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In vitro toxicity of piperazine-derived designer drugs in differentiated neural cell lines
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Piperazine derivatives are common ingredients in recreational “party pills” which are used to provide a stimulant, euphoric effect akin to that of methylenedioxymethamphetamine (MDMA, “ecstasy”). There is a potential for significant toxicity associated with the use of these compounds, and the aim of the present study was to investigate if the common piperazine derivatives N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP), 1-(4-methoxyphenyl)piperazine (MeOPP) and 1-(4-fluorophenyl)piperazine (pFPP), were toxic to retinoic acid-treated neuronally differentiated mouse P19 embryonic carcinoma stem cells and differentiated human neuroblastoma SH-SY5Y cells. Immunofluorescence of the neuron-specific protein βIII-tubulin, fluorescence of intracellular calcein, assays of mitochondrial membrane potential (∆ψm), MTT reduction and extracellular levels of LDH were used to estimate concentration-dependent cell toxicity of the piperazine derivatives and MDMA. All piperazine derivatives were toxic to the P19 neurons, but TFMPP was the most potent cytotoxic compound, producing a major decrease in mitochondrial membrane potential, cellular MTT reduction and fluorescence of calcein and βIII-tubulin, with a simultaneous increase in LDH release. The toxicity of piperazine derivatives is not restricted to differentiated P19 cells, since BZP and TFMPP were also cytotoxic in SH-SY5Y cells and human colon adenocarcinoma Caco-2 cells.

Keyword [en]
piperazine derivatives, MDMA, neuronal cell cultures, neurotoxicity
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
URN: urn:nbn:se:umu:diva-133028OAI: oai:DiVA.org:umu-133028DiVA: diva2:1085433
Available from: 2017-03-29 Created: 2017-03-29 Last updated: 2017-03-31
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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