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Genetic Studies
Project Summary

Title: The Importance of Hydrogen Peroxide Detoxification in Cellular Protection
Synopsis: This study attempts to determine the importance of hydrogen peroxide detoxification in protecting mice against certain environmental hazards (such as ionizing radiation and paraquat).
Overall Project Objective: This project will test the hypothesis that alterations in catalase levels will alter the sensitivity of mice to agents that induce oxidative stress. Mice with a targeted mutation in the catalase gene will be generated. The sensitivity of these mice to gamma-irradiation and paraquat, will be compared to wild type mice and transgenic mice that overexpress catalase.
Status/Results to Date: We have measured the expression (mRNA level) and activity of catalase in a variety of tissues from wild type and catalase transgenic mice. Catalase mRNA is increased 2 to 8-fold in all tissues we have examined in the transgenic mice and the increase in mRNA is accompanied by a 2 to 4 increase in catalase activity. The activities of the other major antioxidant enzymes, glutathione peroxidase and Mn and CuZn superoxide dismutase are not altered (data not shown). Therefore the increase in catalase activity does not result in compensatory changes in other major antioxidant enzymes. Catalase knockout mice will be produced by targeted deletion of the region from 600bp upstream of the transcription start site to intron 1. This region contains the promoter, transcription and translation start sites. The targeting construct to produce the catalase knockout mice has been generated and the linearized targeting construct has been currently introduced into embryonic stem (ES) cells by electroporation. We are currently screening the clones for targeting by Southern blot analysis. We have compared the sensitivity of hepatocytes isolated from wild type and catalase transgenic mice to induction of oxidative stress by several agents, i.e. hydrogen peroxide, t-butylhydrogenperoxide, and paraquat. In contrast, hepatocytes isolated from transgenic and wild type mice show no difference in sensitivity following treatment with t-butylhydroperoxide. In response to 24 hour treatment with paraquat, which is a superoxide anion generator, the hepatocytes from the catalase transgenic mice exhibit increased sensitivity (decreased viability) when viability is assessed using an assay that measures the metabolic integrity of the cell (neutral red). However, if the viability is assayed using the activity of lactate dehydrogenase related into the media, an indicator of loss of cell membrane integrity and cell death, there is no difference between the hepatocytes isolated from the wild type and the catalase transcription mice.
Project:VA-65C
Agency:Department Of Veterans' Affairs
Location:San Antonio Environmental
P.I. Name:J Chen, Holly Van Remmen
Research Type:Mechanistic
Research Focus:Environmental Toxicology
Focus Category:Genetic Studies
Status:Ongoing
Study Start Date:January 07,2000
Estimated Completion Date:January 07,2005
Specific Aims: Our hypothesis is that mice with increased expression of catalase activity will be more resistant to oxidative damage to lipids, proteins and DNA caused by exposure to the oxidative insults of gamma-irradiation and paraquat while mice with reduced expression of catalase will show increased sensitivity to these insults.
Methodology: To test this hypothesis, we are currently breeding mice in which the activity of catalase is increased several fold in a variety of tissues in order to examine the effect of increased protection against hydrogen peroxide generation. When we have bred these mice to generate homozygous catalase transgenic mice, in which the human catalase gene is over-expressed in a variety of tissues, we will characterize the antioxidant status of these mice by measuring the activity of catalase and also the activity of the other major antioxidant enzymes (glutathione peroxidase and CuZn-and Mn-superoxide dismutase) to determine whether there is compensation in response to the increased catalase activity in other components of the antioxidant system. Glutathione levels will also be compared in wild-type and catalase transgenic mice. To study the effect of reduced detoxification of hydrogen peroxide, catalase knockout mice will be generated by targeted disruption of the catalase gene by deleting the region 600bp upstream of the transcription start site to intron 1. These mice should have no detectable catalase activity. Finally, to determine whether the changes in catalase activity result in altered sensitivity to oxidative insults, we will measure the survival of the two mouse models in response to an acute whole-body exposure to gamma-irradiation and treatment with paraquat. The extent of oxidative damage will be determined in various tissues in mice by measuring lipid peroxidation, protein oxidation and DNA mutations.
Most Recent Publications:

Van Remmen H, Chen J, Mele J, Richardson A. Aging and Oxidative Stress in Transgenic Mice. Oxidative Stress and Aging: Advances in Basic Scienc, Diagnostics, and Interentions, In press, 2001. Article

Guo ZM, Van Remmen H, Yang H, Chen X, Mele J, Vijg J, Epstein CJ, Ho YS, Richardson A. Changes in expression of antioxidant enzymes affect cell-mediated LDL oxidation and oxidized LDL-induced apoptosis in mouse aortic cells. Arterioscler Thromb Vasc Biol, 21(7):1131-8, Jul 2001. Abstract

Van Remmen H, Williams MD, Guo ZM, Estlack L, Yang H, Carlson EJ, Epstein CJ, Huang TT, Richardson A. Knockout mice heterozygous for Sod2 show alterations in cardiac mitochondrial function and apoptosis. Am J Physiol Heart Circ Physiol, 281(3):H1422-32, Sep 2001. Abstract

Van Remmen H, Richardson A. Oxidative damage to mitochondria and aging. Exp Gerontol, 36(7):957-68, Jul 2001. Abstract

Van Remmen H, Guo ZM, Richardson A. The anti-ageing action of dietary restriction. Novartis Found Symp, 235:221-30; discussion 230-3, 2001. Abstract