Pentoxyresorufin O-dealkylation Research Articles

Effectiveness of the liver micronucleus assay using juvenile mice.

This study investigated the effectiveness of the liver micronucleus (MN) assay using juvenile mice. Therefore, we analyzed various hepatic cytochrome P450 (CYP)- mediated activities of ethoxyresorufin O-deethylation, pentoxyresorufin O-dealkylation, tolbutamide hydroxylation, bufuralol 1’-hydroxylation, aniline hydroxylation and midazolam 4-hydroxylation by CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A, respectively, in non-treated male ICR mice aged between 3 and 8 weeks. The enzyme efficiency levels in 3- and 4-week-old mice were approximately similar to or higher than those in 8-week-old mice, except for CYP1A and CYP2E in 3- and 4-week-old mice, respectively. Since these results suggest that juvenile mice have sufficient activities for most CYP enzymes, we also conducted a liver MN assay using diethylnitrosamine (DEN), a rodent hepatocarcinogen, on male ICR mice aged between 3 and 6 weeks. A peripheral blood (PB) MN assay was performed simultaneously in 4-week-old mice. Assays incorporating DEN produced positive results in 3- and 4-week-old mice and showed a dose-dependent increase in the micronucleated hepatocyte frequencies at 4 weeks. Both the liver MN assay in 5- and 6-week-old mice and the PB MN assay had negative results when using DEN. These results suggest that 3- and 4-week-old mice have micronuclei-inducing potential in the liver to detect genotoxic compounds using the liver MN assay.

P.3.c.005 - Potent inhibition of CYP1A2 and CYP2D6 by the novel antipsychotic drug asenapine in human liver

In this study, we performed a screening of the specificities of rat cytochrome P450 (CYP) isoforms for metabolic reactions known as the specific probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation (DFH), dextromethorphan O-demethylation (DMOD) and midazolam 4-hydroxylation were specifically catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresorufin O-dealkylation and pentoxyresorufin O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 1′-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples.

Expression of microRNAs, CYP1A1 and CYP2B1 in the livers and ovaries of female rats treated with DDT and PAHs

AimsIn this study, we determined the expression level of miRNAs and the induction of CYP1A1 and CYP2B1 in the livers and ovaries of female Wistar rats treated with DDT, benzo[a]pyrene (BP), and 3-methylcholanthrene (MC). This study compared CYP1A/2B induction and miRNA expression levels to cast light on a possible role of miRNA in the tissue-specific induction of CYPs. Main methodsThe induction of CYP1A1/2B1 enzymes was detected by ethoxy-, pentoxyresorufin O-dealkylation and Western blot analysis. The CYP1A1/2B1 gene expression was determined by RT—real time PCR. Relative levels of expression for selected in silico miR species were determined by real time PCR with small nuclear U6 RNA employed as a reference gene. Key findingsAfter bioinformatic analysis, miR-21, 221, 222, and 429 were chosen as potential post-transcriptional regulators of rat CYP1A and CYP2B. It was shown that miR-21, 221, 222, and 429 expression levels decreased in the liver of DDT-, BP-, and MC-treated rats, whereas increases were observed in CYP1A1 and CYP2B1 mRNA expression levels and protein content, and EROD and PROD activities. Conversely, a tendency for elevated levels of miRNAs in the ovaries of inducer-treated female rats was observed. In the ovaries, a high level of CYP1A1 and CYP2B1 mRNA expression was observed, although protein content and enzyme activity were not visible. SignificanceThese data suggest a potential involvement of miRNA in the post-transcriptional regulation of CYP1A and CYP2B in the livers and ovaries of chemically induced rats.

