Regulation Of Cytochrome P450 Genes Research Articles

The Polymorphic Nuclear Factor NFIB Regulates Hepatic CYP2D6 Expression and Influences Risperidone Metabolism in Psychiatric Patients.

The genetic background for interindividual variability of the polymorphic CYP2D6 enzyme activity remains incompletely understood and the role of NFIB genetic polymorphism for this variability was evaluated in this translational study. We investigated the effect of NFIB expression in vitro using 3D liver spheroids, Huh7 cells, and the influence of the NFIB polymorphism on metabolism of risperidone in patients in vivo. We found that NFIB regulates several important pharmacogenes, including CYP2D6. NFIB inhibited CYP2D6 gene expression in Huh7 cells and NFIB expression in livers was predominantly nuclear and reduced at the mRNA and protein level in carriers of the NFIB rs28379954 T>C allele. Based on 604 risperidone treated patients genotyped for CYP2D6 and NFIB, we found that the rate of risperidone hydroxylation was elevated in NFIB rs28379954 T>C carriers among CYP2D6 normal metabolizers, resulting in a similar rate of drug metabolism to what is observed in CYP2D6 ultrarapid metabolizers, with no such effect observed in CYP2D6 poor metabolizers lacking functional enzyme. The results indicate that NFIB constitutes a novel nuclear factor in the regulation of cytochrome P450 genes, and that its polymorphism is a predictor for the rate of CYP2D6 dependent drug metabolism in vivo.

Differential regulation of cytochrome P450 genes associated with biosynthesis and detoxification in bifenthrin-resistant populations of navel orangewom (Amyelois transitella)

Pyrethroid resistance was first reported in 2013 for the navel orangeworm, Amyelois transitella, but the genetic underpinnings of pyrethroid resistance are unknown. We investigated the role of cytochrome P450 monooxygenases (P450s) belonging to the CYP3 and CYP4 clans using colonies derived from individuals collected in 2016 from almond orchards in two counties. One colony (ALM) originated from an almond orchard in Madera County with no reported pyrethroid resistance and the second colony (R347) originated from the same Kern County orchard where pyrethroid resistance was first reported. We used high-throughput quantitative real-time PCR (qRT-PCR) analyses of 65 P450s in the CYP3 and CYP4 clans of A. transitella to identify P450s induced by bifenthrin and associated with pyrethroid resistance. Nine P450s were constitutively overexpressed in R347 compared to ALM, including CYP6AE54 (11.7-fold), belonging to a subfamily associated with metabolic pesticide detoxification in Lepidoptera and CYP4G89 (33-fold) belonging to a subfamily associated with cuticular hydrocarbon (CHC) synthesis and resistance via reduced pesticide penetrance. Cuticular hydrocarbons analysis revealed that R347 produced twice as many total CHCs in the egg and adult stages as ALM. Topical toxicity bioassays for R347 determined that egg mortality was reduced at low bifenthrin concentrations and larval mortality was reduced at high concentrations of bifenthrin compared to ALM. Our discovery of both changes in metabolism and production of CHCs for R347 have implications for the possible decreased efficacy of other classes of insecticide used to control this insect. The threat of widespread pyrethroid resistance combined with the potential for cross-resistance to develop through the mechanism of reduced penetrance warrants developing management strategies that facilitate insecticide passage across the cuticle.

Understanding the Mechanisms Involved in the Regulation of Cytochrome P450 Gene Expression in Drosophila melanogaster (Diptera: Drosophilidae)

