AJP - Renal Physiology, Vol 271, Issue 1 209-F215, Copyright © 1996 by American Physiological Society

ARTICLES

Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells

H. Hagar, N. Ueda and S. V. Shah
Division of Nephrology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.

Hypoxia is considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, generally considered a feature of apoptosis, to be almost entirely responsible for DNA damage in hypoxic injury to renal tubular epithelial cells. The role of reactive oxygen metabolites in endonuclease-induced DNA damage and cell death in chemical hypoxic injury has not been previously examined. LLC-PK1 cells exposed to chemical hypoxia with antimycin A resulted in enhanced generation of intracellular reactive oxygen species as measured by oxidation of a sensitive fluorescent probe, 2',7'-dichlorofluorescin diacetate. Superoxide dismutase, a scavenger of superoxide radical, significantly reduced the fluorescence induced by antimycin A and provided significant protection against chemical hypoxia-induced DNA strand breaks (as measured by the alkaline unwinding assay). Pyruvate, a scavenger of hydrogen peroxide, provided significant protection against chemical hypoxia-induced DNA strand breaks and DNA fragmentation (as measured by agarose gel electrophoresis). The interaction between superoxide anion and hydrogen peroxide in the presence of a metal catalyst leads to generation of other oxidant species such as hydroxyl radical. Hydroxyl radical scavengers, dimethylthiourea, salicylate, and sodium benzoate, and two metal chelators, deferoxamine and 1,10-phenanthroline, also provided marked protection against DNA strand breaks and DNA fragmentation. These scavengers of reactive oxygen metabolites and metal chelators provided significant protection against cell death as measured by trypan blue exclusion and lactate dehydrogenase release. Taken together, these data indicate that reactive oxygen species play an important role in the endonuclease activation and consequent DNA damage, as well as cell death in chemical hypoxic injury to renal tubular epithelial cells.

This article has been cited by other articles:

				A. Kulkarni and K. C. Das Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L998 - L1006. [Abstract] [Full Text] [PDF]

				G. P. Kaushal, L. Liu, V. Kaushal, X. Hong, O. Melnyk, R. Seth, R. Safirstein, and S. V. Shah Regulation of caspase-3 and -9 activation in oxidant stress to RTE by forkhead transcription factors, Bcl-2 proteins, and MAP kinases Am J Physiol Renal Physiol, December 1, 2004; 287(6): F1258 - F1268. [Abstract] [Full Text] [PDF]

				N. UEDA, S. M. R. CAMARGO, X. HONG, A. G. BASNAKIAN, P. D. WALKER, and S. V. SHAH Role of Ceramide Synthase in Oxidant Injury to Renal Tubular Epithelial Cells J. Am. Soc. Nephrol., November 1, 2001; 12(11): 2384 - 2391. [Abstract] [Full Text] [PDF]

				C. Rota, Y. C. Fann, and R. P. Mason Phenoxyl Free Radical Formation during the Oxidation of the Fluorescent Dye 2',7'-Dichlorofluorescein by Horseradish Peroxidase. POSSIBLE CONSEQUENCES FOR OXIDATIVE STRESS MEASUREMENTS J. Biol. Chem., October 1, 1999; 274(40): 28161 - 28168. [Abstract] [Full Text] [PDF]

				J. Bai, A. M. Rodriguez, J. A. Melendez, and A. I. Cederbaum Overexpression of Catalase in Cytosolic or Mitochondrial Compartment Protects HepG2 Cells against Oxidative Injury J. Biol. Chem., September 10, 1999; 274(37): 26217 - 26224. [Abstract] [Full Text] [PDF]

				L. R. Feldenberg, S. Thevananther, M. del Rio, M. de Leon, and P. Devarajan Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells Am J Physiol Renal Physiol, June 1, 1999; 276(6): F837 - F846. [Abstract] [Full Text] [PDF]

				K. Machida, T. Tanaka, K.-i. Fujita, and M. Taniguchi Farnesol-Induced Generation of Reactive Oxygen Species via Indirect Inhibition of the Mitochondrial Electron Transport Chain in the Yeast Saccharomyces cerevisiae J. Bacteriol., September 1, 1998; 180(17): 4460 - 4465. [Abstract] [Full Text]

				M. D. Aleo, J. A. Morgan, and R. G. Schnellmann Recovery of cellular functions following oxidant injury Am J Physiol Renal Physiol, March 1, 1998; 274(3): F509 - F515. [Abstract] [Full Text] [PDF]

				J. Bai and A. I. Cederbaum Overexpression of Catalase in the Mitochondrial or Cytosolic Compartment Increases Sensitivity of HepG2 Cells to Tumor Necrosis Factor-alpha -induced Apoptosis J. Biol. Chem., June 16, 2000; 275(25): 19241 - 19249. [Abstract] [Full Text] [PDF]