Rat Hepatic Cytochrome P450 Enzymes Research Articles

Evaluation of the impact of vindoline, an active components of Catharanthus roseus, on rat hepatic cytochrome P450 enzymes by using a cocktail of probe drugs.

The objection of this study was to investigate the effects of vindoline(VDL) on the cytochrome P450 (CYP 450) isoforms (CYP1A2, 2B, 2C11, 2D1 and 3A) in rats. Firstly, the rats were randomly divided into VDL pretreatment group and blank group, each group had six rats. VDL pretreatment group was administrated VDL (20 mg·kg-1) by oral gavage for fifteen days consecutively, and the equivalent CMC-Na solution without VDL was given to the blank group by gavage. Secondly, a cocktail of caffeine, bupropion, diclofenac, dextromethorphan and midazolam was then administered on the sixteenth day. Finally, blood samples were collected at the specified time point, and the plasma concentration of the probe drug was determined by UHPLC-QTOF-MS/MS. The effects of VDL on the activity of these CYP enzymes in rats were evaluated by pharmacokinetic parameters. VDL pretreatment group compared with the blank group, accelerated the metabolism of diclofenac, and weakened the metabolism of caffeine. These results suggested that VDL could induce the activity of CYP2C11, and inhibits the activity of CYP1A2, but had no significant effects on CYP2B, CYP2D1 and CYP3A. The results in this study can provide beneficial information for the later clinical application of VDL.

Modulatory effects of Benjakul extract on rat hepatic cytochrome P450 enzymes

Benjakul, a traditional Thai formulation, has been used as a carminative and adaptogenic drug. It consists of five plants, Piper chaba Hunter, Piper sarmentosum Roxb., Piper interruptum Opiz., Plumbago indica Linn., and Zingiber officinale Roscoe, in equal ratios. Some individual herbs present in Benjakul were reported to modulate cytochrome P450 (CYP) enzymes. This study aimed to investigate the effects of Benjakul extract on the activities and mRNA expression levels of hepatic CYP2C11 and CYP3A1 in rats. Adult male rats were orally administered 200, 400, or 600 mg/kg BW Benjakul extract for 28 days. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine levels were assayed. CYP2C11 and CYP3A1 activities were analyzed using cytochrome P450 assay kits. The mRNA expression of CYP2C11 and CYP3A1 was measured using a quantitative real-time PCR assay. Benjakul treatment significantly increased the serum ALT and BUN levels. At doses of 200, 400, and 600 mg/kg BW, Benjakul treatment increased hepatic CYP3A1 activity and CYP3A1 mRNA expression. CYP2C11 mRNA expression was unchanged by treatment with Benjakul extract; however, treatment with the high and middle doses of Benjakul extract increased CYP2C11 activity. Treament with Benjakul extract induced CYP2C11 and CYP3A1 activity in rats. Concurrent use of Benjakul with conventional drugs should be considered to potentially induce herb-drug interactions.

Evaluation of the impact of 16-dehydropregnenolone on the activity and expression of rat hepatic cytochrome P450 enzymes

16-dehydropregnenolone (DHP) is a promising novel antihyperlipidemic agent developed and patented by Central Drug Research Institute (CDRI), India. The purpose of the present study was to investigate whether DHP influences the activities and mRNA expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D2, CYP2E1 and CYP3A1) in Sprague-Dawley (SD) rats. A cocktail suspension of CYP probe substrates which contained caffeine (CYP1A2), tolbutamide (CYP2C11), dextromethorphan (CYP2D2), chlorzoxazone (CYP2E1) and dapsone (CYP3A1) was administered orally on eighth- or fifteenth-day to rats pre-treated with DHP intragastrically at a dose of 36 and 72mg/kg for one week and two weeks. The concentrations of probe drugs in plasma were estimated by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Alongside, the effect of DHP on CYPs activity and mRNA expression levels were assayed in isolated rat liver microsomes and by real-time reverse transcription-polymerase chain reaction (RT-PCR), respectively. DHP had significant inducing effects on CYP1A2, 2C11, 2D2 and 2E1 with no effect on CYP3A1 in dose- and time-dependent manner, as revealed from the pharmacokinetic profiles of the probe drugs in rats. In-vitro microsomal activities and mRNA expression results were in good agreement with the in-vivo pharmacokinetic results. Collectively, the results unveiled that DHP is an inducer of rat hepatic CYP enzymes. Hence, intense attention should be paid when DHP is co-administered with drugs metabolized by CYP1A2, 2C11, 2D2 and 2E1, which might result in drug-drug interactions and therapeutic failure.