Cytochrome P450 monooxygenases (P450s) are known to play a central role in the adaptive response of insects and other animals to chemicals in the environment. Drosophila spp. P450s are known to be regulated by Cap ‘n’ collar isoform C (CnCC) and/ Spineless (ss) which are orthologs to Nuclear factor erythroid-2 factor 2 (Nrf2)/Aryl hydrocarbon receptor (AhR) in higher mammals. However, the mechanism underpinning this regulation in insects including fruit fly, Drosophila melanogaster is poorly understood. Understanding the constitutive and inducible patterns of expression requires knowledge about the signalling pathways that control insect P450 expression, which is still lacking for most identified insect P450s. D. melanogaster, because of its longstanding use as a genetic model insect, is a powerful tool for identifying possible regulatory mechanisms and for following expression through to function. Here, we describe the roles played by the cis-acting elements and the Transcription Factors (TFIF) mechanisms involved in the regulation of cytochrome P450 genes in D. melanogaster in response to xenobiotic compounds. These cis-acting elements include; promoters, enhancers, repressors, silencers and insulators. The regulatory mechanisms involved in the regulation of the P450s by the spineless (ss)/tango (Tgo) and CnCC/dKeap1 (Drosophila Kelch-like ECH-associated protein 1) signalling pathways in insecticide resistance were also extensively discussed. This review increases our understanding of the regulatory mechanisms involved in the insecticide metabolism in Drosophila melanogaster.

Pregnane X receptor and aryl hydrocarbon receptor both participate in regulation of cytochrome P450 genes (CYP2s and CYP3s) in developing zebrafish

Pregnane X receptor and aryl hydrocarbon receptor both participate in regulation of cytochrome P450 genes (CYP2s and CYP3s) in developing zebrafish

Roles of Mitogen-Activated Protein Kinases in the Regulation of CYP Genes

Cells undergo phenotypic changes after exposure to a wide range of exogenous stimuli that include growth factors, proinflammatory cytokines and environmental chemicals. Such stimuli may arise as components of disease pathogenesis and cellular injury, or as a result of exposure to environmental chemicals and radiation. These stimuli modulate the proliferation and differentiation of cells by altering the regulation of genes that control homeostasis. A generalized response appears to be a decline in the expression and function of many cytochrome P450 (CYP) genes in liver and other tissues. Thus, individuals who have been exposed to such exogenous stimuli often exhibit a decreased capacity for drug clearance, which has important consequences for concurrent drug therapy. Several signaling pathways transduce exogenous stimuli within cells, with the mitogen-activated protein kinases (MAPKs) being one of the most important. Evidence is increasing that MAPKs may impair the expression of multiple CYP genes by modulating the activity of transcription factors, including nuclear receptors, the aryl hydrocarbon receptor, and the activator protein-1 complex. MAPKs catalyze the phosphorylation of transcription complexes that incorporate these factors, which modulates their capacity to transactivate target genes, including CYPs. An understanding of the mechanisms that account for the regulatory impact of MAPKs on the transcriptional factors that regulate CYP genes will provide critical insight into the consequences from exposure to injurious stresses that impact cellular function.

Transcriptional Regulation of Cytochrome P450 Genes by the Nuclear Receptor Hepatocyte Nuclear Factor 4-Alpha

Hepatocyte nuclear factor 4-alpha (HNF4alpha, NR2A1) is a nuclear receptor (NR) required for liver development and for controlling the expression of many hepatic-specific genes associated with important metabolic pathways. Many studies have also identified HNF4alpha as a direct transactivator of numerous xenobiotic-metabolizing cytochrome P450 (CYP) genes, suggesting that this factor is a global regulator which supports CYP transcription in the liver. Moreover, HNF4alpha expression displays a significant variability in human liver which may account for a proportion of the inter-individual variability in the expression of drug-metabolism genes and the clearance rate of a wide variety of prescribed drugs. In the last few years, a number of complex interactions and cross-talks between HNF4alpha and other transcription factors and coregulators have also surfaced, and the impact on CYP gene expression has been demonstrated. Thus, it is now clear that HNF4alpha modulates CYP expression in the liver by interacting with the xenosensor receptors (PXR and CAR), the glucocorticoid receptor (GR), the feeding-fasting cycle target PGC-1alpha, the sexual-dimorphism factor Stat5b, and other liver-enriched factors, such as C/EBPs. In addition to regulating drug elimination pathways, HNF4alpha also triggers pleiotropic effects on cholesterol and fatty acid metabolism, glucose homeostasis and inflammation. As a whole, current evidence indicates that HNF4alpha is a central regulator in the network of NRs that integrates drug-metabolism not only with the liver intermediate metabolism, but also with a number of patho-physiological conditions where the CYP expression is altered. The purpose of this review is to summarize and discuss these studies and their conclusions, with particular emphasis on the role of HNF4alpha in the regulation of drug-metabolizing CYP genes in the human liver.

