ABSTRACT
This study explored the protective effects of endoplasmic reticulum (ER) stress preconditioning induced by 2-deoxy-d-glucose (2-DG) or oxidized dithiothreitol (DTTox) on acrylonitrile (AN)-induced cytotocity in primary rat astrocytes. Cells were pretreated with 2-DG or DTTox for different times at various concentration. Next, astrocytes were treated with 2.5mM AN for an additional 12h. Cell viability and cytotoxicity were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) leakage, respectively. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were determined. Expression of glucose-regulated protein 78 (GRP78), phosphorylated-eukaryotic translation initiation factor 2α (p-eIF2α), microtubule-associated protein light chain 3 (LC3), P62, and Beclin1 were used to assess autophagy. In addition, 3-methyadenine (3-MA), an autophagy-specific inhibitor, was used to assess the role of autophagy in ER stress preconditioning-induced protection against AN cytotoxicity. The results showed that AN alone significantly decreased astrocytic viability and enhanced cytotoxicity. Compared to the AN-alone group, preconditioning with 2-DG or DTTox significantly increased cell viability and reduced cytotoxicity to indistinguishable levels. Decreased ROS generation and increased ΔΨm were also inherent to ER stress preconditioning with these compounds. Furthermore, autophagy was activated by both 2-DG and DTTox. Blockage of autophagy attenuated the protection afforded by 2-DG or DTTox preconditioning in AN-treated astrocytes. These results establish that ER stress preconditioning affords cellular protection against AN, and that activation of autophagy mediates the cytoprotection. Modulation of ER stress and resultant activation of autophagy may be a novel target for to ameliorate AN toxicity.
Subject(s)
Acrylonitrile/toxicity , Astrocytes/drug effects , Autophagy/physiology , Carcinogens/toxicity , Cerebral Cortex/cytology , Endoplasmic Reticulum Stress/drug effects , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Animals, Newborn , Astrocytes/ultrastructure , Autophagy/drug effects , Cells, Cultured , Deoxyglucose/pharmacology , Dithiothreitol/pharmacology , Glial Fibrillary Acidic Protein/metabolism , Heat-Shock Proteins/metabolism , Membrane Potential, Mitochondrial/drug effects , Microtubule-Associated Proteins/metabolism , Oxidoreductases/metabolism , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolismABSTRACT
Autophagy is an evolutionarily conserved process in which cytoplasmic proteins and organelles are degraded and recycled for reuse. There are numerous reports on the role of autophagy in cell growth and death; however, the role of autophagy in methylmercury (MeHg)-induced neurotoxicity has yet to be identified. We studied the role of autophagy in MeHg-induced neurotoxicity in astrocytes. MeHg reduced astrocytic viability in a concentration- and time-dependent manner, and induced apoptosis. Pharmacological inhibition of autophagy with 3-methyladenine or chloroquine, as well as the silencing of the autophagy-related protein 5, increased MeHg-induced cytotoxicity and the ratio of apoptotic astrocytes. Conversely, rapamycin, an autophagy inducer, along with as N-acetyl-L-cysteine, a precursor of reduced glutathione, decreased MeHg-induced toxicity and the ratio of apoptotic astrocytes. These results indicated that MeHg-induced neurotoxicity was reduced, at least in part, through the activation of autophagy. Accordingly, modulation of autophagy may offer a new avenue for attenuating MeHg-induced neurotoxicity.
