Allopregnanolone is required for prepulse inhibition deficits induced by D1 dopamine receptor activation.

INTRODUCTION: The extraction of salient information from the environment is modulated by the activation of dopamine receptors. Using rodent models, we previously reported that gating deficits caused by dopamine receptor activation - as measured by the prepulse inhibition (PPI) of startle - are effectively opposed by inhibitors of the steroidogenic enzyme 5α-reductase (5αR). The specific 5αR isoenzyme and steroids implicated in these effects, however, remain unknown. METHODS: The effects of the selective D1 dopamine receptor agonist SKF-82958 (SKF, 0.3 mg/kg, IP) and D2 receptor agonist quinpirole (QUIN, 0.5 mg/kg, IP) were tested in the startle reflex and PPI of knockout (KO) mice for either 5αR type 1 (5αR1) or type 2 (5αR2). Furthermore, we established whether these effects may be modified by the 5α-reduced steroids dihydroprogesterone (DHP), allopregnanolone (AP), dihydrotestosterone (DHT), 5α-androstane-3α,17ß-diol (3α-diol), or androsterone. To test the mechanisms whereby 5αR products may alter the PPI-disrupting properties of D1 agonists, we studied the involvement of GABA-A and PXR, two receptors targeted by neuroactive steroids. Specifically, we tested the effects of SKF in combination with the GABA-A antagonist bicuculline, as well as in KO mice for the GABA-A δ subunit and PXR. RESULTS: 5αR1, but not 5αR2, knockout (KO) mice were insensitive to the PPI-disrupting effects of SKF. This sensitivity was reinstated by AP (3 mg/kg, IP), but not other 5α-reduced steroids. The PPI deficits induced by SKF were not modified by bicuculline, δ-subunit KO mice and PXR KO mice. CONCLUSIONS: These results collectively suggest that 5αR1 enables the negative effects of D1 dopamine receptor activation on information processing via production of AP. The contribution of AP to the PPI-disrupting mechanisms of D1 receptor agonists, however, do not appear to be mediated by either GABA-A or PXR receptors.

Protein targets of thioacetamide metabolites in rat hepatocytes.

Thioacetamide (TA) has long been known as a hepatotoxicant whose bioactivation requires S-oxidation to thioacetamide S-oxide (TASO) and then to the very reactive S,S-dioxide (TASO2). The latter can tautomerize to form acylating species capable of covalently modifying cellular nucleophiles including phosphatidylethanolamine (PE) lipids and protein lysine side chains. Isolated hepatocytes efficiently oxidize TA to TASO but experience little covalent binding or cytotoxicity because TA is a very potent inhibitor of the oxidation of TASO to TASO2. However, hepatocytes treated with TASO show extensive covalent binding to both lipids and proteins accompanied by extensive cytotoxicity. In this work, we treated rat hepatocytes with [(14)C]-TASO and submitted the mitochondrial, microsomal, and cytosolic fractions to 2DGE, which revealed a total of 321 radioactive protein spots. To facilitate the identification of target proteins and adducted peptides, we also treated cells with a mixture of TASO/[(13)C2D3]-TASO. Using a combination of 1DGE- and 2DGE-based proteomic approaches, we identified 187 modified peptides (174 acetylated, 50 acetimidoylated, and 37 in both forms) from a total of 88 nonredundant target proteins. Among the latter, 57 are also known targets of at least one other hepatotoxin. The formation of both amide- and amidine-type adducts to protein lysine side chains is in contrast to the exclusive formation of amidine-type adducts with PE phospholipids. Thiobenzamide (TB) undergoes the same two-step oxidative bioactivation as TA, and it also gives rise to both amide and amidine adducts on protein lysine side chains but only amidine adducts to PE lipids. Despite their similarity in functional group chemical reactivity, only 38 of 62 known TB target proteins are found among the 88 known targets of TASO. The potential roles of protein modification by TASO in triggering cytotoxicity are discussed in terms of enzyme inhibition, protein folding, and chaperone function, and the emerging role of protein acetylation in intracellular signaling and the regulation of biochemical pathways.

Liver protein targets of hepatotoxic 4-bromophenol metabolites.

