Injurious effects of oxygen free radicals on human intestinal epithelial cells and their prevention

Abstract

Although the importance of oxygen radicals in tissue injury has become more apparent, the deleterious direct actions of these radicals on human intestinal epithelial cells (IEC) and their prevention have not been investigated. In this study, by introducing different combinations of the oxygen metabolite-producing xanthine/xanthine oxidase (X/XO) system and superoxide dismutase (SOD), allopurinol (ALLO), adenosine triphosphate (ATP), and ATP-MgCl₂ (AM) to cultures of human IEC, the injurious effects of the reactive oxygen species and their prevention were evaluated. The extent of cellular injury was assessed using an MTT assay and measuring the release of creatine kinase (CK). In addition, the possible mechanism responsible for the beneficial effects of ATP and AM on IEC subjected to oxidant attack was also investigated by inhibiting the autoxidation of pyrogallol. The results showed that 30 min after introducing X/XO, the cell survival fraction sharply decreased from 100% to 65% while CK activity significantly increased. Pretreatment with ATP, AM, SOD, or ALLO can all markedly reduce or prevent radical-induced cell injury. Autoxidation of pyrogallol observed by chemoluminescence was efficiently inhibited by the addition of ATP and AM, but not by MgCl₂ alone. It may be concluded that radicals derived from the X/XO system severely damage human IECs, while ATP, AM, SOD, and ALLO are all protective against X/XO-induced cell injury. Antioxidant capability may be one of the mechanisms by which AM significantly attenuates radical-induced injury following tissue ischemia and reperfusion.