Hepatic P450s Research Articles

Role of Small Intestinal Cytochromes P450 in the Bioavailability of Oral Nifedipine

To determine the effect of intestinal cytochrome P450 (P450) enzymes on the bioavailability of oral drugs, we have examined the metabolism of nifedipine, an antihypertensive drug and a model substrate of CYP3A4, in mouse models having deficient expression of the NADPH-cytochrome P450 reductase. Initial studies were performed on Cpr-low (CL) mice, which have substantial decreases in Cpr expression in all tissues examined, including the small intestine. In CL mice, area under the concentration-time curve (AUC) values for blood nifedipine after intraperitoneal and oral dosing were 1.8- and 4.0-fold, respectively, higher than in wild-type mice, despite increased expression of multiple P450 enzymes in both liver and intestine. The greater extent of the increase in the AUC value for oral than for intraperitoneal nifedipine suggested that intestinal P450s influence the bioavailability of oral nifedipine, a notion supported by results from further studies on LCN and CL-LCN mice. The LCN mice, which have liver-specific Cpr deletion, had 6.9-fold higher AUC values and 2.2-fold higher C(max) values for blood nifedipine than did wild-type mice after oral nifedipine, consistent with the critical role of hepatic P450s in systemic nifedipine clearance. However, in the CL-LCN mice, which have global decreases in Cpr expression in all tissues examined and Cpr deletion in the liver, AUC and C(max) values for oral nifedipine were, respectively, 2.2- and 1.8-fold higher than in LCN mice, confirming the fact that P450-catalyzed metabolism in the small intestine, the portal-of-entry organ for oral drugs, plays an important role in the first-pass clearance of oral nifedipine.

Shosaikoto (a Kampo medicine) modulates changes in cytochrome P450 caused by Mycobacterium butyricum injection

A large number of infectious or inflammatory agents are well known to reduce cytochrome P450-catalyzed drug metabolism in the liver. Changes in the levels of hepatic P450s in infectious or inflammatory diseases can lead to alteration in physiology, therapeutic failure, and disease development. This study was therefore designed to examine the effect of shosaikoto, a Kampo medicine used for treatment of chronic hepatitis and acute flu, on the changes in P450 activity and protein levels following intravenous injection of Mycobacterium butyricum. Intravenous injection of M. butyricum into rats reduced hepatic P450: CYP1A1, 1A2, 2B, and 3A activities and CYP2E1 and 3A2 protein levels. When administered orally to rats for 2 weeks since 1 week before M. butyricum injection, shosaikoto prevented the reduction in CYP1A1, 1A2, 2B, and 3A activities dose dependently and further abrogated the repression of CYP3A2 protein but not CYP2E1 protein. Serum C-reactive protein levels in this rat model were not highly elevated, and neither were significantly influenced by shosaikoto treatment. Taken together, these results suggest that shosaikoto would be capable of modulating the change in hepatic P450 caused by M. butyricum injection and the effect, which might not be brought about by its anti-inflammatory activity but by translational or posttranslational regulation, appears to differ in each P450 isoform.

Conditional Deletion of Cytochrome P450 Oxidoreductase in the Liver and Gastrointestinal Tract: A New Model for Studying the Functions of the P450 System