Cytochrome P450 gene regulation and physiological functions mediated by the aryl hydrocarbon receptor

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that functions as an intracellular mediator in the xenobiotic signaling pathway. Although a number of studies have examined AhR-mediated CYP1A1 induction in detail, recent studies of AhR-null mice have revealed that AhR plays important regulatory roles in the normal homeostasis and development of animals. In this short review, we summarize the present state of knowledge about the molecular mechanisms of AhR-mediated CYP1 induction, and we also focus on recent advances in the study of the physiological functions of AhR.

In the regulation of cytochrome P450 genes, phenobarbital targets LKB1 for necessary activation of AMP-activated protein kinase

Transcriptional activation of cytochrome P450 (CYP) genes and various drug metabolizing enzymes by the prototypical inducer phenobarbital (PB) and many other drugs and chemicals is an adaptive response of the organism to exposure to xenobiotics. The response to PB is mediated by the nuclear receptor constitutive androstane receptor (CAR), whereas the chicken xenobiotic receptor (CXR) has been characterized as the PB mediator in chicken hepatocytes. Our previous results suggested an involvement of AMP-activated protein kinase (AMPK) in the molecular mechanism of PB induction. Here, we show that the mechanism of AMPK activation is related to an effect of PB-type inducers on mitochondrial function with consequent formation of reactive oxygen species (ROS) and phosphorylation of AMPK by the upstream kinase LKB1. Gain- and loss-of-function experiments demonstrate that LKB1-activated AMPK is necessary in the mechanism of drug induction and that this is an evolutionary conserved pathway for detoxification of exogenous and endogenous chemicals. The activation of LKB1 adds a proximal target to the so far elusive sequence of events by which PB and other drugs induce the transcription of multiple genes.

Hepatic Cytochrome P450 Gene Regulation during Endotoxin-Induced Inflammation in Nuclear Receptor Knockout Mice

Inflammatory agents such as lipopolysaccharide (LPS) down-regulate the hepatic expression of many cytochrome P450 (P450) mRNAs and proteins. Previous studies suggested that suppression of some P450 mRNAs could involve the regulation or modulation of the nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) or pregnane X receptor (PXR). To determine the involvement of these receptors in P450 down-regulation, PPARalpha knockout (KO), PXR KO, and appropriate wild-type (WT) mice were administered either saline or 1 mg/kg LPS. Hepatic mRNA and protein expression of several P450 isoforms, interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF) alpha, alpha1-acid glycoprotein (AGP), and fibrinogen (FBG) were examined 16 h later. LPS administration significantly decreased the hepatic expression of CYP1A2, 2A5, 2C29, 2E1, 3A11, 4A10, and 4A14 mRNAs in both groups of PPARalpha and PXR mice, whereas CYP3A13 mRNA was increased slightly in PPARalpha WT and KO mice, but not in PXR mice. Effects of LPS administration on mouse hepatic P450 proteins (probed using rat P450 2C, 3A, 4A, and 2E antibodies) were consistent with mRNA results in most cases. LPS treatment significantly increased IL-1beta, IL-6, TNFalpha, AGP, and FBG mRNA in both PPARalpha and PXR mice, with the greatest effect observed with TNFalpha. Because decreases in P450 mRNA expression were essentially identical in both WT and KO mice for both nuclear receptors, these data indicate that down-regulation of P450 during inflammation does not require the nuclear receptors PPARalpha and PXR.

Transfection of Liver In Vivo by Biolistic Particle Delivery: Its Use in the Investigation of Cytochrome P450 Gene Regulation

We describe the delivery of reporter gene constructs to rat liver through the use of the Helios Gene Gun system. The effectiveness of this transfection method is illustrated by describing its use for determining in vivo the role of a DNA element that regulates cytochrome P450 2B1 (CYP2B1) gene expression in response to xenobiotics. DNA was delivered to the liver of an anesthetized animal via DNA-coated gold microcarriers. The highest level of reporter gene expression was obtained about six hours posttransfection; however, at this time endogenous CYP2B1 mRNA is transiently induced by the anesthetic treatment. The optimal time for investigating expression of a reporter gene under the control of CYP2B1 regulatory elements was 24 h after transfection, by which time the inductive effect of the anesthetic had ceased. Reporter gene expression subsequently declined rapidly to a low level by 48 h. In the transfected liver the heterologous SV40 promoter was about eight-fold stronger than the minimal CYP2B1 promoter. However, when attached to the phenobarbital response element both promoters give the same fold-induction of reporter activity in response to phenobarbital.

