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In vitro cellular models for neurotoxicity studies: neurons derived from P19 cells
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
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 [en]
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: urn:nbn:se:umu:diva-133030ISBN: 978-91-7601-659-6 (print)OAI: oai:DiVA.org:umu-133030DiVA: diva2:1085486
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
List of papers
1. A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin
Open this publication in new window or tab >>A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin
2014 (English)In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 28, no 3, 411-418 p.Article in journal (Refereed) Published
Abstract [en]

The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10 days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity. In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity.

Place, publisher, year, edition, pages
Elsevier, 2014
Keyword
neurotoxicity, cell culture, βIII-tubulin, beta III-tubulin, calcein-AM, fluorescence, microplate assay
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-87628 (URN)10.1016/j.tiv.2013.12.009 (DOI)000332053800010 ()
Available from: 2014-04-10 Created: 2014-04-07 Last updated: 2017-03-29Bibliographically approved
2. Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity
Open this publication in new window or tab >>Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Exposure to chemicals might be toxic to the developing brain. There is a need for simple and robust in vitro cellular models for evaluation of chemical-induced neurotoxicity as a complement to traditional studies on animals. In this study, neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) were compared with human neuroblastoma SH-SY5Y cells and rat adrenal pheochromocytoma PC12 cells for their ability to detect toxicity of methylmercury (MeHg), okadaic acid and acrylamide.

Methods: Retinoic acid-treated P19 and SH-SY5Y cells and nerve growth factor-stimulated PC12 cells, allowed to differentiate for six days, were exposed to MeHg, okadaic acid and acrylamide for 48 h. Cell survival and neurite outgrowth were assessed with the calcein-AM assay and fluorescence detection of antibodies against the cytoskeletal neuron-specific protein βIII-tubulin, respectively. The effects of glutathione (GSH) and the potent inhibitor of GSH synthesis buthionine sulfoximine (BSO) on the MeHg induced-toxicity were assessed using the PrestoBlue™ cell viability assay and the TMRE mitochondrial membrane potential assay.

Results: Differentiated P19 cells developed the most extensive neuronal network among the three cell models and were the most sensitive neuronal model to detect neurotoxic effects of the test compounds. MeHg produced a concentration-dependent toxicity in differentiated P19 cells and SH-SY5Y cells, with statistically significant effects at concentrations from 0.1 µM in the P19 neurons and 1 µM in the SH-SY5Y cells. MeHg induced a decrease in the cellular metabolic activity and mitochondrial membrane potential (ΔΨm) in the differentiated P19 cells and SH-SY5Y cells, that were attenuated by GSH. Okadaic acid and acrylamide also showed statistically significant toxicity in the P19 neurons, but not in the SH-SY5Y cells or the P12 cells.

Conclusions: P19 neurons are more sensitive to detect cytotoxicity of MeHg, okadaic acid and acrylamide than retinoic acid-differentiated SH-SY5Y cells and nerve growth factor-treated PC12 cells. P19 neurons are at least as sensitive as differentiated SH-SY5Y cells to detect the loss of mitochondrial membrane potential produced by MeHg and the protective effects of extracellular GSH on MeHg toxicity. P19 neurons may be a useful model to study neurotoxic effects of chemicals.

Keyword
neuronal cell cultures, neurotoxicity, methyl mercury, okadaic acid, acrylamide
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
urn:nbn:se:umu:diva-133027 (URN)
Available from: 2017-03-29 Created: 2017-03-29 Last updated: 2017-03-31
3. Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells
Open this publication in new window or tab >>Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells
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.

National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
urn:nbn:se:umu:diva-128533 (URN)10.1371/journal.pone.0166750 (DOI)000388350300099 ()27861613 (PubMedID)
Available from: 2016-12-06 Created: 2016-12-06 Last updated: 2017-03-29Bibliographically approved
4. In vitro toxicity of piperazine-derived designer drugs in differentiated neural cell lines
Open this publication in new window or tab >>In vitro toxicity of piperazine-derived designer drugs in differentiated neural cell lines
(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
piperazine derivatives, MDMA, neuronal cell cultures, neurotoxicity
National Category
Pharmacology and Toxicology
Research subject
Toxicology
Identifiers
urn:nbn:se:umu:diva-133028 (URN)
Available from: 2017-03-29 Created: 2017-03-29 Last updated: 2017-03-31

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