P-450 Content Research Articles

Tertiary butyl alcohol in drinking water induces phase I and II liver enzymes with consequent effects on thyroid hormone homeostasis in the B6C3F1 female mouse

Tertiary butyl alcohol (TBA) was administered to groups of 15 female B6C3F1 mice in drinking water at concentrations of 0, 2.0 or 20 mg TBA ml(-1), for 14 days, for assessment of gross and histological changes in the liver and thyroid, thyroid hormones (T3, T4, and TSH), total hepatic cytochrome P450 (Cyp) content, specific Cyp activities and quantitative PCR analysis of specific Cyp enzymes (Cyp1a1, Cyp2b9, Cyp2b10, Cyp3a11), sulfuryltransferases (ST1a1, ST2a2, and STn) and glucuronyltransferases (UGT1a1, UGT2b1, and UGT2b5). Phenobarbital (PB) was administered to a positive control group by oral gavage at a daily dose of 80 mg kg(-1). TBA caused, on day 14, a reduction in circulating T3 (12-15% decrease) and a dose-dependent reduction in T4 (13-22% decrease), with no evidence of thyroid pathology. Two of five livers examined in the 20 mg TBA ml(-1) dose group showed mild, diffuse centrilobular hypertrophy. On day 14, Cyp 7-benzoxyresorufin-O-debenzylase activity was significantly induced 12-fold by TBA at 20 mg ml(-1), and 1.8-fold at the 2.0 mg TBA ml(-1) concentration. Cyp 7-pentoxyresorufin-O-dealkylase activity was slightly induced (2.1-fold) by 20 mg TBA ml(-1) on day 14. Quantitative PCR analysis of gene transcripts showed a significant induction of Cyp2b10 and ST1a1 with both TBA concentrations, and a slight induction of Cyp2b9 at 20 mg TBA ml(-1) only. PB induced all phase I and phase II gene transcripts except for Cyp1a1 and Cyp2b9. These findings suggest that TBA, at and below doses used in chronic studies, is an inducer of phase I and phase II liver enzymes, with resulting decreases in circulating thyroid hormones in B6C3F1 mice.

Effects of atrazine on cytochrome P450 enzymes of zebrafish ( Danio rerio)

In this study, the effects of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in males and females of adult zebrafish ( Danio rerio) were studied. The liver microsomal cytochrome P450 content, NADPH-P450 reductase, aminopyrine N-demethylase (APND), and erythromycin N-demethylase (ERND) activity were measured. Zebrafish were exposed to control and 3 treatments (0.01, 0.1, and 1 mg L −1) of atrazine for 5, 10, 15, 20, and 25 days. The results indicated that, within the range of test atrazine concentrations, either P450 content or P450 isozyme activities could be induced by atrazine. Compared to controls, P450 content was significantly increased at all atrazine concentrations at days 10, 15, and 20; thereafter, at day 25, all concentrations decreased to approximately the control levels, both in males and females. In addition, the strongest induction of P450 content was observed on day 15 in males and day 10 in females at treatment concentrations of 1 mg L −1. NADPH-P450 reductase activities showed mild increase in males; however, the females exhibited significant induction on days 15, 20, and 25; especially, at concentrations of 0.01 mg L −1, the induction level was consistently increased during the experiment. The inducements of APND and ERND in males were mainly observed on the days 5, 10, and 15, which showed less distinct induction, while significant induction was observed in cases of treatments during all days in females. In conclusion, atrazine induces P450 enzymes in zebrafish, and the effects may function as significant toxicity mechanisms in zebrafish. Additionally, it also confirms the importance of using a combined multi-time and multi-index diagnostic method to enhance the sensitivity and effectiveness of the indices adopted.

