Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells.

Abstract

Hydrogen peroxide (H202)-induced DNA damage and cell death have been attributed to the direct cytotoxicity of H202 and other oxidant species generated from H202. We examined the possibility that oxidants activate endonucleases leading to DNA damage and cell death in renal tubular epithelial cells, similar to that described for apoptosis. Within minutes, H202 caused DNA strand breaks in a dose-dependent manner, followed by cell death. DNA fragmentation was demonstrated both by the release of [3H]thymidine in 27,000-g supernatant as well as the occurrence of low molecular weight DNA fragments on agarose gel electrophoresis, characteristic of endonuclease cleavage. Endonuclease inhibitors, aurintricarboxylic acid, Evans blue, and zinc ion prevented H202-induced DNA strand breaks, fragmentation, and cell death. Inhibitors of protein or mRNA synthesis had only minor protection against H202-induced DNA damage in contrast to complete protection reported in apoptotic thymocytes. Micrococcal endonuclease induced similar DNA strand breaks in LLC-PKI cells, and the endonuclease inhibitors prevented the events confirming the ability of endonucleases to induce DNA damage. The protective effect of aurintricarboxylic acid was not due to the prevention of the rise in intracellular free calcium. We conclude that endonuclease activation occurs as an early event leading to DNA damage and cell death in renal tubular epithelial cells exposed to oxidant stress and, in contrast to apoptotic thymocytes, does not require macromolecular synthesis. (J. Clin. Invest. 1992.

Evaluation of anti–apoptotic activity of different dietary antioxidants in renal cell carcinoma against hydrogen peroxide

“…As is cited in the literature, H 2 O 2 induces oxidative nucleosomal and chromosomal damage through ROS [3,23], and these ROS are scavenged by polyphenolic antioxidants [2] . In the current study the protective effect of different antioxidants against H 2 O 2 mediated nucleosomal damage in cancer cell lines especially RCC-26 was investigated.…”

“…Cultured RCC-26 cells (2×10 6 ) were seeded and allowed to adhere in a 96-well plate using RPMI-1640 medium. Then cells were treated with various antioxidants (20 and 50 毺 g/mL) followed by 2 h incubation and 3.5 mM H 2 O 2 was added for another 3 h. After the incubation 25 毺 L of 5 mg/mL MTT (dissolved in PBS) was added to each well followed by incubation for 4 h at 37 曟 . Thereafter, formazan crystals were dissolved in 150 毺 L DMSO followed by the quantitative measurement of formazan purple by the absorbance at 540 nm.…”

“…H 2 O 2 belongs to the ROS, and is known to modulate diverse cellular functions in vivo by producing hydroxyl radicals through the interaction with metal ions near DNA. Also, H 2 O 2 induces DNA damage and cell damage by lipid peroxidation [3] . Although apoptosis and necrosis have different impacts on cellular physiology, the cellular response to H 2 O 2 is continuing from apoptosis to necrosis [4] i.e.…”

“…Although apoptosis and necrosis have different impacts on cellular physiology, the cellular response to H 2 O 2 is continuing from apoptosis to necrosis [4] i.e. high concentration induces necrosis converse is applicable for apoptosis [3][4][5] .…”

Prevention of necrosis and activation of apoptosis in oxidatively injured human myeloid leukemia U937 cells

“…That H202 can trigger apoptotic cell death has been clearly demonstrated in a number of reports [9][10][11][12] and it is just as clear that the mode of cell death switches from apoptosis to necrosis when the concentration of the oxidant is increased.…”