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ARS Home » Northeast Area » Leetown, West Virginia » Cool and Cold Water Aquaculture Research » Research » Publications at this Location » Publication #228464
Title: Urate oxidase knockdown decreases oxidative stress in a murine hepatic cell line

Author
item Cleveland, Beth
item BLEMINGS, KENNETH - WEST VIRGINIA UNIVERSTIY
item LEONARD, STEPHEN - NIOSH
item KLANDORF, HILLAR - West Virginia University

Submitted to: Oxidative Medicine and Cellular Longevity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2009
Publication Date: 4/1/2009
Citation: Cleveland, B.M., Blemings, K., Leonard, S.S., Klandorf, H. 2009. Urate oxidase knockdown decreases oxidative stress in a murine hepatic cell line. Oxidative Medicine and Cellular Longevity. 2(2):93-98.

Interpretive Summary: Although both eukaryotes and prokaryotes carry the gene for the uric acid degrading enzyme, urate oxidase, not all organisms express the 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 the terminal product of purine degradation and, as a result, they have plasma uric acid concentrations ten to twenty times that of urate oxidase expressing animals. A widely accepted theory describes the adaptive advantage of this silencing as an enhanced antioxidant defense that the uric acid affords. However, in humans high uric acid concentrations have been associated with kidney and cardiovascular disease. Additionally, knockout mice that lack urate oxidase expression develop severe complications resulting from urate nephropathy and die within weeks after birth. Therefore, a more appropriate mouse model of hyperuricemia is needed. Our objective was to evaluate the use of RNA interference to knockdown urate oxidase expression in a mouse hepatic cell line and determine the effect on the cellular oxidative stress response. A 66% reduction of the urate oxidase expression resulted in the cells producing less free radicals when when exposed to chromium (VI), and less cell death and DNA damage when exposed to the reactive nitrogen species, peroxynitrite. These results are consistent with an increased intracellular uric acid concentration and an increased defense against oxidative stress. Additionally, they demonstrate that RNA interference can be used in place of the knockout technique to study the effects of hyperuricemia.

Technical 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 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.