AZPB showed no effect in the induction of CYP2B1/2. To assess the specificity of the effects of substituents, we compared the inducing activities of p/m-nitro-PBs, p/m-amino-PBs, and p/m-hydroxy-PBs with those of AZPBs. The results showed that p-nitro-PB, m-amino-PB, and p-hydroxy-PB were also potent inducers for CYP2B1/2, with lower activity than that of unmodified PB, whereas the other three isomers had no effect. These results suggest that 1) the absence of any substituents on the aromatic ring of PB is needed for maximal inducing activity and 2) substitution at the meta-position of the PB aromatic ring tends to reduce effectiveness as an inducer more than does substitution at the para-position. Because p-amino-PB and p-acetylamino-PB, the minor and major metabolites of p-AZPB, respectively, were without effect in the induction of CYP2B1/2, the effect of p-AZPB was considered to be due to the unchanged compound itself. The present study demonstrates that, based on the weak but positive ability to induce CYP2B1/2, p-AZPB may be a useful tool for identifying the putative PB receptor.

Deamination or oxidative cleavage of the carbon-nitrogen bond in various phenylisopropylamines was examined in liver microsomes from rabbits and rats, and in reconstituted systems containing CYP2C subfamily isozymes. Kinetic studies of phenylacetone formation from six amphetamine (AP) derivatives, catalyzed by rabbit liver microsomes, indicated that AP had the highest apparent affinity (lowest K(m)) and increasing the size of the substituent on the nitrogen atom decreased the affinity. The values of maximal velocity increased with increasing size of the substituent. Experiments with purified CYP2C3 from rabbit liver gave similar results: this enzyme showed the highest activity for phenylacetone formation from benzphetamine (BZP) and showed lower activities with compounds having smaller nitrogen substituents. Based on these results, we conclude that among a series of AP derivatives, the parent phenylisopropylamine has the highest affinity for rabbit liver deaminase, where as BZP has the highest turnover. However, the intrinsic clearance (Vmax/K(m)) values for the individual reactions tended to be comparable. The rates of BZP and deprenyl N-demethylation by rat CYP2C11 and 2C13 were far greater than those of the reactions at other N-alpha-positions. This result indicated that rat CYP2C enzymes have a more rigid regioselectivity than rabbit CYP2C3 for the deamination/N-dealkylation of phenylisopropylamines.

Metabolism of 2,5,2',5'-tetrachlorobiphenyl (TCB) was studied using liver microsomes of hamsters and two hamster P450 isoforms, CYP1A2 and 2A8. CYP2A8 catalyzed selectively 4-hydroxylation of 2,5,2',5-TCB at a rate of 21.7 pmol/min/nmol P450. In contrast, CYP1A2 showed no activity for hydroxylation of 2,5,2',5'-TCB. Immunological study revealed that rabbit antiserum against CYP2A8 almost completely inhibited the microsomal 4-hydroxylation but that against CYP1A2 did not. It was also shown that the induction pattern of CYP2A8 protein by P450 inducer was similar to that of the 4-hydroxylase activity in hamster liver microsomes. These results suggest that CYP2A8 plays a major role in the 4-hydroxylation of 2,5,2',5'-TCB in hamster liver.

1. The role of cytochrome b5 in the cytochrome P450-dependent hydroxylation of tetrachlorobiphenyl (TCB) isomers was examined using a reconstituted system consisting of CYP2B1 and CYP1A1 and rat liver microsomes. 2. By addition of cytochrome b5 to the reconstituted system containing CYP2B1, the 3-hydroxylation of 2,5,2,'5'- and 2,5,3',4'-TCB was increased about six-fold, but the 3- and 5-hydroxylation of 2,4,3',4'-TCB was decreased by about 50% 3. All hydroxylations of 3 ,4,3',4'-,2,5, 3,4'- and 2,4,3',4'-TCBs were decreased by addition of cytochrome b5 to the reconstituted system containing CYPlA1. 4. In stoichiometry measurements, changes in NADPH oxidation and coupling efficiency by addition of cytochrome b5 was observed and these differed according to the position of chlorine atoms of TCBs and cytochrome P450 isoforms used in the systems.

