Cytochrome P450 Isozymes In Rats Research Articles

Study on the effects of Zhuanggu Guanjie Pill, a modern Chinese medicine formula, on the activities and mRNA expression of seven CYP isozymes in rats

Ethnopharmacological relevanceZhuanggu Guanjie Pill (ZGGJP), a modern Chinese medicine formula, is composed of 12 herbs and has been used to treat osteoporosis in China for almost 30 years. However, no in vivo study of the influences of ZGGJP on the cytochrome P450 (CYP) activities have been reported. Aim of the studyThe aim of this study was to evaluate the effects of ZGGJP on the activities and the mRNA expression of CYP enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A) and their corresponding nuclear receptor levels in rats. Materials and methodsAfter 7 days oral treatment of ZGGJP at low- and high-dose, cocktail solution was given to rats. Blood samples were collected at series of time points. The plasma concentrations of probe drugs and their corresponding metabolites were determined by UPLC-MS/MS. The influence of ZGGJP on the activities of seven CYPs were evaluated the metabolic ratios (Cmax and AUC0-t) for metabolites/probe drugs. In addition, the effects of ZGGJP on the mRNA expression of CYPs and their corresponding nuclear receptors in rat liver were evaluated by real-time PCR. ResultsZGGJP showed significant inductive effects on CYP1A2 and CYP2B6 of both male and female rats. The influence of ZGGJP on CYP2C9 and CYP3A showed gender difference. ZGGJP could induce the activities of CYP2C9 and CYP3A in female rats, but have no influence on the activities in male rats. ZGGJP had no effects on CYP2D6, CYP2C19 and CYP2E1. The mRNA expression results of CYPs were in accordance with the pharmacokinetic results. The mRNA expression levels of constitutive androstane receptor (CAR) and vitamin D receptor (VDR) were increased significantly in female rats at high dosage, but no significant changes were observed in male rats. ConclusionZGGJP had inductive effects on CYP1A2 and CYP2B6 in both male and female rats. The results showed that ZGGJP could induce the activities of CYP2C9 and CYP3A in female rats, but had no effect in male rats. This may suggest that the influence of ZGGJP on CYP2C9 and CYP3A exhibit gender difference. The inductive effects of ZGGJP on the activities of CYPs, exhibiting gender difference, may be regulated by CAR and VDR. Therefore, co-administration of ZGGJP with other drugs, especially using CYP2C9 and CYP3A substrates in females, may need dose adjustment to avoid herb-drug interaction.

Effect of You-Gui Yin on the Activities of Seven Cytochrome P450 Isozymes in Rats.

You-Gui Yin (YGY) is a traditional Chinese medicine (TCM) decoction composed of eight Chinese herbs. The interaction between TCM and Western medicine has attracted much attention nowadays. It is therefore necessary to study the clinical application of YGY in combination with Western medicine from the perspective of metabolic enzymes. This study aims to investigate the effect of YGY on the activities of seven CYP450 isozymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) in rats. Twenty-four Sprague-Dawley (SD) rats were randomly divided into four groups: high, middle, and low-dose YGY-treated groups and the control group. They were given 13.78, 20.67, and 31 g/kg/d YGY decoction by oral administration and normal saline (10 mL/kg), respectively, for 14 days. Half an hour after the last administration, a mixed probe substrate (1 mg/kg) was administered by tail vein injection. Then, blood was taken from the venous plexus at different time points. The protein expression level of the CYP450 enzymes in the control and treatment groups was determined by western blot. The effect of YGY on the activity of CYP isoenzymes was studied by comparing the plasma pharmacokinetics between the control and treatment groups. Compared with the control group, YGY at a high (31 g/kg) dosage could decrease AUC(0–t), AUC(0–∞) and Cmax of diclofenac, omeprazole, and midazolam by at least 35.4%, while increase CL by at least 88.9%; this revealed that YGY could induce CYP2C9, CYP2C19, and CYP3A4. The results show that when we use You-Gui Yin decoction in combination with other drugs, especially drugs metabolized by CYP2C9, CYP2C19, and CYP3A4 enzymes, the interaction between drugs needs special attention.

