Microsomal epoxide hydrolase (EPHX1) biotransforms epoxide derivatives of pharmaceuticals, including metabolites of certain antiepileptic medications, such as phenytoin and carbamazepine, and many environmental epoxides, such as those derived from butadiene, benzene, and carcinogenic polyaromatic hydrocarbons. We previously identified a far upstream promoter region, designated E1-b, in the EPHX1 gene that directs expression of an alternatively spliced EPHX1 mRNA transcript in human tissues. In this investigation, we characterized the structural features and expression character of the E1-b promoter region. Results of quantitative real-time polymerase chain reaction analyses demonstrated that the E1-b variant transcript is preferentially and broadly expressed in most tissues, such that it accounts for the majority of total EPHX1 transcript in vivo. Comparative genomic sequence comparisons indicated that the human EPHX1 E1-b gene regulatory region is primate-specific. Direct sequencing and genotyping approaches in 450 individuals demonstrated that the E1-b promoter region harbors a series of transposable element cassettes, including a polymorphic double Alu insertion. Results of reporter assays conducted in several human cell lines demonstrated that the inclusion of the Alu(ϩ/ϩ) insertion significantly decreases basal transcriptional activities. Furthermore, using haplotype block analyses, we determined that the E1-b polymorphic promoter region was not in linkage disequilibrium with two previously identified nonsynonomous single nucleotide polymorphisms (SNPs) in the coding region or with functional SNPs previously identified in the proximal promoter region of the gene. These results demonstrate that the upstream E1-b promoter is the major regulator of EPHX1 expression in human tissues and that polymorphism in this region may contribute an interindividual risk determinant to xenobiotic-induced toxicities.Although xenobiotic metabolism often results in detoxication, in certain instances, both in cases of pharmaceutical and environmental chemical metabolism, bioactivated and highly toxic intermediates are generated. In particular, cellular levels of epoxide moieties resulting from chemical metabolism appear to be critical initiators of toxic damage, including genetic mutation. Frequently reactive and unstable, epoxide metabolites, formed via the action of the cytochrome P450 monooxygenases, have been identified as ultimate carcinogenic and cytotoxic reaction products (Sayer et al., 1985;Thakker et al., 1986;Fretland and Omiecinski, 2000). Ultimately, the overall balance between bioactivation and detoxication pathways will determine the kinetics and fate of reactive intermediates within target cells. It seems likely that interindividual differences in susceptibility to toxic sequelae, including cancer incidence, may be associated with an altered genetic predisposition to detoxify epoxides. Tissues have developed the capacity to metabolize xenobiotic epoxides through several pathways. Prominent among these is ...

ABSTRACT:We tested the hypothesis that primary cultures of human hepatocytes could predict potential drug interactions with methadone and buprenorphine. Hepatocytes (five donors) were preincubated with dimethyl sulfoxide (DMSO) (vehicle), rifampin, or nelfinavir before incubation with methadone or buprenorphine. Culture media (0-60 min) was analyzed by liquid chromatography-tandem mass spectrometry for R-and S-methadone and R-and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) or for buprenorphine, norbuprenorphine, and their glucuronides [buprenorphine-3-glucuronide (B-3-G) and norbuprenorphine-3-glucuronide (N-3-G)]. R-and S-EDDP were detected in three of five, four of five, and five of five media from cells pretreated with DMSO, nelfinavir, and rifampin. R-EDDP increased 3.1-and 26.5-fold, and S-EDDP increased 2.5-and 21.3-fold after nelfinavir and rifampin. The rifampin effect was significant. B-3-G production was detected in media of all cells incubated with buprenorphine and accounted for most of the buprenorphine loss from culture media; it was not significantly affected by either pretreatment. Norbuprenorphine and N-3-G together were detected in three of five, four of five, and five of five donors pretreated with DMSO, nelfinavir and rifampin, and norbuprenorphine in one of five, one of five, and two of five donors. Although there was a trend for norbuprenorphine (2.8-and 4.9-fold) and N-3-G (1.7-and 1.9-fold) to increase after nelfinavir and rifampin, none of the changes were significant. To investigate low norbuprenorphine production, buprenorphine was incubated with human liver and small intestine microsomes fortified to support both N-dealkylation and glucuronidation; N-dealkylation predominated in small intestine and glucuronidation in liver microsomes. These studies support the hypothesis that methadone metabolism and its potential for drug interactions can be predicted with cultured human hepatocytes, but for buprenorphine the combined effects of hepatic and small intestinal metabolism are probably involved.

The limited availability of hepatic tissue suitable for the treatment of liver disease and for research makes the generation of hepatic‐like cells from alternative sources an attractive proposition. Although a number of studies have demonstrated that human embryonic stem cells (hESCs) are capable of differentiating into hepatic precursors, the methodology has yet to be fully elucidated. To this end, we cultured hESCs for 10 days on collagen substrata in our primary hepatocyte maintenance media comprised of William's E media supplemented with 25 nM dexamethasone, 10 nM insulin, 5 ng/ml selenium, 5 μg/ml transferrin, and 1% linoleic acid. Using quantitative RT‐PCR assays, we found that hESCs cultured under these conditions exhibited a 2‐fold decrease in mRNA levels of the stem cell pluripotency marker, Oct4, a ~30,000‐fold increase in levels of the endodermal marker, α‐fetoprotein, and a ~475‐fold increase in the hepatic marker, albumin. Further, levels of the constitutive androstane receptor, CAR, were increased ~530‐fold, to levels comparable to those in primary cultures of human hepatocytes. These results define a novel, straightforward methodology that may be useful in facilitating hESC differentiation along the hepatic lineage within a relatively short period of time and provide a framework for subsequent studies seeking to develop a renewable supply of human hepatocytes for research applications.Supported by NIH grant, GM66411.