A morphine UDP-glucuronosyltransferase (UGT) which could belong to the UGT2B subfamily was isolated from liver microsomes of a male beagle dog treated with phenobarbital. Glucuronidation toward morphine in the dog liver microsomes was increased threefold by the treatment. The microsomes were solubilized with Emulgen 911 and applied on a column of hemisuccinate derivative of Sepharose 4B column which has been developed in our laboratory. An isoform of UGT in the eluate was purified further by chromatofocusing and UDP-hexanolamine-affinity chromatography. A purified enzyme, UGTDOG-PB, was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional electrophoresis and exhibited a subunit molecular weight of 50 kDa. This isoform showed activities toward the 3-hydroxyl group of morphine, 4-hydroxybiphenyl, 4-nitrophenol, 4-methylumbelliferone, and testosterone, but not toward chloramphenicol and the 6-hydroxyl group of morphine. The substrate specificity of UGTDOG-PB is similar to that of stably expressed UGT2B1 which is considered a phenobarbital-inducible morphine UGT in the rat except that UGTDOG-PB is capable of glucuronidating 4-nitrophenol but not chloramphenicol. The NH2-terminus until the 30th residue of UGTDOG-PB is highly homologous to UGT2B subfamily, and the NH2-terminal 15 residues of UGTDOG-PB are completely identical to those of UGT2B1, UGT2B8, and UGT2B15. This is the first report describing the UGT isoform of dog and the purification of morphine UGT which may belong to UGT2B subfamily.

In the hamster liver, 2,5,2',5'-tetrachlorobiphenyl (TCB) is metabolized to 3-hydroxy- and 4-hydroxy-2,5,2',5'-TCB to a similar extent, and formation of the former metabolite is stimulated by phenobarbital pretreatment of the animals, while that of the latter metabolite is stimulated by 3-methylcholanthrene pretreatment. In the present study, we identified a new isoform (designated P450HPB-1) of cytochrome P450 which proved to be phenobarbital-inducible and responsible for 3-hydroxylation of this TCB isomer. This isoform was purified from liver microsomes of phenobarbital-treated hamsters and characterized. P450HPB-1 has a molecular mass of 50 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the absorption maxima of the oxidized form at 417 nm and of the reduced CO-complex form at 450 nm. The sequence of 28 amino acids of P450HPB-1 at the amino-terminal has a 68% similarity with those of rat P450 2B1 and mouse P450 2b10, 57% similarity with that of guinea pig P450GP-1, and 54% similarity with that of guinea pig P450GP-1, and 54% similarity with those of rabbit P450 2B4 and dog P450 2B11. P450HPB-1 in the reconstituted system catalyzed the 3- but not 4-hydroxylation of 2,5,2',5'-TCB, at a rate of 19.0 pmol/min/nmol P450. The isoform also has high catalytic activity for 17-oxidation of testosterone but low activity for the N-demethylation of benzphetamine and 16 alpha- and 16 beta-hydroxylations of testosterone. In microsomal metabolism of 2,5,2',5'-TCB, rabbit antiserum against P450HPB-1 almost completely inhibited 3- but not 4-hydroxylation. Immunoblot analysis of hamster liver microsomes revealed that P450HPB-1 was constitutive and phenobarbital-inducible but was decreased by pretreatment with 3-methylcholanthrene or 3,4,5,3',4'-pentachlorobiphenyl. These results suggest that P450HPB-1 belongs in the P450 2B subfamily and apparently plays a major role in the 3-hydroxylation of 2,5,2',5'-TCB, in hamster liver.

A morphine UDP-glucuronyltransferase (morphine UGTPB) was purified from liver microsomes of Sprague-Dawley rats treated with phenobarbital. UDP-glucuronyl-transferases in the liver microsomes were solubilized with Emulgen 911 and separated by omega-(beta-carboxypropionyl-amino)octyl Sepharose 4B column chromatography, which has been developed in our laboratory. Morphine UDP-glucuronyltransferases were eluted into two fractions, Peak I and Peak II, which have different substrate specificities. Morphine UGTPB was purified by two times of Chromato-focusing from Peak II which was more specific to morphine. The purified morphine UGTPB gave an apparent pI of 8.0 on chromatofocusing and displayed a subunit molecular weight of 55 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the glucuronidation of 3-hydroxyl group of morphine and small extent of 4-hydroxybiphenyl, but not of androsterone, bilirubin, chloramphenicol, codeine, 4-methylumbelliferone, 4-nitrophenol, testosterone, and 6-hydroxyl group of morphine. The N-terminal amino acid sequences of morphine UGTPB were identical to those of UGT1*01P which is deficient to homozygous Gunn rat. Peak II was absent from the fraction of omega-(beta-carboxypropionylamino)octyl Sepharose 4B column chromatography of liver microsomes of Gunn rats treated with phenobarbital, whereas morphine UGT in Peak I was PB-inducible in Gunn rats. Present results suggest that an isoform of morphine UDP-glucuronyltransferase belongs to the UGT1 family and is phenobarbital-inducible.