The hepatotoxicity of bromobenzene (BB) is directly related to the covalent binding of both initially formed epoxide and secondary quinone metabolites to at least 45 different liver proteins. 4-Bromophenol (4BP) is a significant BB metabolite and a precursor to reactive quinone metabolites; yet, when administered exogenously, it has negligible hepatotoxicity as compared to BB. The protein adducts of 4BP were thus labeled as nontoxic [Monks, T. J., Hinson, J. A., and Gillette, J. R. (1982) Life Sci. 30, 841-848]. To help identify which BB-derived adducts might be related to its cytotoxicity, we sought to identify the supposedly nontoxic adducts of 4BP and eliminate them from the BB target protein list. Administration of [(14)C]-4BP to phenobarbital-induced rats resulted in covalent binding of 0.25, 0.33, and 0.42 nmol equiv 4BP/mg protein in the mitochondrial, microsomal, and cytosolic fractions, respectively. These values may be compared to published values of 3-6 nmol/mg protein from a comparable dose of [(14)C]-BB. After subcellular fractionation and 2D electrophoresis, 47 radioactive spots on 2D gels of the mitochondrial, microsomal, and cytosolic fractions were excised, digested, and analyzed by LC-MS/MS. Twenty-nine of these spots contained apparently single proteins, of which 14 were nonredundant. Nine of the 14 are known BB targets. Incubating freshly isolated rat hepatocytes with 4BP (0.1-0.5 mM) produced time- and concentration-dependent increases in lactate dehydrogenase release and changes in cellular morphology. LC-MS/MS analysis of the cell culture medium revealed rapid and extensive sulfation and glucuronidation of 4BP as well as formation of a quinone-derived glutathione conjugate. Studies with 7-hydroxycoumarin, (-)-borneol, or D-(+)-galactosamine showed that inhibiting the glucuronidation/sulfation of 4BP increased the formation of a GSH-bromoquinone adduct, increased covalent binding of 4BP to hepatocyte proteins, and potentiated its cytotoxicity. Taken together, our data demonstrate that protein adduction by 4BP metabolites can be toxicologically consequential and provide a mechanistic explanation for the failure of exogenously administered 4BP to cause hepatotoxicity. Thus, the probable reason for the low toxicity of 4BP in vivo is that rapid conjugation limits its oxidation and covalent binding and thus its toxicity.

Covalent modification of lipids and proteins in rat hepatocytes and in vitro by thioacetamide metabolites.

Thioacetamide (TA) is a well-known hepatotoxin in rats. Acute doses cause centrilobular necrosis and hyperbilirubinemia while chronic administration leads to biliary hyperplasia and cholangiocarcinoma. Its acute toxicity requires its oxidation to a stable S-oxide (TASO) that is oxidized further to a highly reactive S,S-dioxide (TASO(2)). To explore possible parallels among the metabolism, covalent binding, and toxicity of TA and thiobenzamide (TB), we exposed freshly isolated rat hepatocytes to [(14)C]-TASO or [(13)C(2)D(3)]-TASO. TLC analysis of the cellular lipids showed a single major spot of radioactivity that mass spectral analysis showed to consist of N-acetimidoyl PE lipids having the same side chain composition as the PE fraction from untreated cells; no carbons or hydrogens from TASO were incorporated into the fatty acyl chains. Many cellular proteins contained N-acetyl- or N-acetimidoyl lysine residues in a 3:1 ratio (details to be reported separately). We also oxidized TASO with hydrogen peroxide in the presence of dipalmitoyl phosphatidylenthanolamine (DPPE) or lysozyme. Lysozyme was covalently modified at five of its six lysine side chains; only acetamide-type adducts were formed. DPPE in liposomes also gave only amide-type adducts, even when the reaction was carried out in tetrahydrofuran with only 10% water added. The exclusive formation of N-acetimidoyl PE in hepatocytes means that the concentration or activity of water must be extremely low in the region where TASO(2) is formed, whereas at least some of the TASO(2) can hydrolyze to acetylsulfinic acid before it reacts with cellular proteins. The requirement for two sequential oxidations to produce a reactive metabolite is unusual, but it is even more unusual that a reactive metabolite would react with water to form a new compound that retains a high degree of chemical reactivity toward biological nucleophiles. The possible contribution of lipid modification to the hepatotoxicity of TA/TASO remains to be determined.

