Neuroscience Letters 416 (2007) 34–38 Hydrogen peroxide mediates damage by xanthine and xanthine oxidase in cerebellar granule neuronal cultures Amos A. Fatokun, Trevor W. Stone, Robert A. Smith ∗ Institute of Biomedical and Life Sciences, Division of Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK Received 30 November 2006; received in revised form 9 January 2007; accepted 14 January 2007 Abstract 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 M) and xanthine oxidase (0.02 U/ml) applied for 1 or 6 h 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 (250U/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. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Xanthine; Xanthine oxidase; Hydrogen peroxide; Cerebellar granule neurons; Free radicals Reactive oxygen species (ROS) induce oxidative stress in bio- logical systems, leading to alterations in the structure and function of lipids, proteins and nucleic acids and subsequently to cell damage and death [10,14,22]. These ROS include the superoxide anion (O 2 •- ), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical ( • OH), which are all produced in a wide variety of pathological conditions, although some may also be produced under physiological conditions, usually as products of oxidative metabolism in cells [2,3]. The brain is exceptionally at risk from oxidative damage because it consumes far more oxygen than any other body tissue, has low levels of antioxidants and possesses a tendency to accumulate metals, which are potent in gener- ating more toxic ROS [9]. The aetiology of most neurological conditions (e.g., stroke) and neurodegenerative diseases (e.g., Alzheimer’s and Parkinson’s) involves substantially raised lev- els of oxidants, which are critical in initiating damage, death and disease progression [1,23]. ∗ Corresponding author. Tel.: +44 141 330 6391; fax: +44 141 330 2923. E-mail address: R.A.Smith@bio.gla.ac.uk (R.A. Smith). The free radical-generating system of xanthine and xanthine oxidase, which often produces the superoxide anion [20,24], but could also generate a combination of superoxide and H 2 O 2 [28], or the • OH radical [17], has been employed in the experi- mental induction of oxidative stress. Xanthine oxidase converts xanthine to hypoxanthine and then to uric acid, liberating super- oxide [11]. The aim of this study was therefore, to investigate the ROS mediating neuronal damage and death induced by xanthine and xanthine oxidase in cerebellar granule neurones (CGNs), a cell type that has been extensively used in studying the neuro- toxic effects of agents, such as glutamate [12] and kainic acid [21] and also in investigating apoptosis [13]. Minimum essential medium (MEM), gentamicin and glutamine were obtained from GIBCO (Paisley, UK). Fetal calf serum (FCS), poly-d-lysine, DNase, trypsin, soybean trypsin inhibitor, xanthine (X), xanthine oxidase (XO), catalase (CAT), superoxide dismutase (SOD) and allopurinol (AP) were obtained from Sigma (UK). Alamar blue (AB) dye was obtained from BioSource (Nivelles, Belgium). Cerebellar gran- ule neurones were prepared from 8-day old Sprague–Dawley rat pups as earlier described [7] and maintained in MEM 0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.01.078