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  • 1.
    Björklund, Emmelie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Larsson, Therese N. L.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Jacobsson, Stig O. P.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Fowler, Christopher J.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Ketoconazole Inhibits the Cellular Uptake of Anandamide via Inhibition of FAAH at Pharmacologically Relevant Concentrations2014Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 9, nr 1, s. e87542-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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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  • 2.
    Fowler, Christopher J
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Gustafsson, Sofia B
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Chung, Sui Chu
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Persson, Emma
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
    Jacobsson, Stig O P
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Bergh, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Targeting the endocannabinoid system for the treatment of cancer: a practical view2010Ingår i: Current Topics in Medicinal Chemistry, ISSN 1568-0266, E-ISSN 1873-4294, Vol. 10, nr 8, s. 814-827Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    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.

  • 3.
    Gustafsson, Sofia B
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Lindgren, Theres
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Jonsson, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Jacobsson, Stig OP
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Cannabinoid receptor-independent cytotoxic effects of cannabinoids in human colorectal carcinoma cells: synergism with 5-fluorouracil2009Ingår i: Cancer Chemotherapy and Pharmacology, ISSN 0344-5704, E-ISSN 1432-0843, Vol. 63, nr 4, s. 691-701Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cannabinoids (CBs) have been found to exert antiproliferative effects upon a variety of cancer cells, including colorectal carcinoma cells. However, little is known about the signalling mechanisms behind the antitumoural effect in these cells, whether the effects are shared by endogenous lipids related to endocannabinoids, or whether such effects are synergistic with treatment paradigms currently used in the clinic. The aim of this preclinical study was to investigate the effect of synthetic and endogenous CBs and their related fatty acids on the viability of human colorectal carcinoma Caco-2 cells, and to determine whether CB effects are synergistic with those seen with the pyrimidine antagonist 5-fluorouracil (5-FU). The synthetic CB HU 210, the endogenous CB anandamide, the endogenous structural analogue of anandamide, N-arachidonoyl glycine (NAGly), as well as the related polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid showed antiproliferative and cytotoxic effects in the Caco-2 cells, as measured by using [3H]-thymidine incorporation assay, the CyQUANT proliferation assay and calcein-AM fluorescence. HU 210 was the most potent compound examined, followed by anandamide, whereas NAGly showed equal potency and efficacy as the polyunsaturated fatty acids. Furthermore, HU 210 and 5-FU produced synergistic effects in the Caco-2 cells, but not in the human colorectal carcinoma cell lines HCT116 or HT29. The compounds examined produced cytotoxic, rather than antiproliferative effects, by a mechanism not involving CB receptors, since the CB receptor antagonists AM251 and AM630 did not attenuate the effects, nor did pertussis toxin. However, α-tocopherol and the nitric oxide synthase inhibitor L-NAME attenuated the CB toxicity, suggesting involvement of oxidative stress. It is concluded that the CB system may provide new targets for the development of drugs to treat colorectal cancer.

  • 4.
    Gustafsson, Sofia B
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Palmqvist, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Henriksson, Maria L
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Dahlin, Anna M
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Edin, Sofia
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap.
    Jacobsson, Stig OP
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Öberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap.
    Fowler, Christopher J
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    High tumour cannabinoid CB(1) receptor immunoreactivity negatively impacts disease-specific survival in stage II microsatellite stable colorectal cancer2011Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 6, nr 8, s. 1-11Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: There is good evidence in the literature that the cannabinoid system is disturbed in colorectal cancer. In the present study, we have investigated whether CB(1) receptor immunoreactive intensity (CB(1)IR intensity) is associated with disease severity and outcome.

    Methodology/Principal Findings: CB(1)IR was assessed in formalin-fixed, paraffin-embedded specimens collected with a consecutive intent during primary tumour surgical resection from a series of cases diagnosed with colorectal cancer. Tumour centre (n = 483) and invasive front (n = 486) CB(1)IR was scored from 0 (absent) to 3 (intense staining) and the data was analysed as a median split i.e. CB(1)IR <2 and >= 2. In microsatellite stable, but not microsatellite instable tumours (as adjudged on the basis of immunohistochemical determination of four mismatch repair proteins), there was a significant positive association of the tumour grade with the CB1IR intensity. The difference between the microsatellite stable and instable tumours for this association of CB(1)IR was related to the CpG island methylation status of the cases. Cox proportional hazards regression analyses indicated a significant contribution of CB(1)IR to disease-specific survival in the microsatellite stable tumours when adjusting for tumour stage. For the cases with stage II microsatellite stable tumours, there was a significant effect of both tumour centre and front CB(1)IR upon disease specific survival. The 5 year probabilities of event-free survival were: 8565 and 66+/-8%; tumour interior, 86+/-4% and 63+/-8% for the CB(1)IR<2 and CB(1)IR >= 2 groups, respectively.

