Rat Hepatic Cytochrome P-450 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.

Effects of paeoniflorin on the activities and mRNA expression of rat CYP1A2, CYP2C11 and CYP3A1 enzymes in vivo

Paeoniflorin is the major constituent in extracts of the paeony root, the purpose of the present study was to assess the effects of paeoniflorin on the activities and mRNA expression of the rat hepatic drug-metabolizing enzymes cytochrome P450 (CYP1A2), CYP2C11 and CYP3A1 in vivo. Sprague-Dawley (SD) male rats were treated with paeoniflorin at the dosage of 25, 50 and 100 mg/kg or 0.9% sodium chloride solution by intragastric administration for 7 days, then were given probe drugs phenacetin (CYP1A2), tolbutamide (CYP2C11), or midazolam (CYP3A1) orally on the eighth day. Blood samples were collected at various times, and the plasma concentrations of the probe drugs were estimated with ultra-high-performance liquid chromatography. The mRNA expression levels of rat hepatic CYP1A2, CYP2C11 and CYP3A1 were analysed with real-time PCR. The pharmacokinetic results indicated that paeoniflorin inhibits the activities of CYP1A2, CYP2C11 and CYP3A1 in vivo. The effect was most pronounced on CYP3A1, according to the United States Food and Drug Administration classification of inhibitors of CYP3A, it reached the category of moderate inhibition. The mRNA expression levels of 3 CYP enzymes were also tended to be inhibited. We conclude that paeoniflorin can inhibit the activities of CYP1A2, CYP2C11 and CYP3A1 in vivo, which may affect the metabolism of drugs that are primarily dependent on these pathways.

Development and validation of a sensitive and specific LC-MS/MS cocktail assay for CYP450 enzymes: Application to study the effect of catechin on rat hepatic CYP activity.

A sensitive and specific liquid chromatography tandem mass spectrometric (LC-MS/MS) method that enables the simultaneous quantification of probe substrates and metabolites of cytochrome P450 (CYP) enzymes was developed and validated. These substrates (metabolites)-coumarin (7-hydroxycoumarin), tolbutamide (4-hydroxytolbutamide), S-mephenytoin (4-hydroxymephenytoin), dextromethorphan (dextrorphan), and testosterone (6β-hydroxytestosterone)-were utilized as markers for the activities of the major human CYP enzymes CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. Analytes were separated on Kinetex C18 column (2.1 × 50 mm, 5 μm) using a binary gradient mobile phase of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Metabolites were detected and quantified by MS using multiple reaction monitoring at m/z 163 → 107.2 for 7-hydroxycoumarin, m/z 235 → 150.1 for 4-hydroxymephenytoin, m/z 287 → 171 for 4-hydroxytolbutamide, m/z 258 → 157.1 for dextrorphan, m/z 305 → 269 for 6β-hydroxytestosterone, and m/z 237 → 194 for the internal standard. The assay exhibited good linearity over a range of 10-500 ng/mL with acceptable accuracy and precision criteria. As a proof of concept, the developed cocktail assay was successfully used to examine the potential impact of catechin on the activity of the major rat liver CYP enzymes.

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.

NADPH- and NADH-dependent metabolism of and DNA adduct formation by benzo[a]pyrene catalyzed with rat hepatic microsomes and cytochrome P450 1A1.

Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. Here we investigated the efficiencies of rat hepatic microsomes and rat recombinant CYP1A1 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase, epoxide hydrolase and/or cytochrome b5 in Supersomes™ to metabolize this carcinogen. We also studied the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate BaP metabolism in these systems. Up to eight BaP metabolites and two DNA adducts were generated by the systems, both in the presence of NADPH and NADH. Among BaP metabolites, BaP-9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, and a metabolite of unknown structure were formed by hepatic microsomes and rat CYP1A1. One of two DNA adducts formed by examined enzymatic systems (rat hepatic microsomes and rat CYP1A1) was characterized to be 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N2-BPDE), while another adduct has similar chromatographic properties on polyethylaneimine–cellulose thin layer chromatography to a guanine adduct derived from reaction with 9-hydroxy-BaP-4,5-oxide. In the presence of either of the reductase cofactors tested, NADPH or NADH, cytochrome b5 stimulated CYP1A1-mediated formation of both BaP-DNA adducts. The results demonstrate that NADH can act as a sole electron donor for both the first and the second reduction of CYP1A1 during its reaction cycle catalyzing oxidation of BaP, and suggest that the NADH:cytochrome b5 reductase as the NADH-dependent reductase might substitute POR in this enzymatic system.Graphical abstract

Chronic administration of caderofloxacin, a new fluoroquinolone, increases hepatic CYP2E1 expression and activity in rats.