A dual function of the furanocoumarin chalepensin in inhibiting Cyp2a and inducing Cyp2b in mice: the protein stabilization and receptor-mediated activation

Chalepensin, a furanocoumarin, is present in several medicinal Rutaceae plants and causes a mechanism-based inhibition of human and mouse cytochrome P450 (P450, CYP) 2A in vitro. To address the in vivo effect, we investigated the effects of chalepensin on multiple hepatic P450 enzymes in C57BL/6JNarl mice. Oral administration of 10mg/kg chalepensin to mice for 7days significantly decreased hepatic coumarin 7-hydroxylation (Cyp2a) and increased 7-pentoxyresorufin O-dealkylation (Cyp2b) activities, whereas activities of Cyp1a, Cyp2c, Cyp2e1, and Cyp3a were not affected. Without affecting its mRNA level, the decreased Cyp2a activity was accompanied by an increase in the immunodetected Cyp2a5 protein level. In chalepensin-treated mice, microsomal Cyp2a5 was less susceptible to ATP-fortified cytosolic degradation than that in control mice, resulting in the elevated protein level. The in vitro inactivation through NADPH-fortified pre-incubation with chalepensin also protected microsomal Cyp2a5 against protein degradation. Using cell-based reporter systems, chalepensin at a concentration near unbound plasma concentration activated mouse constitutive androstane receptor (CAR), in agreement with the observed induction of Cyp2b. These findings revealed that suicidal inhibition of Cyp2a5 and the CAR-mediated Cyp2b9/10 induction concurrently occurred in chalepensin-treated mice.

Different profiles of hepatic alkoxyresorufin O‐dealkylase activities in small rodents

The aims of the present study were to determine cytochrome P450 enzyme activity in six strains of experimental rodents (n = 5/sex/species): ICR, C57BL/6 and DBA/2 mice; Sprague Dawley and Wistar rats; and Dunkin Hartley guinea pigs. After animals were treated with the typical inducers β-naphthoflavone (BNF), dexamethasone (DEX) and phenobarbital (PB), the levels of O-dealkylation of ethoxyresorufin (EROD), methoxyresorufin (MROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) activity were determined using responsive catalytic reactions to study CYP1A1, CYP1A2 and CYP2B, respectively. A maximal induction of EROD and MROD was found in BNF-treated animals from all strains (2.4- to 15.1-fold) except DBA/2 (0.9- to 1.8-fold). C57BL/6 mice had the strongest BNF-induced EROD (15.1-fold) and MROD (8.3-fold) activities. No differences in BNF-induced EROD and MROD activities were observed between males and females. However, the EROD activity of Wistar rats and the MROD activity of Sprague Dawley rats were higher in males than females. DEX induced PROD activity only in mice (1.3- to 7.1-fold), but not in rats and guinea pigs (0.2- to 1.1-fold). However, induction of BROD activity was found in DEX-treated mice and rats (1.5 to 12.5-fold), but not in guinea pigs (0.3 to 0.4-fold). PB caused a significant elevation of PROD (1.7- to 10.4-fold) and BROD (31- to 13.2-fold) activities in all the animals. PB-induced BROD activity was higher in females than males in Sprague Dawley rats. These observations strongly suggest that the choice of experimental animal strain, species and inducer is of critical importance for studies of drug metabolism and interaction.

Modulatory effects of Kaempferia parviflora extract on mouse hepatic cytochrome P450 enzymes

