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A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
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. Vol. 28, no 3, 411-418 p.
Keyword [en]
neurotoxicity, cell culture, βIII-tubulin, beta III-tubulin, calcein-AM, fluorescence, microplate assay
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:umu:diva-87628DOI: 10.1016/j.tiv.2013.12.009ISI: 000332053800010OAI: oai:DiVA.org:umu-87628DiVA: diva2:711635
Available from: 2014-04-10 Created: 2014-04-07 Last updated: 2017-12-05Bibliographically 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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