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Mitochondrial toxicity is an important factor to predict drug-induced liver injury (DILI). Previous studies have focused predominantly on mitochondrial toxicities due to parent forms, and no study has adequately evaluated metabolite-induced mitochondrial toxicity. Moreover, previous studies have used HepG2 cells, which lack many cytochrome P450 (CYP) genes. To overcome this problem, CYP-introduced HepG2 cells were constructed using several gene transfer technologies, including adenoviruses and plasmids. However, these methods only led to a transient expression of CYP genes. In the present study, usefulness of four CYPs introduced-HepG2 (TC-Hep) cells previously constructed through mammalian artificial chromosome technology were examined, especially from the perspective of mitochondrial toxicity. First, we evaluated the effects of known compounds, such as rotenone and flutamide, on mitochondrial toxicity and cell death in TC-Hep cells cultured in galactose conditions. Expectedly, rotenone-induced cell death ameliorated because rotenone was metabolized by CYPs into inactive form(s) and flutamide-induced cell death increased in TC-Hep cells. Second, we evaluated five compounds that caused liver injury in clinical phase and were discontinued during pharmaceutical development. The present in vitro tool suggested that three of the five compounds caused metabolite-induced mitochondrial toxicities. In conclusion, the present in vitro tool could easily and inexpensively detect metabolite-induced mitochondrial toxicity; hence, it can be useful for predicting DILI in preclinical phase., Competing Interests: Declarations of Competing Interest None., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Bioactivation of 5-hydroxy-[carbonyl-(14)C]thalidomide, a known metabolite of thalidomide, by human artificial or native cytochrome P450 3A enzymes, and nonspecific binding in livers of mice was assessed using two-dimensional electrophoresis combined with accelerator mass spectrometry. The apparent major target proteins were liver microsomal cytochrome c oxidase subunit 6B1 and ATP synthase subunit α in mice containing humanized P450 3A genes or transplanted humanized liver. Liver cytosolic retinal dehydrogenase 1 and glutathione transferase A1 were targets in humanized mice with P450 3A and hepatocytes, respectively. 5-Hydroxythalidomide is bioactivated by human P450 3A enzymes and trapped with proteins nonspecifically in humanized mice.

Chimeric mice with humanized liver (PXB mice) have been generated by transplantation of urokinase-type plasminogen activator/severe combined immunodeficiency mice with human hepatocytes. The purpose of the present study was to clarify the protein expression levels of metabolizing enzymes and transporters in humanized liver of PXB mice transplanted with hepatocytes from three different donors, and to compare their protein expressions with those of human livers to validate this human liver model. The protein expression levels of metabolizing enzymes and transporters were quantified in microsomal fraction and plasma membrane fraction, respectively, by means of liquid chromatography-tandem mass spectrometry. Protein expression levels of 12 human P450 enzymes, two human UDP-glucuronosyltransferases, eight human ATP binding cassette (ABC) transporters, and eight human solute carrier transporters were determined. The variances of protein expression levels among samples from mice humanized with hepatocytes from all donors were significantly greater than those from samples obtained from mice derived from each individual donor. Compared with the protein expression levels in human livers, all of the quantified metabolizing enzymes and transporters were within a range of 4-fold difference, except for CYP2A6, CYP4A11, bile salt export pump (BSEP), and multidrug resistance protein 3 (MDR3), which showed 4- to 5-fold differences between PXB mouse and human livers. The present study indicates that humanized liver of PXB mice is a useful model of human liver from the viewpoint of protein expression of metabolizing enzymes and transporters, but the results are influenced by the characteristics of the human hepatocyte donor.

Accurate prediction of pharmacokinetics (PK) parameters in humans from animal data is difficult for various reasons, including species differences. However, chimeric mice with humanized liver (PXB mice; urokinase-type plasminogen activator/severe combined immunodeficiency mice repopulated with approximately 80% human hepatocytes) have been developed. The expression levels and metabolic activities of cytochrome P450 (P450) and non-P450 enzymes in the livers of PXB mice are similar to those in humans. In this study, we examined the predictability for human PK parameters from data obtained in PXB mice. Elimination of selected drugs involves multiple metabolic pathways mediated not only by P450 but also by non-P450 enzymes, such as UDP-glucuronosyltransferase, sulfotransferase, and aldehyde oxidase in liver. Direct comparison between in vitro intrinsic clearance (CL(int,in vitro)) in PXB mice hepatocytes and in vivo intrinsic clearance (CL(int,in vivo)) in humans, calculated based on a well stirred model, showed a moderate correlation (r² = 0.475, p = 0.009). However, when CL(int,in vivo) values in humans and PXB mice were compared similarly, there was a good correlation (r² = 0.754, p = 1.174 × 10⁻⁴). Elimination half-life (t(1/2)) after intravenous administration also showed a good correlation (r² = 0.886, p = 1.506 × 10⁻⁴) between humans and PXB mice. The rank order of CL and t(1/2) in human could be predicted at least, although it may not be possible to predict absolute values due to rather large prediction errors. Our results indicate that in vitro and in vivo experiments with PXB mice should be useful at least for semiquantitative prediction of the PK characteristics of candidate drugs in humans.

Hepatic cytochrome P450 (P450) enzymes are down-regulated during inflammation. In this study, an animal model of inflammatory bowel disease was subjected to characterization of hepatic P450 expression under inflammatory conditions. Rats were treated intracolonically with 100 mg/kg trinitrobenzene sulfonic acid (TNBS) dissolved in 30% ethanol, and homogenates of colonic mucosa and hepatic microsomes of the rats were prepared. The colitis was accompanied by appearance of higher levels of portal endotoxin, interleukin-6, and nitric oxide metabolites and decreases in contents and activities for hepatic CYP3A2, CYP2C11, and, to a lesser extent, CYP1A2 and CYP2E1. Nimesulide, a preferential COX-2 inhibitor, protected rats with TNBS-induced colitis (TNBS-colitis) against the down-regulation of hepatic CYP3A2. Polymyxin B, which neutralizes endotoxin, curcumin, which has anti-inflammatory properties, and gadolinium chloride, which inactivates macrophages, attenuated the down-regulation of CYP3A2. Similar effects were observed in other P450s such as CYP2C11, but the agents were less effective in attenuating the down-regulation. Our data suggest that endogenous substances leaked from damaged colon in the rats with TNBS-colitis activate Kupffer cells, leading to down-regulation of hepatic P450s with differential susceptibility to the inflammatory stimuli. The colitis model, instead of exogenous administration of lipopolysaccharide or cytokines, could be applied to the study on mechanisms for altered hepatic P450 expression and other liver functions under mild inflammatory conditions.