Activated pregnenolone X-receptor is a target for ketoconazole and its analogs.

PURPOSE: Variations in biotransformation and elimination of microtubule-binding drugs are a major cause of unpredictable side effects during cancer therapy. Because the orphan receptor, pregnenolone X-receptor (PXR), coordinately regulates the expression of paclitaxel metabolizing and transport enzymes, controlling this process could improve therapeutic outcome. EXPERIMENTAL DESIGN: In vitro RNA-, protein-, and transcription-based assays in multiple cell lines derived from hepatocytes and PXR wild-type and null mouse studies were employed to show the effects of ketoconazole and its analogues on ligand-activated PXR-mediated gene transcription and translation. RESULTS: The transcriptional activation of genes regulating biotransformation and transport by the liganded human nuclear xenobiotic receptor, PXR, was inhibited by the commonly used antifungal ketoconazole and related azole analogs. Mutations at the AF-2 surface of the human PXR ligand-binding domain indicate that ketoconazole may interact with specific residues outside the ligand-binding pocket. Furthermore, in contrast to that observed in PXR (+/+) mice, genetic loss of PXR results in increased (preserved) blood levels of paclitaxel. CONCLUSIONS: These studies show that some azole compounds repress the coordinated activation of genes involved in drug metabolism by blocking PXR activation. Because loss of PXR maintains blood levels of paclitaxel upon chronic dosing, ketoconazole analogues may also serve to preserve paclitaxel blood levels on chronic dosing of drugs. Our observations may facilitate new strategies to improve the clinical efficacy of drugs and to reduce therapeutic side effects.

Gender dictates the nuclear receptor-mediated regulation of CYP3A44.

The CYP3As are broad-spectrum drug-metabolizing enzymes that are collectively responsible for more than 50% of xenobiotic metabolism. Unlike other CYP3As, murine CYP3A44 is expressed predominantly in the female liver, with much lower levels in male livers and no detectable expression in brain or kidney in either gender. In this study, we examined the role of nuclear hormone receptors in the regulation of Cyp3a44 gene expression. Interestingly, we observed differential effects of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) -mediated activation of Cyp3a44 gene expression, which was gender-specific. For example, activation of PXR by pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone (DEX) induced CYP3A44 mRNA levels in a PXR-dependent fashion in male mice, whereas no induction was detected in female mice. In contrast, PCN and DEX down-regulated CYP3A44 expression in female PXR null animals. Similar to PXR, CAR activation also showed a male-specific induction with no effect on CYP3A44 levels in females. When PXR knockout mice were challenged with the CAR activator phenobarbital, a significant up-regulation of male CYP3A44 levels was observed, whereas levels in females remained unchanged. We conclude that gender has a critical impact on PXR- and CAR-mediated effects of CYP3A44 expression.

Genomic characterization and regulation of CYP3a13: role of xenobiotics and nuclear receptors.

We report that CYP3a13 gene, located on mouse chromosome 5, spans 27.5 Kb and contains 13 exons. The transcription start site is 35 bp upstream of the coding region and results in a 109 bp 5' untranslated region. CYP3a13 promoter shows putative binding sites for retinoid X receptor, pregnane X receptor, and estrogen receptor. CYP3a13 shows a broad tissue distribution with predominant expression in liver. Although CYP3a13 shares 92% nucleotide identity with the female-specific rat CYP3A9, its expression does not exhibit sexual dimorphism. Ligand activation of peroxisomal proliferator-activated receptor-gamma and retinoid X receptor inhibit expression of CYP3a13 at the transcription level in a tissue-specific manner. Another novel finding is hepatic induction of CYP3a13 by dexamethasone occurring only in pregnane X receptor null mice. We also report that pregnane X receptor is essential to maintain robust in vivo basal levels of CYP3a13 in contrast to CYP3a11. CYP3a13 protein expressed in vitro can metabolize clinically active drugs ethylmorphine and erythromycin, as well as benzphetamine. We conclude that CYP3a13 is regulated differentially by various nuclear receptors. In humans this may lead to altered drug metabolism, as many of the newly synthesized ligands/drugs targeted toward these nuclear receptors could influence CYP3A gene expression.