Effects of Baoyuan decoction, a traditional Chinese medicine formula, on the activities and mRNA expression of seven CYP isozymes in rats

Ethnopharmacological relevanceBaoyuan decoction (BYD), a traditional Chinese medicine (TCM) formula, is composed of four herbs and widely used with western drugs to treat coronary heart disease, aplastic anemia and chronic renal failure in clinic. However, no study of the effect of BYD on the cytochrome P450 (CYP) activities has been reported. Aim of the studyThe purpose of the present study was to evaluate the potential influences of BYD on the activities of seven CYP isozymes (CYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) in rats. Materials and methodsA sensitive and selective UPLC-MS/MS method for simultaneous determination of seven probe drugs and internal standard (IS) in rat plasma was developed and validated. The influence of BYD on the activities of CYP isozymes and mRNA expression levels were carried out by comparing plasma pharmacokinetics and real-time reverse transcription-polymerase chain reaction (RT-PCR) of probe drugs between control and BYD treatment groups respectively. ResultsThe calibration curve were linear, with correlation coefficient (r) > 0.99 for seven probe drugs. The intra and inter-assay accuracy and precision of the method were within ± 14.9% and the recoveries ranged from 83.2% to 106.1%. Compared with control group, BYD at low (1.46 g/kg) and high (7.30 g/kg) dosages could significantly increase Cmax and AUC0-t of chlorzoxazone and testosterone, while decrease AUC0-t of phenacetin at high dosage and increase AUC0-t of tolbutamide and metoprolol. Additionally, BYD had increased AUC0-t of bupropion at low dosage and decreased it at high dosage. The mRNA expression results were in accordance with those of pharmacokinetic. ConclusionBYD exhibited inhibitory effects on CYP2C9, CYP2E1, and CYP3A4. Moreover, BYD had induction effects on CYP1A2, and CYP2D6 activities. However, no significant change in CYP2C19 activity was observed. It would be useful for the safe and effective usage of BYD in clinic.

Effects of aqueous extract of Cassiae Semen on activity hepatic microsomal CYP450 isozymes in rats

To study the effect of aqueous extract of Cassiae Semen on the activity, mRNA and protein expressions of cytochrome P450(CYP450) system in rat liver microsomes, microsomes of rat liver were prepared after the oral administration with aqueous extract of Cassiae Semen for 14 days. The enzyme activity was quantified by Cocktail method. Meanwhile, the mRNA and protein expressions of CYP1A2, CYP2B1, CYP2C11, CYP2D2, CYP2E1 and CYP3A1 in the livers were detected by RT-PCR and Western blot. The result of this experiment was that aqueous extract of Cassiae Semen obviously induced the enzyme activities of CYP1A2, CYP2B1, CYP2C11, CYP2D2, CYP2E1 and CYP3A1. Low dose of aqueous extract of Cassiae Semen significantly reduced the activity of CYP2D2, but the activity of CYP2D2 was significantly induced by middle dose and high dose of aqueous extract of Cassiae Semen. These subtypes were increased in a dose-dependent manner except for CYP3A1. The mRNA levels of CYP1A2, CYP2C11, CYP2D2 and CYP2E1 were also induced in rats treated with aqueous extract of Cassiae Semen, but with no significant effect in CYP2B1 and CYP3A1 mRNA expressions. The protein levels of CYP2C11 and CYP2E1 were also induced in rats treated with aqueous extract of Cassiae Semen, but with no significant difference. Since the enzyme activity, mRNA and protein expressions of CYP450, particularly CYP2C11and2E1subtypes, were induced or inhibited by aqueous extract of Cassiae Semen to varing degrees, suggesting the potential drug-drug interactions should be concerned.

Effects of long-term smoking on the activity and mRNA expression of CYP isozymes in rats.

To investigate the effect of long-term smoking on the activity and mRNA expression of cytochrome P450 (CYP) enzymes. Sprague-Dawley rats were exposed to passive smoking 6 cigarettes per day for 180 days. A cocktail solution which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg) was given orally to rats. Blood samples were collected at pre-specified time points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by DAS 3.0. In addition, real-time RT-PCR was used to analyze the mRNA expression of CYP1A2, CYP2C11, CYP2E1 and CYP3A1 in rat liver. There were no significant influences of pharmacokinetic profiles of chlorzoxazone in long-term smoking pretreated rats. But many pharmacokinetic profiles of phenacetin, tolbutamide, and midazolam in long-term smoking pretreated rats were affected significantly (P<0.05). The results suggested that long-term smoking had significant inhibition effects on CYP2C11 and CYP3A1 while CYP1A2 enzyme activity was induced. Furthermore, Long-term smoking had no effects on rat CYP2E1. The mRNA expression results were consistent with the pharmacokinetic results. Alterations of CYP450 enzyme activities may fasten or slow down excretion with corresponding influence on drug efficacy or toxicity in smokers compared to nonsmokers, which may lead to clinical failures of lung cancer therapy or toxicity in smokers.