Hepatic drug metabolism is impaired in experimental animals and humans with renal diseases. An anticancer drug, cisplatin induces acute renal failure (ARF) in rats. Under the same experimental conditions, cisplatin causes down-regulation of hepatic cytochrome P450 (P450) enzymes in an isozyme selective manner. The present study examined the pathological role of ARF in the down-regulation of hepatic P450 enzymes in the cisplatin-treated rats. Male rats with single dose of intraperitoneally cisplatin (5 mg/kg) caused marked changes in renal parameters, BUN and serum creatinine but not hepatic parameters, serum alanine aminotransferase or aspartate aminotransferase. The rats also suffered from down-regulation of hepatic microsomal CYP2C11 and CYP3A2, male specific P450 isozymes, but not CYP1A2, CYP2E1, or CYP2D2. The decrease in serum testosterone level was also observed in injured rats, which was consistent with the selective effects on male specific P450 enzymes. Protection of rats against cisplatin-induced ARF by dimethylthiourea, a hydroxyl radical scavenger, also protected rats against the decrease in serum testosterone levels and the down-regulation of CYP2C11 and CYP3A2. Carboplatin, an analogue to cisplatin but no ARF inducer, did not cause decrease in serum testosterone levels and down-regulation of hepatic male specific P450 enzymes. These results suggest that down-regulation of hepatic P450 enzymes in male rats given cisplatin is closely related to the cisplatin-induced ARF and the resultant impairment of testis function.

The induction and inhibition of human cytochrome P450 (P450) enzymes are clinically responsible for drug interactions. Although the induction of P450s is investigated using human hepatocytes in the drug development process, there are some disadvantages, such as the decline of the enzyme activity during culture. In the present study, we examined the in vivo induction potency in chimeric mice with humanized liver, which was recently established in Japan to clarify whether this chimeric mouse model would be more suitable for human induction studies. Rifampicin and 3-methylcholanthrene (3-MC) were used in vivo as typical P450 inducers in the chimeric mice. The expression levels of human CYP3A4 mRNA and CYP3A4 protein and dexamethasone 6-hydroxylase activity, specific for human CYP3A4, were increased 8- to 22-, 3- to 10-, and 5- to 12-fold, respectively, by treatment with rifampicin. In addition, the expression levels of human CYP1A2 mRNA and CYP1A2 protein were also increased 2- to 9- and 5-fold, respectively, by treatment with 3-MC. Although other human P450s are expressed in the chimeric mice, there were few effects by the treatment of rifampicin and 3-MC on the mRNA, protein, and enzyme activity of those P450s. It was demonstrated that human P450s expressed in the chimeric mice with humanized liver were induced by rifampicin and 3-MC. This chimeric mouse model may be a useful animal model to estimate and predict the in vivo induction of P450s in humans.

Chimeric mice with near-completely humanized liver were constructed by transplantating hepatocytes from a Japanese and Caucasian donor. In the present study, we investigated the induction of human CYP1A2 and CYP3A4 mRNA in a primary culture of the cryopreserved chimeric mouse hepatocytes. beta-naphthoflavone (beta-NF) and rifampicin (Rif) were used as typical cytochrome P450 (CYP) inducers for CYP1A2 and CYP3A4, respectively. Analysis was performed by the real-time reverse-transcription polymerase chain reaction method. CYP1A2 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 3.8-, 6.3-, and 3.3-fold by 5 microM beta-NF exposure, respectively, compared with the 0.1% DMSO treated control (p<0.01). CYP3A4 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 8.4-fold (p<0.001), 2.2-fold (p<0.01), and 2.3-fold (p<0.05) by 50 microM Rif exposure, respectively, compared with the 0.1% DMSO treated control. The present study demonstrated that a primary culture of cryopreserved hepatocytes from chimeric mice with humanized liver could be used for evaluating the induction of drug metabolizing enzymes in human. This in vitro method may be a useful method for screening the induction potency of new drug candidates on drug metabolizing enzymes.

Recently, a chimeric mouse line in which the liver could be replaced by more than 80% with human hepatocytes was established in Japan. Because the chimeric mouse produces human albumin (hAlb), replacement by human hepatocytes could be estimated by the hAlb concentration in the blood of chimeric mice. In this study, we investigated human major cytochrome P450 (P450) in the livers of chimeric mice by mRNA, protein, and enzyme activity using real-time polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Chimeric mice with humanized liver generated using hepatocytes from a Japanese and white donor were used. Human P450 mRNAs were expressed in the liver of chimeric mice, and major human P450 proteins such as CYP1A2, CYP2C9, and CYP3A4 were detected. The expression of P450 mRNA and protein was correlated with the hAlb concentration in the blood. The enzyme activities such as diclofenac 4'-hydroxylase activity, dexamethasone 6-hydroxylase activity, and coumarin 7-hydroxylase activity, activities that are specific to human P450 but not to murine P450, were increased in a hAlb concentration-dependent manner. The chimeric mice with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human P450s and drug-metabolizing enzyme activity as those of the donor. It was confirmed that genomic DNA from the livers of the chimeric mice and that from the liver of the donor exhibited the same genotype. In conclusion, the chimeric mice exhibited a similarly efficient capacity of drug metabolism as humans, suggesting that they could be a useful animal model for drug development.