An epoxide hydrolase (mEH) in liver microsomes was purified to apparent homogeneity from a dog treated with phenobarbital. The purified enzyme had a minimum molecular weight of 47,000 as determined by SDS-PAGE. The dog mEH activity was characterized by use of a substrate, 7-glycidoxycoumarin (GOC), and some effectors of this enzyme. In vitro activators, metyrapone, and isoquinoline, stimulated the microsomal activity, but the former had no such effect on the purified enzyme in case of this substrate. All mEH inhibitors, 1,1,1-trichloropropene 2,3-oxide (TCPO), cyclohexene oxide, and 2-bromo-4'-nitroacetophenone (BrNAP), suppressed hydrolase activity. The NH2-terminal amino acid sequence of the purified enzyme was highly homologous (90%) to the sequences deduced from a cDNA clone of rat enzyme. Antiserum to the purified enzyme raised in rabbits cross-reacted with rat and guinea pig epoxide hydrolases. No gender-difference in this enzyme in liver microsomes was observed in dogs.

The cytochrome P450 isozymes in rat liver microsomes that catalyze the demethylenation of methylenedioxymethamphetamine enantiomers to the corresponding dihydroxymethamphetamine were characterized. Dihydroxymethamphetamine formation in liver microsomes from male Sprague-Dawley rats exhibited multienzyme kinetics, with Km values in the micromolar/millimolar range. The stereoselectivity [(+)-isomer versus (-)-isomer] varied from 0.78 to 1.94 after pretreatment of the rats with phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, or pyrazole, suggesting that different isozymes participate in the reaction. The low-Km demethylenation was not induced by these compounds and was not inhibited by antibodies raised against CYP2C11. Liver microsomes from female Dark-Agouti rats, a strain genetically deficient in CYP2D1, exhibited demethylenation activities that were 9% of those in microsomes from male Sprague-Dawley rats. The low-Km demethylenation was also inhibited by CYP2D substrates such as sparteine, bufuralol, or desipramine and was almost completely inhibited by antibodies against P450 BTL, which belongs to the CYP2D family. The higg-Km demethylation activity was induced by phenobarbital and pregnenolone-16 alpha-carbonitrile and the activity in both untreated and phenobarbital-induced microsomes was suppressed by anti-CYP2B1 IgG. Experiments with IgG raised against cytochrome b5 suggested that the hemoprotein contributed to the low-Km activity but not the high-Km activity. These results indicate that cytochrome P450 isozymes belonging to the CYP2D subfamily catalyze demethylenation with low Km values and that the reaction occurring with high Km values is likely to be mediated by members of the CYP2B family, but with the possible participation of other phenobarbital-inducible isoforms.

We studied in vitro metabolites of 2,4,6,2',4',6'-hexachlorobiphenyl (HCB, IUPAC PCB No. 155) produced by liver microsomes of a phenobarbital (PB)-treated beagle dog. The major metabolites were 3-hydroxy-2,4,6,2',4',6'-HCB (M-1), 4-hydroxy-2,6,2',4',6'-pentachlorobiphenyl (PenCB, M-2) and 3,4-dihydroxy-2,6,2',4',6'-PenCB (M-3). Furthermore, 4-hydroxy-2,3,6,2',4',6'-HCB (M-4), which could be formed via the 3,4-epoxidation and the subsequent NIH-shift of the chlorine from the 4 to the 3 position, was also detected. We found that M-3 is a common secondary metabolite of the two major monohydroxy metabolites, M-1 and M-2. These results indicate that the dog seems to metabolize and eliminate this congener not only by a mechanism involving direct insertion of a hydroxyl group but also via an arene oxide intermediate.

