Endosulfan-alpha Induces CYP2B6 and CYP3A4 via the Pregnane X Receptor

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Title: Endosulfan-alpha Induces CYP2B6 and CYP3A4 via the Pregnane X Receptor
Author: Casabar, Richard
Advisors: Gerald A. LeBlanc, Committee Member
Ernest Hodgson, Committee Member
Andrew D. Wallace, Committee Chair
Abstract: The purpose of this research was to establish the metabolic pathway of endosulfan in humans and to elucidate a potential mechanism for endosulfan's endocrine disruptive effects. We hypothesized that endosulfan may exert its endocrine disrupting effects by activating the pregnane X receptor and/or the constitutive androstane receptor and inducing the expression levels of cytochrome P450 enzymes, thereby increasing metabolic rates and the biotransformation of testosterone. In these studies, we utilized endosulfan alpha, the more predominant isomer in technical-grade endosulfan. In Chapter 1, we determined that endosulfan alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes with a Km = 9.8 micromolar and Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (rCYP) isoforms expressed in baculovirus-infected cells or supersomes, we identified CYP2B6 with a Km = 16.2 micromolar and Vmax = 11.4 nmol/nmol CYP/min, and CYP3A4 with a Km = 14.4 micromolar and Vmax = 1.3 nmol/nmol CYP/min, as the primary enzymes catalyzing the metabolism of endosulfan alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CLint = 0.70 microliter/min/pmol CYP) than CYP3A4 (CLint = 0.09 microliter/min/pmol CYP). Using commercially available individual human liver microsomes (HLM), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r² = 0.79) and a moderate correlation with testosterone 6 beta-hyroxylase activity of CYP3A4 (r² = 0.54). Ticlopidine (5 micromolar), a potent mechanism-based inhibitor of CYP2B6, and ketoconazole (10 micromolar), a selective CYP3A4 inhibitor at low doses, together inhibited approximately 90 percent of endosulfan sulfate formation in HLMs, presenting the possibility of utilizing endosulfan alpha as a simultaneous in-vitro probe for CYP2B6 and CYP3A4 activity. The percent total normalized rate (percent TNR) was calculated to estimate the contribution of each rCYP in the total metabolism of endosulfan alpha in HLMs and was used to validate the percent inhibition (percent I) by ticlopidine and ketoconazole in this study. Five of the six HLMs used in this study showed a good correlation between the percent I with ticlopidine and ketoconazole in the same incubation and the combined percent TNRs for CYP2B6 and CYP3A4. In Chapter 2, we investigated if endosulfan alpha induces CYP3A4 and CYP2B6 by activating PXR and/or CAR. These interactions were explored by transient transfection assays in the HepG2 cell line using CYP3A4-luciferase, CYP2B6-luciferase, human PXR (hPXR), and mouse CAR (mCAR) plasmids. Endosulfan alpha (10 micromolar) treatments resulted in 11-fold induction of CYP3A4 and 16-fold induction of CYP2B6 promoter activities over untreated controls in the presence of hPXR. The metabolite endosulfan sulfate (10 micromolar) also induced CYP3A4 and CYP2B6 promoter activities by 6-fold and 12-fold, respectively. In the presence of the constitutively active mCAR and the transcriptional repressor androstenol, endosulfan alpha only reversed androstenol repression weakly and induced CYP2B6 promoter activity by 3-fold over control. Utilizing S9 samples from primary human hepatocytes for western blotting, it was determined that endosulfan alpha induced CYP2B6 translation in a dose-dependent manner (0.1 to 50 micromolar) and CYP3A4 translation, but only at 50 micromolar dose. Using the same S9 samples for testosterone metabolism assays, an increase in testosterone metabolites was observed, although only at 50 micromolar endosulfan alpha treatment. We conclude that endosulfan alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that this activity can be utilized to simultaneously probe for CYP2B6 and 3A4 catalytic activities. We also demonstrated that endosulfan alpha activates hPXR strongly and mCAR weakly to induce CYP2B6 and CYP3A4 promoter activity and protein expression. Endosulfan alpha may affect endocrine homeostasis by inducing P450s and enhancing testosterone inactivation.
Date: 2006-10-16
Degree: MS
Discipline: Toxicology
URI: http://www.lib.ncsu.edu/resolver/1840.16/2291

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