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Effects of amphetamine on neurotransmitter release in PC12 and chromaffin cells
Abstracts / Toxicology Letters 196S (2010) S37–S351
Exposure of PC12 cells to BDE-47 increased fluctuations in basal [Ca2+]i, whereas the investigated OH-BDEs induced a robust increase in basal [Ca2+]i via Ca2+ release from intracellular stores, in some cases combined with influx of extracellular Ca2+. However, HBCD did not induce any changes in basal [Ca2+]i. After 20-min pre-exposure, BDE-47 moderately increased depolarization-evoked [Ca2+]i, while OH-BDEs dose-dependently inhibited depolarization-evoked [Ca2+]i. This inhibition was potentiated by a preceding increase in basal [Ca2+]i. Large increases in basal [Ca2+]i at high concentrations of OH-BDEs strongly inhibited depolarization-evoked [Ca2+]i. Importantly, exposure of PC12 cells to the technical HBCD mixture also induced a dose-dependent inhibition of depolarization-evoked [Ca2+]i. The ã-HBCD stereoisomer inhibited the depolarization-evoked [Ca2+]i more than the technical mixture or other stereoisomers. Using specific blockers it was shown that the inhibitory effect of HBCD is not specific for a voltagegated Ca2+ channel subtype. The combined data demonstrate that OH-BDEs increase basal [Ca2+]i and reduce depolarization-evoked [Ca2+]i, indicating an increased neurotoxic potency by oxidative metabolism. Although HBCD does not increase basal [Ca2+]i, it reduces depolarization-evoked [Ca2+]i with important consequences for neurotransmission. Considering possible additivity with other environmental chemicals that disturb Ca2+ homeostasis, e.g., OHPBDE and polychlorinated biphenyls, the safety of HBCD as an alternative BFR is questionable. doi:10.1016/j.toxlet.2010.03.747
P207-020 Effects of amphetamine on neurotransmitter release in PC12 and chromaffin cells L. Hondebrink 1 , J.T. Timmerman 1 , M. Berg Van Den 1 , J. Meulenbelt 2 , R. Westerink 1 1 Utrecht University, Institute for Risk Assessment Sciences, Netherlands, 2 The National Poisons Information Centre, The National Institute for Public health and Environment, Bilthoven, Netherlands
Amphetamine increases dopamine brain levels mainly via reversal of the dopamine transporter (DAT). The widely accepted theory that amphetamine also causes leakage of dopamine and Ca2+ from intracellular vesicles is based on a few studies. The object of our study was to verify this theory using amperometry and Fura 2 imaging to measure amphetamine-induced changes in exocytosis and intracellular Ca2+ levels, respectively, in PC12 and chromaffin cells. Unexpectedly, exposure to 20 or 200 M d- or dl-amphetamine for 15 min did not decrease vesicular dopamine content or evoke exocytosis in PC12 cells or chromaffin cells. Exposure to amphetamine for 45 min or at 37 ◦ C resulted in comparable results. Additional [Ca2+]i measurements showed no amphetamine induced vesicular Ca2+ release. L DOPA pre treatment increased vesicle content up to ∼140% and a subsequent saline exposure further increased content with ∼10%. However, when these l-DOPA pre-treated cells were exposed to amphetamine, vesicle content dropped with ∼20%, indicating that under these specific conditions amphetamine can cause vesicle leakage. Thus, amphetamine exposure does neither affect the release frequency nor the amount of dopamine released per vesicle as evidenced by amperometry in both PC12 and chromaffin cells. This is supported by the Ca2+ data which do not show [Ca2+]i changes. In PC12 cells, most dopamine is tightly bound to matrix molecules of the large dense core vesicles. l-DOPA pre treatment likely increases the amount of unbound, freely available dopamine.