Effects of Reduning injection on activity of hepatic microsomal CYP450 isozymes in rats

To research the influence of Reduning injection on the activity and mRNA expression of cytochrome P450 (CYP450) system in rat liver microsomes. Rat liver microsomes were prepared after a seven-days continuous administration of Reduning injection. An HPLC-MS method was applied to determine the specific metabolites of CYP450 probe substrates in rat liver microsomal incubations. The activity of CYP450 isozymes were represented by the formation of metabolites. The Real-time quantitative polymerase chain reaction (Q-PCR) was applied to determine the mRNA expression levels of CYP450. Reduning injection significantly reduced the activity of CYP2B1, 2C12, 2C13 (P < 0.01), but did not affect CYPlA2; low dose and high dose of Reduning injection had an inhibition trend on the activity of CYP2D2, but did not statistically differ from control group; low dose of Reduning injection significantly induced the activity of CYP3A1 (P < 0.01), high dose of Reduning injection had an induce trend on the activity of CYP3A1, but did not statistically differ from control. At the mRNA level, low and high dose of Reduning injection had an induce trend on the expression of CYP1A2, 2C11, 2D1, 2E1, 3A1, but did not statistically differ from control. Reduning injection significantly induced the activity of CYP2B1. Reduning injection significantly induced the activity of CYP3A1 in mRNA expression and enzyme activity levels, which may result adverse drug reaction after being combined with macrolides antibiotics. Reduning injection significantly reduced the activity of CYP2B1, 2C12, 2C13, 2D2 in enzyme activity levels, when combined with other drugs, it should be fully taken into account of the possible drug-drug interaction in order to avoid adverse side effects.

Evaluation of Tetrahydropalmatine Enantiomers on the Activity of Five Cytochrome P450 Isozymes in Rats Using a Liquid Chromatography / Mass Spectrometric Method and a Cocktail Approach.

The aim was to evaluate the effects of tetrahydropalmatine (THP) enantiomers on the activity of five cytochrome P450 (CYP450) isozymes in vivo. A liquid chromatography / mass spectrometric (LC-MS) method was developed for simultaneous determination of five specific probe substrates including metoprolol (2D6), caffeine (1A2), dapsone (3A4), chlorzoxazone (2E1), and tolbutamide (2C9) in rat plasma. Analytes were separated with the mobile phase consisting of 0.1% acetic acid aqueous solution and acetonitrile in a gradient elution. The mass spectrometric detection via selected ion monitoring (SIM) was operated in both positive ion mode (for metoprolol m/z 268, caffeine m/z 195, and dapsone m/z 249) and negative ion mode (for chlorzoxazone m/z 168 and tolbutamide m/z 269) in the same run. Linear correlation was obtained (r(2) > 0.99) over the concentration range of 0.050-25.0 µg/mL for caffeine and dapsone, 0.025-10.0 µg/mL for metoprolol, 0.050-50.0 µg/mL for chlorzoxazone, and 0.25-100.0 µg/mL for tolbutamide. Intra- and interday precision were less than 12.09%. The matrix effect ranged from 87.50% to 109.25% and the absolute recoveries were greater than 70%. The method was successfully applied to evaluate the effect of THP enantiomers on the activity of CYP450 isozymes by a cocktail approach. The pharmacokinetic results of five probe drugs indicated that there were stereoselective differences between the two THP enantiomers, i.e., d-THP had the potential to inhibit the activities of CYP2D6 and CYP1A2 isozymes, while l-THP inhibited CYP1A2 isozyme and induced CYP3A4 and CYP2C9 isozymes.

Effects of vitexin on the pharmacokinetics and mRNA expression of CYP isozymes in rats.

