Neurotoxicity induced by environmental tobacco smoke and anhydroecgonine methyl ester, a cocaine pyrolysis product

Abstract

Neurotoxicity has been defined as any adverse effect on the structure and function of the nervous system. Thus, the aim of this study is to evaluate the effects of environmental tobacco smoke (ETS) in vivo and the effects of anhydroecgonine methyl ester (AEME) in vitro. The focus of the ETS part is synaptic plasticity and the endocannabinoid system. Mice C57/BL will be exposed to a mixture of mainstream and sidestream smoke generated from 3R4F reference research cigarettes from P3 to P14, twice a day. Animals will be euthanized in P15 (childhood), P35 (adolescence) and P65 (adulthood). It will be quantified markers of synaptic and structural plasticity; enzymes and receptors of the endocannabinoid system, in the hippocampus, cerebellum, striatum and brainstem. The project will combine several techniques such as immunoblotting, real-time PCR and immunofluorescence. Moreover, to evaluate the changes caused by exposure to ETS in a dynamic way, it will be used the Positron Emission Tomography (PET) in a longitudinal study. The AEME exposure will occur in vitro, in hippocampus primary cell culture. This study focuses on neuronal death as well as its pathways. Therefore, we will evaluate the neuron morphology with or without autophagic process, DNA damages and both apoptosis pathways of cells exposed to AEME, or cocaine or AEME and cocaine together. Death and autophagic markers will be quantified through immunoblotting and flux cytometry analysis. Furthermore, the oxygen consumption by the mitochondria will be determined using a Clark-type oxygen electrode connected to an oxygen polarograph, the DNA damage will be evaluated by HPLC-DAD and the morphological alterations will be evaluated by spectrophotometer and confocal microscopy. (AU)