Role of CYP2E1 and saturation kinetics in the bioactivation of thioacetamide: Effects of diet restriction and phenobarbital

Thioacetamide (TA) undergoes saturation toxicokinetics in ad libitum (AL) fed rats. Diet restriction (DR) protects rats from lethal dose of TA despite increased bioactivation-mediated liver injury via CYP2E1 induction. While a low dose (50 mg TA/kg) produces 6-fold higher initial injury, a 12-fold higher dose produces delayed and mere 2.5-fold higher injury. The primary objective was to determine if this less-than-expected increase in injury is due to saturation toxicokinetics. Rats on AL and DR for 21 days received either 50 or 600 mg TA/kg i.p. T(1/2) and AUCs for TA and TA-S-oxide were consistent with saturable kinetics. Covalent binding of (14)C-TA-derived-radiolabel to liver macromolecules after low dose was 2-fold higher in DR than AL rats. However, following lethal dose, no differences were found between AL and DR. This lack of dose-dependent response appears to be due to saturation of bioactivation at the higher dose. The second objective was to investigate the effect of phenobarbital pretreatment (PB) on TA-initiated injury following a sub-lethal dose (500 mg/kg). PB induced CYP2B1/2 approximately 350-fold, but did not increase covalent binding of (14)C-TA, TA-induced liver injury and mortality, suggesting that CYP2B1/2 has no major role in TA bioactivation. The third objective was to investigate the role of CYP2E1 using cyp2e1 knockout mice (KO). Injury was assessed over time (0-48 h) in wild type (WT) and KO mice after LD(100) dose (500 mg/kg) in WT. While WT mice exhibited robust injury which progressed to death, KO mice exhibited neither initiation nor progression of injury. These findings confirm that CYP2E1 is responsible for TA bioactivation.     

Saturation toxicokinetics of thioacetamide: role in initiation of liver injury.

Thioacetamide (TA), a potent centrilobular hepatotoxicant, undergoes a two-step bioactivation mediated by microsomal CYP2E1 to TA sulfoxide (TASO), and further to TA-S,S-dioxide (TASO2), a reactive metabolite that initiates cellular necrosis. Our earlier studies showed that bioactivation-mediated liver injury of TA is not dose-proportional. The objective of this study was to examine whether increasing doses of TA lead to enzyme saturation, thereby resulting in lack of dose-response for injury: bioactivation of TA --> TASO --> TASO2 may follow zero-order kinetics. A 12-fold dose range of TA (50, 300, and 600 mg/kg i.p.) was injected into male Sprague-Dawley rats. TA and TASO were quantified in plasma, liver, and urine by high-performance liquid chromatography. With increasing doses, the apparent elimination half-lives of TA and TASO increased linearly, indicating that TA bioactivation exhibits saturation kinetics. Increasing TA dose resulted in greater-than-proportional increases in plasma TA and TASO levels. The TASO/TA ratio was inversely proportional to the dose of TA. Covalent binding of 14C-TA-derived radiolabel to liver macromolecules showed a less-than-dose-proportionate increase with a 12-fold higher dose. Less than dose-proportional covalent binding was confirmed in liver microsomal incubations with 14C-TA. Three-fold higher excretion of TASO was seen in urine at the highest dose (600 mg/kg) compared with the lowest dose (50 mg TA/kg). Incubation of TA with rat liver microsomes and purified baculovirus-expressed rat and human CYP2E1 Supersomes, over a concentration range of 0.01 to 10 mM, revealed saturation of TA conversion to TASO at and above 0.05 mM TA concentration, comparable to in vivo plasma and liver levels achieved upon administration of higher doses. Calculated K(m) values for TA (0.1 mM) and TASO (0.6 mM) suggest that the second step of TA bioactivation is 6-fold less efficient. Collectively, the findings indicate saturation of CYP2E1 at the first (TA to TASO) and second (TASO to TASO2) steps of TA bioactivation.     