In traditional therapy with Chinese medicine, vitexin has several pharmacological properties, including antinociceptive, antispasmodic, antioxidant, antimyeloperoxidase, and α-glucosidase inhibitory activities. Recently, vitexin was shown to protect the heart against ischemia/reperfusion injury in an in vitro model by inhibiting apoptosis. The purpose of this study was to find out whether vitexin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, and CYP3A1) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (10 mg/kg), tolbutamide (1 mg/kg), and midazolam (5 mg/kg), was given as oral administration to rats treated with short or long period of intravenous vitexin via the caudal vein. Blood samples were collected at a series of time points, and the concentrations of probe drugs in plasma were determined by HPLC-mass spectrometry (MS)/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time reverse transcription-polymerase chain reaction was performed to determine the effects of vitexin on the mRNA expression of CYP1A2, CYP2C11, and CYP3A1 in rat liver. Treatment with short or long period of vitexin had no effects on rat CYP1A2. However, CYP3A1 enzyme activity was inhibited by vitexin in a concentration-dependent and time-dependent manner. Furthermore, CYP2C11 enzyme activity was induced after short period treatment but inhibited after long period of vitexin treatment. The mRNA expression results were in accordance with the pharmacokinetic results. In conclusion, vitexin can either inhibit or induce activities of CYP2C11 and CYP3A1. Therefore, caution is needed when vitexin is co-administered with some CYP2C11 or CYP3A1 substrates in clinic, which may result in treatment failure and herb-drug interactions.

Effects of hydroxysafflor yellow A on the activity and mRNA expression of four CYP isozymes in rats

Ethnopharmacological relevanceIn traditional therapy with Chinese medicine, hydroxysafflor yellow A (HSYA), a main active component isolated from the dried flower of Carthamus tinctorius L., is the principal efficiency ingredient of Safflor Yellow Injection. Now HSYA has been demonstrated to have good pharmacological activities of antioxidation, myocardial and cerebral protective and neuroprotective effects. The purpose of this study was to find out whether HSYA influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D4 and CYP3A1) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied. Materials and methodsA cocktail solution at a dose of 5mL/kg, which contained phenacetin (20mg/kg), tolbutamide (5mg/kg), dextromethorphan (20mg/kg) and midazolam (10mg/kg), was given as oral administration to rats treated with short or long period of intravenous HSYA via the caudal vein. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC–MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time RT-PCR was performed to determine the effect of HSYA on the mRNA expression of CYP1A2, CYP2C11, CYP2D4 and CYP3A1 in rat liver. ResultsHSYA had significant inhibition effects on CYP1A2 and CYP2C11 in rats as oriented from the pharmacokinetic profiles of the probe drugs. Furthermore, HSYA had no effects on rat CYP2D4. However, CYP3A1 enzyme activity was induced by HSYA. The mRNA expression results were in accordance with the pharmacokinetic results. ConclusionsHSYA can either inhibit or induce activities of CYP1A2, CYP2C11 and CYP3A1. Therefore, co-administration of some CYP substrates with HSYA may need dose adjustment to avoid an undesirable herb–drug interaction.

Assessment of effects of IR and IPC on activities of cytochrome P450 isozymes in rats by a five-drug cocktail approach

Background and objective: To evaluate the effects of ischemia and reperfusion (IR) and ischemic preconditioning (IPC) on the metabolic activities of cytochrome P450 (CYP) isozymes in rats by a five-drug cocktail approach.Methods: Cocktail approach was used to evaluate the influence of IR and IPC on the activities of CYP1A2, CYP2C9, CYP2E1, CYP2D6 and CYP3A4, which were reflected by the changes of pharmacokinetic parameters of five specific probe drugs: caffeine, chlorzoxazone, tolbutamide, metoprolol and midazolam, respectively. Rats were randomly divided into IR, IPC and sham groups, and then injected the mixture of five probe drugs. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by a HPLC method with UV detection. The pharmacokinetic parameters were calculated by the software of DAS 2.0.Results: The parameters including t1/2β, CLs, AUC, MRT and K10 exhibited a similar tendency for both IR and IPC groups. Compared with sham group, CLs and K10 of five probe drugs were significantly lower (p < 0.05), AUC and t1/2β of five or some probe drugs were significantly increased in IR and IPC groups (p < 0.05). Compared with IPC group, CLs of five probe drugs were decreased and AUC were significantly increased in the IR group (p < 0.05).Conclusion: IR can variably decrease the activities of CYP isozymes in rats and this decrease can be attenuated by IPC.

