Use of Phenoxyaniline Analogues To Generate Biochemical Insights into the Interactio n of Polybrominated Diphenyl Ether with CYP2B Enzymes.

Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.

Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression.

Despite widespread use of bromuconazole as a pesticide for food crops and fruits, limited studies have been done to evaluate its toxic effects. Here, we evaluated the hepatotoxic effect of bromuconazole using classical toxicological (biochemical analysis and histopathological examination) and gene-based molecular methods. Male rats were treated either orally or topically with bromuconazole at doses equal to no observed adverse effect level (NOAEL) and 1/10 LD50 for 90 d. Bromuconazole increased activities of liver enzymes (ALT, AST, ALP, and ACP), and levels of bilirubin. It also induced hepatic oxidative stress as evidenced by significant decrease in the activities of superoxide dismutase (SOD), and significant increase in levels of malondialdehyde (MDA) in liver. In addition, bromuconazole caused an increase in liver weights and necrobiotic changes (vacuolation and hepatocellular hypertrophy). It also strongly induced the expression of PXR and its downstream target CYP3A1 gene as well as the activity of CYP3A1. However, it inhibited the expression of CAR and its downstream target CYP2B1 gene without significant changing in CYP2B1 activity. Overall, the oral route showed higher hepatotoxic effect and molecular changes than the dermal route and all changes were dose dependent. This is the first investigation to report that bromuconazole-induced liver oxidative damage is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1.

Intracerebroventricularly and systemically delivered inhibitor of brain CYP2B (C8-Xanthate), even following chlorpyrifos exposure, reduces chlorpyrifos activation and toxicity in male rats.

Chlorpyrifos is a pesticide that is metabolically activated to chlorpyrifos oxon (acetylcholinesterase inhibitor) primarily by the cytochrome P450 2B (CYP2B) enzyme subfamily in the liver and brain. We have previously shown that intracerebroventricular pretreatment with a CYP2B inhibitor, C8-Xanthate, can block chlorpyrifos toxicity. Here, we assessed whether delayed introduction of C8-Xanthate would still reduce toxicity and whether peripheral administration of C8-Xanthate could also inhibit chlorpyrifos activation in the brain and block toxicity. Male rats (N = 4-5/group) were either pretreated with C8-Xanthate (40 µg intracerebroventricular or 5 mg/kg intraperitoneal), or vehicle (ACSF or saline, respectively), 24 h before chlorpyrifos treatment (125 mg/kg subcutaneous) and then treated daily with inhibitor or vehicle until 7 days post-chlorpyrifos treatment. Additional groups received vehicle pretreatment, switching to C8-Xanthate 1, 2, 3, or 4 days after chlorpyrifos and then continuing with daily C8-Xanthate treatment until 7 days post-chlorpyrifos treatment. Neurotoxicity was assessed at baseline (before chlorpyrifos) and then daily after chlorpyrifos, using behavioral assessments (e.g., gait score). Neurochemical assays (e.g., serum and brain chlorpyrifos) were performed at the end of study. Pretreatment with C8-Xanthate completely prevented chlorpyrifos toxicity, and delayed introduction of C8-Xanthate reduced toxicity, even when started up to 4 days after chlorpyrifos treatment. Discontinuation of C8-Xanthate treatment 7 days post-chlorpyrifos treatment did not result in the reappearance of toxicity, tested through 10 days after chlorpyrifos treatment. These findings suggest that CYP2B inhibitor treatment, even days after chlorpyrifos exposure, and using a peripheral delivery route, may be useful as a therapeutic approach to reduce chlorpyrifos toxicity.

Reduction of thyroid hormones triggers down-regulation of hepatic CYP2B through nuclear receptors CAR and TR in a rat model of acute stroke.