Dexamethasone Enhances Constitutive Androstane Receptor Expression in Human Hepatocytes: Consequences on Cytochrome P450 Gene Regulation

The barbiturate phenobarbital induces the transcription of cytochromes P450 (CYPs) 2B through the constitutive androstane receptor (CAR; NR1I3). CAR is a member of the nuclear receptor family (NR1) mostly expressed in the liver, which heterodimerizes with retinoid X receptor (RXR) and was shown to transactivate both the phenobarbital responsive element module of the human CYP2B6 gene and the CYP3A4 xenobiotic response element. Because previous studies in rodent hepatocyte cultures have shown that the phenobarbital-mediated induction of CYP2B genes is potentiated by glucocorticoids, we examined the role of activated glucocorticoid receptor in this process. We show that submicromolar concentrations of dexamethasone enhance phenobarbital-mediated induction of CYP3A4, CYP2B6, and CYP2C8 mRNA in cultured human hepatocytes. In parallel, we observed that glucocorticoid agonists, such as dexamethasone, prednisolone, or hydrocortisone, specifically increase human car (hCAR) mRNA expression. Accumulation of hCAR mRNA parallels that of tyrosine aminotransferase: both mRNAs reach a maximum at a concentration of 100 nM dexamethasone and are down-regulated by concomitant treatment with the glucocorticoid antagonist RU486. Moreover, the effect of dexamethasone on hCAR mRNA accumulation appears to be of transcriptional origin because the addition of protein synthesis inhibitor cycloheximide has no effect, and dexamethasone does not affect the degradation of hCAR mRNA. Furthermore, dexamethasone increases both basal and phenobarbital-mediated nuclear translocation of CAR immunoreactive protein in human hepatocytes. The up-regulation of CAR mRNA and protein in response to dexamethasone explains the synergistic effect of this glucocorticoid on phenobarbital-mediated induction of CYP2B genes and the controversial role of the glucocorticoid receptor on phenobarbital-mediated CYP gene inductions.

Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes.

Baculovirus transfection strategies have proven successful at transferring foreign DNA into hepatoma cells and primary hepatocytes. When testing the utility of these methodologies in cultured hepatocytes, we discovered that the presence of baculovirus disrupts the phenobarbital (PB) gene induction process, a potent transcriptional activation event characteristic of highly differentiated hepatocytes, and repressed expression of the albumin gene. In concert with previous reports from our laboratory demonstrating that increased cAMP levels can completely repress the induction of specific cytochrome P450 (CYP) genes, cAMP concentrations and PKA activities were measured in the primary hepatocytes subsequent to baculovirus exposure. However, neither parameter was affected by the presence of the virus. To evaluate whether immune response modulation was triggered by baculovirus exposure, RNase protection assays were performed and demonstrated that baculovirus infection activates TNF-alpha, IL-1alpha and IL-1beta expression in the primary hepatocyte cultures. Immunocytochemical experiments indicated that the production of cytokines was likely due to the presence of small numbers of Kupffer cells present in the culture populations. Exogenously added TNF-alpha was also effective in repressing PB induction, consistent with other reports indicating that inflammatory cytokines are capable of suppressing expression of biotransformation enzyme systems. Comparative studies demonstrated the specificity of these effects since exposures of hepatocytes to adenoviral vectors did not result in down-regulation of hepatic gene responsiveness. These results indicate that baculovirus vectors enhance the expression of inflammatory cytokines in primary hepatocyte cultures, raising concerns as to whether these properties will compromise the use of baculovirus vectors for study of cytochrome P450 gene regulation, as well as for liver-directed gene therapy in humans.