Effects of enzyme inducers and inhibitors on the pharmacokinetics of intravenous ipriflavone in rats

In order to find out what types of the hepatic microsomal cytochrome P450 (CYP) isozymes are involved in the metabolism of ipriflavone, ipriflavone at a dose of 20 mg kg(-1) (or 15 mg kg(-1)) was infused in male Sprague-Dawley rats. In rats pretreated with SKF 525-A (a non-specific CYP isozyme inhibitor in rats), the total body clearance (CL) of ipriflavone was significantly slower (29.9% decrease) than that in control rats. This indicates that ipriflavone is metabolized via CYP isozymes in rats, hence various enzyme inducers and inhibitors were used in in-vitro or in-vivo studies in rats. In rats pretreated with 3-methylcholanthrene and phenobarbital (main inducers of CYP1A1/2 and 2B1/2 in rats, respectively), the CL values were significantly higher (153 and 67.2% increases, respectively). In rats pretreated with sulfaphenazole (a main inhibitor of CYP2C11 in rats), the CL was significantly slower (22.5% decrease) than that in control rats. On addition of furafylline (a main inhibitor of CYP1A2 in rats), the in-vitro intrinsic clearance for the disappearance of ipriflavone was significantly slower (50.8% decrease) than that without furafylline. However, the CL values were not significantly different in rats pretreated with orphenadrine and isoniazid (a main inducer of CYP2E1 in rats), and quinine and troleandomycin (main inhibitors of CYP2D1 and 3A23/2 in rats, respectively) compared to controls. These data suggest that ipriflavone could be metabolized mainly via CYP1A1/2, 2B1/2 and 2C11 in rats.

Effect of enzyme inducers and inhibitors on the pharmacokinetics of oltipraz in rats

A series of in-vitro and in-vivo experiments, using various inducers and inhibitors of hepatic microsomal cytochrome P450 (CYP) isozymes, was conducted to study oltipraz pharmacokinetics in rats. In in-vivo studies, oltipraz at a dose of 10 mg kg(-) was administered intravenously to rats. In rats pretreated with SKF 525-A (a nonspecific CYP isozyme inhibitor in rats; n-9), the time-averaged total body clearance (CL) of oltipraz was significantly slower (56.6% decrease) than that in untreated rats (n=9). This indicated that oltipraz is metabolized via CYP isozymes in rats. Hence, various enzyme inducers or inhibitors were used in in-vitro and in-vivo studies in rats. In rats pretreated with 3-methylcholanthrene (n=9 and 8 for untreated and treated groups, respectively), phenobarbital (n=7 and 10 for untreated and treated groups, respectively) or dexamethasone (n=7 and 12 for untreated and treated groups, respectively) (main inducers of CYP1A1/2, 2B1/2 and 3A1/2 in rats, respectively), the CL values were significantly faster (38.4, 94.4 and 33.6% increase, respectively). In rats pretreated with sulfaphenazole (n=8 and 9 for untreated and treated groups, respectively), quinine (n=7 and 9 for untreated and treated groups, respectively) or troleandomycin (n=8 and 9 for untreated and treated groups, respectively) (main inhibitors of CYP2C11, 2D1 and 3A1/2 in rats, respectively), the CL values were significantly slower (31.0, 27.6 and 36.3% decrease, respectively). The in-vivo results with various enzyme inhibitors correlated well with the in-vitro intrinsic clearance for disappearance of oltipraz (CL(int)) (n=5, each). The above data suggested that oltipraz could be metabolized in male rats mainly via CYP1A1/2, 2B1/2, 2C11, 3A1/2 and 2D1.

Hepatotoxicity and gene expression down-regulation of CYP isozymes caused by renal ischemia/reperfusion in the rat