Evaluation of the impact of Polygonum capitatum, a traditional Chinese herbal medicine, on rat hepatic cytochrome P450 enzymes by using a cocktail of probe drugs

Ethnopharmacological relevancePolygonum capitatum is a well-known Miao medicinal plant that has been used for many years for its unique therapeutic effects on various urological disorders, including urinary calculus and urinary tract infections. To investigate the effect of Polygonum capitatum on cytochrome P450 (CYP) isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4) in vivo using a “cocktail” approach by administering five probe drugs to rats. This study assessed the potential of Polygonum capitatum to interact with co-administered drugs. Materials and methodsAn aqueous extract of dried whole Polygonum capitatum was prepared and administered orally to rats at a dose of 0.58g/kg or 1.74g/kg twice daily for 7 or 14 consecutive days. A cocktail of caffeine (1.0mg/kg), tolbutamide (1.0mg/kg), omeprazole (2.0mg/kg), chlorzoxazone (4.0mg/kg) and midazolam (4.0mg/kg) was then administered on the eighth or fifteenth day to evaluate the effects of Polygonum capitatum on CYP1A2, 2C9, 2C19, 2E1, and 3A4, respectively. Blood samples were collected at a range of time-points and the plasma concentrations of the probe drugs were simultaneously quantified using ultra high-performance liquid chromatography–tandem mass spectrometry. Pharmacokinetic parameters were calculated to evaluate the effects of Polygonum capitatum on the activities of these CYP enzymes in rats. ResultsPolygonum capitatum pre-treatment had no significant effect on the pharmacokinetic parameters of caffeine, omeprazole or chlorzoxazone. However, the pharmacokinetics of tolbutamide and midazolam were affected significantly (P<0.05) by Polygonum capitatum, which induced more rapid metabolism of these probe compounds. ConclusionsThese results suggested that Polygonum capitatum could induce CYP2C9 and CYP3A4, and did not influence CYP1A2, CYP2C19 or CYP2E1. Therefore, the clinical dose of drugs metabolized by human CYP2C9 or CYP3A4 may need to be adjusted in patients taking Polygonum capitatum, as this herbal medication may result in reduced effective concentrations of these drugs.

Effects of arecoline on hepatic cytochrome P450 activity and oxidative stress.

Betel-quid use is associated with the risk of liver cirrhosis and hepatocellular carcinoma. The aim of the present work was to evaluate the impact of arecoline on human hepatic cytochrome P450 (CYP) enzymes in vitro and rat hepatic CYP enzymes, as well as the hepatic oxidative stress and liver injury of rats in vivo. The in vitro results indicated that arecoline hydrobromide (AH) has no significant effect on the activities of CYP2B, 2C9, 3A4, 1A2, 2E1 and 2D6 in human liver microsome (HLM). However, oral administration of AH at 4 and 20 mg/kg/d for seven consecutive days significantly increased the activities of rat hepatic CYP2B, 2E1, 2D, 3A, 2C and 1A2. In addition, AH at 100 mg/kg/d significantly increased the levels of ALT, AST and MDA, decreased the levels of SOD, CAT, GSH-Px and GSH, in rat liver. The in vivo induction of AH on rat hepatic CYP isoforms suggested that the high risk of metabolic interaction should be existed when the substrate drugs of the six kinds of CYP isoforms was administered in betel-quid use human. Furthermore, the in vivo results also suggested that AH-induced hepatoxicity should be associated with the induction of AH on rat hepatic CYP2E1 and 2B.

Radix Polygoni Multiflori Praeparata and Dioscorea Bulbifera Rhizome Decoctions Display Combined Effects Detected by a Three-Probe Drug Cocktail with Substrates of Rat Hepatic Cytochrome P450 Enzymes

Objectives: Radix Polygoni Multiflori Praeparata (RPMP) and Dioscorea Bulbifera Rhizomes (DBR) are used in Chinese herbal medicine and have been frequently reported for adverse reactions on liver. In this research, we aimed to evaluate in vivo effects of RPMP and DBR on rat cytochrome P450 enzymes (CYP1A2, CYP2E1 and CYP3A2) with their respective substrates as probes. Methods: Rats were orally administered RPMP, DBR and RPMP/DBR combination at 12, 10 and (12 + 10) g/kg, respectively, or saline as a control, once daily for 7 days. Thereafter, a cocktail containing 10 mg/kg caffeine, 20 mg/kg chlorzoxazone and 10 mg/kg dapsone was tail vein injected to rats. At defined time points, plasma drug concentrations were simultaneously evaluated by HPLC. Pharmacokinetic parameters simulated by DAS software were used to assess RPMP and/or DBR effects on cytochrome P450 enzymes activity. ANOVA and Dunnett’s test were used for data analysis. Results: Caffeine metabolism was enhanced in RPMP animals and reduced after pretreatment with DBR, but no effect was observed in RPMP/DBR combination group. Chlorzoxazone and dapsone metabolism was enhanced in both RPMP and DBR groups and consequently in combination group. The data suggested that RPMP independently induces rat CYP1A2, CYP2E1 and CYP3A2 activity, while DBR independently inhibits activity of rat CYP1A2 and induces that of CYP2E1 and CYP3A2. RPMP/DBR combination showed no significant benefit compared with the two drugs alone and even showed a neutralized effect in CYP1A2 activity. Conclusions: Caution is needed when RPMP and/or DBR are co-administered with drugs metabolized by human CYP1A2, CYP2E1 and CYP3A4.

