Peroxidase-catalyzed effects of indole-3-acetic acid and analogues on lipid membranes, DNA, and mammalian cells in vitro

This study aimed to explore the mechanisms and molecular parameters which c ontrol the cytotoxicity of derivatives of indole-3-acetic acid (IAA) when o xidatively activated by horseradish peroxidase (HRP). Lipid peroxidation wa s measured in liposomes, damage to supercoiled plasmid DNA assessed by gel electrophoresis, free radical intermediates detected by EPR following spin trapping, binding of IAA-derived products demonstrated by H-3 labelling, st able products measured by HPLC, and cytotoxicity in hamster fibroblasts mea sured by clonogenic survival. IAA, and nine analogues more easily oxidized by HRP, caused lipid peroxidation in liposomes, but not detectably in membr anes of hamster fibroblasts, and were cytotoxic after HRP activation to var ying degrees. Cytotoxicity was not correlated with activation rate. The hyd rophilic vitamin E analogue, Trolox, inhibited cytotoxicity, whereas loadin g fibroblasts with vitamin E was ineffective, consistent with an oxidative mechanism in which radical precursors to damage are intercepted by Trolox i n the aqueous phase. However, two known oxidation products were nontoxic (t he 3-carbinol and 3-aldehyde, both probably produced from 3-CH2OO. peroxyl radicals via the 3-CH2. [skatolyl] radical following decarboxylation of the radical cation). The skatolyl radical from IAA was shown by EPR with spin trapping to react with DNA; electrophoresis showed binding to occur. Treatm ent of hamster fibroblasts with 5-H-3-IAA/HRP resulted in intracellular bou nd H-3. Together with earlier results, the new data point to unknown electr ophilic oxidation products, reactive towards intracellular targets, being i nvolved in cytotoxicity of the IAA/HRP combination, rather than direct atta ck of free radicals, excited states, or membrane lipid peroxidation. (C) 19 99 Elsevier Science Inc.