Antioxidative properties of natural coelenterazine and synthetic methyl coelenterazine in rat hepatocytes subjected to tert-butyl hydroperoxide-induced oxidative stress

Coelenterazine (CLZn; 3,7-dihydro-2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8 -benzylimidazolo[1,2-a]pyrazin-3-one), the substrate for bioluminescence re actions in many marine animals, is endowed with high antioxidant: propertie s. This work investigated the antioxidative properties of CLZn in primary c ultures of rat hepatocytes subjected to the oxidant tert-butyl hydroperoxid e (t-BHP). Micromolar concentrations of CLZn increased survival and decreas ed lipid peroxidation in rat hepatocytes subjected for 6 hr to 2.5 x 10(-4) M t-BHP. However, the extent of protection was limited by a strong toxicit y of CLZn (IC50 = 6.9 x 10(-5) M). The presence of t-BHP increased the cell ular toxicity of CLZn. Methyl coelenterazine (CLZm, 3,7-dihydro-2-methyl-6- (p-hydroxyphenyl)-8 benzylimidazolo[1,2-a] pyrazin-3-one), a synthetic anal ogue of CLZn, demonstrated excellent antioxidant properties, even at very l ow (3 x 10(-6) M) concentrations and was not toxic throughout most of its e ffective concentration range. CLZm proved far more effective than reference antioxidants such as Trolox C(R), alpha-tocopherol, BHT, and probucol. The assay of thiobarbituric reactive substances (TBARS) associated with cells and in the culture medium indicated that 10(-5) M CLZm provided a total pro tection against t-BHP-induced lipid peroxidation. This coelenterazine analo gue could be used as a model compound for investigating the action mechanis m of imidazolopyrazinones in mammalian hepatocytes. BIOCHEM PHARMACOL 60;4: 471-478, 2000. (C) 2000 Elsevier Science Inc.