Phenobarbital-treated Rats Research Articles

Ring-oxidative biotransformation and drug interactions of propofol in the livers of rats.

Propofol, an intravenous anesthetic agent, is widely used for inducing and maintaining anesthesia during surgical procedures and for sedating intensive care unit patients. In the clinic, rapid elimination is one of the major advantages of propofol. Meanwhile, the biotransformation and drug interactions of propofol in rat livers are still little known. In this study, we evaluated the ring-oxidative metabolism of propofol in phenobarbital-treated rat livers and possible drug interactions. Administration of phenobarbital to male Wistar rats significantly increased levels of hepatic cytochrome P450 (CYP) 2B1/2 and microsomal pentoxyresorufin O-dealkylase (PROD) activity. Analyses by high-performance liquid chromatography and liquid chromatography mass spectroscopy revealed that propofol was metabolized by phenobarbital-treated rat liver microsomes into 4-hydroxypropofol. In comparison, PROD activity and 4-hydroxy-propofol production from propofol metabolism were suppressed by orphenodrine, an inhibitor of CYP2B1/2, and a polyclonal antibody against rat CYP2B1/2 protein. Furthermore, exposure of rats to propofol did not affect the basal or phenobarbital-enhanced levels of hepatic CYP2B1/2 protein. Meanwhile, propofol decreased the dealkylation of pentoxyresorufin by phenobarbital-treated rat liver microsomes in a concentration-dependent manner. Taken together, this study shows that rat hepatic CYP2B1/2 plays a critical role in the ring-oxidative metabolism of propofol into 4-hydroxypropofol, and this anesthetic agent can inhibit CYP2B1/2 activity without affecting protein synthesis.

Four new koumine metabolites in rat liver microsomes

Four new metabolites M-1 [1,2,18,19-tetradehydro-4-demethyl-3,17-epoxy-7,20(2H,19H)-cyclovobasan], M-2 [1,2,4,21,18,19-hexadehydro-4-demethyl-3,17-epoxy-7,20(2H,19H)-cyclovobasan], M-3 [1,2,18,19-tetradehydro-4-demethyl-4-formaldehyde-3,17-epoxy-7,20(2H,19H)-cyclovobasan], and M-4 [1,2,4,21,18,19-hexadehydro-4-demethyl-4-oxy-3,17-epoxy-7,20(2H,19H)-cyclovobasan] were isolated from the chloroform extract of koumine incubated with phenobarbital-treated rat liver microsomes. The structures of M-1, M-2, M-3, and M-4 were elucidated by spectroscopic methods including ESI-TOF-MS, 1D, and 2D NMR experiments. The metabolic pathway of koumine was proposed. The cytotoxic activities between koumine and its metabolites were also compared in the A549 cell line.

2,5-Dihydroxy-4-methoxybenzophenone: a novel major in vitro metabolite of benzophenone-3 formed by rat and human liver microsomes

1. When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP.2. The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole.3. The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats.4. When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.

Probiotic strain Lactobacillus casei does not markedly alter rat liver microsomal cytochromes P450

The metabolism of trimethadione (TMO), a useful indicator of hepatic drug-oxidizing capacity in rats and humans, was studied using 14 different forms of rat cytochrome P450 (CYP1A1,1A2, 2A1, 2A2,2B1,2B2,2C6,2C7, 2C11,2C12,2C13,2E1,3A2 and 4A2) and three forms of human cytochrome P450 (CYP1A2,2C and 3A4). TMO N-demethylation was increased by treating rats with phenobarbital. CYP2C11 and 2B1 had high TMO N-demethylase activity, but 1A1 and 1A2 had low activity. Antibodies raised to CYP2C11 and 2B1/2 inhibited TMO N-demethylation in hepatic microsomes of untreated and phenobarbital-treated rats, respectively. In a reconstituted system, human CYP3A4 and 2C produced efficiently dimethadione (DMO), but CYP1A2 did not catalyse TMO N-demethylation. Antibodies raised to CYP3A2 and 2C11 inhibited TMO N-demethylation in human hepatic microsomes. These results indicated that the N-demethylation of TMO is catalysed mainly by CYP2C11 and 2B1 in rat hepatic microsomes, and that human CYP3A4 and an unspecified isoform of the 2C subfamilies contribute to TMO N-demethylation in human liver.

