Peroxisome Proliferation Induced Oxidative Stress

Holmes, Earle

Abstract

This proposal is focused on determining whether the four PPs studied increase oxidative stress or oxidative damage in rat (responsive species) versus hamster (nonresponsive species) liver, and whether the species difference in sensitivity reflects differences in redox (antioxidant) status during PP treatment. The time points to be examined are days 6 and 34 of treatment for all 4 PPs (to minimize artifacts due to secondary effects), and only the untreated and highest doses will be analyzed. The protein levels to be measured in Aim #1 by semiquantitative immunoblotting as measures of oxidative stress induction include: a) DNA repair enzymes (pol beta and Ref-1); b) heme oxygenase 1; c) apoptosis-related proteins, Bcl-2 and Bax-1; and d) c-fos. Protein levels will be measured by slot immunoblot with chemiluminescent detection and densitometric quantitation of slot intensities on film. Actual indices of oxidative damage will include: e) protein nitrotyrosine, f) protein 4-HNE adducts, and g) protein carbonyl groups. These indices will be analyzed initially by slot immunoblot, using monoclonal antibodies against nitrotyrosine or 4-HNE-histidine. Protein carbonyl groups will be derivatized on slot blots with DNP-hydrazine and stained with rabbit polyclonal anti-DNP antibody. Detection will be by ECL/densitometry as above. These methods will also be used to identify specific proteins preferentially modified, by SDS-PAGE and western blotting.
In Aim #2, total content of the antioxidants glutathione (GSH), ascorbate, and uric acid will be measured using frozen liver. The total GSH and the GSH/GSSG (oxidized glutathione) ratio will be measured by the reductase cycling method of Tietze. Detailed statistical procedures are planned for data analysis, including ANOVA for the main effects of species, dose, and treatment time; pair-wise companions of group means using Bonferroni's test; and Spearman rank correlation coefficients for relationships between markers of oxidative stress and tissue antioxidant levels. The sample requirements for this study are 400 - 500 mg.