Stroke is a neurological condition and may cause changes in hepatic drug-metabolizing enzymes. Hepatic CYP2B is involved in the metabolism of a variety of centrally active substances. The purpose of this study was to investigate the possible down-regulation mechanism of hepatic CYP2B after acute stroke. Using a rat model of acute stroke induced by middle cerebral artery occlusion, we studied the influence of brain ischemia/reperfusion (I/R) injury on CYP2B expression. Effects of 3,5,3'-triiodo-L-thyronine (T3) treatment on constitutive androstane receptor (CAR) and thyroid hormone receptors (TRs, including TRα and TRß) proteins were detected in Huh7 cells. We found dramatic decreases in the levels of plasma free triiodthyronine, free thyroxine and hepatic CYP2B expression. Both CAR and retinoid X receptor alpha (RXRα) were significantly dissociated from the phenobarbital-responsive enhancer module (PBREM) of the CYP2B1 promoter in the early stages of the acute stroke. The levels of the polymer of TRs, CAR, and RXRα were time-dependently decreased after brain I/R injury. T3 regulated the CAR expression at the transcriptional level, and facilitated the translocation of TRα/ß proteins as well as the binding of TRs, RXRα, and CAR to PBREM region. The reduction of thyroid hormone levels after a brain I/R injury may be the initial trigger for the down-regulation of hepatic CYP2B1 via induction of the dissociation of CAR from the TRs and from the PBREM region. Our data suggest that patients with acute ischemic stroke may have a decreased CYP2B-mediated metabolism of exogenous and endogenous compounds because of the low level of thyroid hormones.

Anticarcinogenic activity of meso-zeaxanthin in rodents and its possible mechanism of action.

Anticarcinogenic activity of meso-zeaxanthin (MZ), a xanthophyll carotenoid with profound antioxidant activity, was evaluated against 3-methylcholanthrene (3-MC)-induced sarcoma in mice. Oral administration of MZ at different doses significantly increased tumor latency period. In 3-MC control group, animals started developing sarcoma on 6th week. However animals treated with 3-MC and MZ (50 and 250 mg/kg b.wt) started developing sarcoma only on 15th and 18th week, respectively. Survival of tumor-bearing mice was significantly increased by MZ treatment. Animals in 3-MC control group started dying due to tumor burden from 8th week. All animals treated with MZ (50 and 250 mg/kg b.wt) along with 3-MC were found to be alive even after 16 and 20 wk, respectively. Oral administration of MZ inhibited different CYP450 isoenzymes like CYP1A1 (PROD), CYP1A2 (MROD), and CYP2B1/2 (EROD), which are involved in carcinogen metabolism in a dose-dependent manner. Moreover, levels of phase II enzymes like UDP-glucuronyl transferase and glutathione-S-transferase, which are involved in detoxification of carcinogens, were significantly increased by MZ treatment. Results indicated that mode of action of MZ may be through inhibition of carcinogen activation coupled with enhancement of detoxification process. MZ may also inhibit promotion phases of carcinogenesis by its antioxidant activity.

Comparison of three fluorescent CYP3A substrates in two vertebrate models: pig and Atlantic salmon.

We investigated in vitro inhibitory effects of ketoconazole (KTZ) on cytochrome P450 activity in microsomes from pigs and Atlantic salmon. The following enzymatic reactions were studied: 7-benzyloxyresorufin and 7-ethoxyresorufin O-dealkylation (BROD and EROD, respectively), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylation (BFCOD) and 7-benzyloxyquinoline O-debenzylation (BQOD). KTZ was a potent non-competitive inhibitor of BROD and BQOD in the microsomes from pigs, whereas in the microsomes from Atlantic salmon, these reactions were competitively inhibited by KTZ. BFCOD activity was inhibited by KTZ in a non-competitive manner in both species. KTZ non-competitively inhibited EROD in Atlantic salmon, but not in porcine microsomes. The activity of BROD and BQOD was higher in male than that in female pigs, but the activity of BFCOD showed no sex-related differences.