Comparative Oxidative Metabolism of BDE-47 and BDE-99 by Rat Hepatic Microsomes

Polybrominated diphenyl ethers (PBDEs) are flame-retardant chemicals that have become ubiquitous environmental pollutants. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) are among the most prevalent PBDEs detected in humans, wildlife, and abiotic environmental matrices. The purpose of this study was to investigate the oxidative metabolism of BDE-47 and BDE-99 in rat hepatic microsomes by comparing metabolite formation rates, kinetic parameters associated with metabolite formation, and the effects of prototypical cytochrome P450 (CYP) inducers. The CYP enzymes involved were also identified. Incubation of BDE-47 with hepatic microsomes from phenobarbital-treated rats generated a total of five hydroxylated (OH-BDE) metabolites, among which 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether (4'-OH-BDE-49) and 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (3-OH-BDE-47) were the major metabolites, as identified using authentic standards and quantified by liquid chromatography/mass spectrometry. Incubations of BDE-99 with hepatic microsomes from dexamethasone-treated rats produced a total of seven hydroxylated metabolites, among which 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90) and 6'-hydroxy-2,2',4,4',5-pentabromodiphenyl ether (6'-OH-BDE-99) were the major metabolites. Although the overall rate of oxidative metabolism of BDE-99 by hepatic microsomes was greater than that of BDE-47, para-hydroxylation involving a National Institutes of Health shift mechanism represented a major metabolic pathway for both PBDE congeners. Among the rat recombinant CYP enzymes tested, CYP2A2 and CYP3A1 were the most active in BDE-47 and BDE-99 metabolism, respectively. However, CYP1A1 exhibited the highest activity for 4'-OH-BDE-49 and 6'-OH-BDE-99 formation, and CYP3A1 exhibited the highest activity for 3-OH-BDE-47 and 4-OH-BDE-90 formation. Collectively, the results demonstrate that oxidative metabolism of BDE-47 and BDE-99 is mediated by distinct but overlapping sets of CYP enzymes and represents a key process that determines the bioaccumulation of BDE-47 and BDE-99 in mammals.

A Validated Liquid Chromatography-Mass Spectrometry Method for the Detection and Quantification of Oxidative Metabolites of 2,2',4,4'-Tetrabromodiphenyl Ether in Rat Hepatic Microsomes

In the present study, we developed and validated an analytical method using ultra performance liquid chromatography-mass spectrometry (UPLC/MS) for the quantitative determination of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) metabolism by rat hepatic microsomes. BDE-47 is a brominated flame retardant that was widely used in a variety of consumer products and has subsequently been identified as a ubiquitous environmental contaminant. Hydroxy-bromodiphenyl ethers (OH-BDEs) were isolated from rat hepatic microsomes by liquid-liquid extraction. Chromatographic separation was achieved by UPLC on a C18 column with gradient elution using a mobile phase consisting of methanol and water, each containing 0.1% formic acid, at a flow rate of 0.2 mL/min. Detection and quantification were performed using a mass spectrometer in single ion recording mode with negative electrospray ionization. The UPLC/MS method was validated for linearity, limit of quantification (LOQ), accuracy, precision and recovery. The weighted calibration curves (1/X2) were linear over a concentration range of 5 - 250 nM with LOQ values between 5 nM and 50 nM for the individual OH-BDEs. Intra- and inter- day accuracy (%DEV) and precision (%RSD) values ranged from –11.7% to 9.5% and 5.9% to 16.5%, respectively. Recovery values of 70% to 90% were obtained for all OH-BDEs. The validated method allowed us to successfully analyze metabolite formation following incubation of BDE-47 with hepatic microsomes prepared from phenobarbital-treated rats. Results demonstrate that the UPLC/MS method has sufficient sensitivity and reproducibility to fully characterize the in vitro metabolism of BDE-47 and possibly other PBDEs.

