Methamphetamine (METH) is a powerfully addictive psychostimulant that has a pronounced effect on the central nervous system (CNS). The present study aimed to assess METH toxicity in differentiated C6 astroglia-like cells through biochemical and toxicity markers with acute (1 h) and chronic (48 h) treatments. In the absence of external stimulants, cellular differentiation of neuronal morphology was achieved through reduced serum (2.5%) in the medium. The cells displayed branched neurite-like processes with extensive intercellular connections. Results indicated that acute METH treatment neither altered the cell morphology nor killed the cells, which echoed with lack of consequence on reactive oxygen species (ROS), nitric oxide (NO) or inhibition of any cell cycle phases except induction of cytoplasmic vacuoles. On the other hand, chronic treatment at 1 mM or above destroyed the neurite-like processors and decreased the cell viability that paralleled with increased levels of ROS, lipid peroxidation and lactate, depletion in glutathione (GSH) level and inhibition at G0/G1 phase of cell cycle, leading to apoptosis. Pre-treatment of cells with N-acetyl cysteine (NAC, 2.5 mM for 1 h) followed by METH co-treatment for 48 h rescued the cells completely from toxicity by decreasing ROS through increased GSH. Our results provide evidence that increased ROS and GSH depletion underlie the cytotoxic effects of METH in the cells. Since loss in neurite connections and intracellular changes can lead to psychiatric illnesses in drug users, the evidence that we show in our study suggests that these are also contributing factors for psychiatric-illnesses in METH addicts.

Scientific advances have been made in various areas of medicine. However, morphine remains the leading opioid analgesic of choice for the treatment of moderate and severe pain. Although morphine has very potent analgesic actions, it also has adverse side effects which include the development of tolerance which is the reason for its limited use. The exact mechanisms underlying the development of opioid tolerance is unclear, but in recent years studies have shown that the phosphorylation of G‐coupled protein receptors by various kinases has been linked to receptor internalization. Preliminary studies in our lab yielded results indicative of a significant decrease in the gene expression of Src and EGFR after chronic 24 hrs morphine treatment in differentiated SH‐SY5Y human neuroblastoma cells. The present study was conducted to validate the expression of Src and EGFR by western blot analysis after chronic morphine exposure (10 μM) in the absence and presence of naloxone (10 μM) for 1 hr. Additional studies will determine the involvement of Src and EGFR in morphine‐induced MOR‐1 gene expression. The overall results from this study will provide an understanding on the molecular mechanisms of opioid tolerance and the role that the AKT pathway plays in the development of morphine tolerance. Research supported by NCRR/RCMI G12 RR03020 and G12 MD007582–28

The development of opioid tolerance is a barrier to its clinical use. Continuous exposure to opioids, like morphine, induces several molecular and cellular changes at the receptor and signal transduction pathway. Chronic opioid treatment of differentiated SH‐SY5Y human neuroblastoma cells has been shown to be an excellent in vitro model for studying cellular changes observed after opioid exposure. The present study was conducted to gain a better understanding of alterations in protein expression induced after opioid treatment, using mass spectrometry based proteomics, which in recent years has had a major impact on investigating the molecular and cellular mechanism of protein‐protein interactions. Cells were treated with morphine (10 μM), naloxone (10 μM), or 1 hr naloxone (10 μM) pre‐treatment followed by morphine (10 μM) for 24 hours, harvested, and separated by 2‐D gel electrophoresis. Analysis of these gels revealed alterations in protein expression induced by morphine treatment. Proteins with the most significant changes following drug treatment were identified using MALDI‐TOF. Calmodulin, alpha‐enolase, stathmin, nucleoside diphosphate kinase A, and nucleophosmin were identified. These results form the basis for the analysis of differential protein expression in SH‐SY5Y cells. The proteins detected will further be investigated to determine their role in morphine tolerance. Grant Funding Source: NIH‐NIMHD G12MD007582

No abstract