Ethnopharmacological relevanceKaempferia parviflora is a herbal plant, the extracts of which are commonly used as alternative medicines. It widely uses as aphrodisiac, anti-inflammation, anti-microbacterial, and anti-peptic ulcer. Aim of the studyIn order to obtain an effective utilization and safety of the herb, the influence of Kaempferia parviflora on hepatic CYP450 metabolizing enzymes including CYP1A1, CYP1A2, CYP2B, CYP2E1, and CYP3A was investigated. Materials and methodsThe impact of Kaempferia parviflora on CYP450 both in vitro and in vivo was examined by using ethoxyresorufin O-dealkylation, methoxyresorufin O-dealkylation, pentoxyresorufin O-dealkylation, p-nitrophenol hydroxylation, and erythromycin N-demethylation assays, respectively. ResultsIn vitro studies using non-induced mouse hepatic microsomes in the presence or absence of Kaempferia parviflora extract showed that Kaempferia parviflora extract altered CYP1A1, CYP1A2, CYP2B, and CYP2E1 activities by non-competitive, mixed-competitive, competitive, and uncompetitive mechanisms, respectively. Among these enzymes, CYP1A2 was affected by Kaempferia parviflora based on the highest value of Vmax (15.276±0.206nmol/min) and lowest of Ki value (0.008±0.002μg/ml). In addition, the plant extract also modulated CYP2B activity based on the low Km value (1.599±0.147pmol). For in vivo studies, mice were orally treated with 250mg/kg of Kaempferia parviflora extract for 7, 14, and 21 days. The results demonstrated that Kaempferia parviflora extract significantly induced CYP1A1, CYP1A2 enzyme activities following short-term treatment. CYP2B enzyme activities were markedly increased all Kaempferia parviflora extract treatment timepoints, whereas Kaempferia parviflora extract significantly enhanced CYP2E1 activity only after long-term treatment. However, Kaempferia parviflora extract did not affect the CYP3A enzyme activity. ConclusionsKaempferia parviflora extract modulated several CYP450 enzyme activities, thus, its utilization with drugs or other herbs should raise concern for potential drug–herb interactions.

Verapamil does not modify catalytic activity of CYP450 in rainbow trout after long-term exposure

Little is known about the effects of the cardiovascular drug verapamil (VRP) on metabolic processes in fish. Most calcium channel blockers including VRP are metabolized by cytochrome P450 (CYP450) enzymes. In this study we investigated the in vivo effect of VRP on some CYP450-mediated reactions in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270μgl−1) for 0, 21, and 42 day. The following CYP450-mediated reactions were studied in hepatic microsomes: O-dealkylation of ethoxyresorufin, methoxyresorufin, and pentoxyresorufin, hydroxylation of coumarin, tolbutamide, and p-nitrophenol, and O-debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin. The amounts of products of these reactions did not differ among fish exposed to different levels of VRP and control fish. This suggests that the levels of VPR used did not alter catalytic activity of the selected CYP450 enzymes. In conclusion, none of the investigated CYP450-mediated reactions has potential as a biomarker to monitor VRP contamination of the aquatic environment.

Differential inductive profiles of hepatic cytochrome P450s by the extracts of Sophora flavescens in male and female C57BL/6JNarl mice

Aim of the study Sophora flavescens has been used as an antipyretic and analgesic agent. To assess the possible herb–drug interaction, effects of S. flavescens extracts on hepatic cytochrome P450 (P450, CYP) enzymes were studied. Materials and methods Effects of the extracts prepared by three different pharmaceutical companies on P450 enzymes were investigated in male and female C57BL/6JNarl mice. Results In male mice, extract 1 caused a dose- and time-dependent increase of 7-ethoxyresorufin O-deethylation (EROD) activity. Three-day treatment with 3 g/kg extracts 1–3 elevated EROD, 7-pentoxyresorufin O-dealkylation (PROD), coumarin hydroxylation, and nifedipine oxidation (NFO) activities. In female mice, extracts 1 and 2 increased EROD and PROD activities without affecting coumarin hydroxylation and NFO activities. However, extract 3, which lacked prenylated flavonoids, caused an induction profile in females the same as in males. Treatment with extract 3 fortified with prenylated flavonoids restored the gender difference. An alkaloid, oxymatrine was present in all extracts and increased EROD and PROD activities. At a human equivalent dose (0.18 g/(kg day)), all extracts increased EROD activity. Conclusions These results revealed that Cyp1a had a lower induction response threshold. Oxymatrine contributed at least partly to the P450 induction by S. flavescens. At a higher dose, Cyp2a, Cyp2b, and Cyp3a could be induced and the male-specific induction of Cyp2a and Cyp3a was associated with the presence of prenylated flavonoids.