S223
We propose that amphetamine can induce vesicular dopamine leakage only when dopamine is not bound to the intravesicular matrix. Though the evidence for amphetamine-mediated DAT reversal appears solid, the present findings argue for re-evaluation of the widely accepted amphetamine hypothesis with respect to vesicular dopamine leakage. doi:10.1016/j.toxlet.2010.03.748
P207-021 Glycogen synthase kinase 3 beta (GSK3beta) as a promising target to prevent beta-amyloide and tau toxicity in Alzheimer disease B. Fauconneau, A. Noel, L. Barrier, G. Prie, S. Ingrand University of Poitiers, France Alzheimer disease (AD) is the most common form of dementia affecting approximately 5% of adults over 65 years. According to the aging of the population, this disease has alarming social and economic consequences. Thus, currently, one of the most important challenges is to find a way to treat this pathology. Glycogen synthase kinase 3beta; (GSK3beta) represents a promising target in the therapeutic strategy of AD. Indeed, GSK3beta is a serine/threonine kinase implicated in AD’s hallmarks: (i) Tau abnormal and hyperphosphorylation which composes neurofibrillary tangles, (ii) beta amyloid (abeta) peptide production, the chief constituent of amyloid plaques and (iii) neuronal death by apoptosis. Lithium chloride (LiCl) at millimolar range was largely used for GSK3beta inhibition. To date, more selective and potent inhibitors are available. In this work, we compared the effects of AR-A014418, a selective GSK3beta inhibitor, with those of LiCl, on GSK3beta expression in both neuroblastoma cell line SH-SY5Y exposed to abeta 1-42 and SH-SY5Y overproducing human APP695. We also determined the effects of this inhibition on Tau phosphorylation, Amyloid precursor protein (APP) degradation and cell death. Contrary to LiCl, we found that AR-A014418 decreases phosphorylation of GSK3beta both at the S9 and Y216 epitopes. Concerning the physiopathological hallmarks of AD, we found that AR-A014418 and LiCl have complementary actions. Indeed, AR-A014418 inhibits pathological Tau phosphorylation and APP processing whereas LiCl prevents apoptosis better than AR-A014418. doi:10.1016/j.toxlet.2010.03.749
P207-022 Neurobehavioural toxicity of a 14-day exposure to the airborne polycyclic aromatic hydrocarbon fluorene in adult Wistar male rats J. Peiffer 1 , F. Cosnier 2 , S. Wagner 1 , M.J. Decret 2 , B. Cossec 2 , G. Rychen 1 , H. Schroeder 1 1 UR AFPA - Unité de Recherche Animal et Fonctionnalités des Produits Animaux, France, 2 INRS - Institut National de Recherche et de Sécurité, France
Fluorene is a volatile polycyclic aromatic hydrocarbon (PAH) with an airborne concentration ranging a level of 5 ng/m3 . Since no data on fluorene neurotoxicity are available, a study on adult rats was conducted in order to evaluate the neurobehavioural effects of a repeated inhalation exposure. For that, male rats (n = 12/group) were exposed nose-only to 1.5 (10 g/m3 ) or 150 (1 mg/m3 ) ppb
Exposure of PC12 cells to BDE-47 increased fluctuations in basal [Ca2+]i, whereas the investigated OH-BDEs induced a robust increase in basal [Ca2+]i via Ca2+ release from intracellular stores, in some cases combined with influx of extracellular Ca2+. However, HBCD did not induce any changes in basal [Ca2+]i. After 20-min pre-exposure, BDE-47 moderately increased depolarization-evoked [Ca2+]i, while OH-BDEs dose-dependently inhibited depolarization-evoked [Ca2+]i. This inhibition was potentiated by a preceding increase in basal [Ca2+]i. Large increases in basal [Ca2+]i at high concentrations of OH-BDEs strongly inhibited depolarization-evoked [Ca2+]i. Importantly, exposure of PC12 cells to the technical HBCD mixture also induced a dose-dependent inhibition of depolarization-evoked [Ca2+]i. The ã-HBCD stereoisomer inhibited the depolarization-evoked [Ca2+]i more than the technical mixture or other stereoisomers. Using specific blockers it was shown that the inhibitory effect of HBCD is not specific for a voltagegated Ca2+ channel subtype. The combined data demonstrate that OH-BDEs increase basal [Ca2+]i and reduce depolarization-evoked [Ca2+]i, indicating an increased neurotoxic potency by oxidative metabolism. Although HBCD does not increase basal [Ca2+]i, it reduces depolarization-evoked [Ca2+]i with important consequences for neurotransmission. Considering possible additivity with other environmental chemicals that disturb Ca2+ homeostasis, e.g., OHPBDE and polychlorinated biphenyls, the safety of HBCD as an alternative BFR is questionable. doi:10.1016/j.toxlet.2010.03.747