Cancer prevention mediated by caffeic acid phenethyl ester involves cyp2b1/2 modulation in hepatocarcinogenesis.

Studies of cancer chemoprevention with caffeic acid phenethyl ester (CAPE) in the resistant hepatocyte model of hepatocarcinogenesis have shown the participation of CYP drug metabolizing enzymes. To prevent neoplastic and preneoplasic lesions, we must specifically identify which CYP activities are modified in the mechanism of action of CAPE. Male Fischer-344 rats were pretreated with CAPE twelve hours before administration of diethylnitrosamine (DEN) and were sacrificed twelve hours after CAPE and twelve hours, twenty-four hours, twenty-four days, and twelve months after DEN. Other rats were treated with the CYP inhibitors α-naphthoflavone or SKF525A and sacrificed twenty-four hours and twenty-four days after DEN. Microsomes were obtained from livers to quantify protein using Western blot. Diethylnitrosamine metabolism was measured based on nitrite formation and liver histology using GGT histochemistry. Caffeic acid phenethyl ester diminished the protein levels of CYP1A2 and CYP2B1/2. The inhibition of CYP2B1/2 prevented the appearance of preneoplastic lesions. Microsomal assays demonstrated that CAPE interfered with DEN activation diminishing nitrites similar to SKF525A and probably mediated by CYP2B1/2 inhibition. A single dose of CAPE before DEN treatment reduced the appearance of tumors by 43%. These results confirmed that CAPE is a promising agent to confer chemoprotection in liver cancer and should be considered for human therapies.

Bupropion hydroxylation as a selective marker of rat CYP2B1 catalytic activity.

Benzyloxyresorufin-O-dealkylation (BROD) is usually used as a marker of cytochrome P450 (P450) 2B1 in rat. However, some reports show that CYP1A2 is also highly implicated. The purpose of the present study was to establish bupropion (BUP) hydroxylation, but not BROD, as a selective in vitro marker of CYP2B1 catalytic activity. IC(50) for BROD and BUP hydroxylation were equivalent (40.8 ± 4.6 and 41.8 ± 3.4 µM, respectively) when using liver microsomes from ß-naphthoflavone-pretreated rats in the presence of metyrapone (CYP2B1 inhibitor). When using the same microsomes in the presence of CYP1A1/2-selective inhibitor α-naphthoflavone, we found an IC(50) of 2.5 × 10(-3) ± 0.8 × 10(-3) µM for BROD and >100 µM for BUP hydroxylation. These results suggest that CYP2B1 is similarly involved in both activities, whereas CYP1A2 is involved in BROD activity but not in BUP hydroxylation. BUP hydroxylation was assessed in microsomes from baculovirus-infected insect cells coexpressing NADPH-P450 oxidoreductase, and 14 rat P450s and kinetic parameters (K(m) and V(max)) were determined. BUP hydroxylation was predominantly catalyzed by CYP2B1 (75% of total hydroxybupropion formation), low activity was detected with CYP2E1 and CYP2C11 (10.9 and 8.7% of total hydroxybupropion, respectively), and activity was almost undetectable with the other P450 isoforms at saturating substrate concentrations (2500 µM), thereby validating the use of BUP as a diagnostic in vitro marker of CYP2B1 catalytic activity in rat.

Cytochrome P450 2B1 mediates complement-dependent sublytic injury in a model of membranous nephropathy.

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.

Sex differences in subacute toxicity and hepatic microsomal metabolism of triptolide in rats.