A new method of assaying deaminase activity was established in which methylamine and/or dimethylamine formed from drugs containing N,N-dimethyl or N-methyl group were derivatized with phenylisothiocyanate to phenylthiourea derivatives. After purification with Sep-PAK C18 cartridge, the derivatives were separated by a reversed phase high-performance liquid chromatography monitored by ultraviolet absorption. The recoveries and determination limits of methylamine and dimethylamine were over 55% and about 0.4 nmol/ml of incubation mixture, respectively. The method was used to measure the deaminase activities of liver microsomes of rats, rabbits and guinea pigs for 11 drugs. Of the compounds tested, diphenhydramine and diltiazem are deaminated with microsomes from all the above animal species; rat and rabbit liver microsomes also well deaminated promethazine. Most other drugs such as chlorpromazine, promazine, imipramine, amitriptyline and tetracaine were found to be poor substrates. In general, dimethylamine but not methylamine was the predominant metabolite formed from drugs containing N,N-dimethylamino group. The results also suggested that the deamination of these compounds takes place mainly via a one step mechanism, thus implying that the sequential reaction consisting of N-demethylation and elimination of ammonia is of minor importance. The relation between in vitro deaminase activity and the extent of the in vivo deamination for drugs is discussed.

The induction by the central stimulant picrotoxin of hepatic drug-metabolizing enzymes was studied in rats. The hepatic content of P450 and the activity of benzphetamine N-demethylation increased gradually after administration of picrotoxin dissolved in drinking water (2 mg/mL), to three-times higher levels than the initial values at the third day of treatment. The increase in benzphetamine N-demethylase activity by picrotoxin was somewhat higher than the increase produced by phenobarbital. Supporting these results, immunoblot analysis showed that CYP2B1 and 2B2 proteins in the liver microsomes were increased by picrotoxin Picrotoxinin and picrotin, which are components of the picrotoxin molecule, had the same ability to induce the hepatic activity of benzphetamine N-demethylation. The liver microsomal activities of testosterone 16 alpha- and 16 beta-hydroxylation were enhanced significantly after treatment with picrotoxinin and picrotin. However, benzo[a]pyrene 3-hydroxylation, aniline 4-hydroxylation, and testosterone hydroxylations at the 2 alpha- and 7 alpha-positions were not increased by picrotoxinin and picrotin treatment. In addition to monooxygenase, significant induction of glutathione S-transferase activity for 1-chloro-2,4-dinitrobenzene and UDP-glucuronyltransferase activity for 4-hydroxybiphenyl and 4-nitrophenol was also observed by pretreatment of picrotoxin. These results clearly indicate that picrotoxin is an inducer of phenobarbital-inducible liver enzymes.

The induction of liver microsomal UDP-glucuronyltransferase (UGT) activity toward bilirubin by pretreatment with 3, 4, 3', 4'-tetrachlorobiphenyl (TCB), 3, 4, 5, 3', 4'-pentachlorobiphenyl (PenCB) and 3-methylcholanthrene (MC) was studied in guinea pigs and rats. In addition, microsomal benzo(a)pyrene 3-hydroxylase and cytosolic DT-diaphorase activities were also measured for the comparison. All of the PenCB, TCB and MC significantly induced the bilirubin UGT activity of guinea pig liver microsomes. The highest induction (6-fold over the control) was seen in the PenCB-treated animal, and MC (3.2-fold) and TCB (2.4-fold) were less effective. On the other hand, the induction of benzo(a)pyrene 3-hydroxylase and DT-diaphorase activities of guinea pigs was not so remarkable as that of bilirubin UGT activity. In the guinea pig, the inducibility of bilirubin UGT activity seemed to be correlated with the toxicity induced with PenCB, TCB and MC. In contrast to the guinea pig, UGT activity toward bilirubin in the rat was significantly decreased by the treatment with the inducers described above. These results suggest that bilirubin UGT activity in the guinea pig can be an index for the toxicity of polychlorinated biphenyls as like as benzo(a)pyrene 3-hydroxylase and DT-diaphorase activities in the rat.

1. Metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) was investigated in vitro using liver microsomes of one male beagle dog after phenobarbital treatment. 2. Three major metabolites were isolated and identified as 3-hydroxy-2,4,5,2',4',5'-HCB, 2-hydroxy-4,5,2',4',5'-pentachlorobiphenyl (PenCB), and 2-hydroxy-3,4,5,2',4',5'-HCB, by comparison of g.l.c.-mass spectrometry and 1H-n.m.r. data with those of authentic samples. 3. 2-Hydroxy-3,4,5,2',4',5'-HCB was found as a metabolite of 2,4,5,2',4',5'-HCB for the first time using dog liver microsomes. Present result indicate that this metabolite and the dechlorinated PenCB are derived from a metabolic intermediate, namely, 2,3-epoxy-2,4,5,2',4',5'-HCB. 2,3-Epoxide formation is a new metabolic pathway of PCB.