Evaluation of impact of Herba Erigerontis injection, a Chinese herbal prescription, on rat hepatic cytochrome P450 enzymes by cocktail probe drugs

Ethnopharmacological relevanceHerba Erigerontis injection (HEI), one of the most popular herbal prescription in China, is made from the aqueous extracts of Erigeron breviscapus whole plant. Now HEI is widely used for the treatment of cardiovascular diseases and cerebrovascular diseases such as coronary heart disease, anginapectoris and paralysis. Aim of the studyThe purpose of this study was to investigate the in vivo effect of HEI on rat cytochrome P450 enzymes (CYP1A2, CYP2C11, CYP2D4, CYP2E1 and CYP3A2) to assess its safety through its potential to interact with co-administered drugs. Materials and methodsRats were randomly divided into five groups. Rats were intravenous administrated with HEI via the caudal vein at the dosage of 1.8ml/kg or 7.2ml/kg once daily for consecutive 3 days or 14 days. On the fourth or the fifteenth day, a cocktail solution at a dose of 5ml/kg, which contained caffeine (2.5mg/kg), tolbutamide (2.5mg/kg), chlorzoxazone (5mg/kg), midazolam (5mg/kg) and metoprolol (10mg/kg), was injected via the lingual vein to all rats. Then 0.8ml blood samples were collected at a set of time-points. The plasma concentrations of probe drugs were simultaneously determined by HPLC. Pharmacokinetic parameters simulated by DAS software were used for the evaluation of HEI on the activities of rat CYP1A2, CYP2C11, CYP2D4, CYP2E1 and CYP3A2 enzymes. ANOVA and Dunnett's test was used for data analysis. ResultsThere were no significant influence of pharmacokinetic parameters of caffeine, tolbutamide and chlorzoxazone in HEI pretreated rats. But many pharmacokinetic parameters of metoprolol and midazolam in HEI pretreated rats were affected significantly (P<0.05), which indicated that metabolism of metoprolol and midazolam in these treatment groups was evidently slowed down. ConclusionsThe results from the present in vivo study suggested that HEI showed no effects on rat CYP1A2, CYP2C11 and CYP2E1, however, it demonstrated potential inhibitory effects on rat CYP2D4 and CYP3A2. Therefore, caution is needed when HEI is co-administered with drugs metabolized by human CYP2D6 or CYP3A4 in clinic, which may result in increased concentrations of these drugs and relevant herb–drug interactions.

Rat cytochromes P450 oxidize 3-aminobenzanthrone, a human metabolite of the carcinogenic environmental pollutant 3-nitrobenzanthrone

3-Aminobenzanthrone (3-ABA) is a human metabolite of carcinogenic 3-nitrobenzanthrone (3-NBA), which occurs in diesel exhaust and air pollution. Understanding which cytochrome P450 (CYP) enzymes are involved in metabolic activation and/or detoxication of this toxicant is important in the assessment of an individual's susceptibility to this substance. The aim of this study was to evaluate the efficiency of rat hepatic CYPs to oxidize 3-ABA and to examine the metabolites formed during such an oxidation. The metabolites formed by CYPs in rat hepatic microsomes were separated by high performance liquid chromatography (HPLC). 3-ABA is oxidized by these enzymes to three metabolites, which were separated by HPLC as distinguish product peaks. Using co-chromatography with synthetic standards, two of them were identified to be oxidative metabolites of 3-ABA, N-hydroxy-3-ABA and 3-NBA. The structure of another 3-ABA metabolite remains to be characterized. To define the role of rat hepatic CYP enzymes in metabolism of 3-ABA, we investigated the modulation of its oxidation using different inducers of CYPs for treatment of rats to enrich the liver microsomes with individual CYPs. Based on these studies, we attribute most of 3-ABA oxidation in rat hepatic microsomes to CYP2B, followed by CYP1A, although a role of other hepatic CYPs cannot be ruled out. Inhibition of 3-ABA oxidation by selective inhibitors of individual CYPs, supported this finding.

