Hydrogen peroxide mediates damage by xanthine and xanthine oxidase in cerebellar granule neuronal cultures

The free radical-generating system of xanthine and Xanthine Oxidase is commonly used experimentally as a source of superoxide anion, which can produce oxidative stress, leading to cellular damage and death. Models of oxidative stress are important in elucidating pathologies associated with increased levels of Reactive Oxygen Species, including stroke and neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We therefore, examined the effect of the xanthine/Xanthine Oxidase system on the viability of postnatal cerebellar granule neurones obtained from 8-day old Sprague-Dawley rat pups. Xanthine (100 microM) and Xanthine Oxidase (0.02 U/ml) applied for 1 or 6h reduced the viability of cells at 8 div assessed using the alamar blue assay, and induced morphological changes, such as shrinkage of the cell bodies and neurites. Heat-inactivation of Xanthine Oxidase resulted in complete loss of its activity. Superoxide dismutase (250 U/ml) failed to modify the damage by xanthine and Xanthine Oxidase, while catalase (250 U/ml) completely prevented it. When applied alone, Xanthine Oxidase significantly lowered cell viability, an effect that was blocked by allopurinol and catalase, but not by superoxide dismutase. The results indicate that xanthine and Xanthine Oxidase can produce predominantly hydrogen peroxide instead of the superoxide anion. Cerebellar granule cells in culture may also possess significant levels of endogenous xanthine.