Toxicokinetics and toxicity of thioacetamide sulfoxide: a metabolite of thioacetamide

Thioacetamide (TA) is bioactivated by CYP2E1 to TA sulfoxide (TASO), and to the highly reactive sulfdioxide (TASO₂), which initiates hepatic necrosis by covalent binding. Previously, we have established that TA exhibits saturation toxicokinetics over a 12-fold dose range, which explains the lack of dose–response for bioactivation-based liver injury. In vivo and in vitro studies indicated that the second step (TASO → TASO₂) of TA bioactivation is less efficient than the first one (TA → TASO). The objective of the present study was to specifically test the saturation of the second step of TA bioactivation by directly administering TASO, which obviates the contribution from first step, i.e. TA → TASO. Male SD rats were injected with low (50 mg/kg, ip), medium (100 mg/kg) and high (LD₇₀, 200 mg/kg) doses of TASO. Bioactivation-mediated liver injury that occurs in the initial time points (6 and 12 h), estimated by plasma ALT, AST and liver histopathology over a time course, was not dose-proportional. Escalation of liver injury thereafter was dose dependent: low dose injury subsided; medium dose injury escalated upto 36 h before declining; high dose injury escalated from 24 h leading to 70% mortality. TASO was quantified in plasma by HPLC at various time points after administration of the three doses. With increasing dose (i.e., from 50 to 200 mg/kg), area under the curve (AUC) and C_max increased more than dose proportionately, indicating that TASO bioactivation exhibits saturable kinetics. Toxicokinetics and initiation of liver injury of TASO are similar to that of TA, although TASO-initiated injury occurs at lower doses. These findings indicate that bioactivation of TASO to its reactive metabolite is saturable in the rat as suggested by previous studies with TA.     

Effect of mosquito mats (pyrethroid-based) vapor inhalation on rat brain cytochrome P450s

The effect of transfluthrin (TF) or D-allethrin (DA) pyrethroid (PYR) vapors, often contained as main ingredients in two commercially available mosquito repellent mats, on cytochrome P450 (CYP) enzymes of rat brain and liver was assessed. Immunodetection of CYP2E1 and CYP3A2 proteins revealed their induction in cerebrum and cerebellum, but not in liver microsomes of rats exposed by inhalation to TF or DA. This overexpression of proteins correlated with an increase of their catalytic activities. The specifically increased expression of CYP isoenzymes, due to PYR exposure in the rat brain, could perturb the normal metabolism of endogenous and xenobiotic compounds and leads to increased risks of neurotoxicity by bioactivation, lipid peroxidation and DNA damage.     

Hepatic cytochrome P450 and flavin-containing monooxygenase in male Nts:Mini rat, a transgenic rat carrying antisense RNA transgene for rat growth hormone.

We investigated the characteristics of hepatic cytochrome P450s and flavin-containing monooxygenase 1 (FMO1) in male Nts:Mini rats, a Wistar/Jcl-derived transgenic rat strain showing less plasma GH concentration than the parental strain. The total hepatic P450 contents of Mini rats were significantly reduced. A suppression was observed in the activities and protein expression of male-specific P450s (CYP3A and CYP2C11) and was speculated to be a potential cause of the reduction in total P450 contents. The activity and protein expression of CYP2B1 were suppressed and those of CYP2E1 and CYP2B2 were enhanced. With the exception of our data on CYP2B1, these results largely agreed with previous reports concerning GH-depletion rat models (hypophysectomized rats, rats neonatally treated with glutamate, and dwarf rats), implying that the changes in Mini rats were caused by GH insufficiency. The liver FMO1 protein expression in Mini rats was higher than that in Wistar rats but the activity was comparable, suggesting that GH is not a positive regulator of FMO expression. With their insufficient but not depleted levels of plasma GH, Mini rats may thus become another candidate for use in the investigation of GH regulation of hepatic mixed-function monooxygenases.     

The effect of isoniazid on CYP2E1- and CYP4A-mediated hydroxylation of arachidonic acid in the rat liver and kidney.

