Antioxidants J811 and 17 beta-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death

Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well-known neurotoxicant contaminat ing the environment. In the present study we report that doses of MeHg rang ing from 0.1 muM to 1.5 muM activated apoptosis, as shown by cell shrinkage , nuclear condensation, and formation of high-molecular-weight DNA fragment s. Nevertheless, caspase-3-like activity was not significantly induced, and the broad caspase inhibitor Z-VAD-FMK was not capable of protecting the ce lls. This argues for a minor role of caspases in the intracellular pathways leading to MeHg-induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase-3 and alpha -fodrin wer e increased consistently in samples exposed to MeHg, pointing to a substant ial activation of calpain. Notably, two antioxidants, 17 beta -estradiol (1 0 muM) and the Delta (8,9)-dehydro derivative of 17 alpha -estradiol J811 ( 10 muM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings undersco re the key role of oxidative stress in MeHg toxicity, placing it upstream o f calpain activation. The shielding effect of the 17 alpha -estradiol and t he radical scavenger J811 is potentially relevant for the development of th erapeutic strategies for MeHg intoxication. (C) 2000 Wiley-Liss, Inc.