Regulation Of P450 Research Articles

Humanized Mouse Lines and Their Application for Prediction of Human Drug Metabolism and Toxicological Risk Assessment

Cytochrome P450s (P450s) are important enzymes involved in the metabolism of xenobiotics, particularly clinically used drugs, and are also responsible for metabolic activation of chemical carcinogens and toxins. Many xenobiotics can activate nuclear receptors that in turn induce the expression of genes encoding xenobiotic metabolizing enzymes and drug transporters. Marked species differences in the expression and regulation of cytochromes P450 and xenobiotic nuclear receptors exist. Thus, obtaining reliable rodent models to accurately reflect human drug and carcinogen metabolism is severely limited. Humanized transgenic mice were developed in an effort to create more reliable in vivo systems to study and predict human responses to xenobiotics. Human P450s or human xenobiotic-activated nuclear receptors were introduced directly or replaced the corresponding mouse gene, thus creating "humanized" transgenic mice. Mice expressing human CYP1A1/CYP1A2, CYP2E1, CYP2D6, CYP3A4, CY3A7, pregnane X receptor, and peroxisome proliferator-activated receptor alpha were generated and characterized. These humanized mouse models offer a broad utility in the evaluation and prediction of toxicological risk that may aid in the development of safer drugs.

Clinical versatility of porcine hepatocytes in the light of interspecies differences in cytochrome P450 regulation and expression

In fulminant hepatic failure, the clinical use of bioartifical liver support with porcine hepatocytes is the subject of a controversial debate. Cytochrome P450 (CYP) metabolic functions have relevant implications for drug metabolism and detoxification. In this study, we investigate interspecies differences in CYP gene expression between human and porcine primary hepatocytes and the impact of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) exposition mimicking cytokine release in fulminant hepatic failure. Primary hepatocyte cultures were isolated from human resection specimens and from German landrace pigs. Cell cultures (single and co-cultures) were exposed to porcine vs. human IL-6 and TNF-alpha, respectively. Changes in quantitative CYP gene expression were investigated by semi-quantitative RT-PCR. Significant differences in species-specific CYP gene expression by human and porcine hepatocytes were found after exposure to species-identical IL-6 (10 ng/ml) for CYP 1A1, CYP 2C, CYP 3A (P = 0.002, 0.022, 0.017, respectively) or species-identical TNF-alpha (30 ng/ml) for CYP 1A2 and CYP 2A (P = 0.037, 0.023, respectively). In single vs. co-culture, human hepatocytes demonstrated stronger repression of CYP 1A1, 2C8 and 3A4 expression after dosage with human IL-6 (10 ng/ml) (P = 0.022, 0.031, 0.014, respectively). Our findings demonstrate significant species-specific differences in CYP gene expression and regulation when high doses of IL-6 and TNF-alpha are used (10 and 30 ng/ml, respectively). These findings may point to species-specific physiological incompatibilities of porcine hepatocytes and thus limit their clinical versatility.

Applications and Limitations of Genetically Modified Mouse Models in Drug Discovery and Development

Genetically modified mouse models in which a specific gene is removed or replaced have proven to be powerful tools for identification/validation of target gene and scientific understanding of molecular mechanisms underlying drug-induced toxicity through mechanistic studies. In spite of the advantage, there are significant limitations of genetically modified mouse models. Modification of a given gene does not always result in the anticipated phenotype. In some instances, phenotypes of targeted mouse mutants were not those predicted from the presumed function of the given genes, while other null mutants revealed no apparent defects. Furthermore, the phenotypic outcome can be influenced by many environmental and genetic factors. Therefore, interpretation of the significance of the findings from studies using genetically modified mouse models is not always as straightforward as one would expect, especially when desire is to extrapolate the findings to humans. Interestingly, many humanized mouse models have been generated for evaluating the function and regulation of cytochrome P450 (CYP) enzymes. Our fascination with humanized animals dates back to ancients. For example, the Great Sphinx of Giza, a large half-human and half-lion statue, is believed to have been built by Egyptians about 4500 years ago. Although the creation of humanized animals that carry a particular human CYP gene provides useful tools for scientific understanding of the function and regulation of the CYP enzyme, these humanized mouse models are not so useful in prediction of human pharmacokinetics in a quantitative sense. Accordingly, it is important to keep in mind that an animal engineered to express a human gene and its protein is still an animal.