The cytochrome P450 isozymes catalyzing the oxidation of the methylenedioxyphenyl compounds methylenedioxybenzene (MDB) and methylenedioxyamphetamine (MDA) have been investigated in rabbit liver preparations. The aromatic ring in MDB undergoes both demethylenation to catechol and aromatic hydroxylation to sesamol, whereas that in MDA undergoes only demethylenation to dihydroxyamphetamine. Formation of catechol and sesamol from MDB in microsomal incubation mixtures was enhanced about 5- and 3-fold, respectively, by pretreatment of the rabbits with phenobarbital, which induced CYP2B4 and CYP4B1. The cytochrome P450 isozyme responsible for aromatic hydroxylation of MDB was induced by beta-naphthoflavone and was inhibited by alpha-naphthoflavone. Microsomal demethylenation of MDA was minimally sensitive to pretreatment of the rabbits with phenobarbital, beta-naphthoflavone, pyrazole, or rifampicin. However, MDA competitively inhibited the N-demethylation of erythromycin. Antibodies against CYP2B4, but not those against CYP4B1, caused a marked inhibition of the demethylenation and aromatic hydroxylation of MDB. Antibodies against CYP2C3 did not inhibit the demethylenation of MDA, nor did substrates or inhibitors of the CYP2D family except for bufuralol. MDB and MDA were both capable of forming metabolic intermediate complexes, and the rate of complex formation was accelerated by phenobarbital induction. Reconstitution experiments with CYP2B4 suggested that phenobarbital-inducible complex formation from MDA was not due to the carbene pathway involving the methylenedioxy group but was due to oxidation of the amino group. These results indicate that CYP2B4 oxidizes different regions of methylenedioxyphenyl compounds depending on their structure. MDB undergoes oxidation at the methylenedioxy group (major) and the benzene ring (minor). MDA is oxidized at the alkylamino side chain at the nitrogen and alpha-carbon. The results suggested that one or more constitutive isoforms (probably unknown) of cytochrome P450 present in rabbit liver microsomes are primarily responsible for MDA demethylenation but that CYP3A6 contributes slightly.

1. An unknown metabolite of strychnine termed M-5 was isolated, from an incubation mixture using guinea pig liver microsomes, by repeated preparative t.l.c. 2. The structure of M-5 was shown to be 22-hydroxystrychnine by mass, n.m.r. and i.r. spectrometry. 3. 22-Hydroxystrychnine is a stable enol, a unique metabolite possessing a hydroxyl group on the double bond of the alicylic moiety.

Species difference in codeine uridine diphosphate-glucuronyltransferase (UDPGT) activity was studied in liver microsomes of mice, rats, guinea pigs and rabbits. Codeine UDPGT activity was the highest in guinea pigs, followed by that in rabbits, and the lowest in mice and rats among these four animal species. The specific activities of codeine UDPGT in liver microsomes were not correlated well with those toward morphine, 4-nitrophenol, and 4-hydroxybiphenyl in liver microsomes of each of the species. Inducibility of liver microsomal codeine UDPGT activity in rats was examined by pretreatment with phenobarbital and 3-methylcholanthrene and compared with those of other UDPGT activities. The activity was inducible by phenobarbital pretreatment as the activity toward morphine and 4-hydroxybiphenyl. The inducibility of codeine UDPGT activity by phenobarbital pretreatment was not as high as that of morphine UDPGT activity.

Two distinct forms (FMO-I and FMO-II) of flavin-containing monooxygenase were purified from the liver microsomes of guinea pig. The minimum molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 54,000 for FMO-I and 56,000 for FMO-II, respectively. Tryptic digestion of these enzymes gave different electrophoretic patterns, suggesting that FMO-I and -II have distinct amino acid sequences. The amino terminal sequence of FMO-II could not be estimated probably due to its blocking while that of FMO-I was determined to be highly homologous to the rabbit liver flavin-containing monooxygenase (J. Ozols, 1989, Biochem. Biophys. Res. Commun. 163, 49-55). Absorption maxima of FMO-I and -II were recorded at 368 and 440 nm and 381 and 456 nm, respectively. Molar ratios of FAD to both of these apoenzymes were shown to be one to one. Substrate specificity of FMO-I and -II was determined using 15 compounds as the substrate. The results showed two enzymes that exhibited overlapped but different specificity toward these substrates although FMO-I had lower activity than did FMO-II with all compounds except thiobenzamide. Of particular interest, only FMO-II showed considerably high activities for primary amines, n-octylamine, and n-decylamine. Immunoglobulin G raised against FMO-II could recognize FMO-I as well as FMO-II, but the reactivity of FMO-I toward the antibody was obviously lower than that of FMO-II. Electrophoresis followed by immunostaining revealed that microsomes of lung, kidney, urinary bladder, testis, and spleen contain the same protein as FMO-II and/or FMO-I. Only lung was shown to have an additional isozyme of FAD-monooxygenase with a molecular weight apparently higher than those of FMO-I and -II. These results strongly suggest that at least two forms of flavin-containing monooxygenases distinct from the lung-type isozyme are expressed in liver of guinea pigs.

