Transactivation of a DR-1 PPRE by a human constitutive androstane receptor variant expressed from internal protein translation start sites

Downstream in-frame start codons produce amino-terminal-truncated human constitutive androstane receptor protein isoforms (ΔNCARs). The ΔNCARs are expressed in liver and in vitro cell systems following translation from in-frame methionine AUG start codons at positions 76, 80, 125, 128, 168 and 265 within the full-length CAR mRNA. The resulting CAR proteins lack the N-terminal DNA-binding domain (DBD) of the receptor, yielding ΔNCAR variants with unique biological function. Although the ΔNCARs maintain full retinoid X receptor alpha (RXRα) heterodimerization capacity, the ΔNCARs are inactive on classical CAR-inducible direct repeat (DR)-4 elements, yet efficiently transactivate a DR-1 element derived from the endogenous PPAR-inducible acyl-CoA oxidase gene promoter. RXRα heterodimerization with CAR1, CAR76 and CAR80 isoforms is necessary for the DR-1 PPRE activation, a function that exhibits absolute dependence on both the respective RXRα DBD and CAR activation (AF)-2 domains, but not the AF-1 or AF-2 domain of RXRα, nor CAR's DBD. A new model of CAR DBD-independent transactivation is proposed, such that in the context of a DR-1 peroxisome proliferator-activated response element, only the RXRα portion of the CAR-RXRα heterodimer binds directly to DNA, with the AF-2 domain of tethered CAR mediating transcriptional activation of the receptor complex

An okadaic acid-sensitive pathway involved in the phenobarbital-mediated induction of CYP2B gene expression in primary rat hepatocyte cultures

ABSTRACT We have previously demonstrated that specific activation of a cAMP-dependent protein kinase A (PKA) pathway resulted in complete repression of phenobarbital (PB)-inducible CYP gene expression in primary rat hepatocyte cultures. In the current investigation, we examined the role of protein phosphatase pathways as potential co-regulators of this repressive response. Primary rat hepatocytes were treated with increasing concentrations (0.1-25 nM) of okadaic acid, a potent inhibitor of serine/threonine-specific protein phosphatases PP1 and PP2A. PB induction responses were assessed by use of specific hybridization probes to CYP2B1 and CYP2B2 mRNAs. Okadaic acid completely inhibited the PB induction process in a concentration-dependent manner (IC 50 , ϳ1.5-2 nM). Similar repression was obtained with low concentrations of other highly specific phosphatase inhibitors, tautomycin and calyculin A. In contrast, exposure of hepatocytes to 1-nor-okadaone or okadaol, negative analogs of okadaic acid largely devoid of phosphatase inhibitory activity, was without effect on the PB induction process. At similar concentrations, okadaic acid produced only comparatively weak modulation of the ␤-naphthoflavone-inducible CYP1A1 gene expression pathway. In additional experiments, hepatocytes were treated with suboptimal concentrations of PKA activators together with phosphatase inhibitors. Okadaic acid markedly potentiated the repressive effects of dibutyryl-cAMP on the PB induction process. Together, these results indicate that both PKA and protein phosphatase (PP1 and/or PP2A) pathways exert potent and complementary control of the intracellular processes modulating the signaling of PB in cultured primary rat hepatocytes

Epxoide hydrolase -polymorphism and role in toxicology

Abstract Microsomal epoxide hydrolase is a critical biotransformation enzyme that catalyzes the conversion of a broad array of xenobiotic epoxide substrates to more polar diol metabolites. The gene has been shown previously to exhibit polymorphism, including variation in the coding region leading to amino acid substitutions at positions 113 (Y/H) and 139 (H/R). To better evaluate the phenotype associated with the structural region genetic polymorphisms associated with mEH, we performed enzymatic analyses using purified mEH proteins that were expressed using a baculovirus system, or with microsomal preparations obtained from liver tissues that were derived from individuals with homozygous mEH allelic status. Benzo[a]pyrene-4,5-oxide and cis-stilbene oxide were employed as substrates for the enzymatic determinations. Results obtained with the purified enzymes suggested that the reaction velocity catalyzed by the wild type (Y113/H139) protein was approximately two-fold greater than the corresponding velocities for the variant forms of the enzyme. However, when reaction rates were analyzed using human liver microsomal preparations, the maximal velocities generated among the variant mEH proteins were not statistically different. Collectively, these results indicate that the structural differences coded by the mEH genetic variants may have only modest impact on the enzyme's specific activity in vivo