We have previously described a mouse model, where hepatic cytochrome P450 oxidoreductase (POR) expression has been deleted, resulting in almost complete ablation of hepatic P450 function [Hepatic P450 Reductase Null (HRN)]. HRN mice grow normally but develop fatty livers, and they have increased cytochrome P450 levels. Associated with the hepatic lipid accumulation are significant changes in the expression of genes controlling lipid homeostasis. We have characterized this model extensively and demonstrated its value in drug efficiency testing, in toxicokinetics, and in evaluating the role of the hepatic P450 system in drug pharmacokinetics. To extend the deletion of POR, and P450 inactivation, to other tissues, and to develop the utility of this model, we have generated a mouse where POR can be deleted conditionally in the liver and gastrointestinal tract using the rat cytochrome P450 CYP1A1 promoter to drive Cre recombinase expression. Administration of the CYP1A1 inducers tetrachlorodibenzo-p-dioxin or beta-naphthoflavone resulted in both hepatic and gastrointestinal deletion of POR, whereas administration of 3-methylcholanthrene resulted specifically in loss of hepatic POR expression. In all cases, the resulting hepatic phenotype seemed identical to that of the HRN model, including increased cytochrome P450 expression. Hepatic deletion of POR and the subsequent increase in P450 expression were dependent on inducer dose, with maximal POR deletion occurring at a single dose of 3-methylcholanthrene of 40 mg/kg. This model provides a powerful approach for studying the functions of POR as well as in the evaluation of the role of hepatic and gastrointestinal P450s in drug deposition and chemical toxicity.

Effect of Perinatal Low Protein Diets on the Ontogeny of Select Hepatic Cytochrome P450 Enzymes and Cytochrome P450 Reductase in the Rat

In the present study, we administered two low protein diets (LPDs) to rats during pregnancy and lactation and determined their effect on the ontogeny of select hepatic cytochrome P450 (P450) isoforms in their offspring. The L93 and LM76 LPDs were derived from the American Society of Nutrition recommended AIN93G and a modified version of the AIN76A purified control diets, respectively. The LPDs contained 8% crude protein in the form of casein, whereas the purified control diets contained 19% casein. A regular cereal-based diet (NP) was also included, and, therefore, a total of five groups were tested. Pups in all five groups were weaned onto a regular NP diet on postnatal day 28. Perinatal LPD altered the activities of a number of P450 isoforms in 28-day-old male and female offspring. However, nutritional rehabilitation abolished most of these changes as evidenced by lack of differences between the five groups in the activities of P450 isoforms in either 65- or 150-day-old offspring. Interestingly, 58-day-old female offspring in the LM76 group but not those in the L93 group exhibited shorter hexobarbital sleep time than the purified control group. However, hexobarbital hydroxylase activity and the amount of CYP2C12 protein, an important P450 isoform involved in hexobarbital metabolism in females, were unchanged. This suggests that the decrease in hexobarbital sleep time in this group is not due to an increase in the activity of hexobarbital-metabolizing enzymes. In summary, perinatal LPDs produced transient alterations in activities of select hepatic P450s and resulted in a gender- and diet-dependent long-term alteration in hexobarbital pharmacodynamics.

Regulation of Constitutive Mouse Hepatic Cytochromes P450 and Growth Hormone Signaling Components by 3-Methylcholanthrene

3-Methylcholanthrene (MC) activates the aryl hydrocarbon receptor and increases expression of cytochrome P450 (P450) enzymes such as CYP1A1. MC also decreases expression of CYP2C11, the major hepatic P450 in male rats that is regulated by pulsatile growth hormone (GH) secretion via a pathway partially dependent on signal transducer and activator of transcription 5b (STAT5b). If disruption of this GH signaling pathway is important for MC's ability to suppress CYP2C11 transcription, we hypothesize that MC suppresses other male-specific genes (e.g., mouse Cyp2d9) regulated by pulsatile GH with STAT5b dependence. We examined the time course of MC's effects on hepatic P450s and GH signaling components in male C57BL/6 mice. P450 content, heme content, and NADPH P450 oxidoreductase activity were induced 2.3-, 1.8-, and 1.3-fold, respectively, by MC. MC dramatically induced CYP1A1 mRNA, protein, and catalytic activity. MC caused a 42% decrease in CYP2D9 protein, a 28% decrease in CYP2D9 mRNA, and a 27% decrease in testosterone 16alpha-hydroxylation activity. MC caused a pronounced decrease in CYP3A protein; however, there was no apparent change in testosterone 6beta-hydroxylation activity, and changes in mRNA levels for CYP3A forms were relatively small. Expression of GH receptor and major urinary protein 2, a gene regulated by GH with STAT5b dependence, was decreased by MC at the mRNA level. These results show that MC suppresses mouse Cyp2d9, a pulsatile GH- and STAT5b-dependent male-specific gene, via a pretranslational mechanism that may involve disrupted GH signaling. Mouse CYP3A protein levels are dramatically decreased by MC via a mechanism that is not yet understood.