Cytochrome P450 (P450) bioactivation of arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) has been reported to be isoform- and tissue-specific. To determine whether altered P450 expression affects the production of these metabolites, the formation of HETEs after isoniazid-mediated CYP2E1 induction was evaluated in the rat liver and kidney. Male Sprague-Dawley rats received isoniazid (200 mg/kg) or saline intraperitoneally once daily for 5 days. Chlorzoxazone, lauric acid, and arachidonic acid hydroxylation was measured in liver and kidney microsomes with and without preincubation with the specific CYP2E1 inhibitor, trans-1,2-dichloroethylene (DCE). P450 isoform content and tissue HETE metabolite concentrations were also determined. Isoniazid increased CYP2E1 protein, and the 6-hydroxychlorzoxazone formation rate was increased by 2.7 +/- 0.3- and 2.2 +/- 0.5-fold in liver and kidney, respectively. Formation of 19-HETE and 11-hydroxylauric acid was induced 2.3 +/- 0.6-fold and 2.2 +/- 0.4-fold in the liver, respectively, with no difference in the kidney. All of the induced activities were attenuated by DCE. An unanticipated decrease in liver CYP4A expression and in vitro 20-HETE formation rate was observed after isoniazid administration. Isoniazid decreased liver and kidney 20-HETE content to 34 +/- 10% and 15.6 +/- 5.3% of control, respectively, without significantly altering tissue 19-HETE concentration. Based on these findings, we conclude that under induced conditions, CYP2E1 is a primary enzyme involved in liver, but not kidney, formation of 19-HETE. In addition, formation of both CYP4A and 20-HETE is reduced in the liver by isoniazid. It was also demonstrated that tissue concentrations parallel in vitro inhibited formation rates for 20-HETE, but not the induced 19-HETE formation in the liver.     

Role of induction of specific hepatic cytochrome P450 isoforms in epoxidation of 4-vinylcyclohexene.

4-Vinyl-1-cyclohexene (VCH) is ovotoxic in B6C3F(1) mice but not in Fischer-344 rats, which can be partially attributed to greater formation of toxic epoxides from VCH in mice compared with rats. Since repeated exposure to VCH is necessary to cause ovotoxicity in mice, it is important to determine whether repeated exposure results in induction of cytochrome P450 (CYP) enzymes involved in its bioactivation. Hepatic microsomes prepared from mice or rats treated repeatedly with VCH demonstrated significantly increased VCH bioactivation in vitro, as assessed by VCH-1,2-epoxide, VCH-7,8-epoxide, or vinylcyclohexene diepoxide (VCD) formation. Mice and rats were then dosed with VCH, VCH-1,2-epoxide, or VCD for 10 days and measured for increases in hepatic microsomal CYP levels or activities. Total hepatic CYP levels were elevated only in microsomes from mice pretreated with VCH or VCH-1,2-epoxide. Immunoblotting analysis of microsomes from VCH-treated rodents revealed elevated levels of CYP2A and CYP2B in mice but not rats. VCH-1,2-epoxide pretreatment also increased CYP2B levels in the mouse. Activities toward specific substrates for CYP2A and CYP2B (coumarin and pentoxyresorufin, respectively) confirmed that VCH and VCH-1,2-epoxide pretreatments resulted in increased catalytic activities of CYP2A and CYP2B in the mouse but not the rat. Pretreatment with phenobarbital, a known inducer of CYP2A and CYP2B, increased VCH bioactivation in both species. Interestingly, metabolism studies with human CYP "Supersomes" reveal that, of eight isoforms tested, only human CYP2E1 and CYP2B6 were capable of significantly catalyzing VCH epoxidation, whereas CYP2B6, CYP2A6, CYP2E1, and CYP3A4 were capable of catalyzing the epoxidation of the monoepoxides.     

Cytochrome p450-dependent metabolism of trichloroethylene in rat kidney.