P207-020 Effects of amphetamine on neurotransmitter release in PC12 and chromaffin cells L. Hondebrink 1 , J.T. Timmerman 1 , M. Berg Van Den 1 , J. Meulenbelt 2 , R. Westerink 1 1 Utrecht University, Institute for Risk Assessment Sciences, Netherlands, 2 The National Poisons Information Centre, The National Institute for Public health and Environment, Bilthoven, Netherlands
Amphetamine increases dopamine brain levels mainly via reversal of the dopamine transporter (DAT). The widely accepted theory that amphetamine also causes leakage of dopamine and Ca2+ from intracellular vesicles is based on a few studies. The object of our study was to verify this theory using amperometry and Fura 2 imaging to measure amphetamine-induced changes in exocytosis and intracellular Ca2+ levels, respectively, in PC12 and chromaffin cells. Unexpectedly, exposure to 20 or 200 M d- or dl-amphetamine for 15 min did not decrease vesicular dopamine content or evoke exocytosis in PC12 cells or chromaffin cells. Exposure to amphetamine for 45 min or at 37 ◦ C resulted in comparable results. Additional [Ca2+]i measurements showed no amphetamine induced vesicular Ca2+ release. L DOPA pre treatment increased vesicle content up to ∼140% and a subsequent saline exposure further increased content with ∼10%. However, when these l-DOPA pre-treated cells were exposed to amphetamine, vesicle content dropped with ∼20%, indicating that under these specific conditions amphetamine can cause vesicle leakage. Thus, amphetamine exposure does neither affect the release frequency nor the amount of dopamine released per vesicle as evidenced by amperometry in both PC12 and chromaffin cells. This is supported by the Ca2+ data which do not show [Ca2+]i changes. In PC12 cells, most dopamine is tightly bound to matrix molecules of the large dense core vesicles. l-DOPA pre treatment likely increases the amount of unbound, freely available dopamine.
S223
We propose that amphetamine can induce vesicular dopamine leakage only when dopamine is not bound to the intravesicular matrix. Though the evidence for amphetamine-mediated DAT reversal appears solid, the present findings argue for re-evaluation of the widely accepted amphetamine hypothesis with respect to vesicular dopamine leakage. doi:10.1016/j.toxlet.2010.03.748
P207-021 Glycogen synthase kinase 3 beta (GSK3beta) as a promising target to prevent beta-amyloide and tau toxicity in Alzheimer disease B. Fauconneau, A. Noel, L. Barrier, G. Prie, S. Ingrand University of Poitiers, France Alzheimer disease (AD) is the most common form of dementia affecting approximately 5% of adults over 65 years. According to the aging of the population, this disease has alarming social and economic consequences. Thus, currently, one of the most important challenges is to find a way to treat this pathology. Glycogen synthase kinase 3beta; (GSK3beta) represents a promising target in the therapeutic strategy of AD. Indeed, GSK3beta is a serine/threonine kinase implicated in AD’s hallmarks: (i) Tau abnormal and hyperphosphorylation which composes neurofibrillary tangles, (ii) beta amyloid (abeta) peptide production, the chief constituent of amyloid plaques and (iii) neuronal death by apoptosis. Lithium chloride (LiCl) at millimolar range was largely used for GSK3beta inhibition. To date, more selective and potent inhibitors are available. In this work, we compared the effects of AR-A014418, a selective GSK3beta inhibitor, with those of LiCl, on GSK3beta expression in both neuroblastoma cell line SH-SY5Y exposed to abeta 1-42 and SH-SY5Y overproducing human APP695. We also determined the effects of this inhibition on Tau phosphorylation, Amyloid precursor protein (APP) degradation and cell death. Contrary to LiCl, we found that AR-A014418 decreases phosphorylation of GSK3beta both at the S9 and Y216 epitopes. Concerning the physiopathological hallmarks of AD, we found that AR-A014418 and LiCl have complementary actions. Indeed, AR-A014418 inhibits pathological Tau phosphorylation and APP processing whereas LiCl prevents apoptosis better than AR-A014418. doi:10.1016/j.toxlet.2010.03.749
P207-022 Neurobehavioural toxicity of a 14-day exposure to the airborne polycyclic aromatic hydrocarbon fluorene in adult Wistar male rats J. Peiffer 1 , F. Cosnier 2 , S. Wagner 1 , M.J. Decret 2 , B. Cossec 2 , G. Rychen 1 , H. Schroeder 1 1 UR AFPA - Unité de Recherche Animal et Fonctionnalités des Produits Animaux, France, 2 INRS - Institut National de Recherche et de Sécurité, France
Fluorene is a volatile polycyclic aromatic hydrocarbon (PAH) with an airborne concentration ranging a level of 5 ng/m3 . Since no data on fluorene neurotoxicity are available, a study on adult rats was conducted in order to evaluate the neurobehavioural effects of a repeated inhalation exposure. For that, male rats (n = 12/group) were exposed nose-only to 1.5 (10 g/m3 ) or 150 (1 mg/m3 ) ppb