Title

Studies on the prevention of cytochrome P450 inhibition by products of lipid peroxidation in an in vitro microsomal enzyme system

Date of Completion

January 2004

Keywords

Agriculture, Animal Pathology

Degree

Ph.D.

Abstract

An in vitro canine microsomal system was developed to investigate the effects of lipid peroxidation and lipid peroxidation cytotoxic metabolites on cytochrome P450 enzymatic activity. The attempt was to prolong enzymatic activity of cytochrome P450 and produced milligram quantities of metabolites of interest. Cytochrome P450 microsomal systems are currently used as screening procedures for new drugs or compounds of interest. In vitro cytochrome P450 enzymatic activity diminishes relatively quickly (15 to 30 minutes). Products of lipid peroxidation such as HNE are known to cause destruction of lipid bilayers resulting in disruption of the cytochrome P450 membrane association, adduction to the prosthetic heme, and/or reacting with various amino groups on proteins within the microsomal suspension causing secondary structural deformities. Preventing lipid peroxidation or preventing the cytotoxic effects of lipid peroxidation products has been investigated here with the use of various inhibitors of lipid peroxidation, scavengers of lipid peroxidation products and attempting to convert lipid peroxidation products to less cytotoxic products. ^ Anti-oxidants, such as ascorbic acid and α-tocopherol were tested within the canine microsomal system using codeine as a substrate to probe the effects on CYP2D15 and CYP3A12 enzymatic activity (analogs to human CYP2D6 and CYP3A4/5 respectively). Lysine, histidine, N-acetyl cysteine and GSH were utilized in the same microsomal system to test their ability to scavenger lipid peroxidation products that were produced during N-demethylation of codeine. ADH and ALDH were also tested within the same metabolic system to emulate the in vivo metabolism of HNE. Adduct formation of the HNE with the cytochrome P450 was also investigated. Investigative work by others strongly suggests that lipid peroxidation and its cytotoxic metabolite HNE is the primary perpetrator causing diminished cytochrome P450 activity of in vitro reconstituted system. Studies performed here were unable to be implicate or prove that lipid peroxidation or its cytotoxic products are the cause of cytochrome P450 inactivation. ^