Activation of cytochrome P450 gene expression in the rat brain by phenobarbital-like inducers

ABSTRACT Oxidative biotransformation, coupled with genetic variability in enzyme expression, has been the focus of hypotheses interrelating environmental and genetic factors in the etiology of central nervous system disease processes. Chemical modulation of cerebral cytochrome P450 (P450) monooxygenase expression character may be an important determinant of in situ metabolism, neuroendocrine homeostasis, and/or central nervous system toxicity resulting from exposure to neuroactive drugs and xenobiotic substances. To examine the capacity of the rat brain to undergo phenobarbital (PB)-mediated induction, we developed reverse transcription-polymerase chain reaction methods and evaluated the effects of several PB-like inducers on P450 and microsomal epoxide hydrolase gene expression. Animals treated i.p. with four daily doses of PB demonstrated markedly induced levels of CYP2B1, CYP2B2, and CYP3A1 mRNA in the striatum and cerebellum. In contrast, 1 or 2 days of PB treatment resulted in unchanged or even slightly decreased levels of CYP2B1 and CYP2B2 in the brain, although the latter treatments produced marked induction of the corresponding genes in the liver. Only slight increases in epoxide hydrolase RNA levels resulted in brains of PB-treated animals. Substantial activation of cerebral CYP2B1, CYP2B2, and CYP3A1 mRNA levels also resulted when animals were treated with the neuroactive drugs diphenylhydantoin and amitryptiline, and with the potential PB-like xenobiotic inducers trans-stilbene oxide and diallyl sulfide, whereas dichlorodiphenyltrichloroethane was less efficacious. Although the time course of the induction response is delayed in brain relative to that required for the liver, these results clearly establish that brain P450s are markedly PB inducible

Title Page Di(2-ethylhexyl) phthalate is a highly potent agonist for the human constitutive androstane receptor splice variant, CAR2 MOL 53702 2 Running Title Page Running Title: DEHP is a highly potent agonist for the human CAR splice variant, CAR2

Abbreviations: CAR, constitutive androstane receptor; DEHP, di(2-ethylhexyl) phthalate; PXR, pregnane X receptor MOL 53702 3 Abstract The human constitutive androstane receptor (CAR, CAR1) regulates the expression of genes involved in xenobiotic metabolism in the liver. The CAR gene utilizes multiple alternative splicing events during pre-mRNA processing, thereby enhancing the CAR transcriptome. Previous reports have identified two prominent human CAR variants, CAR2 and CAR3, possessing 4-and 5-amino acid insertions in their ligand binding domains, respectively. Unlike the constitutively active reference form of the receptor, we now demonstrate that CAR2 is a ligand activated receptor and comprises approximately 30% of the reference transcript level in human liver tissues in human hepatocytes. Further, we identify the common plasticizer, di(2-ethylhexyl) phthalate (DEHP), as a highly potent and uniquely selective agonist of CAR2. Results from reporter transactivation and mammalian two-hybrid assays reveal that DEHP activates CAR2 at low nanomolar concentrations, results further supported by analysis of CAR target gene expression in primary human hepatocytes. In addition, comparative genomic analysis show that the typical mouse, rat and marmoset models of DEHP toxicity can not accurately profile potential human toxicity due to these species inability to generate a CAR2-like transcript. The discovery that CAR2 is an ultimate human DEHP receptor identifies a novel pathway modulating human DEHP toxicity with potential clinical implications for a subset of patients undergoing critical care medical interventions. MOL 53702