Triptolide, a major active component of Tripterygium wilfordii Hook F (TWHF), has multiple pharmacological activities. However, its clinical use is often limited by its severe toxicity. In the present study, we evaluated the oral toxicity of triptolide in Sprague-Dawley rats for 28 days at the dosages of 0, 200 and 400microg/kg/day, respectively. Significant difference in the toxicity of triptolide at 400microg/kg was found between different sexes. The triptolide-treated female rats showed many abnormalities, including anorexia, diarrhea, leanness, suppression of weight gain and food intake, fatty liver, splenomegaly and atrophy of ovaries. In contrast, no such abnormalities were observed in male rats except for the significant reproductive toxicity. Furthermore, the metabolism of triptolide in liver microsomes from both sexes was investigated by HPLC. A greater rate of triptolide metabolism was observed in male rat hepatic microsomes, suggesting that one of the cytochrome P450s (CYPs) responsible for triptolide metabolism is male-specific or predominant at least. The inhibition experiments with CYP inhibitors showed that CYP3A and CYP2B were mainly involved in the metabolism of triptolide. In addition, since CYP3A2 is a male-predominant form in rats, significant sex difference in the metabolism of triptolide disappeared in vitro after anti-rat CYP3A2 antibody pretreatment. Results suggested that CYP3A2 made an important contribution to the sex-related metabolism of triptolide, which may result in the sex differences in triptolide toxicity.

In vitro metabolism and covalent binding of ethylbenzene to microsomal protein as a possible mechanism of ethylbenzene-induced mouse lung tumorigenesis.

This study was conducted to determine species differences in covalent binding of the reactive metabolites of ethylbenzene (EB) formed in the liver and lung microsomes of mouse, rat and human in the presence of NADPH. These data further the understanding of the mechanism by which EB causes mouse specific lung toxicity and a follow-up to our earlier report of the selective elevation, although minor, of the ring-oxidized reactive metabolites in mouse lung microsomes (Saghir et al., 2009). Binding assays were also conducted with or without 5-phenyl-1-pentyne (5P1P), an inhibitor of CYP 2F2, and diethyldithiocarbamate (DDTC), an inhibitor of CYP 2E1 to evaluate their role in the formation of the related reactive metabolites. Liver and lung microsomes were incubated with (14)C-EB (0.22 mM) in the presence of 1mM NADPH under physiological conditions for 60 min. In lung microsomes, binding activity was in the order of mouse (812.4+/-102.2 pmol/mg protein)>>rat (57.0+/-3.2 pmol/mg protein). Human lung microsomes had little binding activity (15.7+/-1.4 pmol/mg protein), which was comparable to the no-NADPH control (9.9-16.7 pmol/mg protein). In liver microsomes, mouse had the highest activity (469.0+/-38.5 pmol/mg protein) followed by rat (148.3+/-14.7 pmol/mg protein) and human (89.8+/-3.0 pmol/mg protein). Presence of 5P1P or DDTC decreased binding across species and tissues. However, much higher inhibition was observed in mouse (86% [DDTC] and 89% [5P1P]) than rat (56% [DDTC] and 59% [5P1P]) lung microsomes. DDTC showed approximately 2-fold higher inhibition of binding in mouse and human liver microsomes than 5P1P (mouse=85% vs. 40%; human=59% vs. 36%). Inhibition in binding by DDTC was much higher (10-fold) than 5P1P (72% vs. 7%) in rat liver microsomes. These results show species, tissue and enzyme differences in the formation of reactive metabolites of EB. In rat and mouse lung microsomes, both CYP2E1 and CYP2F2 appear to contribute in the formation of reactive metabolites of EB. In contrast, CYP2E1 appears to be the primary CYP isozyme responsible for the reactive metabolites of EB in the liver.

Mechanism-based inactivation of CYP2B1 and its F-helix mutant by two tert-butyl acetylenic compounds: covalent modification of prosthetic heme versus apoprotein.