Biotransformation of PCBs in Relation to Phase I and II Xenobiotic-Metabolizing Enzyme Activities in Ringed Seals (Phoca hispida) from Svalbard and the Baltic Sea

Polychlorinated biphenyls (PCBs) may induce activity of hepatic enzymes, mainly Phase I monooxygenases and conjugating Phase II enzymes, that catalyze the metabolism of PCBs leading to formation of metabolites and to potential adverse health effects. The present study investigates the concentration and pattern of PCBs, the induction of hepatic phase I and II enzymes, and the formation of hydroxy (OH) and methylsulfonyl (CH3SO2=MeSO2) PCB metabolites in two ringed seal (Phoca hispida) populations, which are contrasted by the degree of contamination exposure, that is, highly contaminated Baltic Sea (n=31) and less contaminated Svalbard (n=21). Phase I enzymes were measured as ethoxyresorufin-O-deethylation (EROD), benzyloxyresorufin-O-dealkylation (BROD), methoxyresorufin-O-demethylation (MROD), and pentoxyresorufin-O-dealkylation (PROD) activities, and phase II enzymes were measured as uridine diphosphophate glucuronosyl transferase (UDPGT) and glutathione-S-transferase (GST). Geographical comparison, multivariate, and correlation analysis indicated that sigma-PCB had a positive impact on Phase I enzyme and GST activities leading to biotransformation of group III (vicinal ortho-meta-H atoms and < or =1 ortho-chlorine (Cl)) and IV PCBs (vicinal meta-para-H atoms and < or =2 ortho-Cl). The potential precursors for the main OH-PCBs detected in plasma in the Baltic seals were group III PCBs. MeSO2-PCBs detected in liver were mainly products of group IV PCB metabolism. Both CYP1A- and CYP2B-like enzymes are suggested to be involved in the PCB biotransformation in ringed seals.

Purification of CYP2B‐like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.

Toxicity Profiles in Rats Treated with Tumorigenic and Nontumorigenic Triazole Conazole Fungicides: Propiconazole, Triadimefon, and Myclobutanil

Conazoles are a class of azole based fungicides used in agriculture and as pharmaceutical products. They have a common mode of antifungal action through inhibition of ergosterol biosynthesis. Some members of this class have been shown to be hepatotoxic and will induce mouse hepatocellular tumors and/or rat thyroid follicular cell tumors. The particular mode of toxic and tumorigenic action for these compounds is not known, however it has been proposed that triadimefon-induced rat thyroid tumors arise through the specific mechanism of increased TSH. The present study was designed to identify commonalities of effects across the different conazoles and to determine unique features of the tissue responses that suggest a toxicity pathway and a mode of action for the observed thyroid response for triadimefon. Male Wistar/Han rats were treated with triadimefon (100, 500, 1800 ppm), propiconazole (100, 500, 2500 ppm), or myclobutanil (100, 500, 2000 ppm) in feed for 4, 30, or 90 days. The rats were evaluated for clinical signs, body and liver weight, histopathology of thyroid and liver, hepatic metabolizing enzyme activity, and serum T3, T4, TSH, and cholesterol levels. There was a dose-dependent increase in liver weight but not body weight for all treatments. The indication of cytochrome induction, pentoxyresorufin O-dealkylation (PROD) activity, had a dose-related increase at all time points for all conazoles. Uridine diphopho-glucuronosyl transferase (UDPGT), the T4 metabolizing enzyme measured as glucuronidation of 1-naphthol, was induced to the same extent after 30 and 90 days for all three conazoles. Livers from all high dose treated rats had centrilobular hepatocyte hypertrophy after 4 days, while only triadimefon and propiconazole treated rats had hepatocyte hypertrophy after 30 days, and only triadimefon treated rats had hepatocyte hypertrophy after 90 days. Thyroid follicular cell hypertrophy, increased follicular cell proliferation, and colloid depletion were present only after 30 days in rats treated with the high dose of triadimefon. A dose-dependent decrease in T4 was present after 4 days with all 3 compounds but only the high doses of propiconazole and triadimefon produced decreased T4 after 30 days. T3 was decreased after high-dose triadimefon after 4 days and in a dose-dependent manner for all compounds after 30 days. Thyroid hormone levels did not differ from control values after 90 days and TSH was not increased in any exposure group. A unique pattern of toxic responses was not identified for each conazole and the hypothesized mode of action for triadimefon-induced thyroid gland tumors was not supported by the data.