Hepatic CYP3A Expression is Attenuated in Obese Mice Fed a High-Fat Diet

Changes in physiological, pathophysiological, and/or nutritional conditions often alter the expression of drug-metabolizing enzymes. In this study, we investigated obesity-induced changes in hepatic cytochrome P450 (P450) levels using nutritionally obese mice. To induce obesity, mice were fed a high-fat diet or treated with gold thioglucose, which impairs ventromedial hypothalamus. Total RNAs and microsomal and nuclear proteins were prepared from the liver of these mice, and mRNA and protein levels of P450s and transcription factors were determined. Among P450s examined, the constitutive expression of CYP3As was drastically reduced at both mRNA and protein levels by nutrition-induced obesity. One-week administration of a high-fat diet also reduced hepatic CYP3As. However, changes in nuclear receptors involved in the transcriptional regulation of CYP3A genes were not correlated with that of CYP3As. Obese mice induced by gold thioglucose exhibited a different expression profile of hepatic P450s with no significant change in CYP3As. High-fat diet-induced changes in energy metabolism, which eventually result in obesity, modulate the hepatic expression profile of P450s, particularly CYP3As. Alternatively, the accumulation of a certain component in a high-fat diet may directly attenuate the CYP3A expression, suggesting a clinically important drug-diet interaction.

INDUCTION OF CYP1A AND CYP2-MEDIATED ARACHIDONIC ACID EPOXYGENATION AND SUPPRESSION OF 20-HYDROXYEICOSATETRAENOIC ACID BY IMIDAZOLE DERIVATIVES INCLUDING THE AROMATASE INHIBITOR VOROZOLE

Cytochrome P450 (P450) enzymes metabolize the membrane lipid arachidonic acid to stable biologically active epoxides [eicosatrienoic acids (EETs)] and 20-hydroxyeicosatetraenoic acid (20-HETE). These products have cardiovascular activity, primarily acting as vasodilators and vasoconstrictors, respectively. EET formation can be increased by the prototype CYP1A or CYP2 inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or phenobarbital (PB), respectively. We report here that imidazole derivative drugs: the anthelminthics, albendazole and thiabendazole; the proton pump inhibitor, omeprazole; the thromboxane synthase inhibitor, benzylimidazole; and the aromatase (CYP19) inhibitor vorozole (R76713, racemate; and R83842, (+) enantiomer) increased hepatic microsomal EET formation in a chick embryo model. Albendazole increased EETs by transcriptional induction of CYP1A5 and the others by combined induction of CYP1A5 and CYP2H, the avian orthologs of mammalian CYP1A2 and CYP2B, respectively. All inducers increased formation of the four EET regioisomers, but TCDD and albendazole had preference for 5,6-EET and PB and omeprazole for 14,15-EET. Vorozole, benzylimidazole, and TCDD also suppressed 20-HETE formation. Vorozole was a remarkably effective and potent inducer of multiple hepatic P450s at a dose range which overlapped its inhibition of ovarian aromatase. Increased CYP1A activity in mouse Hepa 1-6 and human HepG2 cells by vorozole and other imidazole derivatives demonstrated applicability of the findings to mammalian cells. The findings suggest that changes in P450-dependent arachidonic acid metabolism may be a new source of side effects for drugs that induce CYP1A or CYP2. They demonstrate further that in vivo induction of multiple hepatic P450s produces additive increases in arachidonic acid epoxygenase activity and can occur concurrently with inhibition of ovarian aromatase activity.