The mechanism-based inactivation of cytochrome CYP2B1 [wild type (WT)] and its Thr205 to Ala mutant (T205A) by tert-butylphenylacetylene (BPA) and tert-butyl 1-methyl-2-propynyl ether (BMP) in the reconstituted system was investigated. The inactivation of WT by BPA exhibited a k(inact)/K(I) value of 1343 min(-1)mM(-1) and a partition ratio of 1. The inactivation of WT by BMP exhibited a k(inact)/K(I) value of 33 min(-1)mM(-1) and a partition ratio of 10. Liquid chromatography/tandem mass spectrometry analysis (LC/MS/MS) of the WT revealed 1) inactivation by BPA resulted in the formation of a protein adduct with a mass increase equivalent to the mass of BPA plus one oxygen atom, and 2) inactivation by BMP resulted in the formation of multiple heme adducts that all exhibited a mass increase equivalent to BMP plus one oxygen atom. LC/MS/MS analysis indicated the formation of glutathione (GSH) conjugates by the reaction of GSH with the ethynyl moiety of BMP or BPA with the oxygen being added to the internal or terminal carbon. For the inactivation of T205A by BPA and BMP, the k(inact)/K(I) values were suppressed by 100- and 4-fold, respectively, and the partition ratios were increased 9- and 3.5-fold, respectively. Only one major heme adduct was detected following the inactivation of the T205A by BMP. These results show that the Thr205 in the F-helix plays an important role in the efficiency of the mechanism-based inactivation of CYP2B1 by BPA and BMP. Homology modeling and substrate docking studies were presented to facilitate the interpretation of the experimental results.

Docosahexaenoic acid downregulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes via the c-Jun NH2-terminal kinase mitogen-activated protein kinase pathway.

Mitogen-activated protein kinase (MAPK) pathways play central roles in the transduction of extracellular stimuli into cells and the regulation of expression of numerous genes. Docosahexaenoic acid (DHA) was shown to be involved in the regulation of expression of drug metabolizing enzymes (DMEs) in rat primary hepatocytes in response to xenobiotics. Cytochrome P450 2B1 (CYP 2B1) is a DME that is dramatically induced by phenobarbital-type inducers. The constitutive androstane receptor (CAR) plays a critical role in regulating the expression of DMEs, and the phosphorylation/dephosphorylation of CAR is an important event in CYP 2B1 expression. In the present study, we determined the effect of DHA on MAPK transactivation and its role in CYP 2B1 expression induced by phenobarbital. c-Jun NH2-terminal kinase (JNK) JNK1/2 and ERK1/2 were activated by phenobarbital in a dose-dependent manner. DHA (100 muM) inhibited JNK1/2 and ERK2 activation induced by phenobarbital in a time-dependent manner. Both SP600125 (a JNK inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited CYP 2B1 protein and mRNA expression induced by phenobarbital. SB203580 significantly increased the intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) concentration compared with a control group (p < 0.05). Our results suggest that inhibition of JNK activation by DHA is at least part of the mechanisms of DHA's downregulation of CYP 2B1 expression induced by phenobarbital.

Brain drug-metabolizing cytochrome P450 enzymes are active in vivo, demonstrated by mechanism-based enzyme inhibition.

Individuals vary in their response to centrally acting drugs, and this is not always predicted by drug plasma levels. Central metabolism by brain cytochromes P450 (CYPs) may contribute to interindividual variation in response to drugs. Brain CYPs have unique regional and cell-type expression and induction patterns, and they are regulated independently of their hepatic isoforms. In vitro, these enzymes can metabolize endogenous and xenobiotic substrates including centrally acting drugs, but there is no evidence to date of their in vivo function. This has been difficult to demonstrate in the presence of hepatically derived metabolites that may cross the blood-brain barrier. In addition, because of the membrane location of brain CYPs and the rate limiting effect of endogenous heme levels on the activity and appropriate membrane insertion of some induced CYPs, it has been unclear whether sufficient cofactors and coenzymes are present for constitutive and induced CYP forms to be enzymatically active. We have developed a method using a radiolabeled mechanism-based inhibitor of CYP2B1, (3)H-8-methoxypsoralen, to demonstrate for the first time that both the constitutive and induced forms of this enzyme are active in situ in the living rat brain. This methodology provides a novel approach to assess the function of enzymes in extrahepatic tissues, where expression levels are often low. Selective induction of metabolically active drug metabolizing enzymes in the brain may also provide ways to control prodrug activation in specific brain regions as a novel therapeutic avenue.