The metabolism of trichloroethylene (Tri) by cytochrome P450 (P450) was studied in microsomes from liver and kidney homogenates and from isolated renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. Chloral hydrate (CH) was the only metabolite consistently detected and was used as a measurement of P450-dependent metabolism of Tri. Pretreatment of rats with pyridine increased CH formation in both liver and kidney microsomes, whereas pretreatment of rats with clofibrate increased CH formation only in kidney microsomes. Pyridine increased CYP2E1 expression in both liver and kidney microsomes, whereas clofibrate had no effect on hepatic but increased renal CYP2E1 and CYP2C11 protein levels. These results suggest a role for CYP2E1 in both the hepatic and renal metabolism of Tri and a role for CYP2C11 in the renal metabolism of Tri. Studies with the general P450 inhibitor SKF-525A and the CYP2E1 competitive substrate chlorzoxazone provided additional support for the role of CYP2E1 in both tissues. CH formation was higher in PT cells than in DT cells and was time and reduced nicotinamide adenine dinucleotide phosphate (NADPH) dependent. However, pretreatment of rats with either pyridine or clofibrate had no effect on CYP2E1 or CYP2C11 protein levels or on CH formation in isolated cells. These data show for the first time that Tri can be metabolized to at least one of its P450 metabolites in the kidneys and quantitate the effect of P450 induction on Tri metabolism in the rat kidney.     

Cytochrome CYP sources of N-alkylprotoporphyrin IX after administration of porphyrinogenic xenobiotics to rats.

Cytochrome P-450 (CYP) 3A2 and CYP2C11 are sources of 70 and 30%, respectively, of N-vinylprotoporphyrin IX (N-vinylPP) formation after administration of 3-[(arylthio)ethyl]sydnone (TTMS) to rats. Female rats receiving TTMS were pretreated with dexamethasone, which induces CYP3A1 preferentially to CYP3A2. The resulting 12-fold increase in N-vinylPP formation showed that CYP3A1 was also a source of N-vinylPP. Phenobarbital (PB) pretreatment, which induces CYP2B1/2 and 3A1/2 in male rats, increased N-vinylPP formation after TTMS administration. Troleandomycin, a selective CYP3A inhibitor, was unable to decrease TTMS-mediated N-vinylPP formation in PB-treated male rats, indicating that CYP2B1/2 were sources of N-vinylPP. This conclusion was supported by demonstrating a 15-fold increase in TTMSinduced N-vinylPP formation in female rats after CYP2B1/2 induction with PB pretreatment. Allylispropylacetamide (AIA) inactivates rat CYP2B1/2, 2C6, 2C7, 2C11, and 3A1/2. Troleandomycin was unable to decrease N-AIA protoporphyrin IX adduct (N-AIAPP) formation, showing that CYP3A1/2 were not susceptible to AIA-mediated N-alkylation. N-AIAPP formation in females was approximately 30% of that in males, and thus we attribute 30% of N-AIAPP formation in males to the non-gender-specific isozymes (CYP2C6, 2C7, and/or 2B1/2), whereas approximately 70% originates from CYP2C11. PB treatment in female rats resulted in a 5-fold increase in N-AIAPP formation, showing that CYP2B1/2 were also susceptible to N-alkylation mediated by AIA. 1-Aminobenzotriazole elicited formation of equivalent amounts of N'N-aryl bridged protoporphyrin IX in male and female rat liver, demonstrating that nonselective mechanism-based inactivation is accompanied by nonselective conversion of the CYP heme moieties to N'N-aryl bridged protoporphyrin IX.     

Mechanisms of increased liver tissue repair and survival in diet-restricted rats treated with equitoxic doses of thioacetamide.