Regulation of liver cytochrome P450 by activation of brain dopaminergic system: Physiological and pharmacological implications

The aim of the present study was to investigate the influence of activation of brain dopaminergic system by different dopaminomimetics on the level and activity of liver cytochrome P450 (CYP) isoforms. Studies into the identification of hormones and cytokines which are known to mediate liver CYP expression were also simultaneously carried out. Stimulation of dopaminergic receptors in the pituitary, a target for the tuberoinfundibular pathway, by dopamine (a D 1/D 2 receptor agonist) administered intraperitoneally caused a significant increase in the activities and protein levels of CYP2B, CYP2C11 and CYP3A, a substantial increase in the blood plasma level of growth hormone (GH) and a significant decrease in triiodothyronine (T 3) level. Local stimulation of dopaminergic receptors in the nucleus accumbens, a target for the mesolimbic pathway, by apomorphine (a D 1/D 2 receptor agonist), amphetamine (an indirect D 1/D 2 dopaminemimetic) and quinpirole (a D 2 receptor agonist) produced a substantial rise in CYP3A activity and protein level, caused a large increase in corticosterone concentration and a moderate drop in T 3 level. SKF82958 (a D 1 receptor agonist) did not significantly affect the CYP isoforms or hormones studied. In both cases (activation of the tuberoinfundibular or mesolimbic pathway), the activity and the protein level of CYP1A considerably decreased. Plasma levels of thyroxine, testosterone, interleukin-2 and interleukin-6 were not changed after activation of the two pathways. The obtained results establish the brain dopaminergic system as a physiological centre regulating cytochrome P450 (engaging D 2 receptors and pituitary hormones) and demonstrate new pharmacological aspects of neuroactive drugs that affect this system.

Sterol regulatory element binding protein 1 interacts with pregnane X receptor and constitutive androstane receptor and represses their target genes

Sterol regulatory element binding protein 1 (SREBP-1) is a lipogenic transcription factor of the basic helix-loop-helix family. SREBP-1 binds to sterol regulatory elements (SREs) in the promoter of lipogenic genes and induces fatty acid and triglyceride synthesis. Decreased drug clearance has been observed in obese and other dyslipidemic rodents as well as in diabetic, obese or overfed rodents. A hallmark of these conditions is increased expression of SREBP-1 in the liver. We therefore searched for a possible link between regulation of cytochromes P450 (CYPs) and SREBP-1. We combined gene expression analysis, lipid analysis, effects of high levels of SREBP-1 in hepatocyte cultures to characterize the effects and protein interaction and chromatin immunoprecipitation assays to define the underlying mechanism. Finally, mice were fed a diet enriched in cholesterol to demonstrate the relevance of our data in vivo. By analyzing gene expression and lipids in cholesterol-fed mice or transfection of recombinant SREBP-1 in hepatocyte cultures the effect on CYPs was characterized. By use of protein interaction assays and chromatin immunoprecipitation the underlying mechanism was defined. We observed that SREBP-1 represses drug-mediated induction of hepatic CYPs, mainly members of the 2B and the 3A subfamilies. These drugs induce transcription of CYPs and other drug metabolizing enzymes via activation of the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Here we report that the activation of SREBP-1 by insulin or cholesterol in mouse liver and primary human hepatocytes inhibits the transcriptional effects in PXR and CAR. Our results suggest that SREBP-1 functions as a non-DNA binding inhibitor and blocks the interaction of PXR and CAR with cofactors such as steroid receptor coactivator 1. Consequently, mRNA induction of CYPs by drugs and other xenochemicals is impaired. We conclude that PXR and CAR respond to lipid accumulation by direct interaction with SREBP-1 and show that drug metabolism and lipid metabolism are interconnected within a complex network of transcriptional regulators.