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

Mixed reconstituted systems containing CYP2B4, CYP1A2, and NADPH-cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin O-dealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2-CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin O-deethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1-CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450-P450 complexes. The synergistic stimulation observed with the reductase-CYP1A2-CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2-CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2-CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.

Propofol metabolism is enhanced after repetitive ketamine administration in rats: the role of cytochrome P-450 2B induction

In a series of ex vivo and in vivo studies we investigated the ability of repetitive ketamine administration to alter the metabolism and anaesthetic effect of propofol and the role of ketamine-mediated P-450 2B induction in rats. Male Wistar rats were pretreated with 80 mg kg(-1) ketamine i.p. twice daily for 4 days. Pentoxyresorufin O-dealkylation (PROD), P-450 2B protein and mRNA were determined. Residual propofol concentration was measured after incubating hepatic microsomes with 100 muM propofol. Sleeping times induced by i.p. 80 mg kg(-1) propofol were determined. Orphenadrine, a P-450 2B inhibitor, was added in both ex vivo and in vivo studies. Finally, serial whole blood propofol concentrations were determined after i.v. infusion of 15 mg kg(-1) propofol. Ketamine pretreatment produced 5.4-, 3.4- and 1.7-fold increases in hepatic PROD activity, P-450 2B protein and mRNA, respectively. Residual propofol concentration was 46% lower after incubation with microsomes from ketamine-pretreated rats than in the control group. The addition of orphenadrine to ketamine-pretreated microsomes produced an increase in residual propofol concentration in a concentration-dependent manner. Ketamine pretreatment reduced propofol sleeping time to 12% of the control, which was reversed by orphenadrine. The whole blood propofol concentration in ketamine-pretreated rats was significantly lower than that of control rats at 1, 2, 4 and 8 min after cessation of propofol infusion. Repetitive ketamine administration enhances propofol metabolism and reduces propofol sleeping time in rats. We suggest that P-450 2B induction may produce ketamine-propofol interaction in anaesthetic practice.

The effect of fenbuconazole on cell proliferation and enzyme induction in the liver of female CD1 mice

Fenbuconazole, a triazole fungicide, has been associated with an increase in the incidence of liver adenomas in female mice following long-term dietary exposure. The aim of this study was to evaluate whether the mode of action for liver tumor formation by fenbuconazole is similar to that of phenobarbital. Treatment of CD1 mice with 0, 20, 60, 180 or 1300 ppm fenbuconazole for up to 4 weeks caused a dose-dependent increase in liver weight that was associated with centrilobular hepatocellular hypertrophy, cytoplasmic eosinophilia and panlobular hepatocellular vacuolation, as well as an initial increase in the cell proliferation labeling index. Fenbuconazole also caused a dose-dependent increase in liver microsomal cytochromes b(5) and P450 and the levels of immunoreactive CYP2B10 and its associated activity 7-pentoxyresorufin O-dealkylation (PROD). Treatment of mice with 1000 ppm phenobarbital elicited the same effects as treatment of mice with 1300 ppm fenbuconazole, except that phenobarbital was more effective than fenbuconazole at inducing PROD activity, even though fenbuconazole induced CYP2B10 to the same extent as did phenobarbital. This difference was attributed to the ability of fenbuconazole to bind tightly to CYP2B10 and partially mask its catalytic activity in liver microsomes, which is characteristic of several azole-containing drugs. All hepatocellular changes and induced enzyme activity returned to control levels within 4 weeks of discontinuing treatment with fenbuconazole or phenobarbital, indicating that the observed changes were fully reversible. We conclude that fenbuconazole is a phenobarbital-type inducer of mouse liver cytochrome P450, and the mode of action by which fenbuconazole induces liver adenomas in mice is similar to that of phenobarbital.