Caderofloxacin is a new fluoroquinolone that is under phase III clinical trials in China. Here we examined the effects of caderofloxacin on rat hepatic cytochrome P450 (CYP450) isoforms as well as the potential of caderofloxacin interacting with co-administered drugs. Male rats were treated with caderofloxacin (9 mg/kg, ig) once or twice daily for 14 consecutive days. The effects of caderofloxacin on CYP3A, 2D6, 2C19, 1A2, 2E1 and 2C9 were evaluated using a "cocktail" of 6 probes (midazolam, dextromethorphan, omeprazole, theophylline, chlorzoxazone and diclofenac) injected on d 0 (prior to caderofloxacin exposure) and d 15 (after caderofloxacin exposure). Hepatic microsomes from the caderofloxacin-treated rats were used to assess CYP2E1 activity and chlorzoxazone metabolism. The expression of CYP2E1 mRNA and protein in hepatic microsomes was analyzed with RT-PCR and Western blotting, respectively. Fourteen-day administration of caderofloxacin significantly increased the activity of hepatic CYP2E1, leading to enhanced metabolism of chlorzoxazone. In vitro microsomal study confirmed that CYP2E1 was a major metabolic enzyme involved in chlorzoxazone metabolism, and the 14-d administration of caderofloxacin significantly increased the activity of CYP2E1 in hepatic microsomes, resulting in increased formation of 6-hydroxychlorzoxazone. Furthermore, the 14-d administration of caderofloxacin significantly increased the expression of CYP2E1 mRNA and protein in liver microsomes, which was consistent with the pharmacokinetic results. Fourteen-day administration of caderofloxacin can induce the expression and activity of hepatic CYP2E1 in rats. When caderofloxacin is administered, a potential drug-drug interaction mediated by CYP2E1 induction should be considered.

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.

In pursuit of scientific excellence: sex matters

In pursuit of scientific excellence: sex matters

In pursuit of scientific excellence: sex matters

In pursuit of scientific excellence: sex matters

In pursuit of scientific excellence: sex matters

In pursuit of scientific excellence: sex matters

In pursuit of scientific excellence: sex matters

In pursuit of scientific excellence: sex matters

Effects of silver nanoparticles on rat hepatic cytochrome P450 enzyme activity

Silver nanoparticles (AgNPs) are increasingly used in various products and consequentially the potential adverse effects associated with exposure to them are of concern. This study investigated the effects of AgNPs on the hepatic drug-metabolizing enzymes of the cytochrome P450 (CYP) families 1, 2 and 3, using both in vitro and in vivo biological assays. AgNPs were orally administered to Sprague-Dawley rats at various concentrations (0–1000 mg/kg body weight/day) for 2 weeks. No effect was found on the plasma levels of ALT, AST and ALP in all treated rat groups, and no significant change in the activities of CYP1A, CYP2C, CYP2D, CYP2E1 and CYP3A was observed for all tested AgNP doses. The results correlated with the observation that no AgNPs were detected in the liver sections of the tested rats. However, the in vitro system using rat liver microsomes demonstrated a strong inhibition of CYP2C (IC50 = 28 µg/mL) and CYP2D (IC50 = 23 µg/mL) activities, but not of CYP1A, CYP2E1 and CYP3A activities (IC50 > 100 µg/mL) at concentrations up to 100 µg/mL of AgNPs. The inhibitory effect of AgNPs on these CYPs indicates the possibility of the AgNP-drug interaction when co-administered with some medicines and this may cause adverse effects to patients.

In pursuit of scientific excellence: sex matters

the year 2012 marks the 125th anniversary of the American Physiological Society (APS). Throughout its history, APS has been a leader in innovation and excellence in scientific technology, discovery, education, and publication. Consistent with this history, APS is leading the scientific publication

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 hepatic cytochrome P450 oxidoreductase is up‐regulated by low dose dexamethasone and down‐regulated by adrenalectomy