Background/aims: Repetitive indomethacin administration induces down-regulation of hepatic cytochrome P450 (CYP) enzymes. We tested the hypothesis that an increase in intestinal permeability by indomethacin-induced intestinal injury leads to entry of bacterial endotoxin and reaching into liver via the portal vein, resulting in down-regulations of CYPs., Methods: C3H/HeJ mice, which are resistant to endotoxin, have a mutation in Toll-like receptor 4 gene. The sensitivity to indomethacin-induced impairment of hepatic CYPs in the lipopolysaccharide (LPS)-resistant mice was examined along with LPS-sensitive (C3H/He) mice., Results: Treatment of the LPS-sensitive mice with intraperitoneal indomethacin (5 mg/kg per day, 3 days) significantly decreased enzyme activities for CYP3A11, CYP2D9 and CYP1A2 but not CYP2E1. The LPS-resistant mice were resistant to the indomethacin-induced impairment of CYP2D9. The mice were also less sensitive to the effects on CYP3A11 and CYP1A2, but the activities for these isozymes in the indomethacin-treated mice were still lower than in untreated mice. Immunoblot analysis with anti-CYP3A2 and anti-CYP2D2 sera indicated that indomethacin-induced decreases in expression of the proteins recognized by the antibodies were attenuated in the LPS-resistant mice., Conclusions: We conclude that Toll-like receptor 4 is involved in the indomethacin-induced down-regulation of hepatic CYP enzymes, indicating the pivotal role of gut-derived endotoxin in the hepatic effects.

Incubation of human liver microsomes with diclofenac in the presence of NADPH resulted in a decrease in testosterone 6 beta-hydroxylation activity. The decrease in the activity followed time- and concentration-dependent kinetics, required oxidative metabolism, and was resistant to reduced glutathione, suggesting that diclofenac causes a mechanism-based inactivation of cytochrome p450 (p450) 3A4 (CYP3A4). The inactivation was reproduced by using microsomes from B-lymphoblastoid cell lines expressing CYP3A4 instead of human liver microsomes. No other monooxygenase activities measured as indexes of p450 enzymes; CYP2C8, CYP2C9, or CYP2C19 was inactivated by the same incubation procedure. Quinidine, a stimulant of CYP3A4-mediated diclofenac 5-hydroxylation, did not affect the inactivation of CYP3A4 assessed by testosterone 6 beta-hydroxylation activity but accelerated the inactivation assessed by diazepam 3-hydroxylation activity. These results supported the idea that diclofenac 5-hydroxylation is involved in the inactivation of CYP3A4 and described for the first time a stimulation of mechanism-based inactivation attributable to CYP3A4 heterotropic cooperativity. Preincubation of human liver microsomes with 5-hydroxydiclofenac instead of diclofenac did not cause the inactivation of CYP3A4, suggesting that 5-hydroxydiclofenac is not a precursor of a postulated reactive metabolite that inactivates CYP3A4, and thus 5-hydroxylation step is critical to inactivation of CYP3A4.

Background & Aims: Indomethacin impairs liver microsomal monooxygenase activities mediated by cytochrome P450 (CYP). We investigated the inhibition mechanism and the isoform selectivity in vitro and in vivo., Methods: In an in vitro study, liver microsomes from male Wistar rats were preincubated with indomethacin and a reduced nicotinamide adenine dinucleotide phosphate-generating system, followed by assay of monooxygenase activities indicative of several CYP isoforms. In an in vivo study, rats were intraperitoneally treated with indomethacin, followed by preparation of microsomes and the enzyme assays., Results: The preincubation of microsomes with indomethacin and reduced nicotinamide adenine dinucleotide phosphate decreased CYP3A2 activity but not any other isoforms. Kinetic analysis showed the mechanism-based inactivation of CYP3A2. The metabolism of [14C]indomethacin resulted in covalent binding to microsomal protein, which was diminished by inhibiting CYP3A enzyme. Administration of indomethacin caused impairment of not only CYP3A2 but also other CYP isoforms. Rats were protected from the impairment of the CYP enzymes except CYP3A2 by depleting macrophages and inhibiting inducible nitric oxide synthase., Conclusions: Metabolism of indomethacin causes inactivation of CYP3A2, which is the result of the covalent binding of its metabolite, whereas partially selective in vivo impairment of CYP isoforms is suggested to be indirect inhibition by inflammatory mediators probably released from Kupffer cells.

Repetitive administration of propranolol (PL) in rats decreases the activities of cytochrome P450 (CYP) 2D enzyme(s) in hepatic microsomes. We examined the properties of 4-hydroxypropranolol (4-OH-PL) as an inactivator of rat liver microsomal CYP2D enzyme(s) using bunitrolol (BTL) 4-hydroxylation and PL 5- and 7-hydroxylations as indices of CYP2D enzyme activity. Rat microsomal BTL 4-hydroxylase activity was inhibited by the addition of 4-OH-PL to the incubation medium. The inhibition was greater after preincubation of microsomes with 4-OH-PL in the presence of NADPH than in its absence. The type of inhibition kinetics of BTL 4-hydroxylase by 4-OH-PL was changed from a competitive type to a noncompetitive type by the preincubation. The inhibition of rat liver microsomal PL 5- and 7-hydroxylases by 4-OH-PL was blocked efficiently by co-incubation with quinine, a typical inhibitor of rat CYP2D enzyme(s), or to a lesser extent by BTL. However, quinidine, a diastereomer of quinine, did not significantly protect against the enzyme inactivation. The protective capacities of the substrate and inhibitors reflected their affinities for rat CYP2D enzyme(s). BTL hydroxylase was not affected by either 1,4-naphthoquinone or 1,4-dihydroxynaphthalene which are possible metabolites of 4-OH-PL. These results provide further evidence to support the notion that PL is biotransformed by rat CYP2D enzyme(s) to 4-OH-PL, which is further oxidized to a chemically reactive metabolite in the active site. The inactivation of CYP is likely the result of covalent binding of the reactive species to an amino acid residue of the active site.