Fluconazole-induced hepatic cytochrome P450 gene expression and enzymatic activities in rats and mice

This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague–Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods were used as measures of Cyp enzyme activities. Western analyses identified specific Cyp isoforms. Quantitative real-time reverse-transcription polymerase chain reaction (quantitative real time-RT-PCR) assays were used to quantitate the mRNA expression of specific Cyp genes induced by this conazole. Rats and mice were administered fluconazole 2, 25, or 50 mg/kg bw/d by gavage daily for 14 days. In rats, fluconazole treatment (50 mg/kg bw/d) significantly induced pentoxyresorufin O-dealkylation (PROD), benzyloxyresorufin O-dealkylation (BROD), and ethoxyresorufin O-dealkylation (EROD) hepatic microsomal activities. Fluconazole treatment significantly increased rat hepatic mRNA expression of CYP2B1 and CYP3A23/3A1 with dose-related responses. The highest dose of fluconazole gave a 128-fold induction of CYP2B1 and a 4.6-fold induction of CYP3A23/3A1 mRNA. CYP3A2 mRNA levels were also overexpressed 5.6–7.2-fold depending on dose. Western immunoblots of rat hepatic microsomal proteins identified Cyp isoforms: CYP1A1, CYP1A2, CYP2B1/2, CYP3A23/3A1, and Cyp3A2 with increased levels of CYP2B1/2 and CYP3A23/3A1 proteins. In mice, fluconazole induced BROD, PROD, EROD, and methoxyresorufin O-dealkylation hepatic microsomal activities after treatment with 25 and 50 mg/kg bw/d. Fluconazole increased mouse hepatic mRNA expression of Cyp2b10 (1.9-fold) and Cyp3a11 (2.6-fold) in the 50 mg/kg bw/d treatment group. In summary, these results indicated that fluconazole, a triazole-containing conazole, clearly induced CYP2B and CYP3A families of isoforms in rat liver and Cyp2b and Cyp3a families of isoforms in mouse liver.

Induction of Rat Hepatic Cytochrome P-450 by Ketamine and its Toxicological Implications

Ketamine is a common intravenous anesthetic and a frequent drug of abuse, alone or in combination with cocaine. However, the pharmacokinetic effects of ketamine have not been fully investigated. This study determined the effects of ketamine on cytochrome P-450 (P-450)-dependent catalytic activities, protein levels, and hepatotoxicity using male Wistar rats treated with 10, 20, 40, or 80 mg/kg ketamine intraperitoneally twice daily for 4 d. Treatment with ketamine produced a dose-dependent increase of pentoxyresorufin O-dealkylation activity of liver microsomes. Treatment with 80 mg/kg ketamine resulted in 14-, 3-, and 2-fold rise in O-dealkylation of pentoxyresorufin, ethoxyresorufin, and methoxyresorufin of rat liver microsomes, respectively. The treatment produced 31% and 86% increases in 7-ethoxycoumarin O-deethylation and erythromycin N-demethylation, respectively. In addition, aniline hydroxylation activity was elevated by 62%. Protein blot analysis of liver microsomal proteins revealed that 80 mg/kg ketamine induced P-450 1A, 2B, 2E1, and 3A proteins by 2-, 13-, 2-, and 2-fold, respectively. In reversibility study, ketamine-induced pentoxyresorufin O-dealkylation, 7-ethoxycoumarin O-deethylation, erythromycin N-demethylation, and methoxyresorufin O-demethylation activities of liver microsomes prepared from rats 4 d after ketamine treatment were 75%, 48%, 29%, and 38% lower than the respective activities of liver microsomes prepared from rats 1 d after treatment. Protein blot analysis showed that ketamine-induced P-450 2B1/2 proteins also decreased in a time-dependent manner in 4 d. In hepatotoxicity study, treatment of rats with 1 ml/kg CCl4 produced a 7-fold increase in serum alanine aminotransferase activity level and a 17-fold rise in rats pretreated with 80 mg/kg ketamine for 4 d. Treatment of ICR mice with 120 mg/kg cocaine produced a 17% mortality, whereas the same dose of cocaine produced a 50% mortality in mice pretreated with ketamine. Treatment of mice with 100 mg/kg cocaine produced a 76-fold increase in serum alanine aminotransferase activity level and a 260-fold rise in mice pretreated with 80 mg/kg ketamine for 4 d. The present study shows that ketamine induces the expression of multiple forms of P-450 in rat liver microsomes and increases CCl4-induced liver toxicity and cocaine-mediated acute toxicity. Other potential pharmacological or toxicological events related to ketamine use need to be further explored.