Renal ischemia/reperfusion (I/R) occurs in many clinical scenarios, including trauma, elective surgery, and transplantation. Events initiated by this process can lead to inflammation in the kidneys, culminating in local injury as well as distant organ dysfunction. The objectives of this study were to investigate the changes in the functions of the liver and the regulation of gene expression of cytochrome P450 (CYP) isozymes after renal I/R. Hepatoxocity was assessed by serum alanine aminotransferase (sALT), serum aspartate aminotransferase (sAST) and liver glutathione-S-transferase (GST) activities, liver glutathione (GSH) level, and histopathological examination. Hepatic cytochrome P4503A1 (CYP3A1) and cytochrome P4502E1 (CYP2E1) activities were measured by erythromycin N-demethylase (ERD) and aniline hydroxylase (ANH) activities, respectively. CYP3A1 and CYP2E1 mRNA expression was determined by RT-PCR. Results showed that activities of sALT and sAST were significantly increased, while hepatic CYP3A1and CYP2E1 activities as well as their respective mRNA levels were significantly decreased after renal I/R. Moreover, hepatic tissue congestion, degeneration, and local necrosis were observed in rats after 1, 4, and 8 h renal reperfusion following 2 h renal ischemia. In conclusion, the present study suggests that renal I/R can cause hepatotoxicity and gene expression down-regulation of CYP isozymes in rats.

Effect of biliary obstruction and internal biliary drainage on hepatic cytochrome P450 isozymes in rats

To investigate the total cytochrome P450 (CYP) content, microsomal mixed-function oxidase (MFO) activity, and expression of mRNAs for various CYP isozymes in a simple rat model of reversible obstructive jaundice. Obstructive jaundice was created in male rats by causing bile duct obstruction with polyester tape. In another group of rats, bile duct obstruction was followed by internal biliary drainage after releasing the tape. The expression of various CYP isozyme mRNAs was semi-quantitatively assessed by competitive RT-PCR. The total CYP content and microsomal MFO activity showed a significant decrease after biliary obstruction, but returned to respective control levels after biliary drainage. A marked reduction in the expression of CYP1A2, 2B1/2, 2C11, 2E1, 3A1, and 3A2 mRNA was detected during biliary obstruction, while expression increased significantly toward the control level after biliary drainage. Although expression of CYP4A1 mRNA showed no reduction during biliary obstruction, it still increased significantly after biliary drainage. These results suggest that not only obstructive jaundice, but also the subsequent internal biliary drainage may affect regulatory medications of the synthesis of individual CYP isozymes differently.

Effects of enzyme inducers and inhibitors on the pharmacokinetics of intravenous omeprazole in rats

A series of experiments using various inducers and inhibitors of the hepatic microsomal cytochrome P450 (CYP) isozymes were conducted to find CYP isozymes responsible for the metabolism of omeprazole in male Sprague-Dawley rats. Omeprazole, 20 mg/kg, was administered intravenously. In rats pretreated with SKF 525-A (a nonspecific CYP isozyme inhibitor in rats), the time-averaged nonrenal clearance (Cl(nr)) was significantly slower (77.1% decrease) than that in untreated rats. This indicated that omeprazole is metabolized via CYP isozymes in rats. Hence, rats were pretreated with various enzyme inducers and inhibitors. In rats pretreated with 3-methylcholanthrene and dexamethasone (main inducers of CYP1A1/2 and 3A1/2 in rats, respectively), the Cl(nr) values were significantly faster (43.8% and 26.3% increase, respectively). In rats pretreated with troleandomycin and quinine (main inhibitors of CYP3A1/2 and 2D1 in rats, respectively), the Cl(nr) values were significantly slower (20.9% and 12.9% decrease, respectively). However, the Cl(nr) values were not significantly different in rats pretreated with orphenadrine, isoniazid and sulfaphenazole (main inducers of CYP2B1/2 and 2E1, and a main inhibitor of 2C11, respectively, in rats) compared with those of respective control rats. The above data suggested that omeprazole could be mainly metabolized via CYP1A1/2, 3A1/2 and 2D1 in male rats.

Selective protection of curcumin against carbon tetrachloride-induced inactivation of hepatic cytochrome P450 isozymes in rats

We investigated the effects of curcumin, a major antioxidant constituent of turmeric, on hepatic cytochrome P450 (CYP) activity in rats. Wistar rats received curcumin-containing diets (0.05, 0.5 and 5 g/kg diet) with or without injection of carbon tetrachloride (CCl 4). The hepatic CYP content and activities of six CYP isozymes remained unchanged by curcumin treatment, except for the group treated with the extremely high dose (5 g/kg). This suggested that daily dose of curcumin does not cause CYP-mediated interaction with co-administered drugs. Chronic CCl 4 injection drastically decreased CYP activity, especially CYP2E1 activity, which is involved in the bioactivation of CCl 4, thereby producing reactive free radicals. Treatment with curcumin at 0.5 g/kg alleviated the CCl 4-induced inactivation of CYPs 1A, 2B, 2C and 3A isozymes, except for CYP2E1. The lack of effect of curcumin on CYP2E1 damage might be related to suicidal radical production by CYP2E1 on the same enzyme. It is speculated that curcumin inhibited CCl 4-induced secondary hepatic CYPs damage through its antioxidant properties. Our results demonstrated that CYP isozyme inactivation in rat liver caused by CCl 4 was inhibited by curcumin. Dietary intake of curcumin may protect against CCl 4-induced hepatic CYP inactivation via its antioxidant properties, without inducing hepatic CYPs.