Moderate dietary or caloric restriction (DR) modulates animal physiology in a beneficial fashion. Previously, we have reported an equitoxic dose experiment where liver injury in DR male Sprague-Dawley rats exposed to a low dose of thioacetamide (TA, 50 mg/kg) was similar to that observed in ad libitum fed (AL) rats exposed to a 12-fold higher dose (600 mg/kg). Paradoxically, the AL rats experienced 90% mortality while all of the DR rats, with the same amount of initial bioactivation-mediated liver injury, survived. The protection observed in the DR rats was due to efficient compensatory liver tissue repair, which was delayed and attenuated in the AL rats, leading to progression of liver injury. The objective of the present study was to investigate the molecular mechanisms of the enhanced tissue repair in the DR rats upon equitoxic challenge with TA. Promitogenic mechanisms and mediators such as proinflammatory cytokines (TNF-alpha and IL-6), growth factors (TGF-alpha and HGF), and inducible nitric oxide synthase (iNOS) were estimated over a time course after equitoxic challenge (50 mg/kg to DR vs. 600 mg/kg to AL rats). Except for TNF-alpha, all other molecules were expressed earlier and in greater amount in the DR rats. IL-6 was 10-fold greater and peaked 12 h earlier; HGF also peaked 12 h sooner in the DR rats, when it was 2.5-fold greater than the value in the AL rats. TGF-alpha expression in livers of DR rats increased after TA administration and peaked at 24 h. In the AL rats, it was lower and peaked at 36 h. Diet restriction alone induced iNOS 2-fold in the DR rats and remained elevated until 12 h after TA administration, then declined thereafter. The lower iNOS activity in the AL rats further decreased after TA injection. DR rats exhibited higher apoptosis after thioacetamide administration, which further increased the efficiency of tissue repair. Taken together, these data indicate that even though the liver injury is near equal in AL and DR rats, sluggish signal transduction leads to delayed liver regeneration, progression of liver injury, and death in the AL rats. The equitoxic dose experiment indicates that stimulation of tissue repair is independent of the extent of initial liver injury and is governed by physiology of diet restriction. DR stimulates promitogenic signaling leading to a quick and timely response upon liver injury, arrest of progressive injury on one hand, and recovery from injury on the other, paving the way for survival of the DR rats.     

Identification of the cytochromes P450 that catalyze coumarin 3,4-epoxidation and 3-hydroxylation.

Coumarin, a widely used fragrance ingredient, is a rat liver and mouse lung toxicant. Species differences in toxicity are metabolism-dependent, with injury resulting from the cytochrome P450-mediated formation of coumarin 3,4-epoxide (CE). In this study, the enzymes responsible for coumarin activation in liver and lung were determined. Recombinant human and rat CYP1A forms and recombinant human CYP2E1 readily catalyzed CE production. Coinhibition with CYP1A1/2 and CYP2E1 antibodies blocked CE formation by 38, 84, and 67 to 92% (n = 3 individual samples) in mouse, rat, and human hepatic microsomes, respectively. Although CYP1A and 2E forms seem to be the most active catalysts of CE formation in liver, studies conducted with the mechanism-based inhibitor 5-phenyl-pentyne demonstrated that CYP2F2 is responsible for up to 67% of CE formation in whole mouse lung microsomes. In contrast to the CE pathway, coumarin 3-hydroxylation is a minor product of coumarin in liver microsomes from mice, rats, and humans and is catalyzed predominately by CYP3A and CYP1A forms, confirming that CE and 3-hydroxycoumarin are formed via distinct metabolic pathways.     

Investigation of antioxidant,anti-inflammatory and DNA-protective properties of eugenol in thioacetamide-induced liver injury in rats

The present study investigated the preventive effect of eugenol, a naturally occurring food flavouring agent on thioacetamide (TA)-induced hepatic injury in rats. Adult male Wistar rats of body weight 150–180 g were used for the study. Eugenol (10.7 mg/kg b.w./day) was administered to rats by oral intubation for 15 days. TA was administered (300 mg/kg b.w., i.p.) for the last 2 days at 24 h interval and the rats were sacrificed on the 16th day. Markers of liver injury (aspartate transaminase, alanine transaminase, alkaline phosphatase, γ-glutamyl transferase and bilirubin), inflammation (myeloperoxidase, tumor necrosis factor-α and interleukin-6), oxidative stress (lipid peroxidation indices, protein carbonyl and antioxidant status) and cytochrome P4502E1 activity were assessed. Expression of cyclooxygenase-2 (COX-2) and the extent of DNA damage were analyzed using immunoblotting and comet assay, respectively. Liver injury and collagen accumulation were assessed using histological studies by hematoxylin and eosin and Masson trichrome staining. Rats exposed to TA alone showed increased activities of hepatocellular enzymes in plasma, lipid peroxidation indices, inflammatory markers and pro-inflammatory cytokines and decreased antioxidant status in circulation and liver. Hepatic injury and necrosis were also evidenced by histology. Eugenol pretreatment prevented liver injury by decreasing CYP2E1 activity, lipid peroxidation indices, protein oxidation and inflammatory markers and by improving the antioxidant status. Single-cell gel electrophoresis revealed that eugenol pretreatment prevented DNA strand break induced by TA. Increased expression of COX-2 gene induced by TA was also abolished by eugenol. These findings suggest that eugenol curtails the toxic effects of TA in liver.     