Species-dependent hepatic metabolism of immunosuppressive agent tacrolimus (FK-506)

The current study aims to investigate species-related differences in the in-vitro hepatic metabolism of tacroliums using liver microsomes obtained from rat, hamster, guinea pig, rabbit, pig, dog, baboon and humans.Tacrolimus metabolism was characterized using high-performance liquid chromatography- ultraviolet light (HPLC-UV) and two soft ionization mass spectrometric techniques; matrix-assisted lasers desorption/ionization (MALDI) and time-of-flight-secondary ion mass spectrometry (TOF-SIMS).The extent of tacrolimus metabolism, when normalized to the cytochrome P-450 content, was in the order: rat < hamster < rabbit < pig < guinea pig < dog < human < baboon. Tacrolimus metabolism exhibited significant qualitative and quantitative differences between the animal species tested.Desmethyl- (MI–MIII), didesmethyl- (MIV–MVI), monohydroxy- (MVII), dihydroxy- (MVIII), epoxide- (MIX), dihydrodiol- (MX), monodesmethyl and monohydroxy- (MXI–MXIII), and didesmethyl and monohydroxy- (MXIV–MXVI) tacroliums metabolites were identified in the species tested. MI–MX were identified in all the species tested; MXI–MXVI were identified in all species except rat, rabbit and guinea pig; and MXIV–MXVI were identified only in baboon.The current investigation was unable to detect any phase II metabolites due to the limitations of the test system used.The analytical methods were not able to differentiate optical and positional isomers of metabolites due to the nature of the analytical tools used, therefore groups of metabolites were identified based on their molecular weights and available information.From the current in-vitro metabolism studies, the pattern of tacroliums metabolism in baboons closely resembled that in humans and thus it is ideal for studying tacroliums metabolism-related work of clinical relevance.

Effect of antisense oligomer in controlling c-raf.1 overexpression during diethylnitrosamine-induced hepatocarcinogenesis in rat

In ras-mediated signal transduction pathway, c-raf.1 is believed to have predominant oncogenic potential and has been found to be highly expressed in certain human and animal malignancies including hepatocellular carcinoma. In the present study, anticancer efficacy of antisense c-raf.1 oligomer on the inhibition of c-raf.1 mRNA overexpression during hepatocarcinogenesis was determined. Initially antiproliferating effect of the antisense oligomers was studied in vitro by measuring the rate of tritiated thymidine incorporation into DNA in rat hepatocellular carcinoma cells in culture medium. Based on the findings, the antisense treatment was carried out in rat hepatocarcinogenesis model-initiated with diethylnitrosamine and promoted using 2-acetylaminoflourene. Different drug-metabolizing enzymes, lipid peroxidation, liver morphology and histopathological studies along with c-raf.1 gene expression by in situ hybridization were performed. c-raf.1 antisense oligomers exhibited an inhibitory effect (approximately 68%) on cancer cell proliferation in vitro. Gross and microscopic examination of liver showed fewer (29%) and smaller hyperplastic nodules and preneoplastic lesions (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control group. Treatment of antisense c-raf.1 oligomers enhanced cytochrome P-450 content (81%) and reduced glutathione S-transferase activity (33%), UDP glucuronosyltransferase activity (74%) and MDA concentration (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control animals. The oligomer treatment also resulted in less expression in terms of c-raf.1 expressed lesion count as compared to carcinogen control group. The study demonstrates that the antisense oligomer targeted against c-raf.1 mRNA inhibits the overexpression of c-raf.1 gene during hepatocellular carcinoma in rats.

The Effect of Infliximab on Hepatic Cytochrome P450 and Pharmacokinetics of Verapamil in Rats with Pre-Adjuvant Arthritis: A Drug-Disease and Drug-Drug Interaction

Inflammatory conditions result in increased concentration but reduced potency of some cardiovascular drugs. This is associated with increased levels of pro-inflammatory mediators. Infliximab reduces pro-inflammatory mediators and reverses the diminishing effect of inflammation on response in the rat. We suggested that infliximab treatment would also reverse the effects of inflammation on drug metabolism and clearance. We examined hepatic cytochrome P450 content and the pharmacokinetics of verapamil in pre-adjuvant arthritic rats treated with infliximab. Pre-adjuvant arthritis was induced in male Sprague-Dawley rats with a tail base injection of Mycobacterium butyricum. Animals were monitored for symptoms of arthritis, serum nitrite and C-reactive protein. On day 6, rats were administered with single s.c. doses of infliximab (10 mg/kg). On day 14, a single i.v. dose of racemic verapamil (2 mg/kg) was administered, and S- and R-verapamil concentrations were determined by high performance liquid chromatography. Hepatic cytochrome P450 content and verapamil protein binding were also measured. Serum nitrite levels were significantly elevated in pre-adjuvant arthritis. Infliximab did not affect mean nitrite concentrations but there was a significant correlation between nitrite and S-verapamil concentrations as well as cytochrome P450, CYP3A, and CYP1A contents. Infliximab increased cytochrome P450 enzymes content that had been diminished by pre-adjuvant arthritis but had no significant effect on verapamil protein binding. Infliximab partially restores hepatic cytochrome P450 enzyme contents. The effect of infliximab on the mean verapamil clearance was not significantly affected due, likely, to the lack of effect on plasma protein binding.