Enzymatic formation of desipramine (DMI) and imipramine N-oxide (IMINO) was kinetically characterized in rat liver microsomes at pH 8.5 and 7.5. The formation of IMINO was quickly suppressed by the preincubation of microsomes at 37 degrees C at pH 8.5, but the suppression was comparatively gentle at pH 7.5. In kinetic studies, the formation of DMI was monophasic at the two pH points, and a substrate inhibition was observed at pH 8.5, but not at pH 7.5. In contrast, the formation of IMINO was biphasic at both pH points, Le., the summation of a low-Km phase and a high-Km phase. Methimazole (MZ), an inhibitor of flavin-containing monooxygenase (FMO), markedly suppressed the low-Km phase of IMINO formation at both pH points. MZ also suppressed DMI formation at pH 8.5, but it elevated DMI formation at pH 7.5. SKF 525-A, an inhibitor of cytochrome P450 (CYP), markedly suppressed DMI formation at both pH points. The inhibitor suppressed IMINO formation in the high-Km phase of the biphasic kinetics at both pH points, whereas it stimulated the activity of the low-Km phase at pH 7.5. These results suggest that CYP enzyme(s) are mainly responsible for DMI formation at pH 8.5 and 7.5, and FMO enzyme(s) also are involved in IMI N-demethylation at a higher pH range in rat liver microsomes, at least in part. In the formation of IMINO, FMO is a major enzyme at both pH points, and CYP may also contribute to the N-oxide formation to some extent at pH 8.5.

Organ and cellular distribution and expression constancy of microsomal cytochrome P450 (CYP) 2C and 3A in humans were studied with new polyclonal antibodies to CYP2C (MP-1) and 3A (NF-2) active in formalin-fixed, paraffin-embedded tissues. Antibodies were raised against purified human CYP2C9 and CYP3A4. On western blotting, MP-1 reacted with 2C8, 2C9, 2C18 and 2C19, and NF-2 with 3A4. In both frozen and paraffin sections, hepatocytes showed diffuse immunoreactivity with MP-1 and centrilobular staining with NF-2. In-paraffin sections of 40 kinds of nonneoplastic tissues, epithelium of the small and large intestine, bile duct, nasal mucosa, kidney and adrenal cortex stained positively with both MP-1 and NF-2 antibodies. Epithelium of gastric fundic glands, salivary glands, tracheobronchial glands, Brunner's glands, the prostate, uterine cervix and nasopharynx showed definite reactivity with MP-1. Epithelium of the gastric mucosa with intestinal metaplasia, duodenum, gallbladder and intercalated ducts of the pancreas and chief cells of the parathyroid and the corpus luteum of the ovary reacted with NF-2. Among the neoplastic tissues, MP-1 reacted with pleomorphic adenoma of the salivary gland and carcinomas of six different organs, and NF-2 with those of 7 different organs. These results indicate that CYP2C and CYP3A are distributed widely and organ specifically, as well as being variably expressed in neoplastic and normal states.

The antidepressants, imipramine and mianserin, have been reported to cause liver damage. We investigated a role of cytochrome P450 (CYP)-mediated formation of a reactive metabolite in antidepressant-induced acute cell injury using hepatocytes isolated from male and female Wistar rats, and male Dark Agouti rats, which have different relative abundance of CYP enzymes. Culture of the hepatocytes with imipramine and mianserin caused a marked decrease in glutathione followed by protein thiol, which preceded lactate dehydrogenase leakage. The decreases in glutathione and protein thiol contents by imipramine were significantly slower in hepatocytes from male Dark Agouti rats than those from male Wistar rats, whereas no significant sex difference in Wistar rats was observed. The decrease in thiol by mianserin was significantly slower in hepatocytes from female Wistar than those from male Wistar rats, whereas no significant differences were found between Wistar and Dark Agouti males. Results consistent with alteration of the thiols were obtained for lactate dehydrogenase leakage induced by imipramine and mianserin. These findings indicated that CYP-mediated metabolic activation was involved in acute cell injury induced by the antidepressants; namely a CYP2D enzyme(s), which is deficient in Dark Agouti rats, and a male specific CYP enzyme(s) were suggested to be responsible for the cytotoxicity of imipramine and mianserin, respectively.

Trimethadione (TMO) has the properties required of a probe drug for the evaluation of hepatic drug-oxidizing capacity and, in this study, we have summarized the in vivo and in vitro metabolism of TMO in various animal species including mouse, hamster, rat, rabbit, dog, monkey and human. In the in vivo study, the plasma TMO level was measured after intravenous or oral (human) administration of TMO at a dose of 4 mg/kg to various animal species. The rate of TMO metabolic clearance in these animal species in vivo was in the order mouse > hamster > rat > rabbit > dog > monkey > human. In the in vitro study, species differences were observed in the cytochrome P450 (P450) content and drug-oxidizing enzyme activity. The content of P450 was monkey> mouse > dog > rabbit > hamster > rat > human. On the other hand, TMO N-demethylation was in the order mouse > hamster > rat > rabbit > dog > monkey > human. There was a good correlation between the mean total body clearance of TMO (in vivo) and the mean TMO N-demethylase activity (in vitro) (y=1.7 x +0.11, r=0.965, P < 0.001). These results show that TMO is a probe agent with metabolic and pharmacokinetic characteristics making it attractive for the in vivo and in vitro characterization of metabolic activity in various animal species.