Antitumor activity of two gelsemine metabolites in rat liver microsomes

Gelsemine is one of the major alkaloids from Gelsemium elegans Benth., which has been used as an antitumor remedy in clinic. In this paper, metabolism of gelsemine has been investigated in vitro in phenobarbital-treated rat liver microsomes. The metabolites of gelsemine were separated and evaluated using the flash silica gel column, preparative HPLC, using NMR and MS methods. According to the spectral data, two metabolites, M1 and M2, were identified as 4-N-demethylgelsemine and 21-oxogelsemine, respectively. By the MTT method in vitro, the antitumor activities between gelsemine and its metabolites were compared in the HepG2 and HeLa cell lines. Moreover, the main metabolic pathway was further proposed.

Oxidative Metabolism of Epothilones A and B (Patupilone) by Cytochromes P450: Involvement of CYP3A and CYP2C

Epothilones are natural macrolides displaying potent antiproliferative properties against various cell lines and capable to bind tubulin and acting as microtubule-stabilizing agents like taxoids. We intended to isolate and characterize epothilone metabolites and identify enzymes implicated in the biotransformation process. In the presence of NADPH, liver microsomes from phenobarbital-treated rats produce two metabolites resulting from the oxidation of either epothilone A or B by CYP isoforms. Similarly, the oxidative biotransformation of epothilones A and B by human liver microsomes generates three metabolites with Km values ranged from 61 to 86µM. The two major metabolites (m1 and m2) are hydroxylated on the macrolide ring essentially by CYP3A4, whereas 3A5, 3A7 and 2B6 are minor contributors to the reaction. M3 is formed by CYP2C19 and 2C9 and results from the hydroxylation of the methyl on carbon 17 of the lateral chain. Inhibition of CYP3A almost completely abolished the formation of m1 and m2, whereas inhibition of CYP2C19 substantially reduced the production of m3. Collectively these data suggest that the oxidative metabolism of epothilones is principally mediated by CYP3A4 and CYP2C19. Epothilone B was found to be an in vitro inhibitor of CYP2C9 [IC50≈25µM], CYP2C19 [Ki≈1.7µM] and CYP3A4/5 [Ki≈1.85µM] whereas conversely taxanes or Vinca alkaloids significantly reduced oxidation of epothilone B. However, clinically relevant inhibition in patients undergoing chemotherapy is unlikely due to low therapeutic epothilone B blood concentrations.

Sedative and anticonvulsant drugs suppress postnatal neurogenesis

Sedative and anticonvulsant drugs, which inhibit N-methyl-D-aspartate receptor-mediated excitation or enhance GABA-mediated action, may cause apoptotic neurodegeneration in the developing mammalian brain. Here we explored whether such agents influence early postnatal neurogenesis. The N-methyl-D-aspartate antagonist MK801 and the GABA subtype A agonists phenobarbital and diazepam were administered to infant rats, and cell proliferation and neurogenesis were studied in the brain using 5-bromo-2'-deoxyuridine and doublecortin immunohistochemistry and stereology. Using confocal microscopy, we quantified neurogenesis in the dentate gyrus on postnatal day 15 (P15) after treatment with MK801 or phenobarbital on P6 to P10. Learning and memory were assessed at the age of 6 months after early postnatal treatment with phenobarbital. MK801, phenobarbital, and diazepam reduced numbers of newly born cells in the brain. We found no evidence that these agents caused apoptosis of 5-bromo-2'-deoxyuridine-positive cells. In the dentate gyrus, many of the newly formed cells differentiated toward a neuronal phenotype. Phenobarbital and MK801 reduced numbers of newly formed neurons in the dentate gyrus. At the age of 6 months, phenobarbital-treated rats had fewer neurons in the dentate gyrus and performed worse than saline-treated littermates in water maze learning and memory task. These findings show that blockade of N-methyl-D-aspartate receptor-mediated excitation and enhancement of GABA subtype A receptor activation impair cell proliferation and inhibit neurogenesis in the immature rat brain. Because many sedative and antiepileptic drugs used in pediatric medicine act via these mechanisms, our findings raise concerns about their potential impact on human brain development.