PULMONARY BIOACTIVATION OF TRICHLOROETHYLENE TO CHLORAL HYDRATE: RELATIVE CONTRIBUTIONS OF CYP2E1, CYP2F, AND CYP2B1

Pulmonary cytotoxicity induced by trichloroethylene (TCE) is associated with cytochrome P450-dependent bioactivation to reactive metabolites. In this investigation, studies were undertaken to test the hypothesis that TCE metabolism to chloral hydrate (CH) is mediated by cytochrome P450 enzymes, including CYP2E1, CYP2F, and CYP2B1. Recombinant rat CYP2E1 catalyzed TCE metabolism to CH with greater affinity than did the recombinant P450 enzymes, rat CYP2F4, mouse CYP2F2, rat CYP2B1, and human CYP2E1. The catalytic efficiencies of recombinant rat CYP2E1 (V(max)/K(m) = 0.79) for generating CH was greater than those of recombinant CYP2F4 (V(max)/K(m) = 0.27), recombinant mouse CYP2F2 (V(max)/K(m) = 0.11), recombinant rat CYP2B1 (V(max)/K(m) = 0.07), or recombinant human CYP2E1 (V(max)/K(m) = 0.02). Decreases in lung microsomal immunoreactive CYP2E1, CYP2F2, and CYP2B1 were manifested at varying time points after TCE treatment. The loss of immunoreactive CYP2F2 occurred before the loss of immunoreactive CYP2E1 and CYP2B1. These protein decreases coincided with marked reduction of lung microsomal p-nitrophenol hydroxylation and pentoxyresorufin O-dealkylation. Rates of CH formation in the microsomal incubations were time-dependent and were incremental from 5 to 45 min. The production of CH was also determined in human lung microsomal incubations. The rates were low and were detected in only three of eight subjects. These results showed that, although CYP2E1, CYP2F, and CYP2B1 are all capable of generating CH, TCE metabolism is mediated with greater affinity by recombinant rat CYP2E1 than by recombinant CYP2F, CYP2B1, or human CYP2E1. Moreover, the rates of CH production were substantially higher in murine than in human lung.

Propiconazole-induced cytochrome P450 gene expression and enzymatic activities in rat and mouse liver

Propiconazole is a N-substituted triazole used as a fungicide on fruits, grains, seeds, hardwoods, and conifers. In the present study, propiconazole was examined for its effects on the expression of hepatic cytochrome P450 genes and on the activities of P450 enzymes in male Sprague-Dawley rats and male CD-1 mice. Rats and mice were administered propiconazole by gavage daily for 14 days at doses of 10, 75, and 150 mg/kg body weight/day. Quantitative real time RT-PCR assays of rat hepatic RNA samples from animals treated at the 150 mg/kg body weight/day dose revealed significant mRNA overexpression of the following genes compared to control: CYP1A2 (1.62-fold), CYP2B1 (10.8-fold), CYP3A1/CYP3A23 (2.78-fold), and CYP3A2 (1.84-fold). In mouse liver, propiconazole produced mRNA overexpression of Cyp2b10 (2.39-fold) and Cyp3a11 (5.19-fold). mRNA expression of CYP1A1 was not detected in liver tissues from treated or controls animals from either species. Propiconazole significantly induced both pentoxyresorufin O-dealkylation (PROD) and methoxyresorufin O-dealkylation (MROD) activities in both rat and mouse liver at the 150 mg/kg body weight/day and 75 mg/kg body weight/day doses. In summary, these results indicated that propiconazole induced CYP1A2 in rat liver and CYP2B and CYP3A families of isoforms in rat and mouse liver.