Effect of total parenteral nutrition and choline on hepatic flavin-containing and cytochrome P-450 monooxygenase activity in rats.

Total parenteral nutrition provides nutrition by infusion into the systemic circulation. Bypassing the intestine and processes associated with absorption can cause additional pathophysiological changes to occur. For example, in rats, normal gut and pancreatic cell function may change, absorptive capacity may be altered, and enzyme functional activity including drug metabolism may be affected. The objective of this study was to examine the effects of a control diet or a diet of total parenteral nutrition in the presence or absence of choline on urinary biomarkers and hepatic microsome functional activity from rats. Selective functional markers of cytochrome P-4502E1 (CYP2E1) and flavin-containing monooxygenase (FMO) were examined in vitro. The N-oxygenation of trimethylamine was used as an in vivo selective functional marker for FMO. After the administration of total parenteral nutrition plus choline for 5 days, the urinary excretion of trimethylamine and trimethylamine N-oxide declined approximately 7- and 3-fold, respectively, compared with rats treated with control diet. The concentration of urinary biogenic amines was also significantly affected by total parenteral nutrition. Compared with control animals, rats administered total parenteral nutrition plus choline for 5 days showed a decrease of approximately 5- and 2-fold in urinary dopamine and norepinephrine concentration, respectively. To examine a molecular basis for the influence of total parenteral nutrition +/- choline on monooxygenase regulation, hepatic microsomal activity of the FMO and CYP2E1 was examined. Compared with animals treated with a control diet, total parenteral nutrition plus choline in rats caused a 3-fold increase in hepatic microsomal FMO and a 2-fold increase in hepatic cytochrome CYP2E1 functional activity, respectively. Although the data did not reach statistical significance, selective immunoblot studies using hepatic microsomes from rats treated with total parenteral nutrition + choline showed that compared with controls, FMO1 protein was decreased 1.4-fold and FMO3 increased 1.3-fold, respectively. In hepatic microsomes from rats treated with total parenteral nutrition + choline, compared with control animals, FMO4 immunoreactivity was increased 1.6-fold. The data suggest that total parenteral nutrition has a detectable effect on modulating rat FMO3, FMO4, and CYP2E1 monooxygenase functional activity. The clinical relevance of these results is unknown but may be of significance for individuals receiving total parenteral nutrition and those afflicted with trimethylaminuria.     

In vitro sulfoxidation of thioether compounds by human cytochrome P450 and flavin-containing monooxygenase isoforms with particular reference to the CYP2C subfamily.

Cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes are major catalysts involved in the metabolism of xenobiotics. The sulfoxidation of the thioether pesticides, phorate, disulfoton, sulprofos, and methiocarb, was investigated. Using pooled human liver microsomes (HLMs), thioether compounds displayed similar affinities; however, phorate and disulfoton displayed higher intrinsic clearance rates than either sulprofos or methiocarb. The sulfoxidation of thioethers by HLMs was found to be predominantly P450-driven (85-90%) compared with FMO (10-15%). Among 16 cDNA-expressed human P450 isoforms and 3 human FMO isoforms examined, the following isoforms and their polymorphisms had the highest rates for sulfoxidation, as follows: phorate, CYP1A2, 3A4, 2B6, 2C9*1, 2C18, 2C19, 2D6*1, and FMO1; disulfoton, CYP1A2, 3A4, 2B6, 2C9*1, 2C9*2, 2C18, 2C19, 2D6*1, and FMO1; sulprofos, CYP1A1, 1A2, 3A4, 2C9*1, 2C9*2, 2C9*3, 2C18, 2C19, 2D6*1, and FMO1; methiocarb, CYP1A1, 1A2, 3A4, 2B6, 2C9*1, 2C19, 2D6*1, and FMO1. Among these isoforms, members of the CYP2C subfamily often had the highest affinities and clearance rates. Moreover, sulfaphenazole, a CYP2C9 competitive inhibitor, inhibited disulfoton sulfoxidation by CYP2C9 (IC50 0.84 microM) as well as in HLMs. Ticlopidine, a CYP2C19 mechanism-based inhibitor, inhibited disulfoton sulfoxidation by CYP2C19 (IC50 after coincubation, 43.5 microM; IC50 after preincubation, 4.3 microM) and also in HLMs. Our results indicate that current models of the substrate binding site of the CYP2C subfamily would not effectively predict thioether pesticide metabolism. Thus, the substrate specificity of CYP2Cs is more extensive than is currently believed, and some reevaluation of structure-activity relationships may be required.     