Induction of cytochrome P450 by Nrf2 activators: Implication for aldehyde metabolism

Acrolein is an environmental pollutant and product of lipid peroxidation in vivo. Acrolein mediates the activation of Nrf2, so we sought to determine whether Nrf2 regulates clearance of acrolein by inducing specific hepatic P450s. The routes of metabolism of acrolein and α,β-unsaturated aldehydes include carboxylic acid and alcohol formation. C57BL/6 mice were fed either 5 mg/kg of acrolein by gavage for 72 h or AIN76 diet containing 0.45 % BHA, a Nrf2 activator, for 7 days. Mouse liver was frozen at −80 ° and after extraction, the RNA was reverse transcribed to cDNA. Cyp2c29 mRNA levels were analyzed by QRT-PCR. Liver microsomal protein fractions were prepared from control or BHA-fed mouse liver. Our results indicate that hepatic Cyp2c29 mRNA levels were induced 5-fold in BHA-treated mice and 2-fold in acrolein-treated mice. Likewise, there was a two-fold higher activity for 9-anthracene aldehyde oxidation to 9-anthracene carboxylic acid and reduction to 9-anthracene methanol with BHA-treated mouse liver microsomes, reactions catalyzed by Cyp2c29 expressed in E. coli. These reactions were inhibited 90% by 0.5 mM miconazole, a general P450 inhibitor, but only slightly inhibited by troleandomycin, a Cyp3a11-specific inhibitor or cyanamide, an aldehyde dehydrogenase inhibitor. Cyp3A11 mRNA levels remained unchanged upon BHA feeding. This suggests that the aldehyde oxidation and reduction activity present in murine hepatic microsomes is predominantly mediated by Cyp2c29. Supported by USPHS P01 ES11860.

OXIDATIVE METABOLISM OF THE ALKALOID RUTAECARPINE BY HUMAN CYTOCHROME P450

Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa. To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by alpha-naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had V(max) values of 1,388 to approximately 1,893 pmol/min/nmol P450, K(m) values of 4.1 to approximately 9.5 microM, and K(i) values of 45 to approximately 103 microM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC(50) values of 2.56 +/- 0.04, 2.57 +/- 0.11, and 0.09 +/- 0.01 microM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa.

Hepatic cytochrome P-450 reductase-null mice show reduced transcriptional response to quercetin and reveal physiological homeostasis between jejunum and liver

Using mice deficient in hepatic cytochrome P-450 oxidoreductase (POR), which disables the liver cytochrome P-450 system, we examined the metabolism and biological response of the anticarcinogenic flavonoid, quercetin. Profiling circulating metabolites revealed similar profiles over 72 h in wild-type (WT) and POR-null (KO) mice, showing that hepatic P450 and reduced biliary secretion do not affect quercetin metabolism. Transcriptional profiling at 24 h revealed that two- to threefold more genes responded significantly to quercetin in WT compared with KO in the jejunum, ileum, colon, and liver, suggesting that hepatic P450s mediate many of the biological effects of quercetin, such as immune function, estrogen receptor signaling, and lipid, glutathione, purine, and amino acid metabolism, even though quercetin metabolism is not modified. The functional interpretation of expression data in response to quercetin (single dose of 7 mg/animal) revealed a molecular relationship between the liver and jejunum. In WT animals, amino acid and sterol metabolism was predominantly modulated in the liver, fatty acid metabolism response was shared between the liver and jejunum, and glutathione metabolism was modulated in the small intestine. In contrast, KO animals do not regulate amino acid metabolism in the liver or small intestine, they share the control of fatty acid metabolism between the liver and jejunum, and regulation of sterol metabolism is shifted from the liver to the jejunum and that of glutathione metabolism from the jejunum to the liver. This demonstrates that the quercetin-mediated regulation of these biological functions in extrahepatic tissues is dependent on the functionality of the liver POR. In conclusion, using a systems biology approach to explore the contribution of hepatic phase 1 detoxification on quercetin metabolism demonstrated the resiliency and adaptive capacity of a biological organism in dealing with a bioactive nutrient when faced with a tissue-specific molecular dysfunction.

