The Metabolic Rate Constants and Specific Activity of Human and Rat Hepatic Cytochrome P-450 2E1 Toward Toluene and Chloroform

Abstract

Chloroform (CHCl₃) is a near-ubiquitous environmental contaminant, a by-product of the disinfection of drinking water sources and a commercially important compound. Standards for safe exposure have been established based on information defining its toxicity, which is mediated by metabolites. The metabolism of CHCl₃ is via cytochrome P-450 2E1 (CYP2E1)-mediated oxidation to phosgene, which is known to obey a saturable mechanism. CYP2E1 is a highly conserved form, expressed in all mammalian systems studied, and is responsible for the metabolism of a great many low-molecular-weight (halogenated) compounds. However, the Michaelis–Menten rate constants for CHCl₃ oxidation have not been derived in vitro, and the specific activity of CYP2E1 toward CHCl₃ has not been reported. In this investigation with microsomal protein (MSP), apparent V_max values of 27.6 and 28.3 nmol/h/mg MSP and apparent K_m values of 1 and 0.15 μM in rats and human organ donors, respectively, were demonstrated. The specific activity of CYP2E1 toward CHCl₃ in rats and humans was 5.29 and 5.24 pmol/min/pmol CYP2E1, respectively. Toluene metabolism to benzyl alcohol (BA), another CYP2E1-dependent reaction, was also highly dependent on CYP2E1 content in humans, and was more efficient than was CHCl₃ metabolism. The specific activity of human CYP2E1 toward toluene metabolism in human MSP was 23 pmol/min/pmol CYP2E1. These results demonstrate that differences in CYP2E1 content of MSP among individuals and between species are largely responsible for observed differences in toluene and CHCl₃ metabolism in vitro.

The views expressed in this article are those of the authors and do not necessarily reflect the views and policies of the U.S. Environmental Protection Agency. This article (NCEA-C-1429) has been reviewed and cleared for public release in accord with the policies of the U.S. EPA, Office of Research and Development, National Center for Environmental Assessment. The mention of trade names does not imply endorsement. The authors are grateful to Cindy Linsky of the North Carolina State University for undertaking critical initial studies and to Dr. Harlal Choudhury for critical comments during manuscript preparation. The authors express their sincere gratitude to organ donors and their families, for without their generous contributions, studies like these would not be possible.

Present address for Sattar Alcasey is Access Business Group, ILL, Ada, MI, USA.