In vitro metabolism of polychlorinated biphenyls and cytochrome P450 monooxygenase activities in dietary-exposed Greenland sledge dogs

The in vitro metabolism of a polychlorinated biphenyl (PCB) mixture was examined using hepatic microsomes of dietary-exposed Greenland sledge dogs ( Canis familiaris) to an organohalogen-rich diet (Greenland minke whale blubber: EXP cohort) or a control diet (pork fat: CON cohort). The associations between in vitro PCB metabolism, activity of oxidative hepatic microsomal cytochrome P450 (CYP) isoenzymes and concentrations of PCBs and hydroxylated metabolites were investigated. The CON dogs exhibited a 2.3-fold higher depletion percentage for the PCB congeners having at least two pairs of vicinal meta-para Cl-unsubstituted carbons (PCB-18 and -33) relative to the EXP dogs. This depletion discrepancy suggests that there exist substrates in liver of the organohalogen-contaminated EXP dogs that can competitively bind and/or interfere with the active sites of CYP isoenzymes, leading to a lower metabolic efficiency for these PCBs. Testosterone (T) hydroxylase activity, determined via the formation of 6β-OH-T, 16α-OH-T, 16β-OH-T and androstenedione, was strongly correlated with the depletion percentages of PCB-18 and -33 in both cohorts. Based on documented hepatic microsomal CYP isoenzyme substrate specificities in canines, present associations suggest that primarily CYP2B/2C and CYP3A were inducible in sledge dogs and responsible for the in vitro metabolism of PCB-18 and -33.

About mechanisms of formation of multinuclear hepatocytes during toxic action of N-nitrosodimethylamine on rats

The processes of hepatocyte multinucleation were studied in rats exposed to N-nitrosodimethylamine (NDMA). Using the immunohistochemical reaction to γ-tubulin, it was established that the number of cells containing three or more centrosomes increased 48 h after the NDMA injection. The formation of additional centrosomes in hepatocytes was shown to be based on the oxidative stress induced by NDMA metabolism with the participation of the cytochrome P450 superfamily. The administration of NDMA led to a sharp increase in the cytochrome P450 content in liver, especially 24 and 48 h (3.3 and 2.8 times, respectively) after the NDMA injection. The immunohistochemical reaction for cytochrome P4502E1 revealed an intensive staining of the cytoplasm of centrilobular hepatocytes 24 and 48 h after the NDMA action. In the same time period, a 1.1-2.0-fold increase occurs in the concentration of malonic dialdehyde (MDA) (a derivative of lipid peroxidation) and a 1.1-1.3-fold decrease in catalase activity (an enzyme of the cell antioxidative system). At a later time (72–120 h) after the NDMA action, the number of cells with three or more centrosomes, the intensity of cell cytoplasmic staining for cytochrome P450 2E1, and the concentrations of P450 and MDA in the liver decreased, whereas catalase activity increased. After 48 h of NDMA treatment, the incorporation of binuclear hepatocytes with various 3H-thymidines into nuclei occured, which indicates asynchronous DNA synthesis. The immunohistochemical reaction for pKi-67, nuclear protein that is a marker of cell proliferation, has established that the asynchronicity of nuclear proliferative activity in binuclear cells is not only characteristic of the S phase, but also of other cell cycle phases, including G1, G2, and M. Thus, the main mechanisms of hepatocyte multinucleation under the influence of NDMA are as follows: (1) increased hyperamplification of centrosomes as a consequence of oxidative stress and (2) asynchronous DNA synthesis in nuclei of binuclear hepatocytes with subsequent asynchronous acytokinetic mitosis.