ONTOGENY OF HEPATIC AND PLASMA METABOLISM OF DELTAMETHRIN IN VITRO: ROLE IN AGE-DEPENDENT ACUTE NEUROTOXICITY

Deltamethrin (DLM) is a relatively potent and widely used pyrethroid insecticide. Inefficient detoxification has been proposed to be the primary reason for the greater sensitivity of immature rats to the acute neurotoxicity of DLM. The objective of this study was to test this hypothesis by characterizing the age dependence of DLM metabolism in vitro, as well as toxic signs and blood levels of the neurotoxic parent compound following administration of 10 mg DLM/kg p.o. in glycerol formal. Metabolism was quantified in vitro by monitoring the disappearance of the parent compound from plasma [via carboxylesterases (CaEs)] and liver microsomes [via CaEs and cytochromes P450 (P450s)] obtained from 10-, 21-, and 40-day-old male Sprague-Dawley rats. Mean (+/-S.E.) intrinsic clearances (Vmax/Km) in these respective age groups by liver P450s (4.99+/-0.32, 16.99+/-1.85, and 38.45+/-7.03) and by liver CaEs (0.34+/-0.05, 1.77+/-0.38, and 2.53+/-0.19) and plasma CaEs (0.39+/-0.06, 0.80+/-0.09, and 2.28+/-0.56) increased significantly (p<or=0.05) with age, because of progressive increases in Vmax. Intrinsic clearance of DLM by plasma CaEs and liver P450s reached adult levels by 40 days, but clearance by liver CaEs did not. Hepatic P450s played the predominant role in DLM biotransformation in young and adult rats. The incidence and severity of neurotoxic effects varied inversely with age. Correspondingly, blood DLM areas under the concentration versus time curve (AUCs) and Cmax values progressively decreased with increasing age. Internal exposure to DLM (blood AUCs) was closely correlated with toxic signs (salivation and tremors). The present study provides evidence that the limited metabolic capacity of immature rats contributes to elevated systemic exposure and ensuing neurotoxic effects of DLM.

Hepatic Gene Expression Changes in Mouse Models with Liver-specific Deletion or Global Suppression of the NADPH-Cytochrome P450 Reductase Gene: MECHANISTIC IMPLICATIONS FOR THE REGULATION OF MICROSOMAL CYTOCHROME P450 AND THE FATTY LIVER PHENOTYPE

NADPH-cytochrome P450 reductase (CPR) is an essential component for the function of many enzymes, including microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. In liver-Cpr-null (with liver-specific Cpr deletion) and Cpr-low (with reduced CPR expression in all organs examined) mouse models, a reduced serum cholesterol level and an induction of hepatic P450s were observed, whereas hepatomegaly and fatty liver were only observed in the liver-Cpr-null model. Our goal was to identify hepatic gene expression changes related to these phenotypes. Cpr-lox mice (with a floxed Cpr gene and normal CPR expression) were used as the control. Through microarray analysis, we identified many genes that were differentially expressed among the three groups of mice. We also recognized the 12 gene ontology terms that contained the most significantly changed gene expression in at least one of the two mouse models. We further uncovered potential mechanisms, such as an increased activation of constitutive androstane receptor and a decreased activation of peroxisomal proliferator-activated receptor-alpha by precursors of cholesterol biosynthesis, that underlie common changes (e.g. induction of multiple P450s and suppression of genes for fatty acid metabolism) in response to CPR loss in the two mouse models. Additionally, we observed model-specific gene expression changes, such as the induction of a fatty-acid translocase (Cd36 antigen) and the suppression of carnitine O-palmitoyltransferase 1 (Cpt1a) and acyl-CoA synthetase long chain family member 1 (Acsl1), that are potentially responsible for the severe hepatic lipidosis and an altered fatty acid profile observed in liver-Cpr-null mice.