Regulation of Drug-Metabolizing Enzymes and Transporters in Infection, Inflammation, and Cancer

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 07 meeting in Washington, DC. The presentations discussed the phenomenology, clinical consequences, and underlying mechanisms of cytochrome P450 and drug transporter regulation by inflammatory and infectious stimuli. Although considerable insights into the links between inflammatory mediators and altered hepatic drug clearance pathways have been gained from previous studies with acute inflammatory stimuli, this symposium highlighted recent advances in understanding how these processes operate in other organs and chronic inflammatory states relevant to human diseases. The development of mouse models of live bacterial infection provides excellent opportunities to explore the impact of infection on drug metabolism beyond the well characterized effects of bacterial endotoxin. Altered levels of cytochromes P450 and especially drug transporters due to inflammation in brain, intestine, and placenta have significant implications for the use of many drugs in diverse clinical settings. The consequences of inflammatory cytokine production by tumors for drug safety and efficacy in cancer patients were outlined. Repression of drug clearance pathways by tumor-derived cytokines may result in extreme toxicity to chemotherapy, compromising treatment of many cancers. It is fitting that, in honoring the career contributions and achievements of Dr. Kenneth W. Renton, this symposium reinforced the clinical relevance of this field.

Cytochrome P450 Expression and Regulation in CYP3A4/CYP2D6 Double Transgenic Humanized Mice

Analysis of the developmental and sexual expression of cytochrome P450 drug-metabolizing enzymes is impeded by multiple and varied external factors that influence its regulation. In the present study, a CYP2D6/CYP3A4-double transgenic (Tg-CYP2D6/CYP3A4) mouse model was employed to investigate hepatic CYP2D6 and CYP3A4 ontogeny and sexual dimorphism. Both age and sex have considerable effects on hepatic CYP3A4 protein expression in 3- to 8-week-old transgenic mice, whereas neither factor alters CYP2D6 content. Constitutive CYP2D6 expression resulted in 2- to 3-fold higher dextromethorphan O-demethylase activity in Tg-CYP2D6/CYP3A4 mouse liver microsomes compared with wild-type mice. In contrast, expression of CYP3A4 in transgenic mouse livers did not increase dextromethorphan N-demethylase and midazolam 1'-hydroxylase activities. Pretreatment with pregnenolone 16alpha-carbonitrile (PCN) and 1,4-bis-2-(3, 5-dichloropyridyloxy)-benzene (TCPOBOP) elevated CYP3A4 expression in double transgenic mice. Interestingly, induction of hepatic CYP3A4 was greater in females than age- and treatment-matched males. Consequently, the increase in midazolam 1'-hydroxylase activity was markedly higher in 8-week-old female mice than in corresponding males (8-fold versus 6-fold for PCN treatment and 6-fold versus 5-fold for TCPOBOP). Furthermore, increases in testosterone 6beta-hydroxylase activity after CYP3A induction were relatively lower compared with those in midazolam 1'-hydroxylation for age-, sex-, and treatment-matched mice. The difference in CYP3A4 expression and induction between male and female mice suggests that women may be more susceptible to CYP3A4-mediated drug-drug interactions, and the extent of drug-drug interactions could be substrate dependent.

D 2-receptor-linked signaling pathways regulate the expression of hepatic CYP2E1

This study investigated the role of catecholamine-related signaling pathways in the regulation of hepatic cytochrome P450 (CYP2E1). Central and peripheral catecholamine depletion with reserpine down-regulated CYP2E1. On the other hand, selective peripheral catecholamine depletion with guanethidine increased CYP2E1 apoprotein levels. Enrichment of peripheral catecholamines with adrenaline suppressed p-nitrophenol hydroxylase activity (PNP). PNP activity was also markedly suppressed by l-DOPA. Stimulation of D 2-receptors with bromocriptine up-regulated CYP2E1, as assessed by enzyme activity and protein levels, whereas blockade of D 2-dopaminergic receptors with sulpiride down-regulated this isozyme. These findings indicate that central and peripheral catecholamines have different effects on CYP2E1. Central catecholamines appear related to the up-regulation, whereas the role of peripheral catecholamines is clearly related to the type and location of adrenoceptors involved. D 2-receptor-linked signaling pathways have an up-regulating effect on CYP2E1, while D 1-receptor pathways may down-regulate this isozyme. It is worth noting that the widespread environmental pollutant benzo(α)pyrene (B(α)P) altered the modulating effect of catecholaminergic systems on CYP2E1 regulation. In particular, whereas stimulation or blockade of adrenoceptors had no effect on constitutive PNP activity, exposure to B(α)P modified the impact of central and peripheral catecholamines and α 2-adrenoceptors on CYP2E1 expression. It appears that under the influence of B(α)P, α 2-adrenergic receptor-linked signaling pathways increased CYP2E1 apoprotein levels. Given that a wide range of xenobiotics and clinically used drugs are activated by CYP2E1 to toxic metabolites, including the production of reactive oxygen species (ROS), it is possible that therapies challenging dopaminergic receptor- and/or alpha 2-adrenoceptor-linked signaling pathways may alter the expression of CYP2E1, thus affecting the progress and development of several pathologies.