Isolation and Functional Characterization of CYP71AJ4 Encoding for the First P450 Monooxygenase of Angular Furanocoumarin Biosynthesis

The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi majus. Using cDNA probes inferred from CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa (CYP71AJ3 and -4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, whereas CYP71AJ4 only catalyzed the conversion of (+)-columbianetin to angelicin and negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin synthase exhibited an apparent K(m) of 2.1 +/- 0.4 microm for (+)-columbianetin and a k(cat) of 112 +/- 14 min(-1). Moreover, the use of 3'-deuterated (+)-columbianetin as substrate led to an almost complete "metabolic switch," resulting in the synthesis of anti-3'-hydroxy-3'-deuterated(+)-columbianetin. This confirms that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3'. Sequence comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown functionality by gene duplication and subsequent molecular evolution.

Mode of Action Analysis for the Synthetic Pyrethroid Metofluthrin-Induced Rat Liver Tumors: Evidence for Hepatic CYP2B Induction and Hepatocyte Proliferation

Two-year treatment with high doses of Metofluthrin produced hepatocellular tumors in both sexes of Wistar rats. To understand the mode of action (MOA) by which the tumors are produced, a series of studies examined the effects of Metofluthrin on hepatic microsomal cytochrome P450 (CYP) content, hepatocellular proliferation, hepatic gap junctional intercellular communication (GJIC), oxidative stress and apoptosis was conducted after one or two weeks of treatment. The global gene expression profile indicated that most genes with upregulated expression with Metofluthrin were metabolic enzymes that were also upregulated with phenobarbital. Metofluthrin induced CYP2B and increased liver weights associated with centrilobular hepatocyte hypertrophy (increased smooth endoplasmic reticulum [SER]), and induction of increased hepatocellular DNA replication. CYP2B1 mRNA induction by Metofluthrin was not observed in CAR knockdown rat hepatocytes using the RNA interference technique, demonstrating that Metofluthrin induces CYP2B1 through CAR activation. Metofluthrin also suppressed hepatic GJIC and induced oxidative stress and increased antioxidant enzymes, but showed no alteration in apoptosis. The above parameters related to the key events in Metofluthrin-induced liver tumors were observed at or below tumorigenic dose levels. All of these effects were reversible upon cessation of treatment. Metofluthrin did not cause cytotoxicity or peroxisome proliferation. Thus, it is highly likely that the MOA for Metofluthrin-induced liver tumors in rats is through CYP induction and increased hepatocyte proliferation, similar to that seen for phenobarbital. Based on analysis with the International Life Sciences Institute/Risk Science Institute MOA framework, it is reasonable to conclude that Metofluthrin will not have any hepatocarcinogenic activity in humans, at least at expected levels of exposure.

Effect of Aquilegia vulgaris (L.) ethyl ether extract on liver antioxidant defense system in rats

The ethyl ether extract from Aquilegia vulgaris (L.) (Ranunculaceae) contains a lot of phenolic acids. Their hydroxyl groups are capable of donating hydrogen atoms at the initial stage of lipid peroxidation (LPO), which inactivates hydroxyperoxides formed from polyunsaturated fatty acids (PUFAs) and leads to breakdown of the propagation chain. Rats pretreated with acetaminophen (APAP) (600 mg/kg b.w., p.o.) were given ethyl ether extract (100 mg/kg b.w., p.o.) obtained from A. vulgaris herb. The study parameters measured were microsomal lipid peroxidation, reduced glutathione, and the activity of hepatic antioxidant enzymes and some drug metabolizing enzymes. The treatment with ethyl ether extract of the herb produced a 87-95% decrease in uninduced and Fe2+/ascorbate-stimulated microsomal lipid peroxidation in the liver of rats receiving APAP. Hepatic glutathione level depleted by APAP increased significantly (by 18%) after the extract treatment. Antioxidant enzyme activity in the liver, inhibited by APAP, was found to increase after administration of the extract: catalase by about 36%, glutathione reductase by 27% and glutathione S-transferase by 29%. Glucose-6-phosphate dehydrogenase, which decreased after APAP administration, increased again by 26% after extract treatment. The extract tested did not affect the activity of DT-diaphorase. The cytochrome P450 content, depleted by APAP, increased as much as by 100% after the treatment. The activities of NADPH-cytochrome P450 reductase, aniline hydroxylase and aminopyrine N-demethylase were not affected. The protective effect of the Aquilegia vulgaris extract in APAP-induced liver injury was mediated by its antioxidant activity. The extract did not inhibit the formation of reactive intermediate metabolites of APAP.