Inhibitory effects of a dietary phytochemical 3,3'-diindolylmethane on the phenobarbital-induced hepatic CYP mRNA expression and CYP-catalyzed reactions in female rats.

3,3'-diindolylmethane (DIM), derived from indole-3-carbinol (I3C), is used as a dietary supplement for its putative anticancer effects that include suppression of mammary tumor growth in female rats. The mechanism of action DIM may involve its interaction(s) with hepatic cytochromes P450 (CYPs) catalyzing oxidations of 17beta-estradiol (E2). Our study showed that DIM added to hepatic microsomes of female Sprague-Dawley rats was primarily a competitive inhibitor of beta-naphthoflavone (beta-NF)- or I3C-induced CYP1A1 probe activity, and a potent mixed or uncompetitive inhibitor of phenobarbital (PB)-induced CYP2B1 or CYP2B2 probe activity, respectively. Microsomal metabolites of DIM were tentatively identified as two mono-hydroxy isomers of DIM, each formed preferentially by CYP1A1- or CYP2B1/2-catalyzed reaction. Evaluation of the effects of co-treatment of rats with PB and DIM by a full factorial ANOVA showed that DIM decreased the PB-induced CYP2B1 and CYP2B2 mRNA expression levels, and the rates of 2- and 4-hydroxylation of E2, and total E2 metabolite formation. The results suggest that interactions of DIM, and/or its mono-hydroxy metabolites, with CYP2B1 and CYP2B2 found to occur in hepatic microsomes upon addition of DIM or co-treatment of rats with DIM affect the rates of relevant oxidations of E2, and potentially protect against estrogen-dependent tumorigenesis.

Effect of selected antidepressant drugs on cytochrome P450 2B (CYP2B) in rat liver. An in vitro and in vivo study.

The aim of the present study was to investigate the influence of antidepressants with different chemical structures and mechanisms of action affecting serotonergic and/or noradrenergic systems - tricyclic antidepressant drugs (TAD), selective serotonin reuptake inhibitors (SSRIs) and novel antidepressants (mirtazapine, nefazodone) - on the activity of rat CYP2B measured as the rate of 16beta-hydroxylation of testosterone. The reaction was studied in control liver microsomes in the presence of antidepressants, as well as in microsomes of rats treated intraperitoneally for one day or two weeks (twice a day) with pharmacological doses (mg/kg) of the drugs (imipramine, amitriptyline, clomipramine, nefazodone 10; desipramine, fluoxetine, sertraline 5; mirtazapine 3). The obtained K(i) values indicated that nefazodone and the SSRIs sertraline and fluoxetine were the most potent inhibitors of the studied reaction (K(i) = 10-20 microM). The inhibitory effects of TADs were modest (K(i) = 62-85 microM), while mirtazapine was a very weak inhibitor of CYP2B activity (K(i) = 286 microM). After a one-day exposure of rats to the investigated antidepressants, a significant increase in CYP2B activity was only observed after sertraline exposure (300% of the control). Chronic treatment with the antidepressants led to a significant enhancement of CYP2B activity after sertraline, fluoxetine and desipramine (580, 200 and 150% of the control, respectively) treatment, which positively correlated with the observed elevation in CYP2B protein levels. In summary, two different mechanisms of the antidepressant-CYP2B interaction are postulated: 1) a direct inhibition of CYP2B shown in vitro by nefazodone, SSRIs and TADs; 2) in vivo induction of CYP2B produced by prolonged administration of SSRIs and desipramine, which suggests their influence on enzyme regulation. The marked CYP2B-induction produced by SSRIs corresponds with their selective serotonin reuptake inhibition, while the effect of desipramine corresponds with its selective inhibition of noradrenaline reuptake.

Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine.

Aconitine, a major Aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. The current study investigated the metabolism of aconitine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of aconitine in rat liver microsomes. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS(n)) and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. Various selective inhibitors of CYP were used to identify the isoforms of CYP, that involved in the metabolism of aconitine. A total of at least six metabolites were found and characterized in rat liver microsomal incubations. Result showed that the inhibitor of CYP 3A had an inhibitory effect on aconitine metabolism in a concentration-dependant manner, the inhibitor of CYP1A1/2 had a modest inhibitory effect, whereas inhibitors of CYP2B1/2, 2D and 2E1 had no obvious inhibitory effects on aconitine metabolism. Aconitine might be metabolized by CYP 3A and CYP1A1/2 isoforms in rat liver microsome.

RAT CYP3A and CYP2B1/2 were not associated with nevirapine-induced hepatotoxicity.

Nevirapine is an antiretroviral drug that is used for treatment as well as for the prevention of mother-to-child transmission of the human immunodeficiency virus (HIV). Unfortunately, its adverse effects, mainly hypersensitivity skin reactions and hepatotoxicity, have hampered the use of nevirapine. Since nevirapine-induced hepatotoxicity commonly occurs between 2-12 weeks of treatment, and nevirapine is a known inducer of human CYP3A and CYP2B6 isozymes, it was envisaged that the hepatotoxicity was due to activation of nevirapine to toxic metabolites by the induced enzymes. Therefore, the aim of this study was to use a rat model and determine the role of the rat analogues, rat CYP3A and CYP2B1/2, in nevirapine-induced hepatotoxicity. This was tested by the extent at which hepatotoxicity could be prevented when ketoconazole or thiotepa, known inhibitors of CYP3A and CYP2B1/2, respectively, were given one hour prior to administration of a hepatotoxic dose of nevirapine. It was shown here that nevirapine-induced hepatotoxicity only occurred in animals that were pretreated with an enzyme inducer (dexamethasone or nevirapine); that ketoconazole and thiotepa did not prevent the occurrence of nevirapine-induced hepatotoxicity; and that histopathologic examinations were more accurate than the use of liver enzymes in detecting the liver damage. This suggested that nevirapine-induced hepatotoxicity is closely associated with enzyme induction, and that liver function tests alone might not be good markers for determining nevirapine-induced hepatotoxicity. In conclusion, rat CYP3A and CYP2B1/2 may not be involved in the pathogenesis of nevirapine-induced hepatotoxicity, suggesting that a different enzyme inducible by nevirapine or dexamethasone may be responsible. However, this is yet to be proven in humans.

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

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

Identification of 17-alpha-ethynylestradiol-modified active site peptides and glutathione conjugates formed during metabolism and inactivation of P450s 2B1 and 2B6.

The oral contraceptive 17-alpha-ethynylestradiol (17EE) is a mechanism-based inactivator of cytochrome P450s (P450s) 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [3H]17EE resulted in labeling of the P450 apoprotein. Mass spectral analysis of 17EE-inactivated P450 2B1 showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus one oxygen atom. P450s 2B1 and 2B6 were inactivated with [3H]17EE and digested with CNBr. Separation of these peptides resulted in the identification of one major labeled peptide for each enzyme. N-Terminal sequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) and PYTEAV (for P450 2B6) that corresponded to amino acids P347-M376 and P347-M365 in P450s 2B1 and 2B6, respectively. Electrospray ionization (ESI)-liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization (MALDI)-MS analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da consistent with the mass of the P347-M376 peptide (3385 Da) plus a 268 Da 17EE adduct. Chemically reactive intermediates of 17EE that were generated during the metabolism of 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of 17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated different reactive 17EE intermediates that were responsible for the inactivation and protein modification or the formation of GSH conjugates by these two enzymes.