Item – Theses Canada

OCLC number
46586703
Link(s) to full text
LAC copy
LAC copy
Author
Anari, Mohammad Reza,1964-
Title
Cytochrome P450 peroxidaseperoxygenase-dependent metabolic activation of xenobiotics.
Degree
Ph. D. -- University of Toronto, 1997
Publisher
Ottawa : National Library of Canada = Bibliothèque nationale du Canada, [1999]
Description
2 microfiches.
Notes
Includes bibliographical references.
Abstract
Cytochrome P450 (P450) peroxidase and peroxygenase activities have been well documented with microsomal enzymes and purified P450 preparations, however, the biological significance of these pathways in the bioactivation of xenobiotics and induction of oxidative stress in intact cells has not been demonstrated. The following summarizes our new findings regarding the role of P450 peroxidase/peroxygenase pathways in intact cells: (1) tert-butylhydroperoxide (tBHP) at non-toxic concentrations markedly enhanced up to 20 fold the cytotoxicity of various aromatic hydrocarbons and their phenolic metabolites towards isolated rat hepatocytes. The enhanced cytotoxicity was also accompanied by an increase in the hepatocyte O-demethylation of xenobiotics and xenobiotic reactive metabolites-GSH conjugates. An LC/MS analysis of the GSH conjugates identified hydroquinone:GSH and 4-methoxycatechol:GSH conjugates as the predominant adducts for 4-hydroxyanisole (4-HA). Pretreatment of hepatocytes with P450 inhibitors, e.g. phenylimidazole, prevented tBHP-enhanced 4-HA metabolism, GSH depletion and cytotoxicity. Hydroperoxides can therefore be used by intact cells to support the bioactivation of xenobiotics through the P450 peroxygenase system. (2) The naturally occurring hydroperoxide, hydrogen peroxide, was found to be particularly effective at supporting cytochrome P450 1A2-catalyzed activation of the heterocyclic aromatic amine, 2-amino-3-methylimidazo) 4,5-f) quinoline (IQ), to genotoxic metabolites. The addition of hydrogen peroxide or tBHP to rP4501A greatly enhanced the yield of histidine prototrophic (His$\sp+)$ revertants, which was inhibited by a-naphthoflavone, a P450 1A inhibitor. Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with human P450 1A2-containing microsomes (hP4501A2). The hydroperoxide-supported activation of 1Q formed an adduct with 2$\sp\prime$-deoxyguanosine similar to that of the well characterized DNA adduct formed (in vivo or in vitro) by the P450-catalyzed bioactivation system. (3) Organic hydroperoxides are believed to be primarily bioactivated to cytotoxic radical species by non heme iron. However, various P450 inhibitors were found to prevent cumene hydroperoxide (CumOOH) metabolism and subsequent cytotoxic effects including antimycin-A resistant respiration, lipid peroxidation, iron mobilization, ATP depletion, and cell membrane disruption. These results suggest that P450 enzymes in hepatocytes bioactivate CumOOH to form reactive radical metabolites or oxidants that cause lipid peroxidation and cytotoxicity.
ISBN
0612282694
9780612282698