title: Toxicity of the amphetamine metabolites 4-hydroxyamphetamine and 4-hydroxynorephedrine in human dopaminergic differentiated SH-SY5Y cells.
abstract: Amphetamine (AMPH) is a psychostimulant used worldwide by millions of patients in the clinical treatment of attention deficit hyperactivity disorder, narcolepsy or even obesity, and is also a drug of abuse. 4-Hydroxynorephedrine (4-OHNE) and 4-hydroxyamphetamine (4-OHAMPH) are two major metabolites known to persist in the brain longer than AMPH. The contribution of AMPH metabolites for its neurotoxicity is undetermined. We evaluated the toxicity of AMPH and its metabolites 4-OHNE and 4-OHAMPH, obtained by chemical synthesis, in human dopaminergic differentiated SH-SY5Y neurons. Cells were exposed to AMPH (concentration range 0-5mM) or 4-OHAMPH or 4-OHNE (concentration range 0-10mM) for 24 or 48h, and the viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) leakage assays. Results showed that for both AMPH and the metabolites a concentration-dependent toxicity was observed. The toxic concentration 50% (TC50) for AMPH and 4-OHNE following 24h exposure was circa 3.5mM and 8mM, respectively. For 4-OHAMPH the TC50was not reached in the tested concentration range. N-acetyl cysteine, cycloheximide, l-carnitine, and methylphenidate were able to reduce cell death induced by AMPH TC50. Acridine orange/ethidium bromide staining showed evident signs of late apoptotic cells and necrotic cells following 24h exposure to AMPH 3.50mM. The 4-OHAMPH metabolite at 8.00mM originated few late apoptotic cells, whereas 4-OHNE at 8.00mM resulted in late apoptotic cells and necrotic cells, in a scenario similar to AMPH. In conclusion, the AMPH metabolite 4-OHNE is more toxic than 4-OHAMPH, nonetheless both are less toxic than the parent compound in vitro. The most toxic metabolite 4-OHNE has longer permanence in the brain, rendering likely its contribution for AMPH neurotoxicity.
authors.affiliation: UCIBIO/REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Portugal. Electronic address: ritaffeio@gmail.com.
authors.forename: R
authors.initials: R
authors.lastname: Feio-Azevedo
authors.affiliation: UCIBIO/REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Portugal.
authors.forename: V M
authors.initials: VM
authors.lastname: Costa
authors.affiliation: LAQV, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, Portugal.
authors.forename: L M
authors.initials: LM
authors.lastname: Ferreira
authors.affiliation: LAQV, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, Portugal.
authors.forename: P S
authors.initials: PS
authors.lastname: Branco
authors.affiliation: Instituto de Farmacologia e Terapêutica Experimental/Instituto de Imagem Biomédica e Ciências da Vida (IBILI), Faculdade de Medicina, Universidade de Coimbra, Portugal.
authors.forename: F C
authors.initials: FC
authors.lastname: Pereira
authors.affiliation: UCIBIO/REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Portugal.
authors.forename: M L
authors.initials: ML
authors.lastname: Bastos
authors.affiliation: UCIBIO/REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Portugal.
authors.forename: F
authors.initials: F
authors.lastname: Carvalho
authors.affiliation: UCIBIO/REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Portugal; FP-ENAS (Unidade de Investigação UFP em Energia, Ambiente e Saúde), CEBIMED (Centro de Estudos em Biomedicina), Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Portugal. Electronic address: joaocapela@ff.up.pt.
authors.forename: J P
authors.initials: JP
authors.lastname: Capela
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000661:Amphetamine / Q000737:chemistry / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D002331:Carnitine / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D059290:Dopaminergic Neurons / Q000166:cytology / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007928:Lethal Dose 50; D008774:Methylphenidate / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000737:chemistry; D010136:p-Hydroxyamphetamine / Q000737:chemistry / Q000633:toxicity*; D010137:p-Hydroxynorephedrine / Q000737:chemistry / Q000633:toxicity*
chemical_list: D017382:Reactive Oxygen Species; D008774:Methylphenidate; D010137:p-Hydroxynorephedrine; D003513:Cycloheximide; D000661:Amphetamine; D010136:p-Hydroxyamphetamine; D002331:Carnitine; D000111:Acetylcysteine
keywords: 4-hydroxyamphetamine; 4-hydroxynorephedrine; Amphetamine; Human dopaminergic differentiated SH-SY5Y cells; Toxicity