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Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 11, e0166750Article in journal (Refereed) Published
Abstract [en]

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) is a commonly abused recreational drug that causes neurotoxic effects in both humans and animals. The mechanism behind MDMA-induced neurotoxicity is suggested to be species-dependent and needs to be further investigated on the cellular level. In this study, the effects of MDMA in neuronally differentiated P19 mouse embryonal carcinoma cells have been examined. MDMA produces a concentration-, time- and temperature-dependent toxicity in differentiated P19 neurons, as measured by intracellular MTT reduction and extracellular LDH activity assays. The P19-derived neurons express both the serotonin reuptake transporter (SERT), that is functionally active, and the serotonin metabolizing enzyme monoamine oxidase A (MAO-A). The involvement of these proteins in the MDMA-induced toxicity was investigated by a pharmacological approach. The MAO inhibitors clorgyline and deprenyl, and the SERT inhibitor fluoxetine, per se or in combination, were not able to mimic the toxic effects of MDMA in the P19-derived neurons or block the MDMA-induced cell toxicity. Oxidative stress has been implicated in MDMA-induced neurotoxicity, but pre-treatment with the antioxidants α-tocopherol or N-acetylcysteine did not reveal any protective effects in the P19 neurons. Involvement of mitochondria in the MDMA-induced cytotoxicity was also examined, but MDMA did not alter the mitochondrial membrane potential (ΔΨm) in the P19 neurons. We conclude that MDMA produce a concentration-, time- and temperature-dependent neurotoxicity and our results suggest that the mechanism behind MDMA-induced toxicity in mouse-derived neurons do not involve the serotonergic system, oxidative stress or mitochondrial dysfunction.

Place, publisher, year, edition, pages
2016. Vol. 11, no 11, e0166750
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
URN: urn:nbn:se:umu:diva-128533DOI: 10.1371/journal.pone.0166750ISI: 000388350300099PubMedID: 27861613OAI: oai:DiVA.org:umu-128533DiVA: diva2:1052478
Available from: 2016-12-06 Created: 2016-12-06 Last updated: 2017-03-29Bibliographically approved
In thesis
1. In vitro cellular models for neurotoxicity studies: neurons derived from P19 cells
Open this publication in new window or tab >>In vitro cellular models for neurotoxicity studies: neurons derived from P19 cells
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Humans are exposed to a variety of chemicals including environmental pollutants, cosmetics, food preservatives and drugs. Some of these substances might be harmful to the human body. Traditional toxicological and behavioural investigations performed in animal models are not suitable for the screening of a large number of compounds for potential toxic effects. There is a need for simple and robust in vitro cellular models that allow high-throughput toxicity testing of chemicals, as well as investigation of specific mechanisms of cytotoxicity. The overall aim of the thesis has been to evaluate neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) as a model for such testing. The model has been compared to other cellular models used for neurotoxicity assessment: retinoic acid-differentiated human neuroblastoma SH-SY5Y cells and nerve growth factor-treated rat pheochromocytoma PC12 cells. The chemicals assessed in the studies included the neurotoxicants methylmercury, okadaic acid and acrylamide, the drug of abuse MDMA (“ecstasy”) and a group of piperazine derivatives known as “party pills”. Effects of the chemicals on cell survival, neurite outgrowth and mitochondrial function have been assessed.

In Paper I, we describe a fluorescence-based microplate method to detect chemical-induced effects on neurite outgrowth in P19 neurons immunostained against the neuron-specific cytoskeletal protein βIII-tubulin. In Paper II, we show that P19 neurons are more sensitive than differentiated SH-SY5Y and PC12 cells for detection of cytotoxic effects of methylmercury, okadaic acid and acrylamide. Additionally, in P19 neurons and differentiated SH-SY5Y cells, we could demonstrate that toxicity of methylmercury was attenuated by the antioxidant glutathione. In Paper III, we show a time- and temperature-dependent toxicity produced by MDMA in P19 neurons. The mechanisms of MDMA toxicity did not involve inhibition of the serotonin re-uptake transporter or monoamine oxidase, stimulation of 5-HT2A receptors, oxidative stress or loss of mitochondrial membrane potential. In Paper IV, the piperazine derivatives are evaluated for cytotoxicity in P19 neurons and differentiated SH-SY5Y cells. The most toxic compound in both cell models was TFMPP. In P19 neurons, the mechanism of action of TFMPP included loss of mitochondrial membrane potential. In conclusion, P19 neurons are a robust cellular model that may be useful in conjunction with other models for the assessment of chemical-induced neurotoxicity.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2017. 67 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1877
Keyword
Neurotoxicity, Neuronal cell culture, P19 cells, SH-SY5Y cells, βIII-tubulin, Methylmercury, Okadaic acid, Acrylamide, MDMA, Piperazine-derived designer drugs.
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-133030 (URN)978-91-7601-659-6 (ISBN)
Public defence
2017-04-28, Hörsal D, Unod T9, byggnad 1D, NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-04-06 Created: 2017-03-29 Last updated: 2017-04-06Bibliographically approved

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