The expression of cytochrome P450 3A4 (CYP3A4) in the foveolar epithelium of the human stomach with intestinal metaplasia was studied using immunohistochemistry, western blotting and reverse transcription polymerase chain reaction (RT-PCR). CYP3A4 was immunohistochemically detected in the foveolar epithelium with intestinal metaplasia, but was not detected in foveolar epithelium without intestinal metaplasia, in the pyloric gland or in the fundic gland of the stomach. Western blotting and RT-PCR demonstrated that CYP3A4 protein and mRNA were expressed in the liver and pyloric gland mucosa with intestinal metaplasia, but not in the fundic gland mucosa without intestinal metaplasia. Possible roles of CYP expression in the gastric mucosa with intestinal metaplasia in human stomach carcinogenesis are briefly discussed.

The contribution of induced cytochrome P450 (P450) isozymes (CMLa; CYP2B, CMLb; CYP2A and CMLc; CYP3A) and related enzymes to trimethadione (TMO) metabolism in phenobarbital-treated rhesus monkey were investigated. The animals received a single dose of TMO (4 mg kg-1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later. Phenobarbital-treatment (20 mg kg-1 day-1 for 3 days; i.p.) significantly increased the plasma dimethadione (DMO)/TMO ratios at 0.08, 0.5, 1 and 2 h one's appropriate controls. Phenobarbital treatment also increased the P450 content (1.7-fold) and activity of aniline p-hydroxylase (1.3-fold), p-nitroanisole O-demethylase (1.8-fold) and benzphetamine N-demethylase (2.3-fold). The content of CMLa, CMLb and CMLc were increased about 12.8, 2.3 and 2.7-fold by phenobarbital pretreatment, respectively. The activity of TMO N-demethylation was inhibited by anti-P450 CMLa and anti-P450 CMLb. However, the anti-P450 CMLc antibody had no effect on this activity in liver microsomes. The results of both in vivo and in vitro studies of the effects of phenobarbital treatment on TMO metabolism indicate that these effects may be attributed to the induction of CMLa. These findings suggest that plasma DMO/TMO ratio in a single blood sampling after TMO administration is very useful for determination the degree of hepatic induction in clinical study.

Studies to assess the enzyme kinetic behavior and to identify the cytochrome P450 (CYP) isoform(s) involved in the major metabolic pathway (N-demethylation) for citalopram (CIT), a selective serotonin reuptake inhibitor, were performed using human liver microsomes and cDNA-expressed human cytochrome P450 isoforms. The N-demethylation activities showed significant correlations with the alpha- and 4-hydroxylation activities of triazolam (r(s) = 0.818 and 0.851, respectively; P < .01) in 10 different human liver microsomes. Anti-CYP3A antibodies and ketoconazole strongly inhibited CIT N-demethylation. In addition, there was a significant correlation between CIT N-demethylation and (S)-mephenytoin 4'-hydroxylation (r(s) = 0.773, P < .05), although little inhibition was observed in the presence of anti-CYP2C antibodies or (S)-mephenytoin. cDNA-expressed CYP3A4 and CYP2C19 catalyzed CIT N-demethylation, whereas no appreciable activities were observed for CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6 and CYP2E1. The percentage contributions of CYP3A4 and CYP2C19 to the overall N-demethylation of CIT in human liver microsomes were estimated using a relative activity factor; respective values of 70% and 7% were calculated for microsomes obtained from livers from putative extensive metabolizers for (S)-mephenytoin 4'-hydroxylation. These results suggest that CYP3A4 is the major isoenzyme and CYP2C19 is the minor form involved in the major metabolic pathway for CIT in human liver microsomes.

Repeated oral administration of propranolol (PL, 100 mg/kg daily, for 5, 10 and 15 days) to male Wistar rats increased PL N-desisopropylase and decreased PL 4-,5- and 7-hydroxylase activities in liver microsomes. The increase was highest at the 10 day time point whereas the decrease was relatively constant over the 15 day treatment period. There were no significant changes in the total content of cytochromes P450 (P450) or cytochrome b5 or in NADPH-cytochrome c reductase activity during the PI, treatment. The enhanced N-desisopropylase activities were markedly inhibited by alpha-naphthoflavone (a P450-1A1/2 inhibitor), and moderately by triacetyloleandomycin (a P450-3A1/2 inhibitor) and diethyldithiocarbamate (a P450-2E1 inhibitor). Phenacetin O-deethylase activity, an index of P450-1A2, was significantly increased on day 5, 10 and 15 of the treatment, whereas p-nitrophenol hydroxylase activity was elevated on day 10 only. The PL N-desisopropylation showed a strong and significant correlation with phenacetin O-deethylation, and a weaker but significant correlation with p-nitrophenol hydroxylation. Immunoblot analysis revealed that a protein band corresponding to P450-1A2 was increased by PL pretreatment, and protein band corresponding to P450-3A tended to be increased slightly, but other protein band corresponding to the subfamily of P450-2B, -2C, or -2E was not changed. Pretreatment of rats with P450 inducers (beta-naphthoflavone, phenobarbital, acetone and dexamethasone) increased PL N-dealkylase activity in liver microsomes. Furthermore, antibodies raised against P450-1A and -3A enzymes suppressed PL N-desisopropylation in a concentration-dependent manner, but P450-2E antibody did not. Reconstitution studies showed that P450-1A1, -1A2, -2E1 and -3A2 exhibited catalytic activities for PL N-dealkylation. These results suggest that P450-1A2 is a major PL N-desisopropylase in the PL-treated rats, and P450-3A related enzyme(s) and P450-2E1 as a moderate or minor enzyme are also involved in PL N-dealkylation in native and PL-treated rats.

Monooxygenase activities in rat renal microsomes were determined with the substrates of hepatic CYP2D enzymes. Seven kinds of CYP2D-mediated monooxygenase activities and immunochemically determined CYP2D contents in kidneys corresponded to approximately 3% of those in livers. Debrisoquine 4-hydroxylase and bunitrolol 4-hydroxylase in renal microsomes were inhibited almost completely by the antibody against a CYP2D enzyme purified from rat liver. A marked strain difference (Wistar > Dark Agouti) in these activities was observed in kidney like in liver. The two hydroxylases were inhibited stereoselectively by quinine and quinidine both in renal and hepatic microsomes. Substrate stereoselectivity in (+)- and (-)-bunitrolol 4-hydroxylase activities in kidneys was also consistent with that in livers. These results suggested that the CYP2D enzyme(s) was expressed in the kidney at levels much less than in the liver but had similar functions to those in the liver.