Microsomal cytochrome P450 (CYP) enzyme activity requires cytochrome P450 oxidoreductase (POR). We studied rat hepatic POR after adrenalectomy (ADX) since some CYP activities are decreased by ADX. The effects of ADX or glucocorticoids on POR are not clear. Male ADX or SHAM‐ADX rats were treated with dexamethasone (DEX) once 6 h before euthanasia, 4 days post‐ADX (acute), or once daily in the third week post‐ADX (subacute). A DEX time‐course study was also performed in intact rats. Hepatic POR was measured by quantitative RT‐PCR and by immunoblot. Hepatic POR protein but not mRNA was decreased at 4 days and 3 weeks post‐ADX. Subacute DEX dosing (1 mg/kg) returned hepatic POR protein in ADX rats to SHAM levels; a single dose (1.5 mg/kg) showed a similar trend. DEX induced hepatic POR mRNA in SHAM and ADX rats in both dosing regimens. DEX is a selective glucocorticoid receptor (GR) agonist at low doses but activates the pregnane X receptor (PXR) at higher doses. CYP3A23, a PXR target gene, was measured to determine whether DEX activated the PXR. Hepatic CYP3A23 mRNA was not induced up to 27 h post‐DEX in the time‐course but showed a modest 30% increase with subacute DEX dosing in SHAM rats. In this study, DEX induced POR mRNA and protein but not CYP3A23 mRNA, suggesting a role for GR in POR regulation. Further studies are needed to determine how DEX at low doses acts to induce the obligate redox partner for all microsomal CYPs. [Support: CIHR]

Induction of rat hepatic cytochrome P-450 3A by zolmitriptan mediated with pregnane X receptor

To investigate whether pregnane X receptor (PXR) is involved in the induction of rat hepatic cytochrome P450 3A1/2 by zolmitriptan. The induction effects of zolmitriptan were investigated in three concentrations (100 micromol L(-1)), 50 micromolL(-1)) and 10 micromol L(-1))) using luciferase report gene method based on PXR. Pregnenolone-16 alpha-carbonitrile (PCN) was used as the positive control and the solvent DMSO as the negative control. Compared with the negative groups, the positive and high level zolmitriptan groups exhibited a significant induction effect on CYP 3A1, the activity increased to 1.93 and 1.96 times, respectively (P<0.05). The positive, middle level and low level zolmitriptan groups exhibited a significant induction effect on CYP 3A2, the activity increase to 2.51, 2.10 and 1.63 times, respectively (P<0.01, <0.05 ). Zolmitriptan can significantly induce CYP 3A2 in low and middle concentrations and induce CYP 3A1 in high concentration.PXR is involved in the induction of CYP 3A1/2 by zolmitriptan.

Differential induction of ethanol-metabolizing CYP2E1 and nicotine-metabolizing CYP2B1/2 in rat liver by chronic nicotine treatment and voluntary ethanol intake

Alcohol and nicotine are frequently co-used and co-abused, and use of both drugs alone can affect hepatic drug metabolism. We investigated the influences of chronic nicotine treatment and voluntary ethanol intake on the induction of rat hepatic cytochrome P450 (CYP) enzymes that metabolize ethanol and nicotine. Rats were trained to voluntarily drink ethanol (6% v/v, 1 h) with nicotine pretreatment for 10 days. Another group of rats were treated with the same nicotine doses alone. Hepatic CYP2E1, CYP2B1/2 and CYP2D1 proteins were assessed by immunoblotting. Nicotine pretreatment (0.4, 0.8 and 1.2 mg/kg) increased voluntary ethanol intake on day 10 by 1.8, 2.0, and 1.4 fold respectively compared to saline pretreatment ( P < 0.01–0.3). CYP2E1 was increased 1.7, 1.8, and 1.4 fold by the three doses of nicotine alone ( P < 0.02–0.21); CYP2E1 levels were increased by voluntary ethanol intake alone and a further 2.4, 2.2, and 1.8 fold by 0.4, 0.8, and 1.2 mg/kg nicotine respectively versus saline pretreatment ( P < 0.002–0.06). CYP2B1/2 proteins were not induced by nicotine alone, but were increased by 2.2–2.5 fold by ethanol drinking ( P < 0.05). CYP2E1 ( r = 0.67, P < 0.001) and CYP2B1/2 levels ( r = 0.49, P = 0.007) correlated with alcohol consumption on day 10. There was no change in CYP2D1. Chronic nicotine increased voluntary ethanol intake thereby enhancing CYP2E1 and CYP2B1/2 levels. Thus CYPs are regulated not only directly by nicotine and ethanol, but also indirectly via an increase in the ethanol consumption in the presence of nicotine pretreatment. Together this may contribute to the co-abuse of these drugs and alter the metabolism of clinical drugs and endogenous substrates.