Modulation of porcine cytochrome P450 enzyme activities by surgical castration and immunocastration

The present study aimed to evaluate some cytochrome P450 metabolic enzyme activities in hepatic microsomes prepared from entire male pigs (uncastrated pigs), surgically castrated pigs and pigs immunized against gonadotropin-releasing hormone (immunocastrated pigs). The activities of the following enzymes were measured: ethoxyresorufin O-deethylase (EROD, CYP1A1/1A2), methoxyresorufin O-deethylase (MROD, CYP1A2), pentoxyresorufin O-depentylase (PROD, CYP2B), coumarin hydroxylase (COH, CYP2A) and p-nitrophenol hydroxylase (PNPH, CYP2A/2E1). The total cytochrome P450 contents were not affected by either surgical or immunocastration. Hepatic microsomal activities for EROD, PROD, COH and PNPH were lower in entire male pigs compared with surgically castrated and immunocastrated pigs (P < 0.05). Surgically and immunocastrated male pigs were similar with respect to EROD, MROD, PROD and COH activities (P > 0.05), whereas surgically castrated pigs exhibited lower PNPH activity compared with immunocastrated pigs (P = 0.029). The effect of different concentrations of testicular steroids – testosterone, 17β-estradiol, free estrone and androstenone – on enzyme activities was evaluated by in vitro microsomal study. Testosterone at the concentration of 8 pmol/ml inhibited EROD activities and estradiol-17β at the concentration of 1.8 pmol/ml inhibited PROD activities in hepatic microsomes from surgically castrated pigs. The highest concentration of androstenone (7520 pmol/ml) inhibited COH activities, whereas a 42-fold lower concentration of androstenone (180 pmol/ml) stimulated COH activities in surgically castrated pigs. Both free estrone (3.5 pmol/ml) and androstenone (55 pmol/ml) inhibited EROD activities in microsomes from entire male pigs. Stimulation of COH activities by the highest dose of free estrone (18 pmol/ml) was recorded in microsomes from entire male pigs. However, these effects of steroids were not concentration-dependent and the maximum extent did not exceed ±15% variation compared with the controls. There was no inhibition of PNPH activities in the hepatic microsomes from either entire or castrated pigs. In conclusion, we showed that EROD, PROD, COH and PNPH activities were lower in entire male pigs compared with those in surgically and immunocastrated pigs. Direct inhibition by the testicular steroids – testosterone, 17β-estradiol, free estrone and androstenone – was not the primary cause of the reduced enzyme activities.

Phenobarbital (PB)-induced changes in blood coagulationrelated parameters in pregnant rats, lactating rats and pups

Effects of repeated administration of phenobarbital (PB) on blood coagulation-related parameters were examined in non-pregnant, pregnant and lactating rats, and also in pups born to PB-treated lactating dams. PB was orally administered at a dose level of 80 mg/kg/day to pregnant (from gestation day (GD) 13), postpartum (from postpartum day (PPD) 7) and non-pregnant rats (from 13 weeks of age) for 7 days. Blood was collected on GD20 or PPD14 to perform blood coagulation examination. Concurrently, the blood coagulation parameters were examined in the pups. Increases in liver weight and/or hepatic cytochrome P450 content were observed in the PB-treated non-pregnant, pregnant and lactating rats. Activated partial thromboplastin time (APTT) was prolonged and anti-thrombin III (ATIII) concentration was increased in the lactating rats, while there were no changes in prothrombin time (PT) or APTT in the non-pregnant and pregnant rats. Moreover, prolongation of PT and APTT and decreases in factors VII and IX activities were observed in their pups. Thus, prolongation of blood coagulation time was confirmed in both dams and their pups following PB-administration to lactating dams. Effects of vitamin K(2) (VK(2)) on PB-induced changes in blood coagulation-related parameters of both dams and their pups were examined by co-administration with PB and VK(2) to lactating dams. PT and APTT were comparable to the control and PB-induced prolongation of blood coagulation time was improved in the pups while APTT was prolonged in dams, suggesting that VK(2) was beneficial to pups but not to dams.