Effects of monocrotaline on energy metabolism in the rat liver

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in the plants of the Crotalaria species that causes cytotoxicity and genotoxicity in animals and humans, and it is hepatically metabolized to the alkylating agent dehydromonocrotaline by cytochrome P-450. The exact cellular and molecular mechanisms of MCT-induced tissue injury remain unclear. We previously demonstrated that dehydromonocrotaline, but not monocrotaline, inhibits the activity of NADH-dehydrogenase at micromolar concentrations in isolated liver mitochondria, an effect associated with significantly reduced ATP synthesis. Impairment of energy metabolism is expected to lead to several alterations in cell metabolism. In this work, the action of different concentrations of monocrotaline (250, 500, and 750 μM) on energy metabolism-linked parameters was investigated in isolated perfused rat livers. In the fed state, monocrotaline increased glycogenolysis and glycolysis, whereas in the livers of fasted rats, it decreased gluconeogenesis and urea synthesis from l-alanine. These metabolic alterations were only found in livers of phenobarbital-treated rats, indicating that active metabolites including dehydromonocrotaline were responsible for the observed activity. Our findings indicate that hepatic metabolic changes may be implicated, partly at least, in the hepatotoxicity of monocrotaline in animals and humans.

Toluene-induced hearing loss in phenobarbital treated rats

Exposure to aromatic organic solvents may induce hearing loss in rats, the cochlea being the primary target. The aim of this study which was carried out in rat, was to evaluate the impact of the hepatic metabolism of toluene on its ototoxic potency. To this end, the solvent hepatic metabolism was shifted by treating the rats with 50 mg/kg/d of phenobarbital (PhB), a potent inducer of the microsomal cytochromes P450 system, alcohol and aldehyde dehydrogenases, and glutathione-S-transferases. The two main urinary metabolites of the oxidative and conjugate pathways [hippuric (HA) and benzyl mercapturic acids (BMA) respectively] confirmed the efficacy of the PhB treatment. For the PhB-induced rats, the amount of excreted HA increased by 43% and the amount of BMA by 35%. Auditory function impairments were assessed using auditory-evoked potentials. On completion of the auditory tests, the organs of Corti were dissected to evaluate hair cell losses. The permanent auditory threshold shifts were approximately 15 dB greater in the toluene-exposed rats than in the PhB-induced rats. Both the functional and morphological data confirmed that PhB treatment can decrease the ototoxic potency of toluene.

Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes

The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the Vmax/Km values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25 nM−1 min−1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a Vmax value of 6.96 nmol min−1 nmol−1 P450 and apparent Km value of 0.09 mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the Km values for the formation of fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03 mM, and Vmax values were 2.94, 6.1 and 13.8 nmol min−1 nmol−1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.

