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Genetic Studies
Project Summary

Title: Does a Variant of the Human SOD2 Gene Increase Sensitivity to Hazards?
Synopsis: This study attempts to assess whether mice with certain gene (SOD2) variants are more sensitive to oxidative stress.
Overall Project Objective: This project will test the hypothesis that mice expressing a Sod2 variant (Ile58Thr: threonine replacing isoleucine) will be more sensitive to oxidative stress. A novel knockout/knockin approach will be used to generate transgenic mice in which the human Ile58Thr variant replaces the wild type murine Sod2 allele. The sensitivity of these mice to gamma-irradiation and paraquat will be compared to wild type mice. Based on our experience with Sod2 mice, we hypothesize that individuals carrying the Thr58 variant of Sod2 are at a greater risk to environmental exposure to hazards because this variant has less biological activity than the wild type enzyme (Ile58) and because our studies with Sod2 mice clearly demonstrate that reduced MnSOD activity is associated with decreased mitochondrial function and increased sensitivity to apoptosis induced by oxidative stress.
Status/Results to Date: In specific aim 1 we are using a KO/KI strategy where the Sod2 locus is disrupted (knockout) in embryonic stem cells by a targeting vector that introduces (knockin) into the locus the tetracycline repressor fused to the promoter of the endogenous Sod2 gene and a suppressible minigene. The targeting construct to produce the catalase knockout mice has been generated and the linearized targeting construct has been introduced to embryonic stem (ES) cells by electroporation. We are currently screening the clones for targeting by Southern blot analysis. In addition to our studies with Sod2 +/- mice, we are also studying the effect of a larger deficit in the mitochondrial antioxidant defense on mitochondrial function and sensitivity to oxidative stress. We have crossed the Sod2 +/- mice to a homozygous GPx1-/- mouse that completely lacks cGPx activity. By crossing these mice to the Sod2+/- mice we have generated a breeding colony of Sod2+/- knockout mice in the C57BL/6 genetic background. These mice show no obvious phenotype; they are able to breed and we have maintained Sod2+/- knockout mice to at least 8 months of age without any observable pathology or deficit. We have characterized the antioxidant status of these mice as compared to wild type mice. As predicted, the Sod2-/- mice have a 50% reduction in MnSOD activity and no cGPx activity in the mitochondria. The activities of the other major antioxidant enzymes (CuZnSOD and catalase) are not altered to compensate for the reduction in mitochondrial antioxidant defense. We have several lines of evidence to show that the Sod2+/- mice are more sensitive to oxidative stress than the Sod2+/- knockout mice. For example, oxidative damage to nuclear DNA in liver is much higher in the Sod2+/- mice or the wild type mice. We have also studied the sensitivity of primary cultures of fibroblasts isolated from 3 to 5 day old pups from wild type, Sod 2+/-, Sod 2-/-, GPx1-/-, and Sod2+/-/GPx1-/- mice to oxidative stress induced by t-butylhydroperoxide, ceramide, and gamma-irradiation. In all cases, the cells from the Sod2+/-/GPx1-/- mice show a sensitivity to oxidative stress greater than the Sod2+/- mice and similar to that of the homozygous MnSOD knockout (Sod2-/-). It should be noted that the homozygous MnSOD mutant is lethal and the mice do not live longer than just a few days.
Project:VA-65A
Agency:Department Of Veterans' Affairs
Location:San Antonio Environmental
P.I. Name:Arlan Richardson, Ph.D.
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: To generate mice that conditionally express the human Thr58 variant and Ile58 wild type MnSOD. (2) To determine if Sod2+/- mice are more sensitive to oxidative stress than their Sod2+/+ wild-type littermates. (3) To determine if the KO/KI mice expressing the Thr58 variant of MnSOD are more sensitive to oxidative stress than mice expressing the wild type (Ile58) MnSOD.
Methodology: See Specific Aims.
Most Recent Publications:

Hamilton M, Guo WD, Fuller J, Van Remmen H, Ward WF, Austad SN, Troyer DA, Thompson I, Richardson A. A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA. Nucleic Acids Res, 15;29(10):2117-26, May 2001. Abstract

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

Hamilton M, Van Remmen H, Drake JA, Yang H, Guo ZM, Kewitt K, Walter CA, Richardson A. Does oxidative damage to DNA increase with age? Pro. Natl Academy of Sciences, 28;98(18):10469-74, Aug 2001. Abstract

Pahlavani MA, Mele J, Richardson A. Effect of overexpression of human Cu/Zn-SOD on activation-induced lymphocyte proliferation and apoptosis. Federal Investigators Meeting, 1;30(11):1319-27, Jun 2001. Abstract

Guo ZM, Yang H, Hamilton M, Van Remmen H, Richardson A. Effects of age and food restriction on oxidative DNA damage and antioxidant enzyme activities in the mouse aorta. Mech Ageing Dev, 122(15):1771-86, Oct 2001. Abstract

Han ES, Hilsenbeck SG, Richardson A, Nelson JF. cDNA expression arrays reveal incomplete reversal of age-related changes in gene expression by calorie restriction. Mech Ageing Dev, 20;115(3):157-74, Jun 2000. Abstract