title: Toxicological impact of JWH-018 and its phase I metabolite N-(3-hydroxypentyl) on human cell lines.
abstract: The emergence and abuse of synthetic cannabinoids has been increasing as an alternative to cannabis, mainly among youth. As their appearance on the drug market has been recent, the pharmacological and toxicological profiles of these psychoactive substances are poorly understood. Current studies suggest that they have stronger effects compared to their natural alternatives and their metabolites retain affinity towards CB1 receptors in CNS. Since studies on its toxicological properties are scarce, the effects of the drug in human derived cell lines were investigated. The present study was designed to explore the toxicological impact of parent drug versus phase I metabolites of synthetic cannabinoids on human cells with and without CB1 receptor. The human cell line of neuroblastoma SH-SY5Y and human kidney cell line HEK-293T were exposed to JWH-018 and to its N-(3-hydroxypentyl) metabolite. Cell toxicity was evaluated using the MTT and LDH assay. Additionally, a dual staining methodology with fluorescent Annexin V-FITC and propidium iodide was performed to address the question of whether JWH-018 N-(3-hydroxypentyl) metabolite is inducing cell death through apoptosis or necrosis, in HEK293T and SH-SY5Y cell lines. The obtained results show that JWH-018 does not cause a statistically significant decrease in cell viability, in contrast to its N-(3-hydroxypentyl) metabolite, which at ≥25μM causes a significant decrease in cell viability. Both cell lines are affected by JWH-018 metabolite. Our results point to higher toxicity of JWH-018 metabolite when compared to its parent drug, suggesting a non-CB1 receptor mediated toxicological mechanism. Comparing the results from Annexin V/PI with MTT and LDH assays of SH-SY5Y and HEK293T in the presence of the synthetic cannabinoid metabolite, emerges the picture that cellular viability decreases and associated death is occurring through necrosis.
authors.affiliation: Centro de Investigação Interdisciplinar Egas Moniz, Instituto Superior de Ciências da Saúde Egas Moniz, 2825-084 Caparica, Portugal; Laboratório de Ciências Forenses e Psicológicas Egas Moniz, Campus Universitário-Quinta da Granja, Monte de Caparica, 2825-084 Caparica, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Couceiro
authors.affiliation: Centro de Investigação Interdisciplinar Egas Moniz, Instituto Superior de Ciências da Saúde Egas Moniz, 2825-084 Caparica, Portugal.
authors.forename: Susana
authors.initials: S
authors.lastname: Bandarra
authors.affiliation: Centro de Investigação Interdisciplinar Egas Moniz, Instituto Superior de Ciências da Saúde Egas Moniz, 2825-084 Caparica, Portugal.
authors.forename: Haider
authors.initials: H
authors.lastname: Sultan
authors.affiliation: C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV, USA.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Bell
authors.affiliation: Centro Cardiovascular da Universidade de Lisboa, Faculdade de Medicina da Universidade de Lisboa, Angiogenesis Unit, Lisboa, Portugal.
authors.forename: Susana
authors.initials: S
authors.lastname: Constantino
authors.affiliation: Centro de Investigação Interdisciplinar Egas Moniz, Instituto Superior de Ciências da Saúde Egas Moniz, 2825-084 Caparica, Portugal; Laboratório de Ciências Forenses e Psicológicas Egas Moniz, Campus Universitário-Quinta da Granja, Monte de Caparica, 2825-084 Caparica, Portugal. Electronic address: lcforenses@egasmoniz.edu.pt.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Quintas
mesh_terms: D001681:Biological Assay / Q000379:methods; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D053593:Forensic Toxicology; D057809:HEK293 Cells; D006801:Humans; D013287:Illicit Drugs / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D009281:Naphthalenes / Q000494:pharmacology*
chemical_list: D013287:Illicit Drugs; D007211:Indoles; D009281:Naphthalenes; C552597:1-pentyl-3-(1-naphthoyl)indole
keywords: HEK293T; JWH-018; JWH-018 N-(3-hydroxypentyl) metabolite; SH-SY5Y; Synthetic cannabinoids; Toxicity