Repetitive oral administration of imipramine (100 mg/kg/day for 5 days) caused a decrease in rat liver microsomal debrisoquine 4-hydroxylase activity, a characteristic reaction catalyzed by cytochrome P450 (CYP) 2D1. Other CYP2D-dependent reactions (such as bunitrolol 4-hydroxylation, lidocaine 3-hydroxylation, and propranolol 4-, 5- and 7-hydroxylations) were also impaired by the treatment, but not those catalyzed by other CYP isozymes. Imipramine pretreatment did not change the immunochemically determined content of the CYP2D protein, suggesting that CYP2D is inactivated. Imipramine pretreatment also resulted in an increase in total CYP content and in formation of a ferrous CYP metabolic intermediate (MI)-complex absorbing at 454 nm. Although the total CYP content was increased by the treatment of these microsomes with ferricyanide to dissociate the MI-complex, the CYP2D-dependent activities were not restored, suggesting that the MI-complex was not the primary cause of CYP2D inhibition. This pretreatment regimen caused marked increases in immunochemically determined levels of CYP2A1, CYP2B1, CYP2B2, CYP2C6, and CYP3A2, and in the activities of 2 alpha-, 2 beta-, 6 beta-, 7 alpha-, 16 alpha-, and 16 beta-hydroxylation and 17-oxidation of testosterone. These results indicate that imipramine has two actions on the liver CYP system (i.e. as an inhibitor of the CYP2D enzyme and as a phenobarbital-type inducer).

We examined the oxidative metabolism of a beta-blocker bunitrolol (BTL) by 10 human cytochromes P450 (CYP) (1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, 3A3, 3A4 or 3A5), which were individually expressed in Hep G2 cells with a vaccinia virus complementary DNA expression system. Among the 10 isozymes, only CYP2D6 and 1A2 at a substrate concentration of 5 microns, and CYP2C8 and 2C9 in addition to the two isozymes at a BTL concentration of 1 mM, exhibited detectable BTL 4-hydroxylase activities. The activities at 1 mM BTL were on the order of CYP2D6 (100% as relative activity) > CYP1A2 (86%) >> CYP2C8 and 2C9 (7-8%). Enzyme kinetic parameters of CYP2D6 were calculated to be 4.41 microns as a Km value and 0.442 nmol min-1 per nmol CYP as a Vmax value. Kinetic parameters of CYP1A2 were calculated as 295 microns and 0.411 nmol min-1 per nmol CYP for Km and Vm values, respectively. These results suggest that both CYP2D6 and 1A2 primarily catalyse BTL 4-hydroxylation, but that the former is a predominant isozyme responsible for the reaction at a low substrate concentration range of BTL in human liver.

We isolated a form of cytochrome P450 (P450) from hepatic microsomes of untreated doguera baboons. The final preparation (referred to as P450 BLa) was apparently homogenous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The estimated minimum molecular weight of P450 BLa was 50 kDa. The N-terminal amino acid sequence of P450 BLa (identified 10 residues) was identical with that of P450 3A8 purified from cynomolgus monkeys. This protein was cross-reactive with antibodies raised against P450 3A4 and P450 CMLc which were P450 3A enzymes purified from hepatic microsomes of humans and cynomolgus monkeys, respectively. P450 BLa was capable of catalyzing testosterone 6 beta-hydroxylation and zonisamide reduction. P450 BLa antibody inhibited the activity of testosterone 6 beta-hydroxylase, but not the activities of testosterone 16 alpha- and 16 beta-hydroxylases in liver microsomes of doguera baboons. From these lines of evidence we conclude that P450 BLa can be classified as part of the P450 3A subfamily and acts as a constitutive testosterone 6 beta-hydroxylase in hepatic microsomes of doguera baboons.

Oxidative metabolic pathways of propranolol consist of naphthalene ring-hydroxylations (at the 4-, 5-, and 7-positions) and side-chain N-desisopropylation in mammals. We characterized cytochrome P450 isozymes responsible for propranolol metabolism, especially N-desisopropylation and 5-hydroxylation, in human liver microsomes. 4-Hydroxy, 5-hydroxy-, and N-desisopropylpropranolol were detected as primary metabolites, whereas 7-hydroxypropranolol was in trace amounts. Good correlations were obtained for activities of propranolol 4- and 5-hydroxylases with immunochemically determined CYP2D6 content, whereas correlations of these activities with CYP1A2, CYP2C, or CYP3A4 content were relatively low. The activities also correlated highly with debrisoquine 4-hydroxylase, compared with other metabolic activities such as phenacetin O-deethylase, hexobarbital 3'-hydroxylase, and testosterone 6 beta-hydroxylase, which are typical reactions for CYP1A2, CYP2C, and CYP3A4, respectively. Propranolol N-desisopropylase activity in the samples highly correlated with CYP1A2 content and phenacetin O-deethylase activity, but not with the other P450 isozyme contents or metabolic activities. Quinidine, a specific inhibitor of CYP2D6, inhibited propranolol 4- and 5-hydroxylase activities selectively and in a concentration-dependent manner. alpha-Naphthoflavone, a potent inhibitor of CYP1A2, inhibited all of the propranolol oxidation activities, and the IC50 value for N-desisopropylase activity was much smaller than the values for ring-hydroxylase activities. Antibody directed to CYP2D inhibited propranolol 4- and 5-hydroxylase activities by 70% at an antibody/microsomal protein ratio of 1.0. Anti-CYP2C9 antibody did not inhibit any activity determined. These results indicate that propranolol 5-hydroxylation, as well as 4-hydroxylation, is mainly catalyzed by CYP2D6 in human liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