PHARMACOKINETICS AND DISPOSITION OF THE KAVALACTONE KAWAIN: INTERACTION WITH KAVA EXTRACT AND KAVALACTONES IN VIVO AND IN VITRO

Reported adverse drug interactions with the popular herb kava have spurred investigation of the mechanisms by which kava could mediate these effects. In vivo and in vitro experiments were conducted to examine the effects of kava extract and individual kavalactones on cytochrome P450 (P450) and P-glycoprotein activity. The oral pharmacokinetics of the kavalactone, kawain (100 mg/kg), were determined in rats with and without coadministration of kava extract (256 mg/kg) to study the effect of the extract on drug disposition. Kawain was well absorbed, with >90% of the dose eliminated within 72 h, chiefly in urine. Compared with kawain alone, coadministration with kava extract caused a tripling of kawain AUC(0-8 h) and a doubling of C(max). However, a 7-day pretreatment with kava extract (256 mg /kg/day) had no effect on the pharmacokinetics of kawain administered on day 8. The 7-day pretreatment with kava extract only modestly induced hepatic P450 activities. The human hepatic microsomal P450s most strongly inhibited by kava extract (CYP2C9, CYP2C19, CYP2D6, CYP3A4) were inhibited to the same degree by a "composite" kava formulation composed of the six major kavalactones contained in the extract. K(i) values for the inhibition of CYP2C9 and CYP2C19 activities by methysticin, dihydromethysticin, and desmethoxyyangonin ranged from 5 to 10 microM. Kava extract and kavalactones (< or =9 microM) modestly stimulated P-glycoprotein ATPase activities. Taken together, the data indicate that kava can cause adverse drug reactions via inhibition of drug metabolism.

Role of Hepatic Cytochrome P450s in the Pharmacokinetics and Toxicity of Cyclophosphamide: Studies with the Hepatic Cytochrome P450 Reductase Null Mouse

Cyclophosphamide (CPA) is an anticancer prodrug that is dependent on cytochrome P450 (CYP) metabolism for its therapeutic effectiveness. In spite of the use of CPA in the clinic for over 50 years, little is known about the relationship between its toxicokinetics and therapeutic response. We have employed a powerful new model, the Hepatic Cytochrome P450 Reductase Null (HRN) mouse, which has almost no hepatic cytochrome P450 activity, to study the toxicokinetics of CPA and to establish in vivo the role of hepatic P450 metabolism in its pharmacokinetics. In HRN mice the in vitro metabolism and intrinsic clearance of CPA was over 6-fold lower than in wild-type animals. This change in CPA metabolism was also reflected in vivo, with a profound difference in the pharmacokinetics of both CPA and its metabolites. At a CPA dose of 100 mg/kg, the Cmax, plasma area under the curve (AUC) and half-life were increased by 2.6-, 6.2-, and 3.2-fold, respectively, in the HRN mice. Similar changes were also observed at a dose of 300 mg/kg. These data confirm that hepatic metabolism is the major route of CPA elimination and disposition. The primary metabolites of CPA, 4-hydroxycyclophosphamide (4-OH-CPA) and 3-dechloroethylcyclophosphamide, were still formed, but at altered rates in the HRN mice. At 100 mg/kg the t1/2 for 4-OH-CPA was increased 1.8-fold, the Cmax reduced 1.7-fold, and the AUC remained unchanged. This latter finding shows that P450-mediated oxidative metabolism is essential for the clearance of this compound. Toxicokinetic analysis of CPA-induced myelosuppression and granulocytopenia showed that at high doses (> or =100 mg/kg) there was no difference in myelotoxicity between the wild-type and HRN mice. However, at lower doses (< or =70 mg/kg) a significant difference was observed, with little toxicity seen in HRN mice but at least a 45% reduction in the bone marrow granulocyte population in wild-type mice. Meta-analysis of the toxicity experiments showed the myelotoxicity of CPA was found to be closely correlated with the Cmax of 4-OH-CPA (r2= 0.80, P = 0.002). As the therapeutic effectiveness of CPA has been linked to the AUC for 4-OH-CPA, the finding that 4-OH-CPA Cmax may determine its level of myelotoxicity indicates that the therapeutic index could be altered by changing the method of CPA administration. Furthermore, monitoring 4-OH-CPA Cmax may identify individuals at most risk of CPA side effects.