The metabolism of strychnine was studied with hepatic microsomes of rats, mice, guinea pigs, rabbits and dogs, using high-performance liquid chromatography. Eight metabolites were found by the incubation with rabbit liver microsomes. Five of them were identified as strychnine N-oxide (St N-oxide), 2-hydroxystrychnine (2-OH St), strychnine 21,22-epoxide, 16-hydroxystrychnine (16-OH St) and 18-oxostrychnine by comparing the chromatographic and spectral data to those of authentic samples. Significant differences in metabolic profiles of strychnine were observed among the above species. The main metabolite in rats and mice was 16-OH St, while in guinea pigs and rabbits, and in dogs, it was 2-OH St, and St N-oxide, respectively. The metabolic activity in guinea pig liver microsomes was much higher than those of other species. There seems to be a fairly good inverse correlation between the metabolic activity and strychnine toxicity in guinea pigs and other animal species.

Relative participation of flavin-containing mono-oxygenase and cytochrome P-450 systems in N-hydroxylation of and formaldehyde release from methamphetamine were studied in vitro using liver microsomes of guinea-pigs and rats. In guinea pigs, only methimazole, an inhibitor of flavin-containing mono-oxygenase, significantly suppressed the above reactions. Formaldehyde release from methamphetamine was significantly inhibited not only by methimazole but also by inhibitors of the cytochrome P-450 system in liver microsomes from rats, but not guinea-pigs. Pretreatment of guinea-pigs with phenobarbital and 3-methylcholanthrene did not enhance the metabolism of methamphetamine. Pretreatment of rats with phenobarbital but not 3-methylcholanthrene increased slightly the N-demethylation of methamphetamine by liver microsomes. The results indicate that a marked species difference exists in the enzymes concerned with N-demethylation of methamphetamine. N-Oxidation predominates in guinea-pigs, whereas in rats, N-oxidation and C-oxidation of the methyl group participate equally as the initial reaction of the N-demethylation pathway.

A cytochrome P450 isozyme responsible for amphetamine deamination was purified from hepatic microsomes of untreated rabbits. The purification procedures consisted of a set of column chromatographies with omega-aminooctyl-Sepharose 4B, DEAE-cellulose, CM-Sephadex C-50, and hydroxyapatite. The deamination activity was determined by measuring the formation of phenylacetone after derivatization to the p-nitrobenzyloxim by HPLC. This isozyme, which was designated P450APD, showed a monomeric molecular weight of 51,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited an absorption maximum of reduced CO complex at 451 nm. On the basis of the specificity toward testosterone metabolism and the N-terminal amino acid sequence, P450APD was attributed to a member of P450 class IIC subfamily, which is identical or closely related to LM3b (D. R. Koop and M. J. Coon (1979) Biochem, Biophys, Res. Commun. 91, 1075-1081), form 3b (E. F. Johnson (1980) J. Biol. Chem. 255, 304-309), and other similar preparations. Antibody against the P450APD inhibited about 80% of the amphetamine deamination activity in rabbit hepatic microsomes as well as in the reconstitution system of this P450. The present results support that P450APD is the major P450 isozyme responsible for amphetamine deamination in rabbit liver.