We isolated a new form of cytochrome P450 (P450) which was able to catalyze S-mephenytoin 4'-hydroxylation from hepatic microsomes of cynomolgus monkeys. The final preparation (referred to as P450 CMLd) was apparently homogenous judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the estimated minimum molecular weight of this protein was 53 kDa. The N-terminal amino acid sequence of P450 CMLd (identified 16 residues) was identical with that of protein encoded by P450 2C9 cDNA. P450 CMLd was cross-reactive with both antibodies raised against P450 2C11 and P450 2C9 which were purified from hepatic microsomes of male rats and humans, respectively. In hepatic microsomes of cynomolgus monkeys, both antibodies recognized two proteins showing different mobilities on SDS-PAGE (50 and 53 kDa). P450 CMLd was a good catalyst for S-mephenytoin 4'-hydroxylation in a reconstituted system. Anti-P450 2C9 antibody inhibited the activity of S-mephenytoin 4'-hydroxylase, but not the activities of R-mephenytoin 4'-hydroxylase and R- and S-mephenytoin N-demethylases in liver microsomes from cynomolgus monkeys. From these lines of evidence we conclude that P450 CMLd is classified into the P450 2C subfamily and acts as one of the S-mephenytoin 4'-hydroxylases in hepatic microsomes of cynomolgus monkeys.

1. We have examined the effects of (S)-(+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8, 11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3':4,5]thieno[3, 2-f][1, 2, 4]triazolo[4, 3-a][1, 4]diazepine (E-6123), a novel thienodiazepine platelet-activating factor antagonist, on drug-oxidizing capacity in beagle dog, using antipyrine (AP) and trimethadione (TMO) as two model substrates. 2. The plasma half-life (t1/2) and area under the curve (AUC) of AP (0.5 mg/kg, i.v. injection) increased in a dose-dependent manner after a single oral dose of E-6123 (0.2, 1 or 10 mg/kg), whereas the total body clearance (Cl) of AP was decreased, and the apparent volume of distribution (Vd) was unchanged. 3. The pharmacokinetic parameters (t1/2, Cl and AUC) of the metabolism of TMO (4 mg/kg, i.v.) after repeated oral administration of E-6123 (10 mg/kg for 7 days) were not significantly changed in comparison with findings in control dog. The ratio of dimethadione (DMO), being the only TMO metabolite, to TMO in plasma after i.v. administration of TMO in E-6123-treated dog was increased only 5 and 15 min after the final dose, but was not changed at other sampling times (0.5, 1, 2 4, 6, 8 and 12 h). 4. The content of b5, the activity of p-nitroanisole O-demethylase and benzphetamine N-demethylase were significantly increased, compared with controls, by repeated E-6123 treatment. However, aniline hydroxylase activity was not significantly changed. 5. Content of P450 2B was significantly increased in E-6123 treated dog, while that of 3A was not.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined the effect of successive administrations of aztreonam, which is used clinically as an antibiotic, on the mixed function oxidase system in non-human primates. Treatment of cynomolgus monkeys with aztreonam at doses of between 40 and 300 mg/kg for 4 weeks resulted in a significant decrease in the content of hepatic microsomal P450. On the other hand, no significant change was observed in hepatic cytochrome b5 content and NADPH-cytochrome c (P450) reductase activity following treatment with aztreonam. The activity of testosterone 6 beta-hydroxylase, but not 2 beta- and 16 alpha-hydroxylases in hepatic microsomes, was decreased following the treatment of cynomolgus monkeys with aztreonam. The content of P450 CMLc, which is classified into the 3A subfamily, was also decreased by aztreonam treatment although the content of P450 CMLb, which is a 2A enzyme in cynomolgus monkeys, was unchanged. From these results, we concluded that aztreonam decreased P450 CMLc in hepatic microsomes of cynomolgus monkeys.

Two forms of cytochrome P450, referred to as P450 CMLb and P450 CMLc, were purified and characterized from hepatic microsomes of untreated cynomolgus monkeys (Macaca irus). The final preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The minimum molecular weights of P450 CMLb and P450 CMLc estimated from the mobilities on the gel were 48 and 50 kDa, respectively. The N-terminal amino acid sequences of P450 CMLb and P450 CMLc (first 13 residues) were identical, respectively, with those of P450 FI isolated from baboons and P450 MK-2, which has been characterized as a P450 3A enzyme in cynomolgus monkeys. P450 CMLb showed coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation activities. This P450 enzyme reacted with anti-P450 2A6 antibody and acts as a coumarin 7-hydroxylase in hepatic microsomes. On the basis of these results P450 CMLb was classified into the 2A subfamily. P450 CMLb is expressed constitutively as one of the minor forms of P450 in liver microsomes of untreated cynomolgus monkeys and is inducible by phenobarbital. P450 CMLc reacted with anti-P450 3A4 antibody and hydroxylated testosterone at the 6 beta-position. The testosterone 6 beta-hydroxylation activities in hepatic microsomes of cynomolgus monkeys, common squirrel monkeys, and humans were strongly inhibited by the anti-P450 CMLc antibody. These results demonstrate that this P450 enzyme is in the P450 3A subfamily.