Functional Regulation of Hepatic Cytochrome P450 Enzymes by Physicochemical Properties of Phospholipids in Biological Membranes

Knowledge regarding the regulation of hepatic cytochrome P450 (P450) is crucial to the fields of drug therapy and drug development, as well as to our understanding of the mechanisms underlying the metabolic activation of toxic and carcinogenic compounds. P450 is a membrane-anchored protein that shows a variety of interaction with membrane phospholipids, which affect the membrane topology and catalytic activities of the protein. In particular, anionic phospholipids, nonbilayer forming lipids, and the degree of saturation of the lipid fatty acyl chain play important roles in the functional regulation of P450, as well as in the bilayer structure of the membrane. However, despite the importance of phospholipids in the regulation of P450s, the interaction of the protein with membrane phospholipids, and the membrane properties induced by phospholipids which regulate P450, are unclear. In this review, we describe the effect of the physicochemical properties of the phospholipid constituents of biological membranes on hepatic P450 catalytic activity, membrane insertion (and/or penetration), and structural changes.

O08 Association between CYP2C9 polymorphism and warfarin dose

The polymorphic nature of the cytochrome P450 (CYP) genes affects individual drug response and adverse reactions to a great extent. This variation includes copy number variants (CNV), missense mutations, insertions and deletions, and mutations affecting gene expression and activity of mainly CYP2A6, CYP2B6, CYP2C9, CYP2C19 and CYP2D6, which have been extensively studied and well characterized. CYP1A2 and CYP3A4 expression varies significantly, and the cause has been suggested to be mainly of genetic origin but the exact molecular basis remains unknown. We present a review of the major polymorphic CYP alleles and conclude that this variability is of greatest importance for treatment with several antidepressants, antipsychotics, antiulcer drugs, anti-HIV drugs, anticoagulants, antidiabetics and the anticancer drug tamoxifen. We also present tables illustrating the relative importance of specific common CYP alleles for the extent of enzyme functionality. The field of pharmacoepigenetics has just opened, and we present recent examples wherein gene methylation influences the expression of CYP. In addition microRNA (miRNA) regulation of P450 has been described. Furthermore, this review updates the field with respect to regulatory initiatives and experience of predictive pharmacogenetic investigations in the clinics. It is concluded that the pharmacogenetic knowledge regarding CYP polymorphism now developed to a stage where it can be implemented in drug development and in clinical routine for specific drug treatments, thereby improving the drug response and reducing costs for drug treatment.

The Regulation of Liver Cytochrome P450 by the Brain Dopaminergic System

Genes encoding different cytochrome P450 (CYP) isoforms are regulated by endogenous hormones (e.g. pituitary hormones, thyroid hormones, glucocorticoids) which are all under control of the central nervous system. The aim of the present study was to investigate the influence of lesions of brain dopaminergic pathways on the level and the activity of CYP isoforms (1A, 2A, 2B, 2C6, 2C11, 2D, 3A) in rat liver. At 48 h after lesion of the tuberoinfundibular pathway, only the activity and the protein level of CYP2B were significantly decreased. Seven days after lesion of the above-mentioned pathway, significant inhibition of CYP2B, CYP2C11 and CYP3A activities and a decrease in CYP protein levels were observed. At the same time, the activity and the protein level of CYP1A considerably increased. Fourteen days after damage of the mesolimbic pathway, the activity and the protein level of CYP3A were significantly reduced, while those of CYP1A were substantially elevated. In contrast, lesion of the nigrostriatal pathway did not affect any CYP isoforms studied. The obtained results provide the first direct evidence for the influence of brain dopaminergic system on the level and the activity of CYP in the liver, which is pathway- and isoform-dependent. Hence stimulation or inhibition of the brain dopaminergic system (e.g. by dopamine receptor-blocking neuroleptics) may cause changes in CYP activity of physiological, pharmacological and toxicological significance, since CYP isoforms that are regulated by the dopaminergic system catalyze the metabolism of endogenous substances (e.g. steroids), clinically important drugs (e.g. psychotropics, calcium channel antagonists, antibiotics) and toxins.