    Conclusions/Significance: The level of CB(1) receptor expression in colorectal cancer is associated with the tumour grade in a manner dependent upon the degree of CpG hypermethylation. A high CB(1)IR is indicative of a poorer prognosis in stage II microsatellite stable tumour patients.

  • 5.
    Gustafsson, Sofia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Ghasimi, Soma
    Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
    Popova, Dina
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Krzemien, J
    Wallenius, Anders
    Jacobsson, Stig OP
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    The effects of cannabinoids on the viability and differentiation of neurons derived from retinoic acid-induced  P19 embryonal carcinoma cellsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Cannabinoids and cannabinoid receptors play an important role in development and differentiation of the nervous system, but the mechanisms behind that role have not been fully elucidated. We have examined the effects of synthetic and endogenous cannabinoids and related polyunsaturated fatty acids upon mouse embryonal carcinoma P19 stem cell viability - before, during and after retinoic acid (RA)-induced neural differentiation. Experiments were also performed to investigate whether the cannabinoids affect the differentiation of P19-derived neurons by measuring the development and growth of neurites and intracellular acetylcholinesterase activity.

    Both synthetic and endogenous cannabinoids as well as related fatty acids produced a concentration-dependent decrease in undifferentiated P19 cell viability, but induction of the neural pathway reduced the sensitivity to the cytotoxic effects, and in differentiated neurons anandamide and related fatty acids showed no cytotoxicity. However, synthetic cannabinoids such as HU 210, HU 211 and WIN 55,212-2 produced cytotoxicity in both undifferentiated and differentiated cells, but there was a right-shifted concentration-effect curve in RA-induced cells and differentiated neurons compared with the undifferentiated cells.

    HU 210 produced a time- and concentration-dependent decrease in cell number, percentage of cells expressing neurites, number of neurites per cell and neurite length. Statistically significant inhibition was seen at a concentration of 1 µM to 3 µM, and this was confirmed by the measurement of intracellular acetylcholinesterase activity, an enzyme that is dramatically increased during the differentiation process, where HU 210 significantly decreased the activity after six and nine days of exposure. However, these effects of HU 210 could only be observed in the same concentration range as those affecting neuronal viability. Anandamide, on the other hand, had modest effect on measured markers of neuronal differentiation but decreased the fraction of neurite expressing cells and neurite length after nine days of exposure at a concentration ≥ 10 µM. No effect on the acetylcholinesterase activity was observed.

    It is concluded that cannabinoids and related fatty acids have cytotoxic effects in undifferentiated P19 embryonal carcinoma cells, but induction of the neuronal pathway reduces the sensitivity to the cytotoxic effects. The synthetic cannabinoids are more potent than the endogenous cannabinoids and fatty acids in causing cytotoxicity in differentiated neurons, but the CB-induced decrease in neurite formation and acetylcholinesterase activity in RA-induced P19-derived neurons occurs only at concentrations that cause measurable neuronal cell death. 

  • 6.
    Gustafsson, Sofia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Jacobsson, Stig OP
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Effects of cannabinoids on the development of chick embryos in ovo2019Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 9, artikel-id 13486Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have examined the effects of the synthetic cannabinoids HU 210 and HU 211, the plant-derived cannabidiol and the endogenous cannabinoid anandamide on the viability and development of chick embryos. Fertilized White Leghorn chicken eggs were injected with the test compounds or carrier vehicle, via a drilled small hole in the egg, directly into the egg yolk. After nine days of exposure, the embryonal viability, length and wet weight of embryos, and wet weight of brains were measured, and the development stages were assessed according to the Hamburger and Hamilton (HH) scale. The potent synthetic cannabinoid receptor agonist HU 210 and the non-psychotropic cannabidiol were embryotoxic at the highest concentrations examined (10µM and 50µM, respectively), with no viable embryos after the HU 210 injection, and 20% viability after the cannabidiol injections. The efects of HU 210 on the chick embryo were attenuated by α-tocopherol and the cannabinoid receptor antagonist AM251, whereas only α-tocopherol gave a statistically signifcant protection against the embryotoxic efects of cannabidiol. This study shows that exposure to plant-derived or synthetic cannabinoids during early embryonal development decreases embryonal viability. Extrapolation of data across species is of course difcult, but the data would argue against the use of cannabinoids, be it recreationally or therapeutically, during pregnancy

