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- Popova, Dina, 1984-
(författare)
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In vitro cellular models for neurotoxicity studies : neurons derived from P19 cells
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Gustafsson, Sofia, 1982-
(författare)
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Cannabinoids as modulators of cancer cell viability, neuronal differentiation, and embryonal development
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cannabinoids (CBs) are compounds that activate the CB1 and CB2 receptors. CB receptors mediate many different physiological functions, and cannabinoids have been reported to decrease tumor cell viability, proliferation, migration, as well as to modulate metastasis. In this thesis, the effects of cannabinoids on human colorectal carcinoma Caco-2 cells (Paper I) and mouse P19 embryonal carcinoma (EC) cells (Paper III) were studied. In both cell lines, the compounds examined produced a concentration- and time-dependent decrease in cell viability. In Caco-2-cells, HU 210 and the pyrimidine antagonist 5-fluorouracil produced synergistic effects upon cell viability. The mechanisms behind the cytocidal effects of cannabinoids appear to be mediated by other than solely the CB receptor, and a common mechanism in Caco-2 and P19 EC cells was oxidative stress. However, in P19 EC cells the CB receptors contribute to the cytocidal effects possibly via ceramide production. In paper II, the association between CB1 receptor immunoreactivity (CB1IR) and different histopathological variables and disease-specific survival of colorectal cancer (CRC) was investigated. In microsatellite stable (MSS) cases there was a significant positive association of the tumor grade with the CB1IR intensity. A high CB1IR is indicative of a poorer prognosis in MSS with stage II CRC patients. Paper IV focused on the cytotoxic effects of cannabinoids during neuronal differentiation. HU 210 affected the cell viability, neurite formation and produced a decreased intracellular AChE activity. The effects of cannabinoids on embryonic development and survival were examined in Paper V, by repeated injection of cannabinoids in fertilized chicken eggs. After 10 days of incubation, HU 210 and cannabidiol (without CB receptor affinity), decreased the viability of chick embryos, in a manner that could be blocked by α-tocopherol (antioxidant) and attenuated by AM251 (CB1 receptor antagonist). In conclusion, based on these studies, the cannabinoid system may provide a new target for the development of drugs to treat cancer such as CRC. However, the CBs also produce seemingly unspecific cytotoxic effects, and may have negative effects on the neuronal differentiation process. This may be responsible for, at least some of, the embryotoxic effects found in ovo, but also for the cognitive and neurotoxic effects of cannabinoids in the developing and adult nervous system.
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