Xanthine oxidase-induced injury to endothelium: role of intracellular iron and hydroxyl radical

Abstract

The major objective of the present study was to characterize the sequence of events leading to endothelial cytotoxicity induced by oxidants generated extracellularly by xanthine oxidase. 51Cr-labeled monolayers of calf pulmonary artery endothelial cells were exposed to a reaction mixture containing hypoxanthine, xanthine oxidase, and chelated iron (HX/XO) and endothelial cell injury was quantitated as 51Cr release into the media. Catalase, but not mannitol or superoxide dismutase, prevented endothelial cell injury induced by HX/XO, indicating that H2O2 was the mediator of the cytotoxicity. Pretreatment of the cells with free deferoxamine (an iron chelator), but not with deferoxamine bound to dextran (mol wt 40,000), prevented endothelial cell injury induced by HX/XO or H2O2. Of the membrane-permeant hydroxyl radical scavengers dimethylsulfoxide and dimethylthiourea, only dimethylthiourea prevented 1) HX/XO or H2O2-induced endothelial cytotoxicity and 2) deoxyribose degradation by hydroxyl radicals (.OH) generated by an iron-catalyzed reaction on the sugar (site-specific reaction). The concentration of ferritin required to produce significant quantities of .OH was much greater than that present in endothelial cells, and ferritin-catalyzed .OH formation was not affected by deferoxamine, indicating that ferritin-bound iron is most likely not the physiologically active catalyst. We conclude that extracellularly generated H2O2 can enter the cell and interact with nonferritin iron to produce the cytotoxic .OH via a site-specific reaction.

Nucleotide Depletion Due to Reactive Oxygen Metabolites in Endothelial Cells: Effects of Antioxidants and 3-Aminobenzamide

“…than SOD at conccntrations from 75 to 500 U/mL (12,39). In our experiments, both cnzymes had only modest effect.…”

“…12,25). It seems that endothelial cells tolerate severe nuclcotidc dcplction without necessarily losing their viability.…”

Mechanisms of H2O2-induced oxidative stress in endothelial cells

“…OH· is highly reactive and damages non-specifically and irreversibly at its site of formation. The formation of OH· results in oxidation of proteins, DNA, and lipids as well as endothelial cell dysfunction and eventual cell death (Kvietys et al 1989;Beckman et al 1990;Visseren et al 2002).…”

“…Within an oxidative environment, Fe 2+ release contributes to the formation of OH· (Kvietys et al 1989;Visseren et al 2002). Intracellular Fe 2+ stores can be released by O 2 ·-and NO stimulation (Beckman et al 1990;Davidson et al 1997;Alderton et al 2001), this likely plays a role in OH· formation with increased metal ion availability during oxidative stress.…”

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

“…It is generally accepted that the DNA damage results from the site-specific generation of hydroxyl radical (or a similar highly reactive oxidant) by the reaction of H202 with metal ions bound upon or very close to the DNA (9)(10)(11)(12)(13). In addition, in in vitro models ofoxidant-induced cell death, hydroxyl radical scavengers and/or iron chelators (presumably because iron is critical in the generation of hydroxyl radicals via the metalcatalyzed Haber-Weiss reaction) have been shown to be protective (14)(15)(16)(17). Thus, both the DNA damage and cell death have been attributed to the direct toxicity of the oxidants.…”