Antiepileptic Drugs Affect Neuronal Androgen Signaling via a Cytochrome P450-Dependent Pathway

Recent data imply an important role for brain cytochrome P450 (P450) in endocrine signaling. In epileptic patients, treatment with P450 inducers led to reproductive disorders; in mouse hippocampus, phenytoin treatment caused concomitant up-regulation of CYP3A11 and androgen receptor (AR). In the present study, we established specific in vitro models to examine whether CYP3A isoforms cause enhanced AR expression and activation. Murine Hepa1c1c7 cells and neuronal-type rat PC-12 cells were used to investigate P450 regulation and its effects on AR after phenytoin and phenobarbital administration. In both cell lines, treatment with antiepileptic drugs (AEDs) led to concomitant up-regulation of CYP3A (CYP3A11 in Hepa1c1c7 and CYP3A2 in PC-12) and AR mRNA and protein. Inhibition of CYP3A expression and activity by the CYP3A inhibitor ketoconazole or by CYP3A11-specific short interfering RNA molecules reduced AR expression to basal levels. The initial up-regulation of AR signal transduction, measured by an androgen-responsive element chloramphenicol-acetyltransferase reporter gene assay, was completely reversed after specific inhibition of CYP3A11. Withdrawal of the CYP3A11 substrate testosterone prevented AR activation, whereas AR mRNA expression remained up-regulated. In addition, recombinant CYP3A11 was expressed heterologously in PC-12 cells, thereby eliminating any direct drug influence on the AR. Again, the initial up-regulation of AR mRNA and activity was reduced to basal levels after silencing of CYP3A11. In conclusion, we show here that CYP3A2 and CYP3A11 are crucial mediators of AR expression and signaling after AED application. These findings point to an important and novel function of P450 in regulation of steroid hormones and their receptors in endocrine tissues such as liver and brain.

Effect of HMGCoA Reductase Inhibitors on Cytochrome P450 Expression in Endothelial Cell Line

Endothelial cells and smooth muscle cells are the major cells that constitute blood vessels, and endothelial cells line the lumen of blood vessels. These 2 types of cells also play an integral role in the regional specialization of vascular structure. On the basis of these observations, we designed our study to investigate the effect of various statins on CYP expression in endothelial cells. 3-hydroxymethyl coenzyme A reductase inhibitors play an important role in vascular function. The majority of the statins available on the market show extensive metabolism by cytochrome P450 (CYP) enzymes. Both cell types are involved in the bioconversion of arachidonic acid into vasoactive compounds. The aim of this study was to demonstrate the effect of statins on cytochrome P450 expression in endothelial cells. Our results show that endothelial cells expressed both CYPs involved in epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) production and the nuclear receptor implicated in cytochrome P450 regulation. Treatment of endothelial cells with lovastatin increased CYP2C9 expression. After 96 hours of treatment, fluvastatin and lovastatin clearly increased CYP2C9 protein level. CAR but not PXR was expressed in endothelial cells, indicating that the upregulating effect of statins on CYP2C9 in endothelial cells could be mediated through CAR only due to the lack of expression of PXR in these cells.

Endo-Xenobiotic Crosstalk and the Regulation of Cytochromes P450

Evolution has provided organisms with an elaborate defense system against foreign compounds and against the accumulation of potentially toxic endogenous molecules, e.g. bile acids. Cytochromes P450 represent an important group of enzymes in this system. This article describes experiments started in the 1970's in Dallas on the coordination of heme and cytochrome P450 synthesis and how these studies evolved over the years into a concept of molecular links between xenobiotic metabolism and endogenous pathways of sterol, lipid, bile acid and energy homeostasis.