Effect of berberine on hepatocyte proliferation, inducible nitric oxide synthase expression, cytochrome P450 2E1 and 1A2 activities in diethylnitrosamine- and phenobarbital-treated rats

This study investigated the effect of berberine on the early phase of hepatocarcinogenesis stimulated by diethylnitrosamine (DEN, 150 mg/kg, 4 weeks) plus phenobarbital (PB, 75 mg/kg, 7 days) in rats. The expressions of proliferating cell nuclear antigen (PCNA) and inducible nitric oxide synthase (iNOS) were evaluated by immunohistochemistry. The activities of CYP isoenzymes were analyzed using different probe drugs including chlorzoxazone (CYP2E1) and phenacetin (CYP1A2) by high-performance liquid chromatography (HPLC) in vivo or in vitro. Results showed that the expressions of PCNA and iNOS were induced by DEN plus PB in liver tissues. Oral administration of berberine (50 mg/kg) inhibited the hepatocyte proliferation and iNOS expression, decreased cytochrome P450 content, inhibited activities of CYP2E1 and CYP1A2 in DEN-plus-PB-treated rats in vivo. Moreover, berberine (10, 50 and 100 μM) inhibited the activities of CYP2E1 and CYP1A2 in microsomes isolated from DEN-plus-PB-treated rats in vitro, suggesting that anti-hepatocarcinogenetic potential of berberine might be due to inhibiting oxidative metabolic activities of CYP 2E1 and CYP1A2, and decreasing NO production in rats.

An autoradiographic study of cellular proliferaton, DNA synthesis and cell cycle variability in the rat liver caused by phenobarbital-induced oxidative stress: the protective role of melatonin.

The protective effect of melatonin against phenobarbital-induced oxidative stress in the rat liver was measured based on lipid peroxidation levels (malondialedyde and 4-hydroxyalkenals). Cellular proliferation, DNA synthesis and cell cycle duration were quantitated by the incorporation of 3H-thymidine, detected by autoradiography, into newly synthesized DNA. Two experiments were carried out in this study, each on four equal-sized groups of male rats (control, melatonin [10 mg/kg], phenobabital [20 mg/kg] and phenobarbital plus melatonin). Experiment I was designed to study the proliferative activity and rate of DNA synthesis, and measure the levels of lipid peroxidation, while experiment II was for cell cycle time determination. Relative to the controls, the phenobarbital-treated rats showed a significant increase (P < 0.01) in the lipid peroxidation levels (30.7%), labelling index (69.4%) and rate of DNA synthesis (37.8%), and a decrease in the cell cycle time. Administering melatonin to the phenobarbital-treated rats significantly reduced (P < 0.01) the lipid peroxidation levels (23.5%), labelling index (38.2%) and rate of DNA synthesis (29.0%), and increased the cell cycle time. These results seem to indicate that the stimulatory effect of phenobarbital on the oxidized lipids, proliferative activity, kinetics of DNA synthesis and cell cycle time alteration in the liver may be one of the mechanisms by which the non-genotoxic mitogen induces its carcinogenic action. Furthermore, melatonin displayed powerful protection against the toxic effect of phenobarbital.

METABOLISM OF THE α,β-UNSATURATED KETONES, CHALCONE AND TRANS-4-PHENYL-3-BUTEN-2-ONE, BY RAT LIVER MICROSOMES AND ESTROGENIC ACTIVITY OF THE METABOLITES

When chalcone and trans-4-phenyl-3-buten-2-one (PBO) were incubated with liver microsomes of untreated rats in the presence of NADPH, 4-hydroxychalcone and trans-4-(4-hydroxyphenyl)-3-buten-2-one (4-OH-PBO), respectively, were formed as major metabolites. Two minor metabolites of chalcone, 4'-hydroxychalcone and 2-hydroxychalcone, were also observed. The oxidase activity affording 4-hydroxychalcone was inhibited by SKF 525-A, disulfiram, ketoconazole, and alpha-naphthoflavone. The oxidase activities leading to 4-hydroxychalcone and 4'-hydroxychalcone were enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats, respectively. The activity generating 2-hydroxychalcone was enhanced in liver microsomes of 3-methylcholanthrene- and dexamethasone-treated rats. The oxidation of PBO to 4-OH-PBO was inhibited by SKF 525-A, ketoconazole, disulfiram, and sulfaphenazole. This activity was enhanced in liver microsomes of 3-methylcholanthrene-, acetone- and phenobarbital-treated rats. 4-Hydroxylation, 4'-hydroxylation, and 2-hydroxylation of chalcone were catalyzed by rat recombinant cytochrome P450 1A1, 1A2, and 2C6; by 1A1 and 2C6; and by 1A1 and 3A1, respectively. PBO was oxidized by cytochrome P450 1A1, 1A2, 2C6, and 2E1. Chalcone and PBO were negative in an estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, 4-hydroxychalcone, 2-hydroxychalcone, 4'-hydroxychalcone, and 4-OH-PBO exhibited estrogenic activity.