Evaluation of 7-benzyloxy-4-trifluoromethylcoumarin, some other 7-hydroxy-4-trifluoromethylcoumarin derivatives and 7-benzyloxyquinoline as fluorescent substrates for rat hepatic cytochrome P450 enzymes

1. The aim of this study was to evaluate a number of derivatives of 7-hydroxy-4-trifluoromethylcoumarin (HFC) and 7-benzyloxyquinoline (7BQ) as novel fluorescent substrates for monitoring rat hepatic cytochrome P450 (CYP) enzyme specificity in a 96-well plate format. The HFC derivatives examined comprised 7-benzyloxy-4-trifluoromethylcoumarin (BFC), 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC), 3,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BTBFC), 2-(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (2TFBFC), 3-(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (3TFBFC) and 3-(trifluoromethoxy)-7-benzyloxy-4-trifluoromethylcoumarin (3TFMeOBFC). 2. The CYP specificity of the fluorescent probe substrates was examined using characterized liver microsomes from male Sprague-Dawley rats treated with β-naphthoflavone (BNF), sodium phenobarbitone (NaPB), isoniazid, pregnenolone-16 α -carbonitrile (PCN), dexamethasone (DEX) and methylclofenapate to induce CYP1A, CYP2B, CYP2E, CYP3A, CYP3A and CYP4A forms, respectively. Studies were also performed with microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A1, CYP1A2, CYP2B1, CYP3A1 and CYP3A2. 3. BFC metabolism was most markedly induced by BNF and NaPB, whereas BFBFC metabolism was most markedly induced by PCN and DEX and BTBFC was not metabolized by rat liver microsomes. BFC was a high-affinity substrate for cDNA-expressed CYP1A1 and CYP2B1, whereas BFBFC exhibited a high affinity for CYP3A1 and CYP3A2. 4. The metabolism of 2TFBFC and 3TFBFC was induced by NaPB, PCN and DEX. 3TFBFC was a relatively specific substrate for cDNA-expressed CYP2B1, whereas 2TFBFC could be metabolized by CYP2B1, CYP3A1 and CYP3A2. 5. 3TFMeOBFC metabolism was markedly induced by BNF treatment and 3TFMeOBFC was extensively metabolized by cDNA-expressed CYP1A1. 6. The metabolism of 7BQ to 7-hydroxyquinoline was induced by treatment with PCN and DEX. 7BQ was a substrate for cDNA-expressed CYP3A2 and to a lesser extent for CYP3A1. 7. In summary, some of the HFC derivatives studied and 7BQ are useful fluorescent probe substrates for rat CYP enzymes. BFC appears to be a probe for CYP1A and CYP2B, 2TFBFC for CYP2B and CYP3A and 3TFBFC for CYP2B. While 3TFMeOBFC appears to be a relatively specific probe for CYP1A1, both BFBFC and 7BQ are good probes for the induction of CYP3A.

Interaction of bisphenol A with rat hepatic cytochrome P450 enzymes

The effect of bisphenol A (BPA) on the kinetics of cytochrome P450 (P450)-dependent monooxygenases in rat liver microsomes was studied. Testosterone 16β-hydroxylase (TS16BH) and testosterone 2α-hydroxylase (TS2AH) activities were extensively inhibited by BPA at 100 μM (69% and 74%, respectively). The inhibition type was mixed for both P450-dependent monooxyganases. The K i of TS16BH and TS2AH from Lineweaver–Burk plots were 25.9 and 24.9 μM, respectively. The activities of acetanilide 4-hydroxylase (AA4H), 7-ethoxycoumarin O-deethylase (ECOD), bufuralol 1′-hydroxylase (BF1′H), chlorzoxazone 6-hydroxylase (CZ6H) and testosterone 6β-hydroxylase (TS6BH) were also effectively inhibited by BPA at 100 μM (43–52%). The inhibition type of these P450-dependent monooxygenases was mixed or uncompetitive, and the K is (50.5–88.5 μM) were higher than those of TS16BH and TS2AH. By contrast, the values of IC 50 and K i of testosterone 7α-hydroxylase (TS7AH) and lauric acid ω-hydroxylase (LAOH) for BPA were >1000 μM. These results suggest that BPA interacts with rat hepatic CYP1A2, CYP2A2, CYP2B2, CYP2C11, CYP2D1, CYP2E1 and CYP3A2 in vitro.

