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 (2 mM), AEME (1 mM) and their combination (C + A), after 1, 3, 6 and 12 h. Cocaine increased LC3 I after 6 h and LC3 II after 12 h, but reduced the percentage of cells with acid vesicles, suggesting failure in the autophagic flux, which activated the extrinsic apoptotic pathway after 12 h. AEME neurotoxicity did not involve the autophagic process; rather, it activated caspase-9 after 6 h and caspase-8 after 12 h leading to a high percentage of cells in early apoptosis. C + A progressively reduced the percentage of undamaged cells, starting after 3 h; it activated both apoptotic pathways after 6 h, and was more neurotoxic than cocaine and AEME alone. Also, C + A increased the phosphorylation of p62 after 12 h, 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.
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Acknowledgements
The authors acknowledge Dr. Sandra Farsky and her students Lorena Pantaleão and Gustavo Rocha from the Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, for their advice on flow cytometry protocols, and Dr. Felipe Augusto Dörr, from the same institution, for his technical assistance with the drugs’ LC–MS/MS analysis.
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This research was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP—student grant: 2013/20952-6 and research support: 2014/08881-9) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq—159886/2013-9). T.M. and S.S.M.E are research fellows of CNPq.
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This study was performed according to National Institutes of Health guidelines and approved by the Animal Experimentation Ethics Committee of the School of Pharmaceutical Sciences (no. 100.2014) and School of Veterinary Medicine and Animal Science (no. 5737050814), at the University of São Paulo.
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Udo, M.S.B., da Silva, M.A.A., de Souza Prates, S. et al. Anhydroecgonine methyl ester, a cocaine pyrolysis product, contributes to cocaine-induced rat primary hippocampal neuronal death in a synergistic and time-dependent manner. Arch Toxicol 95, 1779–1791 (2021). https://doi.org/10.1007/s00204-021-03017-z
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DOI: https://doi.org/10.1007/s00204-021-03017-z