Determination of microsome and hepatocyte scaling factors for in vitro/in vivo extrapolation in the rat and dog

1. In vivo clearance predictions from in vitro assays require scaling factors to relate the concentrations of hepatocytes or microsomal protein to the intact liver.2. The aims were to measure the variability in scaling factors for Wistar rat and beagle dog for which the literature is particularly scarce and determine any sex differences.3. Scaling factors were determined by comparing the cytochrome P450 (P450) content in hepatocytes or microsomes against the P450 content of fresh liver homogenate. The use of fresh homogenate is recommended as freezing can increase contamination and affect the P450 assay.4. Meangeo hepatic microsomal concentrations in Wistar rats were 61 mg g−1 liver (95% confidence interval (CI); 47–75 mg g−1 liver) and in beagle dogs 55 mg g−1 liver (95% CI = 48–62 mg g−1 liver). Meangeo hepatocellularity was 163 × 106 cells g−1 liver for Wistar rats (95% CI = 127–199 × 106 cells g−1 liver) and 169 × 106 cells g−1 liver (95% CI = 131–207 × 106 cells g−1 liver) for beagle dogs. The data generated in this study indicate a consistency in scaling factors between rat and dog. No sex differences were observed.

Changes of midazolam pharmacokinetics in Wistar rats treated with lipopolysaccharide: relationship between total CYP and CYP3A2

It has been reported that infection interferes with drug metabolism, resulting in changes in pharmacokinetics. In this study, we investigated the effects of lipopolysaccharide (LPS) on hepatic total cytochrome P450 (CYP), CYP3A2, and CYP2C11 contents in a transient, LPS-induced, endotoxemia model of rats. In addition, to assess the effects on CYP3A2 activities, the pharmacokinetics of midazolam (CYP3A2 substrate) and 1-OH-midazolam (metabolite of midazolam) were investigated. Hepatic total CYP contents were significantly low until day 3 (P < 0.05) but returned to the control level on day 5. Hepatic CYP3A2 contents were significantly decreased on day 1 until day 5 (P < 0.05) but returned to the control level on day 7. Hepatic CYP2C11 contents were continuously low until day 7, and lowest on day 3. The AUC of 1-OH-midazolam was significantly decreased on day 1 after LPS administration (P < 0.01). In conclusion, LPS (5 mg/kg) challenge decreased hepatic total CYP, CYP3A2, and CYP2C11 contents and also decreased the activities of hepatic CYP3A2. It took at least 7 days for hepatic total CYP and CYP3A2 to recover to control levels, and it was suggested that the changes of hepatic total CYP contents might correlate with those of hepatic CYP3A2 contents and activities. Additionally, it is shown that their changes might reflect the recovery process from inflammation.

CYP2B6: New Insights into a Historically Overlooked Cytochrome P450 Isozyme

Human CYP2B6 has been thought to account for a minor portion (<1%) of total hepatic cytochrome P450 (CYP) content and to have a minor function in human drug metabolism. Recent studies, however, indicate that the average relative contribution of CYP2B6 to total hepatic CYP content ranges from 2% to 10%. An increased interest in CYP2B6 research has been stimulated by the identification of an ever-increasing substrate list for this enzyme, polymorphic and ethnic variations in expression levels, and evidence for cross-regulation with CYP3A4, UGT1A1 and several hepatic drug transporters by the nuclear receptors pregnane X receptor and constitutive androstane receptor. Moreover, 20- to 250-fold interindividual variation in CYP2B6 expression has been demonstrated, presumably due to transcriptional regulation and polymorphisms. These individual differences may result in variable systemic exposure to drugs metabolized by CYP2B6, including the antineoplastics cyclophosphamide and ifosfamide, the antiretrovirals nevirapine and efavirenz, the anesthetics propofol and ketamine, the synthetic opioid methadone, and the anti-Parkinsonian selegiline. The potential clinical significance of CYP2B6 further enforces the need for a comprehensive review of this xenobiotic metabolizing enzyme. This communication summarizes recent advances in our understanding of this traditionally neglected enzyme and provides an overall picture of CYP2B6 with respect to expression, localization, substrate-specificity, inhibition, regulation, polymorphisms and clinical significance. Emphasis is given to nuclear receptor mediated transcriptional regulation, genetic polymorphisms, and their clinical significance.