Intrinsic sex differences determine expression of growth hormone-regulated female cytochrome P450s

The masculine profile of cytochrome P450s found in male liver is determined by the episodic secretion of growth hormone characteristic of males. In turn, the female pattern of P450s observed in female rat liver is regulated by the continuous secretion of growth hormone characteristic of the female. In order to determine if intrinsic and possibly permanent sex differences exist in the response of hepatic P450s to growth hormone regulation, we compared the effects of the episodic and continuous growth hormone profiles on the expression of female-dependent isoforms in cultured hepatocytes isolated from both sexes. We observed that female-specific CYP2C12 as well as female-predominant CYP2A1, 3A1, and 2C6 could be induced by growth hormone concentrations equal to as little as 6, 0.6, and 0.06% of the mean circulating hormone profile found in normal females. Irrespective of sex, all four female-dependent isoforms were far more responsive to the continuous growth hormone profile than the episodic pattern. Lastly, female-derived hepatocytes in general responded with strikingly greater induction levels of P450s than male hepatocytes exposed to the same growth hormone profiles. The present findings demonstrate intrinsic, irreversible sex differences in growth hormone-regulated female-dependent P450s.

CYP2C44, a new murine CYP2C that metabolizes arachidonic acid to unique stereospecific products.

The human CYP2Cs have been studied extensively with respect to the metabolism of clinically important drugs and endogenous chemicals such as arachidonic acid (AA). Five members of the mouse CYP2C family have previously been described that metabolize arachidonic acid into regio- and stereospecific epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids, which have many important physiological roles. Herein, we describe the cloning and characterization of a new mouse cytochrome P450 (P450), CYP2C44, which has the lowest homology with other known mouse CYP2Cs. Western blotting and real-time polymerase chain reaction detected CYP2C44 mRNA and protein in liver >> kidney > adrenals. Kidney contained approximately 10% of the CYP2C44 mRNA content of liver. CYP2C44 metabolized AA to unique stereospecific products, 11R,12S-EET and 8R, 9S-EET, which are similar to those produced by rat CYP2C23. CY2C23 is highly expressed in rat kidney and has been suggested to be important in producing compensatory renal artery vasodilation in response to salt-loading in this species. Immunohistochemistry showed the presence of CYP2C44 in hepatocytes, biliary cells of the liver, and the proximal tubules of the kidney. Unlike mouse CYP2C29, CYP2C38, and CYP2C39, CYP2C44 did not metabolize the common CYP2C substrate tolbutamide. CYP2C44 was not induced by phenobarbital or pregnenolone-16alpha-carbonitrile, two prototypical inducers of hepatic P450s. The presence of CYP2C44 in mouse liver, kidney, and adrenals and the unique stereospecificity of its arachidonic acid metabolites are consistent with the possibility that it may have unique physiological roles within these tissues, such as modulation of electrolyte transport or vascular tone.

Lipopolysaccharide Induces CYP2E1 in Astrocytes through MAP Kinase Kinase-3 and C/EBPβ and -δ

Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide (LPS) or interleukin-1beta. We have studied the mechanism of induction, and found that transcriptional activation of CYP2E1 occurred within 3 h, and CYP2E1 dependent catalytic activity was induced more than 4-fold within 5 h. The induction was sensitive to several tyrosine kinase inhibitors, and was further modulated by inhibitors of p38 MAP kinase. MAP kinase kinase-3 (MKK3) was phosphorylated in response to LPS, and expression of constitutively active MKK3, but not the MAP kinase kinases MEKK1 or MKK1, activated CYP2E1. Transcriptional activation was mediated through a C/EBPbeta and -delta binding element situated at -486/-474, and appeared to involve activation of prebound factors as well as recruitment of newly synthesized C/EBPbeta and -delta. It is thus suggested that LPS induces MKK3 activation in astrocytes, which in turn stimulates a C/EBPbeta and -delta binding element to mediate transcriptional activation of CYP2E1.

