Metabolism of Endosulfan- by Human Liver Microsomes and Its Utility as a Simultaneous in Vitro Probe for CYP2B6 and CYP3A4

نویسندگان

  • Richard C. T. Casabar
  • Andrew D. Wallace
  • Ernest Hodgson
  • Randy L. Rose
چکیده

Endosulfanis metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km 9.8 M, Vmax 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km 16.2 M, Vmax 11.4 nmol/nmol P450/min) and CYP3A4 (Km 14.4 M, Vmax 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CLint 0.70 l/min/pmol P450) than CYP3A4 (CLint 0.09 l/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r 0.79), whereas a moderate correlation with testosterone 6 -hydroxylase activity of CYP3A4 (r 0.54) was observed. Ticlopidine (5 M), a potent CYP2B6 inhibitor, and ketoconazole (10 M), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfanmetabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfanis metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities. Endosulfan is an organochlorine pesticide and a contaminant at toxic superfund sites. It is currently applied as a broad-spectrum insecticide to a variety of vegetables, fruits, cereal grains, and cotton (USEPA, 2002). Endosulfan is sold under the tradename Thiodan and as a mixture of two isomers, namely 70% and 30% -endosulfan (ATSDR, 2000). Endosulfan exposure has been shown to increase rodent liver weights and elevate microsomal enzyme levels (Gupta and Gupta, 1977). In mice, endosulfan exposure resulted in increased testosterone metabolism and clearance (Wilson and LeBlanc, 1998). Studies involving children suggest that long-term environmental exposure to endosulfan causes delayed male sexual maturation and reduced testosterone levels (Saiyed et al., 2003). The mechanism by which endosulfan exerts these effects may involve its ability to activate the human pregnane X receptor and induce the expression levels of cytochrome P450 (P450) enzymes, thereby increasing metabolic rates for steroid hormones. Before beginning an investigation of endosulfan’s possible endocrine-disrupting effects, we wished to examine its metabolic pathway in humans. Until recently, there have been no published data on human metabolism of endosulfan or on the possible contributions of P450 isoforms to its metabolism. Based on animal studies, a proposed metabolic pathway for endosulfan was published by the Agency for Toxic Substances and Disease Registry (ATSDR, 2000) and is shown in Fig. 1. A study using cats reported the immediate presence of endosulfan sulfate in the liver following intravenous administration of endosulfan (Khanna et al., 1979). In rats administered a single oral dose of C-endosulfan, the metabolites sulfate, lactone, ether, and diol were detected in their feces 5 days later (Dorough et al., 1978). Analyses of human adipose tissue, placenta, umbilical cord serum, and milk samples demonstrated the presence of parent compound ( and -endosulfan) and metabolites endosulfan sulfate, diol, lactone, and ether, although the sulfate was the predominant degradation product (Cerrillo et al., 2005). The present study determined that endosulfanis metabolized to a single metabolite, endosulfan sulfate, in human liver microsomes, and its metabolism is primarily mediated by CYP2B6 (at high efficiency) and CYP3A4 (at low efficiency). CYP2B6 is recognized to be expressed at only 3 to 5% of total P450s in human livers (Gervot et al., 1999; Lang et al., 2001, whereas CYP3A4 is known as the most abundant P450 isoform, expressed at 20 to 60% of total P450s in human liver. The respective levels of CYP2B6 and CYP3A4 in human liver microsomes in combination with their strong affinity to endosulfan(Km 16.2 and 14.4 M, respectively) and their corresponding This work was supported by National Institute for Occupational Safety and Health Grant OH 07551-ECU. R.C. was a recipient of the Air Force Institute of Technology scholarship. Results were presented at the 13th annual meeting of ISSX in Maui, HI, Oct 23–27, 2005 (Drug Metab Rev 37:244). 1 This article is dedicated in memory of Dr. Randy Rose, who died in a tragic

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تاریخ انتشار 2006