Developmental Exposure to Di-(2-ethylhexyl) Phthalate Induces Cerebellar Granule Cell Apoptosis via the PI3K/AKT Signaling Pathway

Di-(2-ethylhexyl) phthalate (DEHP) is an ubiquitous environmental contaminant because of its extensive use in plastics and its persistence. As an environmental endocrine disruptor, it is suspected to interfere with neurodevelopment in people. However, evidence of the effects of maternal DEHP exposure on cerebellar development in offspring is scarce. The objective of this study was to investigate maternal exposure to DEHP and its effect on apoptosis of cerebellar granule cells (CGCs) and related mechanisms. Pregnant Wistar rats were administrated DEHP (0, 30, 300 and 750 mg/kg/d) by gavage from gestational day (GD) 0 to postnatal day (PN) 21. Primary CGCs were also exposed to mono-(2-ethylhexyl) phthalate (MEHP), the main metabolite of DEHP, for 24 h with concentrations of 0, 25, 100 and 250 µM. The CGCs of male offspring from 300 and 750 mg/kg/d DEHP exposure groups showed significantly increased apoptosis. In addition, the PI3K/AKT signaling pathway was inhibited in the male offspring of the 300 and 750 mg/kg/d DEHP exposure groups. However, effects on female pups were not obvious. Apoptosis was also elevated and the PI3K/AKT signaling pathway was inhibited after primary CGCs were exposed to MEHP. Furthermore, apoptosis was reduced after treatment with the PI3K/AKT signaling pathway activator, insulin-like growth factor (IGF) 1, and increased after treatment with LY294002, an inhibitor of the PI3K/AKT signaling pathway. These results suggested that maternal DEHP exposure induced apoptosis in the CGCs of male pups via the PI3K/AKT signaling pathway, and the apoptosis could be rescued by IGF1 and aggravated by LY294002.

Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway

Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway.

Chlorination of ortho-position on Polychlorinated Biphenyls Increases Protein Kinase C Activity in Neuronal Cells

Polychlorinated biphenyls (PCBs) are persistent and bioaccumulative environmental pollutants. Recently, it is suggested that neurotoxic effects such as motor dysfunction and impairment in memory and learning have been associated with PCB exposure. However, structure relationship of PCB congeners with neurotoxic effects remains unknown. Since PKC signaling pathway is implicated in the modulation of motor behavior as well as learning and memory and the role of PKC are subspecies-specific, we attempted to study the effects of structurally distinct PCBs on the total PKC activity as well as subspecies of PKC in cerebellar granule cell culture model. Cells were exposed to 0, 25 and 50 microM of PCB-126, PCB-169, PCB-114, PCB-157, PCB-52 and PCB-4 for 15 min. Cells were subsequently analyzed by [3H] phorbol ester binding assay or immunoblotted against PKC-alpha and -epsilon monoclonal antibodies. While non-dioxin-like-PCB (PCB-52 and PCB-4) induced a translocation of PKC-alpha and -epsilon from cytosol to membrane fraction, dioxin-like PCBs (PCB-126, -169, -114, -157) had no effects. [3H] Phorbol ester binding assay also revealed structure-dependent increase similar to translocation of PKC isozymes. While PCB-4 induced translocation of PKC-alpha and -epsilon was inhibited by ROS inhibitor, the pattern of translocation was not affected in presence of AhR inhibitor. It is suggested that PCB-4-induced PKC activity may not be mediated via AhR-dependent pathway. Taken together, our findings suggest that chlorination of ortho-position in PCB may be a critical structural moiety associated with neurotoxic effects, which may be preferentially mediated via non-AhR-dependent pathway. Therefore, the present study may contribute to understanding the neurotoxic mechanism of PCBs as well as providing a basis for establishing a better neurotoxic assessment.

Role of cGMP-dependent protein kinase against ethanol neurotoxicity of cerebellar granule cells / 第三军医大学学报

Objective To analyze the function of cGMP-dependent protein kinase(PKG)in the survival of cultured rat cerebellar granule cells(CGC).Methods CGC culture was incubated for 24 h before addition of ethanol and different reagents,such as Deta-NONOate(a NO donor),Br-cGMP and Rp-8-pCPT-cGMPS(a cGMP-dependent protein kinase inhibitor).After the treatment,samples were incubated for another 24 h,then the cells were collected and counted by using hemocytometer.Results Deta-NONOate and Br-cGMP enhanced the cell survival in the absence of ethanol,indicating their neurotrophic effects.They showed neuroprotective effect on the cultured CGC since they could reduce the ethanol-induced cell death.Inhibiting PKG with Rp-8-pCPT-cGMPS could eliminate both neurotrophic and neuroprotective effects mediated by Deta-NONOate and Br-cGMP.Conclusion cGMP-dependent protein kinase plays a key role in the cultured CGC survival and protects the cells against ethanol neurotoxicity.

Differential effect of homocysteic acid and cysteic acid on changes of inositol phosphates and (Ca2+)i in rat cerebellar granule cells

The present study was undertaken to characterize homocysteic acid (HCA)-and cysteic acid (CA)mediated formation of inositol phosphates (InsP) in primary culture of rat cerebellar granule cells. HCA and CA stimulated InsP formation in a dose-dependent manner, which was prevented by the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphopentanoic acid (APV). CA-, but not HCA-, mediated InsP formation was in part prevented by the metabotropic glutamate receptor antagonist alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG). Both HCA- and CA-mediated increases in intracellular calcium concentration were completely blocked by APV, but were not altered by (+/-)-MCPG. CA-mediated InsP formation was in part prevented by removal of endogenous glutamate. In contrast, the glutamate transport blocker L-aspartic acid-beta-hydroxamate synergistically increased CA responses. These data indicate that in cerebellar granule cells HCA mediates InsP formation wholly by activating NMDA receptor. In contrast, CA stimulates InsP formation by activating both NMDA receptor and metabotropic glutamate receptor, and in part by releasing endogenous glutamate into extracellular milieu.