Toxicologic implications of the metabolism of thiram, dimethyldithiocarbamate and carbon disulfide mediated by hepatic cytochrome P450 isozymes in rats

These studies were conducted to identify the P450 isozymes participating in the metabolism of thiram (tetramethylthiuram disulfide) and its two metabolites DMDC (dimethyldithiocarbamate) and CS 2 (carbon disulfide), and to establish the relationship between the P450-mediated metabolism of these compounds and their possible hepatotoxic effects. These compounds were individually administered i.p. to normal rats at two equimolar doses (0.1 and 0.5 mmol/kg).The animals were sacrificed at 3 h after treatment to identify the inhibited isozymes, and at 24 h to determine the hepatic damage. The results indicated that only the higher dose of thiram inhibited the activities of CYP1A1 and 2B1 significantly at 3 h after treatment whereas CYP2E1 was depressed by both doses and CYP3A2 remained unaffected by either dose. At 24 h after treatment, higher dose of thiram significantly depressed only the CYP1A1 and 2E1 activities. Both CYP1A1 and 2E1 were inhibited by higher dose of DMDC, but there was no significant inhibition of CYP2B1 or 3A2 by DMDC at 3 h following treatment. Unlike thiram, DMDC did not affect the activity of any of the CYP isozymes at 24 h after treatment. At 3 h post-treatment, although the higher dose of CS 2 significantly inhibited CYP1A1, 2B1, 2E1, and 3A2, the lower dose decreased the activities of only CYP1A1, 2B1, and 2E1. On the other hand, CS 2 increased the activities of CYP1A1, 2B1, and 2E1 at 24 h following treatment with no effect on CYP3A2. It is further observed from the results that only thiram at higher dose caused liver damage as measured by a significant increase in the activities of SDH and ALT. The results suggest that CYP1A1, 2B1, and 2E1 appear to be mostly involved in the metabolism of these compounds, and among them only thiram is hepatotoxic.

Catalytic and Immunochemical Characterization of Cytochrome P450 Isozyme Induction in Dog Liver

The purpose of this study was to characterize hepatic cytochrome P450 induction in the dog by phenobarbital, β-naphthoflavone, dexamethasone, and isoniazid using catalytic activities and Western blots with antibodies prepared against rat cytochrome P450 isozymes. Male beagle dogs were treated with phenobarbital (10 mg/kg for 2 days and 30 mg/kg for the following 5 days), β-naphthoflavone (50 mg/kg for 5 days), or isoniazid (10 mg/kg for 2 days and 30 mg/kg for the following 5 days). Female beagle dogs were treated with dexamethasone (50 mg/kg for 5 days). Increases in the liver/body weight ratio were observed after treatment of dogs with phenobarbital (133% of control) and dexamethasone (153%). Total cytochrome P450 content was increased as a percentage of control after treatment with phenobarbital (264%) and β-naphthoflavone (186%), while it slightly decreased after treatment with isoniazid (54%) and dexamethasone (71%). Dog liver microsomes hydroxylated testosterone mainly at the 6β and 16α positions but also at the 6α-, 15β-, 15α-, 16β-, 18-, 2β-, and 17-positions. There were no sex differences in terms of regioselectivity of testosterone metabolism between control male and female dogs. Treatment of dogs with phenobarbital produced increases in 6β- (184%), 16α- (379%), 16β- (210%), 18- (195%), and 2β-testosterone (203%) hydroxylase and pentoxyresorufinO-dealkylase (651%) activities. On Western blots, phenobarbital treatment produced induction of P450 3A- and 2B1-related proteins. Although treatment with dexamethasone resulted in a large increase in liver weight, no significant increase in P450 3A-related protein or 6β-hydroxylase activity was detected. However, dexamethasone and isoniazid treatment produced slight increases in chlorzoxazone hydroxylase activity. Treatment with isoniazid induced a P450 2E1-related protein. Treatment with β-naphthoflavone produced increases that were 689 and 357% of control in ethoxyresorufinO-deethylase and chlorzoxazone hydroxylase activities, respectively. β-Naphthoflavone treatment increased the amount of two proteins immunochemically related to the cytochrome P450 1A subfamily. Thus, although generally similar to other species, the response of the dog to cytochrome P450 inducers differs significantly from the rat and human in some cases.

