ASSESSMENT OF THE ROLE OF THE GLUTATHIONE AND PENTOSE-PHOSPHATE PATHWAYS IN THE PROTECTION OF PRIMARY CEREBROCORTICAL CULTURES FROM OXIDATIVE STRESS

Reactive oxygen species have been implicated in neuronal injury associ ated with various neuropathological disorders. However, little is know n regarding the relationship between antioxidant enzyme capacity and r esultant toxicity, The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coup led to glutathione peroxidase (GPx) detoxification of hydrogen peroxid e (H2O2). PPP activity was quantified from data obtained by gas chroma tography/mass spectrometry analysis of released labeled lactate follow ing metabolic degradation of [1,6-C-13(2),6,6-H-2(2)] glucose by cereb rocortical cultures. The antioxidant capacity of these cultures was sy stematically evaluated using H2O2, tau and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H2O2 caused stimulation of PPP ac tivity in a concentration-dependent fashion from 0.25 to 22.2% and fro m 6.9 to 66.7% of glucose metabolized to lactate through the PPP, resp ectively. In the mixed cultures, chelation of iron before H2O2 exposur e was protective and resulted in a correlation between PPP saturation and toxicity, Conversely, addition of iron, inhibition of GPx, or depl etion of glutathione decreased H2O2-induced PPP stimulation and increa sed toxicity, These results implicate the Fenton reaction, reflect the pivotal role of GPx in H2O2 detoxification, and contribute to our und erstanding of the etiological role of free radicals in neuropathologic al conditions.