The olivacine derivative s 16020 (9-hydroxy-5,6-dimethyl-N-[2-(dimethylamino)ethyl)-6H-pyrido(4,3-B)-carbazole-1-carboxamide) induces CYP1A and its own metabolism in human hepatocytes in primary culture.

The olivacine derivative 9-hydroxy-5,6-dimethyl-N-[2-(dimethylamino)ethyl)-6H-pyrido(4,3-b)-carbazole-1-carboxamide (S 16020) exhibits a potent antitumor activity. However, when administered in cancer patients, its blood clearance increases after repeated administrations, whereas the volume of distribution remains constant, suggesting that the drug is able to induce its own metabolism. The aim of this work was to identify the enzymes involved in S 16020 metabolism and determine whether this molecule is an enzyme inducer in human hepatocytes in primary cultures. Among a battery of cDNA-expressed cytochromes P450 (P450s) and flavin monooxygenase (FMO), only CYP1A1, CYP1A2, and FMO3 were able to generate detectable amounts of metabolites of S 16020. In primary hepatocytes, S 16020 behaved as a CYP1A inducer, producing an increase in CYP1A2 protein, acetanilide 4-hydroxylation, ethoxyresorufin O-deethylation, and chlorzoxazone 6-hydroxylation to an extent similar to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical CYP1A inducer. The levels of other P450 proteins, including CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, and CYP3A4, and related activities were not affected by S 16020. In primary hepatocytes, pretreatment of cells with S 16020 or TCDD produced a significant and similar increase of S 16020 metabolism, consistent with the previous indications on the role of CYP1As. We conclude that CYP1As and FMO3 are the major phase I enzymes involved in the metabolism of S 16020 and that this molecule is a potent hydrocarbon-like inducer able to stimulate its own metabolism in primary human hepatocytes and liver.     

Imidacloprid insecticide metabolism: human cytochrome P450 isozymes differ in selectivity for imidazolidine oxidation versus nitroimine reduction

Many metabolites of imidacloprid (IMI) have been identified, but the enzymatic basis for their formation has not been reported. This study with individual recombinant cytochrome P450 (CYP450) isozymes from human liver shows that the principal organoextractable NADPH-dependent metabolites are the 5-hydroxy (major) and olefin (minor) derivatives from hydroxylation and desaturation of the imidazolidine moiety and the nitrosoimine (major), guanidine (minor) and urea (trace) derivatives from reduction and cleavage of the nitroimine substituent. Isozymes selective for imidazolidine oxidation in order of decreasing overall activity are CYP3A4>CYP2C19 or CYP2A6>CYP2C9, while those selective for nitroimine reduction are CYP1A2, CYP2B6, CYP2D6 and CYP2E1. Three flavin monooxygenase isozymes (FMO1, FMO3, and FMO5) with NADPH are not active as assayed. These observations establish site specificity in IMI metabolism by CYP450 isozymes and that a single enzyme (CYP3A4) both oxidizes and reduces IMI at the imidazolidine and nitroimine moieties, respectively.     

Identification of cytochrome P-450IIB1 as a cocaine-bioactivating isoform in rat hepatic microsomes and in cultured rat hepatocytes.