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.

The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo, an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 mug of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.

The hepatic cytochrome P450 reductase null mouse as a tool to identify a successful candidate entity

Cytochrome P450s (CYP) play a pivotal role in the metabolism of drugs and xenobiotics, and have been intensively studied over many years. Much of the work carried out on the role of hepatic cytochrome P450s in drug metabolism and disposition has been done in vitro, and has yielded vital information on P450 regulation and function. However, additional factors such as route of administration, absorption, drug transporters, renal clearance and extra-hepatic P450s, make it difficult to extrapolate from in vitro data to in vivo pharmacokinetics. A number of cytochrome P450s knockout mice have been generated, although many have been of limited usefulness due to either embryonic/perinatal lethality, or the functional redundancy inevitably found in a large family of isoenzymes. We have developed a mouse line (HRN) in which cytochrome P450 oxidoreductase (POR), the unique electron donor to cytochrome P450s is deleted specifically in the liver, resulting in the loss of essentially all hepatic P450 function. The HRN mouse, although having disturbances in lipid and bile acid homeostasis develops and breeds normally. We have used the HRN mouse as a model to establish the role of hepatic versus extra-hepatic metabolism in drug metabolism and disposition, and also to investigate the relationship between drug toxicokinetics and therapeutic effect, initially with the chemotherapeutic prodrug cyclophosphamide (CPA).

HEPATIC AND RENAL CYTOCHROME P450 GENE REGULATION DURINGCITROBACTER RODENTIUMINFECTION IN WILD-TYPE AND TOLL-LIKE RECEPTOR 4 MUTANT MICE

Citrobacter rodentium is the rodent equivalent of human enteropathogenic Escherichia coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines, and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice [which lack functional toll-like receptor 4 (TLR4)] were infected with C. rodentium by oral gavage and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16 to 55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4 dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5, and 3A13. Hepatic levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNFalpha) mRNAs were significantly increased in infected HeOu mice, whereas only TNFalpha mRNA was significantly increased in HeJ mice. Hepatic alpha1-acid glycoprotein was induced in both groups, whereas alpha-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated.

Human CYP2C8 Is Transcriptionally Regulated by the Nuclear Receptors Constitutive Androstane Receptor, Pregnane X Receptor, Glucocorticoid Receptor, and Hepatic Nuclear Factor 4α

Cytochrome P450 (P450) enzymes play important roles in the metabolism of endogenous and xenobiotic substrates in humans. CYP2C8 is an important member of the CYP2C subfamily, which metabolizes both endogenous compounds (i.e., arachidonic acids and retinoic acid) and xenobiotics (e.g., paclitaxel). Induction of P450 enzymes by drugs can result in tolerance as well as drug-drug interactions. CYP2C8 is the most strongly inducible member of the CYP2C subfamily in human hepatocytes, but the mechanism of induction by xenobiotics has not been delineated. To determine the mechanisms controlling the regulation of this important P450, we cloned the 5'-flanking region of CYP2C8 and investigated its transcriptional regulation by nuclear factors such as the pregnane X receptor (PXR), constitutive androstane receptor (CAR), glucocorticoid receptor (GR), and hepatic nuclear factor 4 (HNF4alpha) that are known to be involved in the induction of other P450 enzymes using both cell lines and primary hepatocyte models. We initially identified a distal PXR/CAR-binding site in the CYP2C8 promoter that confers inducibility of CYP2C8 via the PXR agonist/ligand rifampicin and the CAR agonist/ligand CITCO [6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime]. A glucocorticoid-responsive element was identified that mediates dexamethasone induction via the GR. We finally identified an HNF4alpha-binding site within the CYP2C8 basal promoter region that is cis-activated by cotransfected HNF4alpha. In summary, the present studies show that CAR, PXR, GR, and HNF4alpha can regulate CYP2C8 expression and identify specific cis-elements within the promoter that control these regulatory pathways.

Regulation of Cytochrome P450 by Posttranslational Modification

Regulation of Cytochrome P450 by Posttranslational Modification