The Toxicity of Sodium Arsenite on Fibroblast (BALB/C 3T3) : effect on signal transduction and actin cytoskeleton

Abstract

Contamination of drinking water with arsenic has created health problems in a number of countries including Thailand. Many epidemiological studies have indicated a causal relationship between exposure to Inorganic arsenic via drinking water and not only skin cancer but also intemal cancer. The cytotoxicity-induced by arsenite was though to be based on its reaction with the dihydrolipoyl moiety of pyruvate dehydrogenase leading to the inhibition of carbohydrate metabolism in the citric acid cycle, acetyl coenzyme A depletion, and thereby reduced oxygen consumption and ATP formation. It is recognized that receptor tyrosine kinase (RTK) activation can lead to mitogen-activated protein (MAP) kinase activation and then MAP kinase phosphorylation as well as transcription that causes cell growth and proliferation. The mechanisms of toxicity and cell signaling related to the actin cytoskeleton disruption induced by arsenite were studied. We used tyrosine kinase inhibitor (genistein), epidermal grcmth factor receptor (EGFR) inhibitor (4,5- dianilophthalimide). phosphatidylinositol 3-kinase (PI3K) Inhibitor (wortmannln), RNA synthesis Inhibitor (actinomycin D), protein synthesis Inhibitor (cycloheximide), The effects of arsenite on the actin cytoskeleton and vinculin in mouse fibroblasts (Balb/c 3T3) were studied by immunofluorescence microscopy and confocal microscopy. Lamellipodia were formed (Rae activation) when fibroblasts were exposed to arsenite at 5 µM for 16 hours. At 25 µM arsenite caused a severe loss of filamentous actin (F-actin) and vinculin and most cells became rounded. MAP kinase expression especially SAPKIJNK and p36 MAP kinase which can be induced by arsenite were studied by immunoblotling assay. The Inhibitors listed above can not block the expression of SAPKIJNK, phospho­ SAPKIJNK. p36 MAP kinase and phospho-p36 MAP kinase induced by arsenite. We concluded that the downstream effectors of Rae and Cdc42 did not play an important role In actin cytoskeleton disruption induced by arsenite. The inhibition of Rho kinase is related to loss of cytoplasmic tension and loss of stress fibers and focal adhesions were reported. We suggest that arsenite acts through mechanisms that involve Rho kinase via the tyrosine phosphorylation related to the EGFR and Src. For the study of ftavone apigenin as an antidote for arsenite, male WIstar rats (n=6/group) were exposed to arsenite at 25-10 mg/kg by oral for 24 hours.Arsenite decreased the plasma inter1eukin-6 (ll-6) level in a dose-dependent manner. But when the rats were pretreated with the apigenin at 30 mg/kg by oral for 1 hour and then treated with the arsenite at 10 mglkg by oral for 24 hours, this toxic effect was completely blocked by apigenin (p value < 0.05).