NMR-based metabonomic evaluation of livers from rats chronically treated with tamoxifen, mestranol, and phenobarbital

In this study, we look at the metabolic effects of long-term dosing with tamoxifen, mestranol or phenobarbital on the liver. Tamoxifen, mestranol and phenobarbital have all been reported to act as promoters of hepatic tumors. While tamoxifen and mestranol are known to have estrogenic activity, in the liver phenobarbital is a non-estrogenic compound. Aqueous and lipophilic liver extracts from control and chronically treated Fisher 344 rats were evaluated by nuclear magnetic resonance spectroscopy (NMR). In both the aqueous and lipophilic sample sets, the estrogenic action of mestranol appears to be responsible for the clustering of these samples with those animals treated with tamoxifen. Phenobarbital does not have estrogenic activity and, therefore, clusters away from the estrogenic and control groups. In the lipophilic samples, the fatty acid peak (CH2) n was higher in tamoxifen-treated rats than in control, phenobarbital- or mestranol-treated rats. In the aqueous samples, serine and choline levels were higher in phenobarbital-treated rats than controls, which may be an indication that the folate–homocysteine metabolic pathways were altered.

Reductase‐mediated metabolism of motexafin gadolinium (Xcytrin®) in rat and human liver subcellular fractions and purified enzyme preparations

1The biotransformation of motexafin gadolinium (MGd, Xcytrin®) was investigated in subcellular rat and human liver fractions. Microsomal MGd metabolism was dependent on NADPH in both species. Cytosolic metabolism in rat and human livers was dependent on NADPH or NADH. Under anaerobic conditions, MGd metabolism increased in liver microsomes and purified enzyme preparations. Cytochrome P450 (CYP450) inhibitors ketoconazole, proadifen, and carbon monoxide increased NADPH‐dependent MGd metabolism in microsomes. Treatment of rats with β‐naphthoflavone increased cytosolic metabolism of MGd twofold, but had no effect on microsomal metabolism. Conversely, in liver preparations from phenobarbital treated rats microsomal metabolism of MGd was enhanced twofold, but not in cytosolic preparations. Purified CYP450 reductase from phenobarbital‐treated rabbit or untreated human livers metabolized MGd suggesting involvement of CYP450 reductase. Dicumarol, a potent DT‐diaphorase inhibitor, inhibited MGd metabolism in both rat and human liver cytosol. These data suggest MGd metabolism in rat liver involves CYP450 reductase and/or DT‐diaphorase. In human liver preparations only CYP450 reductase is directly involved in MGd metabolism. A metabolite identified in microsomes and cytosol is a metal‐free, reduced form of MGd, indicating that both enzymes generate metabolite , which appears to be PCI‐0108, a synthetic precursor to MGd. © 2005 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:559–570, 2005

Production of superoxide radical in reductive metabolism of a synthetic food-coloring agent, indigocarmine, and related compounds

