Peroxidase-Mediated Oxidation, a Possible Pathway for Activation of the Fungal Nephrotoxin Orellanine and Related Compounds. ESR and Spin-Trapping Studies

Abstract

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 orellanine leading to kidney impairment is not yet fully understood until now. Electron spin resonance (ESR) spectroscopy has been used to study the activation of orellanine by horseradish peroxidase/H₂O₂ system at physiological pH. Evidence for a one-electron oxidation of the toxin by this enzymatic system to an ortho-semiquinone radical intermediate is presented. The orellanine ortho-semiquinone generated by the peroxidase/H₂O₂ system abstracts hydrogen from glutathione, generating the glutathionyl radical which is spin-trapped by 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) and subsequently detected by ESR spectroscopy. Similarly, the ortho-semiquinone abstracts hydrogen from ascorbic acid to generate the ascorbyl radical which is detected by direct ESR. The peroxidatic oxidation of orellanine to semiquinone followed by its reduction by glutathione or ascorbic acid does not induce dioxygen uptake. The relationship between chemical structure and HRP oxidation of orellanine-related molecules, namely orelline and DHBPO₂ (the parent molecule lacking of hydroxyl groups in 3 and 3′ position) has been investigated in absence or in presence of reducing agents. None of the orellanine-related compounds can be oxidized by the HRP/H₂O₂ system, showing that both catecholic moieties and aminoxide groups are necessary for observing the formation of the ortho-semiquinone form of orellanine. As shown for the (photo)chemical oxidation of orellanine, the mechanism of toxicity could be correlated with a depletion of glutathione and ascorbate levels which are implicated in the defence against oxidative damage.