Interaction of buprenorphine and its metabolite norbuprenorphine with cytochromes p450 in vitro.

Buprenorphine is a thebaine derivative used in the treatment of heroin and other opiate addictions. In this study, the selective probe reactions for each of the major hepatic cytochromes P450 (P450s) were used to evaluate the effect of buprenorphine and its main metabolite norbuprenorphine on the activity of these P450s. The index reactions used were CYP1A2 (phenacetin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (diclofenac 4'-hydroxylation), CYP2C19 (omeprazole 5-hydrxoylation), CYP2D6 (dextromethorphan O-demethylation), CYP2B6 (7-ethoxy-4-trifluoromethyl-coumarin 7-deethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), and CYP3A4 (omeprazole sulfoxidation). Buprenorphine exhibited potent, competitive inhibition of CYP2D6 (Ki 10 +/- 2 microM and 1.8 +/- 0.2 microM) and CYP3A4 (Ki 40 +/- 1.6 microM and 19 +/- 1.2 microM) in microsomes from human liver and cDNA-expressing lymphoblasts, respectively. Compared with buprenorphine, norbuprenorphine demonstrated a lower inhibitory potency with CYP2D6 (22.4% inhibition at 20 microM norbuprenorphine) and CYP3A4 (13.6% inhibition at 20 microM) in microsomes from human cDNA-expressing lymphoblast cells. Furthermore, buprenorphine was shown to be a substrate of CYP2D6 (Km = 600 microM; Vmax = 0.40 nmol/min/mg protein) and CYP3A4 (Km = 36 microM; Vmax = 0.19 nmol/min/mg protein). The present in vitro study suggests that buprenorphine and its major metabolite norbuprenorphine are inhibitors of CYP2D6 and CYP3A4; however, at therapeutic concentrations they are not predicted to cause potentially clinically important drug interactions with other drugs metabolized by major hepatic P450s.

Inactivation of the Hepatic Cytochrome P450 System by Conditional Deletion of Hepatic Cytochrome P450 Reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.

Amodiaquine clearance and its metabolism to N-desethylamodiaquine is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe substrate.

Amodiaquine (AQ) metabolism to N-desethylamodiaquine (DEAQ) is the principal route of disposition in humans. Using human liver microsomes and two sets of recombinant human cytochrome P450 isoforms (from lymphoblastoids and yeast) we performed studies to identify the CYP isoform(s) involved in the metabolism of AQ. CYP2C8 was the main hepatic isoform that cleared AQ and catalyzed the formation of DEAQ. The extrahepatic P450s, 1A1 and 1B1, also cleared AQ and catalyzed the formation of an unknown metabolite M2. The K(m) and V(max) values for AQ N-desethylation were 1.2 microM and 2.6 pmol/min/pmol of CYP2C8 for recombinant CYP2C8, and 2.4 microM and 1462 pmol/min/mg of protein for human liver microsomes (HLMs), respectively. Relative contribution of CYP2C8 in the formation of DEAQ was estimated at 100% using the relative activity factor method. Correlation analyses between AQ metabolism and the activities of eight hepatic P450s were made on 10 different HLM samples. Both the formation of DEAQ and the clearance of AQ showed excellent correlations (r(2) = 0.98 and 0.95) with 6alpha-hydroxylation of paclitaxel, a marker substrate for CYP2C8. The inhibition of DEAQ formation by quercetin was competitive with K(i) values of 1.96 for CYP2C8 and 1.56 microM for HLMs. Docking of AQ into the active site homology models of the CYP2C isoforms showed favorable interactions with CYP2C8, which supported the likelihood of an N-desethylation reaction. These data show that CYP2C8 is the main hepatic isoform responsible for the metabolism of AQ. The specificity, high affinity, and high turnover make AQ desethylation an excellent marker reaction for CYP2C8 activity.