PEROXIDASE-MEDIATED OXIDATION, A POSSIBLE PATHWAY FOR ACTIVATION OF THE FUNGAL NEPHROTOXIN ORELLANINE AND RELATED-COMPOUNDS - ESR AND SPIN-TRAPPING STUDIES

Orellanine is the tetrahydroxylated and di-N-oxidized bipyridine toxin extracted from several Cortinarius mushrooms among them C. orellanus. The pathogenic mechanism involved in the C. orellanus-poisoning by or ellanine leading to kidney impairment is not yet fully understood unti l now. Electron spin resonance (ESR) spectroscopy has been used to stu dy the activation of orellanine by horseradish peroxidase/H2O2 system at physiological pH. Evidence for a one-electron oxidation of the toxi n by this enzymatic system to an ortho-semiquinone radical intermediat e is presented. The orellanine ortho-semiquinone generated by the pero xidase/H2O2 system abstracts hydrogen from glutathione, generating the glutathionyl radical which is spin-trapped by 5,5'-dimethyl-1-pyrroli ne N-oxide (DMPO) and subsequently detected by ESR spectroscopy. Simil arly, the ortho-semiquinone abstracts hydrogen from ascorbic acid to g enerate the ascorbyl radical which is detected by direct ESR. The pero xidatic oxidation of orellanine to semiquinone followed by its reducti on by glutathione or ascorbic acid does not induce dioxygen uptake. Th e relationship between chemical structure and HRP oxidation of orellan ine-related molecules, namely orelline and DHBPO2 (the parent molecule lacking of hydroxyl groups in 3 and 3' position) has been investigate d in absence or in presence of reducing agents. None of the orellanine -related compounds can be oxidized by the HRP/H2O2 system, showing tha t both catecholic moieties and aminoxide groups are necessary for obse rving the formation of the ortho-semiquinone form of orellanine. As sh own for the (photo)chemical oxidation of orellanine, the mechanism of toxicity could be correlated with a depletion of glutathione and ascor bate levels which are implicated in the defence against oxidative dama ge.