Pre-translational regulation of cytochrome P450 genes is responsible for disease-specific changes of individual P450 enzymes among patients with cirrhosis

We have recently reported that disease-specific differential alterations in the hepatic expression of xenobiotic-metabolizing cytochrome P450 (CYP P450) enzymes occur in patients with advanced liver disease. In order to determine whether the observed changes in CYP proteins are modulated at pre- or post-translational levels, we have now examined the hepatic levels of mRNA for CYPs 1A2, 2C9, 2E1 and 3A4 by solution hybridization in the same livers of 20 controls (surgical waste from histologically normal livers), 32 cases of hepatocellular and 18 of cholestatic severe chronic liver disease. CYP1A2 mRNA and CYP1A immunoreactive protein were both reduced in livers with hepatocellular and cholestatic types of cirrhosis. In contrast, CYP3A4 mRNA and protein were reduced only in livers from patients with hepatocellular diseases. For 1A2 and 3A4 there were significant correlations between mRNA species and the respective protein contents ( r S1A2 = 0.74, r S3A4 = 0.64, P < 0.0001). CYP2C9 mRNA was reduced in patients with both cholestatic and hepatocellular types of liver disease, but 2C protein was reduced only in patients with cholestatic dysfunction. The correlation between CYP2C9 mRNA and protein, was also significant ( r s = 0.36, P < 0.005) but mRNA levels accounted for only 13% of the variability in protein rankings. This is probably a consequence of other CYP2C proteins apart from 2C9 being detected by the anti-2C antibody. CYP2E1 mRNA and protein were reduced in patients with cholestatic liver disease, but in hepatocellular disease the expression of only CYP2E1 mRNA was decreased. CYP2E1 mRNA was significantly correlated with CYP2E1 protein but accounted for only 18% of the variability in protein rankings ( r S = 0.43, P < 0.0005). Taken collectively these data indicate that the disease-specific alterations of xenobiotic-metabolizing CYP enzymes among patients with cirrhosis is due, at least in part, to pre-translational mechanisms. The lack of a strong correlation between CYP2E1 mRNA and protein suggests that this gene, like its rat orthologue, may be subject to pre-translational as well as translational and/or post-translational regulation.

Common pathways of cytochrome P450 gene regulation by peroxisome proliferators and barbiturates in Bacillus megaterium ATCC14581.

Bacillus megaterium contains a barbiturate-inducible cytochrome P450BM-3, which catalyzes the hydroxylation of fatty acids. We report the intriguing finding that peroxisome proliferators, a major class of epigenetic carcinogen, are also extremely potent inducers of this enzyme being up to 50-fold more potent than one of the most effective barbiturates, secobarbital. Similar to barbiturates, the mechanism of induction appears to involve the direct binding of the peroxisome proliferator to the transcriptional repressor (Bm3R1), resulting in its dissociation from its DNA operator. These observations provide evidence that peroxisome proliferators can interact with a transcription factor to modulate gene expression. The data also demonstrate that the effects of these compounds are highly conserved through evolution and that there are important common denominators in the regulation of gene expression by peroxisome proliferators and the barbiturates. Evidence is presented to indicate that this may involve effects on unsaturated fatty acid homeostasis.

Differential regulation of cytochrome P-450 genes along rat intestinal crypt-villus axis.

Mammalian small intestine contains cytochrome P-450-dependent monooxygenase enzymes that are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cells lining the small intestine are separated into a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expression and induction by xenobiotics of genes that encode components of the cytochrome P-450-dependent mono-oxygenase system along the rat intestinal crypt-villus axis were investigated using isolated epithelial cells and in situ hybridization. For each gene examined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IA1 mRNA (CYP1A1), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIB1 mRNA (CYP2B1), the major phenobarbital-inducible P-450, were constitutively expressed in villus cells with no detectable mRNA present in crypts. Treatment with several chemical inducers resulted in a marked increase in CYP1A1 mRNA in both crypt and villus cells. In contrast, although CYP2B1 mRNA was inducible in villus cells, CYP2B1 mRNA was not detected in crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes, was expressed constitutively at low levels only in villus cells. Treatment with dexamethasone induced reductase mRNA in both crypt and villus cells. Taken together, these results demonstrate that there is a complex gene-specific pattern of expression of the microsomal monooxygenase system along the crypt-villus axis of rat small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P450 Gene Regulation

Cytochrome P450 Gene Regulation