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| 2. |
- Björklund, Emmelie, et al.
(författare)
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Ketoconazole Inhibits the Cellular Uptake of Anandamide via Inhibition of FAAH at Pharmacologically Relevant Concentrations
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:1, s. e87542-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The antifungal compound ketoconazole has, in addition to its ability to interfere with fungal ergosterol synthesis, effects upon other enzymes including human CYP3A4, CYP17, lipoxygenase and thromboxane synthetase. In the present study, we have investigated whether ketoconazole affects the cellular uptake and hydrolysis of the endogenous cannabinoid receptor ligand anandamide (AEA). Methodology/Principal Findings: The effects of ketoconazole upon endocannabinoid uptake were investigated using HepG2, CaCo2, PC-3 and C6 cell lines. Fatty acid amide hydrolase (FAAH) activity was measured in HepG2 cell lysates and in intact C6 cells. Ketoconazole inhibited the uptake of AEA by HepG2 cells and CaCo2 cells with IC50 values of 17 and 18 mu M, respectively. In contrast, it had modest effects upon AEA uptake in PC-3 cells, which have a low expression of FAAH. In cell-free HepG2 lysates, ketoconazole inhibited FAAH activity with an IC50 value (for the inhibitable component) of 34 mu M. Conclusions/Significance: The present study indicates that ketoconazole can inhibit the cellular uptake of AEA at pharmacologically relevant concentrations, primarily due to its effects upon FAAH. Ketoconazole may be useful as a template for the design of dual-action FAAH/CYP17 inhibitors as a novel strategy for the treatment of prostate cancer.
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| 3. |
- De Lago, Eva, et al.
(författare)
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Acyl-based anandamide uptake inhibitors cause rapid toxicity to C6 glioma cells at pharmacologically relevant concentrations.
- 2006
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Ingår i: J Neurochem. - 0022-3042. ; 99:2, s. 677-88
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Tidskriftsartikel (refereegranskat)abstract
- Compounds blocking the uptake of the endogenous cannabinoid anandamide (AEA) have been used to explore the functions of the endogenous cannabinoid system in the CNS both in vivo and in vitro. In this study, the effects of four commonly used acyl-based uptake inhibitors [N-(4-hydroxyphenyl)arachidonylamide (AM404), N-(4-hydroxy-2-methylphenyl) arachidonoyl amide (VDM11), (5Z,8Z,11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707) and (9Z)-N-[1-((R)-4-hydroxybenzyl)-2-hydroxyethyl]-9-octadecen-amide (OMDM2)] and the related compound arvanil on C6 glioma cell viability were investigated. All five compounds reduced the ability of the cells to accumulate calcein, reduced the total nucleic acid content and increased the activity of lactate dehydrogenase recovered in the cell medium. AM404 (10 microm) and VDM11 (10 microm) acted rapidly, reducing cell viability after 3 h of exposure when cell densities of 5,000 per well were used. In contrast, UCM707 (30 microm), OMDM2 (10 microm) and the related compound arvanil (10 microm) produced a more slowly developing effect on cell viability, although robust effects were seen after 6-9 h of exposure. At higher cell densities, the toxicities of AM404 and UCM707 were reduced. Comparison of the compounds with arachidonic acid, arachidonic acid methyl ester, AEA, arachidonoyl glycine and oleic acid suggested that the toxicity of the arachidonoyl-based compounds was related primarily to the acyl side-chain rather than the head group. A variety of pre-treatments blocking possible metabolic pathways and receptor targets were tested, but the only consistent protective treatment against the effects of these compounds was the antioxidant N-acetyl-L-cysteine. It is concluded that AM404, VDM11, UCM707 and OMDM2 produce a rapid loss of C6 glioma cell viability over the same concentration range as is required for the inhibition of AEA uptake in vitro, albeit with a longer latency. Such effects should be kept in mind when acyl-derived compounds are used to probe the function of the endocannabinoid system in the CNS, particularly in chronic administration protocols.
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| 4. |
- Fowler, Christopher J, et al.
(författare)
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Targeting the endocannabinoid system for the treatment of cancer : a practical view
- 2010
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Ingår i: Current Topics in Medicinal Chemistry. - : Bentham Science Publishers. - 1568-0266 .- 1873-4294. ; 10:8, s. 814-827
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Forskningsöversikt (refereegranskat)abstract
- In recent years, considerable interest has been generated by findings that cannabinoids not only have useful palliative effects, but also can affect the viability and invasivity of a variety of different cancer cells. In the present review, the potential of targeting the cannabinoid system for the treatment of cancer is considered from a practical, rather than a mechanistic viewpoint, addressing questions such as whether human tumour cells express CB receptors; whether the potencies of action of cannabinoids in vitro match the potencies expected on the base of receptor theory; what is known about the in vivo effects of cannabinoids and cancer, and how relevant the experiments undertaken are to the clinical situation; and finally, what approaches can be taken to minimise unwanted effects of cannabinoid treatment. It is concluded that cannabinoids (or agents modulating the endogenous cannabinoid system) are an attractive target for drug development in the cancer area, but that more in vivo studies, particularly those investigating the potential of cannabinoids as an addition to current treatment strategies, are needed.
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| 7. |
- Popova, Dina, et al.
(författare)
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A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin
- 2014
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Ingår i: Toxicology in Vitro. - : Elsevier. - 0887-2333 .- 1879-3177. ; 28:3, s. 411-418
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Popova, Dina, et al.
(författare)
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Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity
- 2017
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Ingår i: BMC Pharmacology & Toxicology. - : BIOMED CENTRAL LTD. - 2050-6511. ; 18
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Tidskriftsartikel (refereegranskat)abstract
- 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 6 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 beta III-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 (TM) 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 mu M in the P19 neurons and 1 mu M in the SH-SY5Y cells. MeHg induced a decrease in the cellular metabolic activity and mitochondrial membrane potential (Delta Psi m) 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.
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| 9. |
- Popova, Dina, 1984-, et al.
(författare)
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Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 10. |
- Popova, Dina, 1984-, et al.
(författare)
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In vitro toxicity of piperazine-derived designer drugs in differentiated neural cell lines
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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