Anhydroecgonine methyl ester, a cocaine pyrolysis product, contributes to cocaine-induced rat primary hippocampal neuronal death in a synergistic and time-dependent manner.

Abstract

Crack cocaine users are simultaneously exposed to volatilized cocaine and to its main pyrolysis product, anhydroecgonine methyl ester (AEME). Although the neurotoxic effects of cocaine have been extensively studied, little is known about AEME or its combination. We investigated cell death processes using rat primary hippocampal cells exposed to cocaine (2mM), AEME (1mM) and their combination (C + A), after 1, 3, 6 and 12h. Cocaine increased LC3 I after 6h and LC3 II after 12h, but reduced the percentage of cells with acid vesicles, suggesting failure in the autophagic flux, which activated the extrinsic apoptotic pathway after 12h. AEME neurotoxicity did not involve the autophagic process; rather, it activated caspase-9 after 6h and caspase-8 after 12h leading to a high percentage of cells in early apoptosis. C + A progressively reduced the percentage of undamaged cells, starting after 3h; it activated both apoptotic pathways after 6h, and was more neurotoxic than cocaine and AEME alone. Also, C + A increased the phosphorylation of p62 after 12h, but there was little difference in LC3 I or II, and a small percentage of cells with acid vesicles at all time points investigated. In summary, the present study provides new evidence for the neurotoxic mechanism and timing response of each substance alone and in combination, indicating that AEME is more than just a biological marker for crack cocaine consumption, as it may intensify and hasten cocaine neurotoxicity.