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  • 7.
    Gustafsson, Sofia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Wallenius, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Zackrisson, H
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Popova, Dina
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Plym Forshell, Linus
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Jacobsson, Stig OP
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells2013Ingår i: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 87, nr 11, s. 1939-1951Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Compounds acting on the cannabinoid (CB) receptors are involved in the control of cell fate, and there is an emerging consensus that CBs have anticancer effects. However, the CB-mediated effects are contradictory since some studies suggest stimulatory effects on cancer cell proliferation, and CBs have been shown to stimulate both proliferation and differentiation of other mitotic cells such as stem and progenitor cells. In this study, the concentration-dependent effects of synthetic and endogenous CBs on the viability of mouse P19 embryonal carcinoma (EC) cells have been examined by using fluorescence assays of cell membrane integrity, cell proliferation, oxidative stress, and detection of apoptosis and necrosis. All compounds examined produced a concentration-dependent decrease in cell viability in the micromolar range, with the potent CB receptor agonist HU 210 and the enantiomer HU 211(with no CB receptor activity) being the most potent compounds examined with apparent IC50 values of 1 µM and 0.6 µM, respectively. The endogenous CB anandamide showed similar potency and efficacy as structurally related polyunsaturated fatty acids with no reported activity at the CB receptors. The rapid (within hours) decrease in cell viability induced by the examined CBs suggests cytocidal rather than antiproliferative effects, and is dependent on the plating cell population density with the highest toxicity around 100 cells/mm2. The CB-induced cytotoxicity, that appears to involve CB receptors and the sphingomyelin-ceramide pathway, is a mixture of both apoptosis and necrosis that can be blocked by the antioxidants α-tocopherol and N-acetylcysteine. In conclusion, both synthetic and endogenous CBs, produce seemingly unspecific cytotoxic effects in the P19 EC cells.

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    Paper III
  • 8.
    Popova, Dina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Forsblad, Andréas
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Hashemian, Sanaz
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Jacobsson, Stig O. P.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells2016Ingår i: PLOS ONE, E-ISSN 1932-6203, Vol. 11, nr 11, artikel-id e0166750Artikel i tidskrift (Refereegranskat)
    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.

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  • 9.
    Popova, Dina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Forsblad, Andréas N. P.
    Umeå universitet.
    Jacobsson, Stig O. P.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    In vitro studies on the neurotoxic effects of piperazine-derived designer drugs and 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy")2013Ingår i: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 221, nr Suppl. S, s. S151-S151Artikel i tidskrift (Övrigt vetenskapligt)
  • 10.
    Popova, Dina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Jacobsson, Stig O. P.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin2014Ingår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 28, nr 3, s. 411-418Artikel i tidskrift (Refereegranskat)
    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.

  • 11.
    Popova, Dina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Karlsson, Jessica
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Jacobsson, Stig O. P.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity2017Ingår i: BMC Pharmacology & Toxicology, E-ISSN 2050-6511, Vol. 18, artikel-id 42Artikel i tidskrift (Refereegranskat)
    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 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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  • 12.
    Popova, Dina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Karlsson, Jessica
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Stig O.P., Jacobsson
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicityManuskript (preprint) (Övrigt vetenskapligt)
    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.

  • 13.
    Popova, Dina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Stig O.P., Jacobsson
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    In vitro toxicity of piperazine-derived designer drugs in differentiated neural cell linesManuskript (preprint) (Övrigt vetenskapligt)
    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.

  • 14.
    Rodriguez-Gaztelumendi, Antonio
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Alvehus, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Andersson, Therése
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Jacobsson, Stig
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
    Comparison of the effects of nicotine upon the transcellular electrical resistance and sucrose permeability of human ECV304/rat C6 co-cultures and human CaCo2 cells2011Ingår i: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 207, nr 1, s. 1-6Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    It is now well established that nicotine adversely affects the integrity of the blood–brain barrier (BBB). In contrast, nicotine has been reported to increase the transendothelial electrical resistance (TEER) of CaCo2 colon cancer cells. In the present study, the effects of nicotine upon the TEER and sucrose permeability of ECV304/C6 co-cultures and, for comparative purposes, CaCo2 cells has been investigated. Neither ECV304 nor C6 cells were found to express measurable membrane levels of nicotinic acetylcholine receptors, as assessed by [3H]–epibatidine binding. Nicotine treatment (0.01–1 µM) for up to 48 h had little or no effect upon the TEER or sucrose permeability of either ECV304/C6 co-cultures or CaCo2 cells. It is concluded that in contrast to the situation for the BBB, ECV304 cells lack nicotinic acetylcholine receptors and the barrier properties of ECV304/C6 co-cultures are not affected to any important extent by nicotine. This study underlines the conclusions made by other authors that the ECV304/C6 co-culture system is of limited validity as a model of the BBB.

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