Induction of cytochrome P-450-dependent monooxygenases with phenobarbital (PB) or other hepatic drug-metabolizing enzyme inducers in the rat is associated with enhanced cocaine hepatotoxicity both in vivo and in cultured rat hepatocytes. To demonstrate whether the major PB-inducible P-450 subfamily (P-450IIB) could be involved in the metabolic activation of cocaine, rates of cocaine N-demethylation (the first step of cocaine bioactivation) and the rate of irreversible (covalent) binding of tritiated cocaine to hepatic microsomal proteins (a measure for the overall bioactivation) were determined in microsomes from saline or PB-pretreated rats. PB pretreatment augmented Vmax (6-fold), but not KM, of cocaine N-demethylation. Similarly, the rate of irreversible protein binding was 3-fold increased in microsomes from PB-pretreated rats as compared with those from saline controls. Addition of benzphetamine, a substrate of P-450IIB, markedly inhibited cocaine irreversible binding. In addition, various concentrations of cocaine inhibited microsomal pentoxyresorufin O-depentylase activity in a competitive-type pattern. A polyclonal antibody raised against purified rat P-450IIB1 markedly inhibited cocaine N-demethylation as compared with control incubations with preimmune IgG. Finally, pretreatment of rats with PB potentiated cocaine-induced cytotoxicity in primary, short-term cultured hepatocytes, assessed as lactate dehydrogenase release into the culture medium. This enhancing effect of PB became even more evident in glutathione-depleted cells. These results suggest that cocaine is metabolized and bioactivated by P-450IIB1 in the rat liver, and that induction of this isoform with various agents may be associated with enhanced lethal hepatocyte injury in the rat.     

罗格列酮对大鼠肝细胞色素P-4502E1活性的影响

目的 :探讨罗格列酮对细胞色素P 4 5 02E1活性的影响。方法 :在正常大鼠肝细胞微粒体中加入浓度为 0 ,0 .10 ,0 .2 5 ,0 .5 0 ,1.0 0mmol·L- 1罗格列酮 ,以N 二甲基亚硝胺为底物 ,测定细胞色素P 4 5 0 2E1活性。另外用 0 ,0 .2 ,2 ,2 0mg·kg- 1剂量的罗格列酮分别对用乙醇诱导和未用乙醇诱导的大鼠灌胃 ,然后取大鼠肝细胞微粒体 ,以N 二甲基亚硝胺为底物 ,测定细胞色素P 4 5 0 2E1活性。结果 :在体外试验中 ,加入不同浓度罗格列酮的大鼠肝微粒体中细胞色素P 4 5 0 2E1活性差异无显著意义。在体内试验中 ,给予不同剂量罗格列酮的大鼠的肝细胞色素P 4 5 0 2E1活性差异也无显著意义 ;结论 :罗格列酮对大鼠的肝细胞色素P 4 5 0 2E1活性无明显影响     

Styrene monomer primarily induces CYP2B1 mRNA in rat liver

To determine the cytochrome P450 (CYP) primarily expressed after styrene exposure, seven forms of hepatic CYP mRNA in rats treated with 600?mg?kg^?1 styrene were examined. CYP1A2, CYP2B1/2, CYP2E1 and CYP3A2 mRNA were observed using real-time LightCycler PCR. The amount of CYP2B1 mRNA was significantly increased, 47-fold compared with controls, suggesting that this CYP is the primary cytochrome P450 in rats exposed to styrene. Significant increases in the amount of CYP2E1, CYP1A2 and CYP2B2 mRNA were also observed after styrene exposure, and their increase levels were 3.1-, 1.7- and 1.7-fold higher than controls, respectively. Western blot analysis also indicated that the protein levels of CYP2B1, CYP2B2, CYP2E1 and CYP1A2 showed clear increases after styrene treatment, corresponding to their mRNA expression. CYP2C11 mRNA decreased significantly in rats after styrene exposure. CYP1A1 was detected at the mRNA level in rat liver, but it was not detected at the protein level. The expression of epoxide hydrolase (EH), involved in Phase I drug metabolism, was also examined. EH mRNA increased 2-fold compared with controls after styrene exposure. Styrene thus appears to be a chemical compound that induces multiple CYPs. The results demonstrate that CYP2B1 is the primarily induced CYP form by styrene treatment to rats at acute toxic level.