Phenobarbital influence on neuromuscular block produced by rocuronium in rats

To evaluate in vitro and in vivo neuromuscular blockade produced by rocuronium in rats treated with Phenobarbital and to determine cytochrome P450 and cytochrome b5 concentrations in hepatic microsomes. Thirty rats were included in the study and distributed into 6 groups of 5 animals each. Rats were treated for seven days with phenobarbital (20 mg/kg) and the following parameters were evaluated: 1) the amplitude of muscle response in the preparation of rats exposed to phenobarbital; 2) rocuronium effect on rat preparation exposed or not to phenobarbital; 3) concentrations of cytochrome P450 and cytochrome b5 in hepatic microsomes isolated from rats exposed or not to phenobarbital. The concentration and dose of rocuronium used in vitro and in vivo experiments were 4 microg/mL and 0,6 mg/kg, respectively. Phenobarbital in vitro and in vivo did not alter the amplitude of muscle response. The neuromuscular blockade in vitro produced by rocuronium was significantly different (p=0.019) between exposed (20%) and not exposed (60%) rats; the blockade in vivo was significantly greater (p=0.0081) in treated rats (93.4%). The enzymatic concentrations were significantly greater in rats exposed to phenobarbital. Phenobarbital alone did not compromise neuromuscular transmission. It produced enzymatic induction, and neuromuscular blockade in vivo produced by rocuronium was potentiated by phenobarbital.

Antihyperalgesic effect of a Cannabis sativa extract in a rat model of neuropathic pain: mechanisms involved

This study aimed to give a rationale for the employment of phytocannabinoid formulations to treat neuropathic pain. It was found that a controlled cannabis extract, containing multiple cannabinoids, in a defined ratio, and other non-cannabinoid fractions (terpenes and flavonoids) provided better antinociceptive efficacy than the single cannabinoid given alone, when tested in a rat model of neuropathic pain. The results also demonstrated that such an antihyperalgesic effect did not involve the cannabinoid CB1 and CB2 receptors, whereas it was mediated by vanilloid receptors TRPV1. The non-psychoactive compound, cannabidiol, is the only component present at a high level in the extract able to bind to this receptor: thus cannabidiol was the drug responsible for the antinociceptive behaviour observed. In addition, the results showed that after chronic oral treatment with cannabis extract the hepatic total content of cytochrome P450 was strongly inhibited as well as the intestinal P-glycoprotein activity. It is suggested that the inhibition of hepatic metabolism determined an increased bioavailability of cannabidiol resulting in a greater effect. However, in the light of the well known antioxidant and antiinflammatory properties of terpenes and flavonoids which could significantly contribute to the therapeutic effects, it cannot be excluded that the synergism observed might be achieved also in the absence of the cytochrome P450 inhibition.

Efeitos neuromusculares in vitro e in vivo do atracúrio e do rocurônio em ratos submetidos a tratamento de sete dias com carbamazepina

