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      Protection against Hydrogen Peroxide Induced Injury in Renal Proximal Tubule Cell Lines by Inhibition of Poly(ADP–Ribose) Synthase

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          Radicals including superoxide anions, hydrogen peroxide or hydroxyl radicals and NO or peroxynitrite cause the breakage of DNA strands and activation of poly–(ADP–ribose) synthase (PARS). Recent studies showed that inhibition of PARS activity reduces the tissue injury after exposure to oxidative stress. However, the role of PARS in renal injury by oxidants has not been examined. In this study effect of a PARS inhibitor, 3–aminobenamide (3–AB), on injury of opossum kidney or LLC–PK<sub>1</sub> cells by hydrogen peroxide or tert–butyl hydroperoxide (t–BHP) was examined. The exposure of opossum kidney cells to hydrogen peroxide activated PARS and decreased cellular adenosine triphosphate levels in a concentration–dependent manner. Inhibition of PARS with 3–AB prevented the cell death induced by hydrogen peroxide and also prevented adenosine triphosphate depletion. 3–AB did not have hydroxyl radical scavenging effect. In contrast, t–BHP did not affect the PARS activity. The decrease in cellular adenosine triphosphate levels by t–BHP was less than that by hydrogen peroxide. 3–AB failed to prevent the cell death induced by t–BHP. PARS activation after exposure of hydrogen peroxide was inhibited by addition of t–BHP. However, t–BHP showed an additive effect on cell death with hydrogen peroxide. These results indicate that activation of PARS plays an important role in hydrogen peroxide induced injury in opossum kidney cells and that hydrogen peroxide and t–BHP induce cell injury by different mechanisms.

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          DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite.

          The free radicals nitric oxide and superoxide anion react to form peroxynitrite (ONOO-), a highly toxic oxidant species. In vivo formation of ONOO- has been demonstrated in shock and inflammation. Herein we provide evidence that cytotoxicity in cells exposed to ONOO- is mediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase (PARS). Exposure to ONOO- (100 microM to 1 mM) inhibited mitochondrial respiration in cultured J774 macrophages and in rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1-3 h after ONOO- exposure, and reversible, with recovery after a period of 6-24 h. The inhibition of mitochondrial respiration was paralleled by a dose-dependent increase in DNA strand breakage, reaching its maximum at 20-30 min after exposure to ONOO-. We observed a dose-dependent increase in the activity of PARS in cells exposed to ONOO-. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellular respiration in cells exposed to ONOO-. Activation of PARS by ONOO--mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by a decline of intracellular NAD+ and ATP content. 3-Aminobenzamide prevented the loss of NAD+ and ATP in cells exposed to ONOO-. In contrast, impairment of cellular respiration by the addition of the nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine or diethyltriamine nitric oxide complex, was not associated with the development of DNA strand breaks, in concentrations up to 1 mM, and was largely refractory to PARS inhibition. Our results suggest that DNA damage and activation of PARS, an energy-consuming futile repair cycle, play a central role in ONOO--mediated cellular injury.
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            Inactivation of the poly(ADP-ribose) polymerase gene affects oxygen radical and nitric oxide toxicity in islet cells.

            Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is an early response of cells exposed to DNA-damaging compounds such as nitric oxide (NO) or reactive oxygen intermediates (ROI). Excessive poly-(ADP-ribose) formation by PARP has been assumed to deplete cellular NAD+ pools and to induce the death of several cell types, including the loss of insulin-producing islet cells in type I diabetes. In the present study we used cells from mice with a disrupted and thus inactivated PARP gene to provide direct evidence for a causal relationship between PARP activation, NAD+ depletion, and cell death. We found that mutant islet cells do not show NAD+ depletion after exposure to DNA-damaging radicals and are more resistant to the toxicity of both NO and ROI. These findings directly prove that PARP activation is responsible for most of the loss of NAD+ following such treatment. The ADP-ribosylation inhibitor 3-aminobenzamide partially protected islet cells with intact PARP gene but not mutant cells from lysis following either NO or ROI treatment. Hence the protective action of 3-aminobenzamide must be due to inhibition of PARP and does not result from its other pharmacological properties such as oxygen radical scavenging. Finally, the use of mutant cells an alternative pathway of cell death was discovered which does not require PARP activation and NAD+ depletion. In conclusion, the data prove the causal relationship of PARP activation and subsequent islet cell death and demonstrate the existence of an alternative pathway of cell death independent of PARP activation and NAD+ depletion.
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              Metabolism of pyridine nucleotides in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide


                Author and article information

                Kidney Blood Press Res
                Kidney and Blood Pressure Research
                S. Karger AG
                15 October 1999
                : 23
                : 1
                : 14-19
                Department of Physiology, College of Medicine, Pusan National University, Pusan, Korea
                25949 Kidney Blood Press Res 2000;23:14–19
                © 1999 S. Karger AG, Basel

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                Figures: 7, References: 27, Pages: 6
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