A cytochrome P450 (P450) (referred to as P450CMLa) was purified and characterized from hepatic microsomes from untreated cynomolgus monkeys (Macaca irus). The final preparation was electrophoretically homogeneous and its estimated minimum molecular mass was 49.5 kDa. The amino-terminal amino acid sequence of the protein (first 34 residues) closely resembled that of the protein encoded by the 2B6 cDNA from humans (94%). This protein was cross-reactive with antibodies raised against P450 2B1 (P450 b), P450 2B11 (P450 PBD-2), and P450GP-1, which were purified from hepatic microsomes from phenobarbital-pretreated rats, beagle dogs, and guinea pigs, respectively. Also, the antibody raised against P450CMLa was able to cross-react with P450 2B1, P450 2B11, and P450GP-1. P450CMLa was capable of catalyzing benzphetamine N-demethylation and testosterone 16 beta-hydroxylation in a reconstituted system. Anti-P450CMLa antibody inhibited the activity of testosterone 16 beta-hydroxylase but not the activities of testosterone 2 beta- and 6 beta-hydroxylases in liver microsomes from cynomolgus monkeys. The content of P450CMLa, as estimated by immunoblot analysis, was 70 pmol/mg (about 5% of total P450). The protein immunoreactive with the anti-P450CMLa antibody was also present in liver microsomes from Japanese monkeys, baboons, common marmosets, and common squirrel monkeys. In liver microsomes from common squirrel monkeys, the content of protein immunoreactive with the anti-P450CMLa antibody and the activity of testosterone 16 beta-hydroxylase were effectively increased by pretreatment with phenobarbital. The antibody against P450CMLa strongly inhibited the activity of testosterone 16 beta-hydroxylase in liver microsomes not only from untreated cynomolgus monkeys but also from phenobarbital- and pregnenolone 16 alpha-carbonitrile-pretreated common squirrel monkeys. These results indicated that the P450CMLa purified here is very similar to the forms of P450 classified into the 2B subfamily, in its amino-terminal amino acid sequence, catalytic activities, and immunochemical properties.

Eisai-hyperbilirubinuria rats (EHBR) is a mutant originated from Sprague Dawley rats. The activities of UDP-glucuronyltransferase and drug metabolizing enzymes in EHBR were compared with those in Sprague Dawley rats as the control. The activity of aniline hydroxylase was significantly increased in liver microsomes of EHBR whereas the activity of ethylmorphine N-demethylase was found to be significantly decreased in EHBR as compared to control rats. In addition, the activity of testosterone 7 alpha-hydroxylase was increased in EHBR whereas the activity of testosterone 6 beta-hydroxylase was significantly decreased in EHBR as compared to control rats. Western blot analysis of liver microsomes of EHBR with antibodies to P-450IA2, P-450IIB1, P-450IIC11 and P-450IIIA2 showed that the amounts of P-450IIB1 and P-450IIIA2 in liver microsomes were significantly lower in EHBR than in control rats. These results indicated the form-specific alteration in the amounts of cytochrome P-450 in liver microsomes of EHBR.

Induction of cytochrome P-450 PCN (P-450 PCN) by non-steroidal compound, M79193 (cyclohexane spiro-2,6-chloro-1'-(3-dimethyl-aminopropyl) spiro(chroman-4,4'-imidazolidine)-2',5'-dione], was investigated in both sexes of rats. The immunohistochemical localization of P-450 PCN was also studied in liver lobules of untreated and M79193-treated male rats. Immunoblot analysis of rat liver microsomes with anti-P-450 PCN indicated that the amount of P-450 PCN increased 5- to 7-fold by the treatment with M79193, but no increase was observed in P-450 PB-1 (P450IIB1) or P-450 MC-1 (P-450IA1). P-450 PCN was uniformly distributed in liver lobule of untreated rats, and was significantly increased by the treatment with M79193 in both the periportal and pericentral regions of the lobules.

The relationship between the structure of spirohydantoin derivatives and their inducing effects on hepatic monooxygenase system was studied. At the dose of 1 mumol/kg (0.3 mg/kg), 6-fluoro-2-methylspiro(chroman-4,4'-imidazolidine)-2',5'-dione (M79175) was found to exhibit an inducing effect on benzphetamine N-demethylase. On the contrary, the inducing effect of Sorbinil, in which the 2-methyl group on the chroman ring of M79175 was replaced by hydrogen atom, on benzphetamine N-demethylase was 100 times less than that of M79175. The substitution of the methyl group of M79175 with the dimethyl group did not affect the inducing effect, whereas the substitution with the hexyl group resulted in the loss of the inducing effect on drug oxidation activities tested at a dose of 10 mg/kg. Furthermore, cyclohexane spiro-2,6-chloro-1'-(3-dimethylaminopropyl)spiro(chroman-4,4'-imidazolid ine)-2' , 5'-dione (M79193) had an inducing effect on total cytochrome P-450 content, but had no inducing effect on benzphetamine N-demethylase. In addition, M79175 was found to induce cytochrome P-450 which is immunochemically related to one of the major forms of phenobarbital-inducible cytochrome P-450 (P-450(PB-1], but not cytochrome P-450 which is immunochemically related to the major form of 3-methylcholanthrene-inducible cytochrome P-450 (P-450(MC-1]. On the other hand, M79193 was found to induce neither P-450(PB-1) nor P-450(MC-1).

Effect of successive administration of Aztreonam on microsomal monooxygenase system was investigated in male and female Sprague-Dawley rats. The activities of benzphetamine N-demethylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes from male rats were decreased dose-dependently by Aztreonam. On the contrary, the activities in liver microsomes from female rats were slightly increased rather than decreased by the administration of Aztreonam. In addition, Aztreonam was found to decrease the specific content of microsomal cytochrome P-450 in male rats but not in female rats. The decreases in the activities observed in male rats were accompanied by a parallel decrease in the specific content of cytochrome P-450. Furthermore, the results of quantitation of P-450 (M-1), one of the male specific forms of cytochrome P-450, indicated that the administration of Aztreonam resulted in a dose-dependent decrease in the content of P-450 (M-1) in liver microsomes from male rats.

The localization of sex-specific cytochrome P-450, P-450 (M-1), was investigated immunohistochemically in the liver of untreated- and Aztreonam-treated male rats. P-450 (M-1) was uniformly distributed in the liver lobule of untreated rats. By treatment with Aztreonam, P-450 (M-1) was decreased more in the periportal region rather than the pericentral region of the lobule. In addition, oxidation of testosterone and 1 alpha-hydroxycholecalciferol catalyzed by P-450 (M-1) was also decreased by Aztreonam administration.

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24 h after the last treatment. 2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene. 3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes. 4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes. 5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30 min after treatment with the drug.