Lipid peroxidation induced by indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacin radicals

Abstract

Some of the side-effects of using indomethacin (IM) involve damage to the gastric mucosa and liver mitochondria. On the other hand, neutrophils infiltrate inflammatory sites to damage the tissues through the generation of reactive oxygen species by myeloperoxidase. The stomach and intestine have large amounts of peroxidase. These findings suggest that peroxidases are involved in tissue damage induced by IM. To clarify the basis for the tissue damage induced by IM in the presence of horseradish peroxidase (HRP) and H₂O₂ (HRP-H₂O₂), lipid peroxidation was investigated. When IM was incubated with liver microsomes in the presence of HRP-H₂O₂ and ADP-Fe³⁺, lipid peroxidation was time-dependent. Catalase and desferrioxamine almost completely inhibited lipid peroxidation, indicating that H₂O₂ and iron are necessary for lipid peroxidation. Of interest, superoxide dismutase strongly inhibited lipid peroxidation, and it also inhibited the formation of bathophenanthroline-Fe²⁺, indicating that reduction of the ferric ion was due to superoxide (O₂⁻). ESR signals of IM radicals were detected during the interaction of IM with HRP-H₂O₂. However, the IM radical by itself did not reduce the ferric ion. These results suggest that O₂⁻ may be generated during the interaction of IM radicals with H₂O₂. Ferryl species, which are formed during the reduction of iron by O₂⁻, probably are involved in lipid peroxidation.

Introduction

IM, one of the most effective NSAIDs, is widely used clinically to treat rheumatic and arthritic disease because it has analgesic, antipyretic, and anti-inflammatory actions; these actions are mediated by inhibiting prostaglandin synthesis by preventing the cyclooxygenase activity of prostaglandin H synthase [1], [2]. However, side-effects of the administration of IM are damage to the gastric mucosa [3], [4] and liver mitochondria [5], [6]. Oxygen radicals and lipid peroxidation may be involved in the gastric mucosal damage induced by IM [7], [8], [9]. However, the exact role of IM in lipid peroxidation remains to be clarified.

Through its cyclooxygenase activity, prostaglandin H synthase catalyzes the bis-dioxygenation of arachidonic acid to form hydroperoxy endoperoxide prostaglandin G₂, while via its hydroperoxidase activity it catalyzes the reduction of the hydroperoxide groups of the endoperoxide. During this enzyme-catalyzed reduction of the endoperoxide, many xenobiotics are oxidized, via the hydroperoxidase activity, through a one-electron transfer [10], [11], [12]. Similarly, other peroxidases, such as lactoperoxidase, myeloperoxidase, and HRP, can also oxidize various drugs [13], [14], [15], [16], [17]. Neutrophils infiltrate inflammatory sites to damage the tissues through the generation of reactive oxygen species by myeloperoxidase, and they have an important role in the pathogenesis of gastric ulcers induced by NSAIDs [18]. The stomach and intestine also have large amounts of peroxidase [19]. These findings suggest that when IM acts on tissues, it may be metabolized to a free radical. The IM free radical is produced during the interaction of IM with lactoperoxidase in the presence of H₂O₂[20]. However, whether the IM radical contributes to the tissue damage induced by IM is not clear.

In this study, we show that lipid peroxidation of microsomes is induced through the formation of IM free radicals generated during the interaction of IM with HRP and H₂O₂ (HRP-H₂O₂).