Mechanism to study 1:1 stoichiometry of NADPH and alkoxyphenoxazones metabolism spectrophotometrically in subcellular biological preparations

Our prior studies have shown that pentoxyresorufin-O-dealkylation (PROD) can be measured spectrophotometrically with simultaneous monitoring of stoichiometry of NADPH/substrate and NADP/product as 10:1:10:1 [Rastogi et al. FEBS Letters 512 (2002) 121-124]. In the present investigation, mechanism of action of other enzymes in modulating the stoichiometry of alkoxyphenoxazones metabolism to 1:1 for electron donor/substrate and oxidized electron donor/product in the same incubation mixture was studied. The spectrophotometric analysis reveals 10:1 ratio between NADPH and pentoxyresorufin (PRF)-ethoxyresorufin (ERF) in microsomal system. The high ratio of electron donor to substrate is due to the presence of the other forms of P-450, which may participate in endogenous metabolism of compounds, thereby reducing the ratio to 4:1 and 7:1 for NADPH/PRF-ERF. Incubation of dicumarol in the microsomal PROD or ethoxyresorufin-O-dealkylase (EROD) assay led to significant decrease in the consumption of NADPH with a ratio of 4:1 and 7:1 for NADPH/PRF-ERF which is due to inhibition of NADPH cytochrome c (P-450) reductase. In post mitochondrial fraction (S-9), the ratio of 11:1 and 15:1 is seen for NADPH/PRF-ERF. The addition of dicumarol in S-9 fraction showed enhanced rate of alkoxyphenoxazone utilization, suggesting the possibility of reduced resorufin product as a feedback inhibitor. Equating the ratio of NADPH/substrate(s) derived after endogenous utilization of NADPH with the ratio after accounting for NADPH consumption following dicumarol addition in either S-9 or microsomal fraction, a 1:1 mol of NADPH/substrate(s) and oxidized electron donor/product is obtained. The results further suggest that cytosolic fraction may interfere in monitoring the formation of resorufin during dealkylation of alkoxyphenoxazones making dicumarol a mandatory cofactor.

Induction of hepatic Cyp2b and Cyp3a subfamily enzymes by nicardipine and nifedipine in mice

Nicardipine (Nic) or nifedipine (Nif) was given to male and female C57BL/6J mice by a single gavage at doses of 100, 200 and 400 µmol kg−1, and changes in the levels of mRNA and apoprotein of hepatic cytochrome P450 (P450) enzymes, including Cyp2b9, Cyp2b10, Cyp3a11 and Cyp3a41, were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Furthermore, hepatic microsomal activities for pentoxyresorufin O-dealkylation (PROD) and nifedipine oxidation, which are mainly mediated by Cyp2b and Cyp3a subfamily enzymes, respectively, were measured.Results from RT-PCR analysis revealed that Nic, but not Nif, showed a capacity for activating the Cyp3a11 gene in either sex of mice and that both chemicals could induce a male-selective activation of Cyp2b10 gene, although they had no capacity for activating the Cyp2b9 and Cyp3a41 genes in either sex.Increased levels of the mRNAs of Cyp2b10 and Cyp3a11 were closely correlated with those of apoprotein and activity of the corresponding P450 subfamily enzymes.The study demonstrated for the first time that Nic, but not Nif, showed the ability to induce Cyp3a11 in both sexes of mice, although both Nif and Nic led to a male-selective induction of Cyp2b10, and that Nic and Nif had no ability to induce Cyp2b9 and Cyp3a41 in either sex.