Indigocarmine, which is widely used as a synthetic colouring agent for foods and cosmetics in many countries, was reduced to its leuco form and decolorized by rat liver microsomes with NADPH under anaerobic conditions. The reductase activity was enhanced in liver microsomes of phenobarbital-treated rats, and inhibited by diphenyliodonium chloride, a NADPH-cytochrome P450 reductase (P450 reductase) inhibitor, but was not inhibited by SKF 525-A or carbon monoxide. Indigocarmine reductase activity was exhibited by purified rat P450 reductase. In contrast, when indigocarmine was incubated with rat liver microsomes and NADPH under aerobic conditions, superoxide radical was produced and its production was inhibited by superoxide dismutase and diphenyliodonium chloride. When indigocarmine was incubated with purified rat P450 reductase in the presence of NADPH, superoxide radical production was enhanced 17.7-fold (similar to the enhancement of indigocarmine-reducing ability) as compared with that of rat liver microsomes. A decrease of one molecule of NADPH was accompanied with formation of about two molecules of superoxide radical. P450 reductase exhibited little reductase activity towards indigo and tetrabromoindigo, which also afforded little superoxide radical under aerobic conditions. These results indicate that indigocarmine is reduced by P450 reductase to its leuco form, and superoxide radical is produced by autoxidation of the leuco form, through a mechanism known as futile redox cycling.

Increased Hepatobiliary Clearance of Unconjugated Thyroxine Determines DMP 904-Induced Alterations in Thyroid Hormone Homeostasis in Rats

4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine (DMP 904) is a potent and selective antagonist of corticotropin releasing factor receptor-1 (CRF1 receptor) with an efficacious anxiolytic profile in preclinical animal models. In subchronic toxicity studies in Sprague-Dawley rats, DMP 904 produced thyroid follicular cell hypertrophy and hyperplasia, and a low incidence of follicular cell adenoma. The current investigations were designed to determine the mode of action by which DMP 904 disrupts thyroid homeostasis in male rats. Five-day treatment with DMP 904 (300 mg/kg/day) dramatically lowered serum thyroxine (T4) to levels below detectable limits (< 1 microg/dl) by 72 h, with concurrent decreases in triiodothyronine (T3, about a 70% decrease) and increases in thyroid stimulating hormone (TSH; about a three-fold increase). DMP 904 increased [125I]T4 total body clearance (Cl tb) (38.21 +/- 10.45 ml/h) compared to control (5.61 +/- 0.59 ml/h) and phenobarbital-treated rats (7.92 +/- 1.62 ml/h). This increase in Cl(tb) was associated with a significant increase in biliary clearance (Cl bile) of unconjugated [125I]T4 (nearly 80-times control rates) and increased liver:blood ratios of T4, suggestive of enhanced hepatic uptake of T4. A single dose of DMP 904 (200 mg/kg) increased mRNA levels of hepatic cytochrome P450s (CYP 3A1 and CYP 2B1) and UDP-glucuronosyltransferases (UGT 1A1 and UGT 1A2). DMP 904 also induced mRNAs of the canalicular transporter, multi-drug resistance protein-2 (Mrp2) and sinusoidal transporters, organic anion transporting proteins (Oatp1 and Oatp2) within 24 h. Western blot analysis confirmed DMP 904 related increases in Oatp2 protein expression. Collectively, these data suggest that DMP 904 is an agonist of the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) and that the decreased serum levels of T4 and T3 resulted from increased hepatobiliary clearance. However, DMP 904 is distinguished from other compounds associated with similar effects on thyroid hormone homeostasis because its effects were primarily related to increased biliary excretion of unconjugated T4.

Roles of human CYP2A6 and 2B6 and rat CYP2C11 and 2B1 in the 10-hydroxylation of (-)-verbenone by liver microsomes.

(-)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (-)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome p450 (p450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and (-)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human samples, indicating that CYP2A6 is a principal enzyme in (-)-verbenone 10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed (-)verbenone 10-hydroxylation at Vmax values of 15 and 21 nmol/min/nmol p450 with apparent Km values of 16 and 91 microM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze (-)-verbenone 10-hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (-)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (-)-verbenone 10-hydroxylation with Vmax and Km ratios (ml/min/nmol p450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (-)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (-)-verbenone by liver microsomes and that there are species-related differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (-)-verbenone.