JUSTIFICATIVA E OBJETIVOS: Trata-se de um estudo experimental que investigou in vitro e in vivo o bloqueio neuromuscular produzido pelo rocurônio e atracúrio em ratos tratados com carbamazepina e determinou as concentrações de citocromo P450 e b5 redutase em microssomos hepáticos. MÉTODO: Ratos foram tratados por sete dias com carbamazepina (CBZ) - 40 mg.kg-1 pelo método de gavagem e sacrificados no oitavo dia sob anestesia com uretana. As preparações in vitro e in vivo foram montadas de acordo com as técnicas de Bulbring e de Leeuwin e Wolters, respectivamente. As concentrações e doses utilizadas dos bloqueadores nas preparações in vitro e in vivo foram, respectivamente, 20 µg.mL-1 e 0,5 mg.kg-1 para atracúrio (ATC); 4 µg.mL-1 e 0,6 mg.kg-1 para rocurônio (ROC). Cada protocolo teve um n = 5 e as respostas foram observadas por 60 minutos. Os efeitos do ATC e ROC foram avaliados nas preparações de ratos tratados (Cbz t) e comparados com os observados nas de ratos não-tratados (CBZst). As concentrações de citocromo P450 e b5 redutase foram determinadas em microssomos isolados de fígados de ratos tratados (CBZt) e comparadas com as obtidas em ratos não tratados (CBZst). RESULTADOS: A carbamazepina não alterou a amplitude das respostas musculares; in vitro e in vivo, não houve diferença entre o bloqueio neuromuscular produzido pelo atracúrio nas preparações CBZt versus CBZst; o bloqueio neuromuscular produzido pelo rocurônio nas preparações CBZt foi potencializado in vitro. A carbamazepina não alterou as concentrações de citocromo P450 e b5. CONCLUSÕES: O tratamento por sete dias com carbamazepina não influenciou no bloqueio produzido pelo atracúrio, e alterou in vitro os efeitos do rocurônio. O tempo de tratamento não foi suficiente para causar indução enzimática e diminuir a sensibilidade ao rocurônio.

Effects of Cu 2+ and Pb 2+ on different fish species: Liver cytochrome P450-dependent monooxygenase activities and FTIR spectra

The effects of Cu 2+-sulfate and Pb 2+-acetate on carp ( Cyprinus carpio L.), silver carp ( Hypopthalmichtys molitrix V.) and wels ( Silurus glanis L.) were studied. The liver microsomal Cyt P450 content, the EROD, ECOD and APND monooxygenase activities were measured. In vivo treatment with 1 mg L − 1 Cu 2+ significantly elevated the activities of these enzymes and Cyt P450 content in silver carp livers. The high-dose Cu 2+ treatment (10 mg L − 1 ) on silver carp caused two-fold higher induction in the P450 dependent monooxygenase isoensymes than in wels. Although the 2 mg kg − 1 treatment with Pb 2+ in carp elevated significantly the P450 content, the EROD isoenzyme activities were significantly decreased after 1 day, showing the destructive effect of metal ion on the enzyme system. In vitro, Cu 2+ and Pb 2+ decreased the Cyt P450 content in the carp liver microsomes and the absorption peak shifted to higher wavelength. Fourier Transform Infrared (FTIR) spectroscopy was used to detect the damaging effects of the heavy metals. According to the inhibitory potency to Cu 2+, the most sensitive isoenzyme was the EROD in wels, the least was the silver carp's isoenzyme. The investigated fish P450 isoenzymes showed, that the Cu 2+ was a stronger inhibitor than Pb 2+.

Potential lipid microdomains of the endoplasmic reticulum: how does this affect the P450 system

Little is known about the organization of NADPH‐cytochrome P450 reductase (CPR) and multiple P450 enzymes in the endoplasmic reticulum (ER). Although these proteins are known to form a 1:1 molar complex in the ER, in most tissues the P450 concentration exceeds that of CPR by about a 20:1. The goal of this study is to examine how components of the P450 system are organized, and to determine if they are heterogeneously distributed in lipid microdomains of the ER. Liver microsomes from untreated rabbits and those pretreated with P450 inducing agents were partially solubilized with 1% Brij 98 at 4°C, and centrifuged, separating the detergent‐resistant (pellet) and ‐soluble (supernatant) membranes. The supernatants and pellets were then examined by immunoblotting to determine the regional distribution of CPR and P450s in the membrane. Both CPR (∼90%) and CYP1A2 (∼50%) remained in a region of the membrane that is resistant to solubilization, whereas CYP2E1 and CYP2B4 were located in a more detergent‐sensitive region. Lipid analysis of these samples illustrated that the pellet was enriched with saturated and mono‐unsaturated fatty acids of phosphatidylcholine and sphingomyelin while the supernatant was enriched with polyunsaturated fatty acids. These results suggest that the proteins of the ER are not randomly distributed in the membrane, but appear to exist in specific lipid microdomains. (supported by NIEHS ES00434)