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

Abstract

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.

Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether

“…Several lines of evidence suggest that plasmalogens, by virtue of their vinyl ether, act as antioxidants. The generation of ROS during chemical hypoxia has been well established [11,30,31]. Our ability to partly rescue cells with the antioxidant Trolox demonstrated that reactive oxygen generation had a role in the chemical hypoxia-induced cytotoxicity.…”

Regulation of caspase-3 and -9 activation in oxidant stress to RTE by forkhead transcription factors, Bcl-2 proteins, and MAP kinases

“…ROS-induced cell death has been reported in a wide variety of cells, including renal tubular epithelial cells (RTE) (1,14,24,31,38,57,58) and nonrenal cells (25,40). Studies from other laboratories and that from ours have shown an important role of caspases in RTE injury (15, 21, 23, 33-36, 38, 41, 44, 46).…”

Role of abscisic acid and drought stress on the activities of antioxidant enzymes in wheat

“…The production of reactive oxygen species is a common phenomenon in plants under drought stress. These reactive oxygen species (ROS) generations led to lipid peroxidation (Sreeni-Nivasuhu et al 1999, Chen et al 2000, protein degradation (Jiang and Zhang 2001) and nucleic acid damages (Hagar et al 1996). To alleviate adverse effects of reactive oxygen species, plants have evolved an antioxidant defense system that include enzymes like superoxide dismutase, peroxidase and catalase etc.…”