Detection of Poly(ADP-ribose) Polymerase Activation in Oxidatively Stressed Cells and Tissues Using Biotinylated NAD Substrate

Abstract

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by DNA damage. Activated PARP cleaves NAD⁺ into nicotinamide and (ADP-ribose) and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP by reactive oxygen and nitrogen intermediates represents a pathogenetic factor in various forms of inflammation, shock, and reperfusion injury. Using a novel commercially available substrate, 6-biotin-17-nicotinamide-adenine-dinucleotide (bio-NAD⁺), we have developed three applications, enzyme cytochemistry, enzyme histochemistry, and cell ELISA, to detect the activation of PARP in oxidatively stressed cells and tissues. With the novel assay we were able to detect basal and hydrogen peroxide-induced PARP activity in J774 macrophages. We also observed that mitotic cells display remarkably elevated PARP activity. Hydrogen peroxide-induced PARP activation could also be detected in wild-type peritoneal macrophages but not in macrophages from PARP-deficient mice. Application of hydrogen peroxide to the skin of mice also induced bio-NAD⁺ incorporation in the keratinocyte nuclei. Hydrogen peroxide-induced PARP activation and its inhibition by pharmacological PARP inhibitors could be detected in J774 cells with the ELISA assay that showed good correlation with the traditional [³H]-NAD incorporation method. The bio-NAD⁺ assays represent sensitive, specific, and non-radioactive alternatives for detection of PARP activation.