title: Neurotoxicity of anhydroecgonine methyl ester, a crack cocaine pyrolysis product.
abstract: Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME).  Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaine-AEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEME's effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaine-AEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.
authors.affiliation: Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, 05508-900, São Paulo/SP, Brazil.
authors.forename: Raphael Caio Tamborelli
authors.initials: RC
authors.lastname: Garcia
authors.forename: Livia Mendonça Munhoz
authors.initials: LM
authors.lastname: Dati
authors.forename: Suelen
authors.initials: S
authors.lastname: Fukuda
authors.forename: Larissa Helena Lobo
authors.initials: LH
authors.lastname: Torres
authors.forename: Sidnei
authors.initials: S
authors.lastname: Moura
authors.forename: Nathalia Delazeri
authors.initials: ND
authors.lastname: de Carvalho
authors.forename: Daniel Carneiro
authors.initials: DC
authors.lastname: Carrettiero
authors.forename: Rosana
authors.initials: R
authors.lastname: Camarini
authors.forename: Adriana Cristina
authors.initials: AC
authors.lastname: Levada-Pires
authors.forename: Mauricio
authors.initials: M
authors.lastname: Yonamine
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Negrini-Neto
authors.forename: Fernando Maurício Francis
authors.initials: FM
authors.lastname: Abdalla
authors.forename: Maria Regina Lopes
authors.initials: MR
authors.lastname: Sandoval
authors.forename: Solange Castro
authors.initials: SC
authors.lastname: Afeche
authors.forename: Tania
authors.initials: T
authors.lastname: Marcourakis
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003042:Cocaine / Q000031:analogs & derivatives* / Q000633:toxicity; D005260:Female; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects*; D007150:Immunohistochemistry; D011247:Pregnancy; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D014316:Tritium
chemical_list: D014316:Tritium; C068464:anhydroecgonine methyl ester; D011813:Quinuclidinyl Benzilate; D003042:Cocaine