Urate Oxidase Knockdown Decreases Oxidative Stress in a Murine Hepatic Cell Line

Beth M Cleveland,Hillar Klandorf,Kenneth P Blemings,Stephen S Leonard

doi:10.4161/oxim.2.2.8372

Abstract

Humans, birds, and some primates do not express the uric acid degrading enzyme urate oxidase (UOX) and, as a result, have plasma uric acid concentrations higher than UOX expressing animals. Although high uric acid concentrations are suggested to increase the antioxidant defense system and provide a health advantage to animals without UOX, knockout mice lacking UOX develop pathological complications including gout and kidney failure. As an alternative to the knockout model, RNA interference was used to decrease UOX expression using stable transfection in a mouse hepatic cell line (ATCC, FL83B). Urate oxidase mRNA was reduced 66% (p < 0.05) compared to wild type, as measured by real time RT-PCR. To determine if UOX knockdown resulted in enhanced protection against oxidative stress, cells were challenged with hexavalent chromium (Cr(VI)) or 3-morpholinosydnonimine hydrochloride (SIN-1). Compared to wild type, cells with UOX knockdown exhibited a 37.2 +/- 3.5% reduction (p < 0.05) in the electron spin resonance (ESR) signal after being exposed to Cr(VI) and displayed less DNA fragmentation (p < 0.05) following SIN-1 treatment. Cell viability decreased in wild type cells (p < 0.05), but not cells with UOX knockdown, after treatment with SIN-1. These results are consistent with an increased intracellular uric acid concentration and an increased defense against oxidative stress.

Highlights

In the genomes of both prokaryotes and eukaryotes lies the gene for the uric acid degrading enzyme, urate oxidase (UOX, Submitted: 02/23/09; Revised: 03/10/09; Accepted: 03/10/09Previously published online as an Oxidative Medicine and Cellular Longevity E-publication: http://www.landesbioscience.com/journals/oximed/article/8372EC 1.7.3.3.), not all organisms express this gene
Cell viability of wild type, negative control sion line to determine if 3-morpholinosydnonimine hydrochloride (SIN-1) exposure had an effect on cell viability. In both the wild type and negative control cell lines, cell viability decreased with increasing concentrations of SIN-1 (p < 0.01)
Short interfering RNAs were used to silence UOX mRNA approximately 66% compared to wild type

Summary

Introduction

EC 1.7.3.3.), not all organisms express this gene Among those organisms in which its expression is silenced are humans, birds, some primates, terrestrial reptiles and most insects. In these organisms, uric acid is excreted as the terminal product of purine degradation. In UOX expressing animals, UOX further degrades uric acid to allantoin, the terminal product of purine degradation. When UOX is expressed, the plasma concentration of uric acid is much lower than in those animals that do not express the enzyme. In humans the normal range of serum uric acid is 200 to 400 μM, which is similar to that found in birds[1] and 10 to 20 times that found in UOX expressing animals. Without UOX, humans have achieved a state of hyperuricemia that approaches the saturation point[2,3] and, as a result, gout is a prevalent disease in humans

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