Interactions of delta 8-tetrahydrocannabinol (delta 8-THC) and its metabolites, 11-hydroxy-delta 8-THC, 11-oxo-delta 8-THC and delta 8-THC-11-oic acid, with hepatic microsomal drug metabolizing enzyme were studied in vitro and in vivo using mice. All the cannabinoids used were shown to produce a type I spectral change of cytochrome P-450 in hepatic microsomes. THe apparent binding affinities (Ks) for delta 8-THC, 11-hydroxy-delta 8-THC, 11-oxo-delta 8-THC and delta 8-THC-11-oic acid were 5.2, 169.6, 33.2 and 148.8 microM, respectively. delta 8-THC, 11-oxo-delta 8-THC and delta 8-THC-11-oic acid (100 microM) caused a significant stimulation of NADPH oxidation in vitro with microsomes. In addition, delta 8-THC (20, 40 and 80 microM) markedly inhibited p-nitroanisole O-demethylase and aniline hydroxylase in microsomes. 11-Hydroxy-delta 8-THC and 11-oxo-delta 8-THC also showed the inhibitory effect, but to lesser extents. Furthermore, single pretreatments with delta 8-THC and 11-oxo-delta 8-THC (30 mg/kg, i.v.) significantly decreased activities of p-nitroanisole O-demethylase and aniline hydroxylase in hepatic microsomes, accompanying a decrease of cytochrome P-450 content in case of delta 8-THC. On the other hand, all these pretreatments except for that with delta 8-THC-11-oic acid showed a stimulatory effect on p-nitrophenol glucuronidation with hepatic microsomes, in contrast to an inhibitory effect in vitro.

UDP-glucuronyltransferase activities for delta 8-tetrahydrocannabinol, 11-hydroxy-delta 8-tetrahydrocannabinol, cannabidiol and cannabinol were examined using liver microsomes of rabbits. The activities for cannabidiol and cannabinol were 2.5 and 19 times higher, respectively, than those for delta 8-tetrahydrocannabinol and 11-hydroxy-delta 8-tetrahydrocannabinol. Thus, the present study supports the view that glucuronide formation plays an important role in the metabolism of cannabinol, but does only a minor role in that of delta 8-tetrahydrocannabinol and 11-hydroxy-delta 8-tetrahydrocannabinol.

The effect of polyriboinosinic acid-polyribocytidylic acid [poly(I).poly(C)] on glucuronyltransferase activities toward 4-nitrophenol and 4-hydroxybiphenyl in liver microsomes of Wistar rats was examined by its single or co-administration with 3-methylcholanthrene and phenobarbital. The increased 4-nitrophenol glucuronyltransferase activity by treatment with 3-methylcholanthrene was significantly suppressed following the co-administration with poly(I).poly(C), although the activity was not affected by the treatment with poly(I).poly(C) alone. In addition, 4-hydroxybiphenyl glucuronyltransferase activity decreased or tended to decrease by the treatment with poly(I).poly(C) alone, and the activity induced by phenobarbital was strikingly decreased following the co-administration with poly(I).poly(C). This result suggested that poly(I).poly(C) comprehensively decrease the induction of glucuronyltransferases regardless of their multiple forms. Furthermore, contents of cytochromes P-450 and b5 were also decreased by the treatment with poly(I).poly(C) alone or the co-administration with the inducers. Concomitantly, arylhydrocarbon hydroxylase and benzphetamine N-demethylase activities were significantly decreased by the treatment alone or the co-administration with the inducers. These findings supported a view that the suppressive effect of poly(I).poly(C) may be derived from the prevention of de novo synthesis of the apoprotein of the enzymes and/or the increased degradation.

The glucuronidation of delta 9-tetrahydrocannabinol (delta 9-THC), cannabidiol (CBD) and cannabinol (CBN) in rat liver microsomes was studied. The enzyme activities for the cannabinoids were 26.3 (delta 9-THC), 52.9 (CBD) and 104.8 (CBN) pmol/min/mg protein. The apparent Km values of UDP-glucuronyltransferase for the cannabinoids were 0.29 (delta 9-THC), 0.18 (CBD) and 2.78 (CBN) mM, while Vmax were 40.3 (delta 9-THC), 104.9 (CBD) and 593.3 (CBN) pmol/min/mg protein. Following treatment of rats with 3-methylcholanthrene, the enzyme activities for delta 9-THC, CBD and CBN were increased 132, 43 and 1198%, respectively, whereas the corresponding increases in microsomes from phenobarbital-treated rats were 127, 13 and 97%, respectively. The cannabinoid glucuronidation was activated 2 to 3 folds by the addition of UDP-N-acetylglucosamine, but not activated by the addition of Triton X-100 in vitro. The properties of cannabinoid UDP-glucuronyltransferase were discussed from the above results.