title: Reactive oxygen injury to cultured pulmonary artery endothelial cells: mediation by poly(ADP-ribose) polymerase activation causing NAD depletion and altered energy balance.
abstract: The vascular endothelium is a significant site for tissue injury following exposure to reactive oxygen species derived from a number of sources. In order to develop a better understanding of the mechanism(s) of oxidative damage, monolayer cultures of endothelial cells obtained from bovine pulmonary arteries were exposed to reactive oxygen species generated from the oxidation of dihydroxyfumarate (DHF) to diketosuccinate. Exposure to oxidizing DHF caused a loss of cell membrane integrity that was delayed in onset; that is, it did not begin until 2 h after the addition of DHF although reactive oxygen species are produced immediately by DHF in solution. Endothelial cell lysis by DHF was prevented by the simultaneous addition of superoxide dismutase (SOD), catalase (CAT), or deferoximine (DFX). This oxidant-induced lysis was unaffected by N,N,-diphenyl-p-phenylenediamine (DPPD), a potent inhibitor of lipid peroxidation. However, simultaneous addition of 3-aminobenzamide (3AB) and nicotinamide (NA), inhibitors of poly(ADP-ribose) polymerase, prevented cell lysis. Oxidant-induced loss of membrane integrity was preceded by the early appearance of DNA strand breaks, by increased levels of poly(ADP-ribose), the product of polymerase activity, and by depletion of NAD+ and ATP, followed by a decline in the energy charge ratio of the cells. None of these intracellular changes occurred when either SOD, CAT, or DFX were added at the same time as DHF, suggesting that O2-., H2O2, and HO. mediated these changes. The O2-. appears to be important in the autoxidation reaction of DHF. The latter two reactive oxygen species may be part of cellular-catalyzed Fenton chemistry. The increase in poly(ADP-ribose), depletion of NAD+, and the decline in ATP were also prevented by the addition of 3AB. The oxidant-induced DNA strand breakage was, however, unaffected by either 3AB or NA. Addition of 3AB immediately prior to the onset of cell lysis (2 h after the addition of DHF), prevented cell lysis, i.e., "rescued" the cells when neither SOD, CAT, nor DFX addition were effective. Concurrent with the "rescue" from lysis by 3AB, there was an increase in NAD+ content and a return of the energy charge ratio to control levels. The data presented in this study suggests that in endothelial cells, DNA is a very sensitive target for reactive oxygen species and HO. is the likely proximal damaging species.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pathology, University of British Columbia, Vancouver, Canada.
authors.forename: R L
authors.initials: RL
authors.lastname: Thies
authors.forename: A P
authors.initials: AP
authors.lastname: Autor
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003676:Deferoxamine / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004734:Energy Metabolism / Q000187:drug effects*; D004789:Enzyme Activation; D005609:Free Radicals; D005650:Fumarates / Q000378:metabolism / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism*; D010100:Oxygen / Q000633:toxicity*; D010655:Phenylenediamines / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011651:Pulmonary Artery; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism
chemical_list: D000227:Adenine Nucleotides; D000975:Antioxidants; D005609:Free Radicals; D005650:Fumarates; D010655:Phenylenediamines; D009243:NAD; D013481:Superoxides; C005998:dihydroxyfumarate; D006861:Hydrogen Peroxide; C004845:N,N'-diphenyl-4-phenylenediamine; D002374:Catalase; D013482:Superoxide Dismutase; D011065:Poly(ADP-ribose) Polymerases; D003676:Deferoxamine; D010100:Oxygen