Effects of musk xylene and musk ketone on rat hepatic cytochrome P450 enzymes

The purpose of the present work was to characterize the effect of musk xylene (MX) and musk ketone (MK) treatment on rat hepatic cytochrome P450 enzymes. Male F344 rats were dosed orally with MX (10, 50 or 200 mg/kg) or MK (20, 100 or 200 mg/kg) for 7 days, after which CYP1A, 2B and 3A enzyme activities and protein levels were determined. MX treatment resulted in a two- to four-fold increase in the activity of CYP1A, 2B and 3A enzymes. For CYP1A and 3A, these changes were consistent with small increases in immunoreactive proteins. However, for CYP2B, despite only a three-fold increase in enzyme activity, protein levels were increased nearly 50-fold relative to control. This induction occurred by transcriptional activation of the CYP2B1 gene as evidenced by increased steady state CYP2B1 mRNA levels. In contrast to MX, MK treatment increased CYP2B activity, protein and mRNA levels. However MK treatment also increased CYP1A enzyme activity nearly 30-fold higher than control rats, a profile that was markedly different from MX, and very different from its effects in mice (Stuard, S.B., Caudill, D., Lehman-McKeeman, L.D., 1997. Characterization of the effects of musk ketone on mouse cytochrome P450 enzymes. Fund. Appl. Toxicol. 40, 264–271). These results indicate that in rats, MX is an inducer of CYP2B enzymes, but these enzymes are not functionally active. In contrast, MK also induces CYP2B enzymes, with no concurrent inactivation. MK also exhibits a unique pattern of cytochrome P450 induction by increasing both CYP1A and CYP2B in rats.

Effect of tamoxifen feeding on metabolic activation of tamoxifen by the liver of the rhesus monkey: does liver accumulation of inhibitory metabolites protect from tamoxifen-dependent genotoxicity and cancer?

Tamoxifen induces hepatocellular carcinomas in rats and is converted by rat hepatic cytochrome P450 enzymes into reactive metabolites capable of forming adducts with nucleic acids, proteins and chromosomal aberrations. In rats tamoxifen has also been shown to induce liver cytochrome P450 enzymes, to stimulate its own metabolism leading to greater covalent binding and to induce a higher degree of unscheduled DNA synthesis. This suggests that, at least in the rat, a sensitive species, tamoxifen may contribute significantly to its genotoxic and carcinogenic potential, by assisting its own metabolic activation. We have now investigated the effect of feeding tamoxifen to male and female Rhesus monkeys. A marked induction of the hepatic cytochrome(s) P450 is found in the monkey but, in spite of this, the in vitro metabolism of 7-ethoxyresorufin by microsomes from treated animals is markedly inhibited and so is the dealkylation of two other 7-alkoxyresorufin substrates. Evidence is presented for the accumulation in the liver of monkeys treated with tamoxifen of a powerful inhibitor of drug metabolism, and the inhibitor is identified as a metabolite of tamoxifen, its N,N-didesmethyl derivative. The level of 32P-postlabelled DNA adducts was considerably higher in rats given tamoxifen than in similarly treated monkeys. Also, whereas rats responded to tamoxifen treatment with a marked increase in covalent binding to microsomal protein, in the monkeys, where accumulation of the inhibitory metabolite in the microsomal fraction was also seen, covalent binding was not greater with microsomes from treated animals than in the corresponding controls. N,N-Didesmethyl-tamoxifen, added in vitro to human and rat microsomes, reduced significantly the extent of covalent binding, suggesting that the accumulation of the metabolite observed in the liver of primates may discourage the cytochrome P450-dependent conversion of tamoxifen into reactive derivatives and in this way protect against the formation of adducts. This mechanism may also contribute to protecting the primate against tamoxifen- induced liver cancer.

Induction of Rat Hepatic Cytochrome P450 Enzymes by Myristicin

The effect of myristicin on the expression of liver cytochrome P450s and its mRNA levels was examined in rats. Treatment of rats with myristicin (i.p., 500 μmol/kg) caused 2-20 fold increases in fiver P450 1A1/2, 2B1/2, and 2E1 enzyme activities relative to controls. Immunoblot analysis using anti-rat liver P450 1A, 2B, and 2E1 showed that the increases in each of P450 protein levels by myristicin were consistent with those in enzyme activity levels. When increased levels of P450 mRNA by myristicin were examined by Northern blot analysis, levels of mRNA of P450 1A1/2 and 2B1/2 also increased. However, the level of P4502E1 mRNA did not increase. The total amount of spectrally detectable P450, also increased about 1.5-fold following treatment of myristicin. These results demonstrate that myristicin is an inducer of rat liver P450 1A1/2, 2B1/2, and 2E1, and that the induction involves increases in mRNA levels except in the case of P4502E1.