Catalytic and Immunochemical Characterization of Cytochrome P450 Isozyme Induction in Dog Liver

The purpose of this study was to characterize hepatic cytochrome P450 induction in the dog by phenobarbital, β-naphthoflavone, dexamethasone, and isoniazid using catalytic activities and Western blots with antibodies prepared against rat cytochrome P450 isozymes. Male beagle dogs were treated with phenobarbital (10 mg/kg for 2 days and 30 mg/kg for the following 5 days), β-naphthoflavone (50 mg/kg for 5 days), or isoniazid (10 mg/kg for 2 days and 30 mg/kg for the following 5 days). Female beagle dogs were treated with dexamethasone (50 mg/kg for 5 days). Increases in the liver/body weight ratio were observed after treatment of dogs with phenobarbital (133% of control) and dexamethasone (153%). Total cytochrome P450 content was increased as a percentage of control after treatment with phenobarbital (264%) and β-naphthoflavone (186%), while it slightly decreased after treatment with isoniazid (54%) and dexamethasone (71%). Dog liver microsomes hydroxylated testosterone mainly at the 6β and 16α positions but also at the 6α-, 15β-, 15α-, 16β-, 18-, 2β-, and 17-positions. There were no sex differences in terms of regio-selectivity of testosterone metabolism between control male and female dogs. Treatment of dogs with phenobarbital produced increases in 6β- (184%), 16α- (379%), 16β- (210%), 18- (195%), and 2β-testosterone (203%) hydroxylase and pentoxyresorufin O-dealkylase (651%) activities. On Western blots, phenobarbital treatment produced induction of P450 3A-and 2B1-related proteins. Although treatment with dexamethasone resulted in a large increase in liver weight, no significant increase in P450 3A-related protein or 6β-hydroxylase activity was detected. However, dexamethasone and isoniazid treatment produced slight increases in chlorzoxazone hydroxylase activity. Treatment with isoniazid induced a P450 2E1-related protein. Treatment with β-naphthoflavone produced increases that were 689 and 357% of control in ethoxyresorufin O-deethylase and chlorzoxazone hydroxylase activities, respectively. β-Naphthoflavone treatment increased the amount of two proteins immunochemically related to the cytochrome P450 1A subfamily. Thus, although generally similar to other species, the response of the dog to cytochrome P450 inducers differs significantly from the rat and human in some cases.

Norfloxacin interferes with cyclosporine disposition in pediatric patients undergoing renal transplantation*

Prophylactic treatment with norfloxacin was initiated in a group of pediatric patients undergoing renal transplantation who were receiving cyclosporine and were susceptible to recurrent urinary tract infections. At discharge from the hospital, the mean daily dose of cyclosporine needed to maintain trough cyclosporine blood levels of 150 to 400 ng/ml was 4.5 mg/kg/day for the patients who received norfloxacin compared with 7.4 mg/kg/day for patients who did not receive the antibiotic. This observation suggested that the clearance of cyclosporine was decreased by the concomitant use of norfloxacin. The effect of norfloxacin on specific drug-metabolizing cytochrome P450 isozymes in vitro was examined to determine if the metabolism and subsequent clearance of cyclosporine and possibly other drugs are altered through a metabolic interaction with norfloxacin. In human liver microsomes, the activity of cytochrome P4503A4, the isozyme that metabolizes cyclosporine in humans, was inhibited by norfloxacin. In rat liver microsomes, norfloxacin inhibited the activity of cytochrome P4503A2, the isozyme responsible for cyclosporine metabolism in this species, but did not alter the activity of the rat cytochrome P450 isozymes 1A, 2E1, and 4A1. The in vitro studies suggest that the lower cyclosporine dose required by pediatric patients who were given norfloxacin was caused by inhibition of the metabolism of cyclosporine.