Toxicity of the amphetamine metabolites 4-hydroxyamphetamine and 4-hydroxynorephedrine in human dopaminergic differentiated SH-SY5Y cells

Rita Feio-Azevedo, M. E V Costa, L. M. Ferreira, P. S. Branco, F. C. Pereira, M. L. Bastos, Henrique F Carvalho, J. P. Capela

Research output: Contribution to journalArticle

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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 0–5 mM) or 4-OHAMPH or 4-OHNE (concentration range 0–10 mM) for 24 or 48 h, 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 24 h exposure was circa 3.5 mM and 8 mM, respectively. For 4-OHAMPH the TC50 was 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 24 h exposure to AMPH 3.50 mM. The 4-OHAMPH metabolite at 8.00 mM originated few late apoptotic cells, whereas 4-OHNE at 8.00 mM 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.

Original languageEnglish
Pages (from-to)65-76
Number of pages12
JournalToxicology Letters
Volume269
DOIs
Publication statusPublished - 5 Mar 2017

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p-Hydroxyamphetamine
Amphetamine
Metabolites
Toxicity
Poisons
Cells
Brain
Acetylcysteine
Narcolepsy
Acridine Orange
Methylphenidate
Ethidium
Carnitine
Street Drugs
Cell death
Cycloheximide
Attention Deficit Disorder with Hyperactivity
L-Lactate Dehydrogenase

Keywords

  • 4-hydroxyamphetamine
  • 4-hydroxynorephedrine
  • Amphetamine
  • Human dopaminergic differentiated SH-SY5Y cells
  • Toxicity

Cite this

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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 0–5 mM) or 4-OHAMPH or 4-OHNE (concentration range 0–10 mM) for 24 or 48 h, 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 24 h exposure was circa 3.5 mM and 8 mM, respectively. For 4-OHAMPH the TC50 was 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 24 h exposure to AMPH 3.50 mM. The 4-OHAMPH metabolite at 8.00 mM originated few late apoptotic cells, whereas 4-OHNE at 8.00 mM 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.",
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Toxicity of the amphetamine metabolites 4-hydroxyamphetamine and 4-hydroxynorephedrine in human dopaminergic differentiated SH-SY5Y cells. / Feio-Azevedo, Rita; Costa, M. E V; Ferreira, L. M.; Branco, P. S.; Pereira, F. C.; Bastos, M. L.; Carvalho, Henrique F; Capela, J. P.

In: Toxicology Letters, Vol. 269, 05.03.2017, p. 65-76.

Research output: Contribution to journalArticle

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T1 - Toxicity of the amphetamine metabolites 4-hydroxyamphetamine and 4-hydroxynorephedrine in human dopaminergic differentiated SH-SY5Y cells

AU - Feio-Azevedo, Rita

AU - Costa, M. E V

AU - Ferreira, L. M.

AU - Branco, P. S.

AU - Pereira, F. C.

AU - Bastos, M. L.

AU - Carvalho, Henrique F

AU - Capela, J. P.

N1 - sem pdf conforme despacho. Fundacao para a Ciencia e Tecnologia - (PTDC/SAU-FCF/102958/2008; PTDC/DTP-FTO/1489/2014) ; European Union (FEDER funds through COMPETE) - (FCOMP-01-0124-FEDER-011079; POCI-01-0145-FEDER-016537) ; FCT - (SFRH/BPD/63746/2009; SFRH/BPD/110001/2015; NORTE-01-0145-FEDER-000024)

PY - 2017/3/5

Y1 - 2017/3/5

N2 - 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 0–5 mM) or 4-OHAMPH or 4-OHNE (concentration range 0–10 mM) for 24 or 48 h, 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 24 h exposure was circa 3.5 mM and 8 mM, respectively. For 4-OHAMPH the TC50 was 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 24 h exposure to AMPH 3.50 mM. The 4-OHAMPH metabolite at 8.00 mM originated few late apoptotic cells, whereas 4-OHNE at 8.00 mM 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.

AB - 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 0–5 mM) or 4-OHAMPH or 4-OHNE (concentration range 0–10 mM) for 24 or 48 h, 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 24 h exposure was circa 3.5 mM and 8 mM, respectively. For 4-OHAMPH the TC50 was 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 24 h exposure to AMPH 3.50 mM. The 4-OHAMPH metabolite at 8.00 mM originated few late apoptotic cells, whereas 4-OHNE at 8.00 mM 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.

KW - 4-hydroxyamphetamine

KW - 4-hydroxynorephedrine

KW - Amphetamine

KW - Human dopaminergic differentiated SH-SY5Y cells

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JF - Toxicology Letters

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