Erk1/2 phosphorylation and reactive oxygen species formation via nitric oxide and Akt-1/Raf-1 crosstalk in cultured rat cerebellar granule cells exposed to the organic solvent 1,2,4-trimethylcyclohexane

Abstract

Previously, we have shown that exposure of cultured rat cerebellar granule cells to the hydrocarbon solvent 1,2,4-trimethylcyclohexane leads to formation of reactive oxygen species (ROS). However, the cellular mechanisms responsible for formation of ROS in these cells after exposure to organic solvents are poorly understood. Here, we found that 1,2,4-trimethylcyclohexane induced a time and concentration dependent dephosphorylation of Akt-1 at Ser-473 and Raf-1 at Ser-259. An increased level of phosphorylated extracellular signal-regulated kinases (Erk1/2) at Tyr-204 was observed. By use of the nitric oxide synthase inhibitors Nω-nitro-L-arginine methylester and diphenyleneiodonium, we found that intracellular formation of nitric oxide was necessary for phosphorylation of Erk1/2 and for the formation of ROS. Furthermore, the ROS formation was inhibited by the Erk1/2 pathway inhibitor U0126. A 1,2,4-trimethylcyclohexane (TMCH)-induced cell death was lowered by U0126 and the free radical scavenger vitamin E. Our results show that Erk1/2 kinases and nitric oxide (NO) may participate in ROS formation induced by 1,2,4-trimethylcyclohexane in cultured rat cerebellar granule cells, and also indicate a crosstalk between Akt and the Raf-Mek-Erk signaling systems. © Society of Toxicology 2004; all rights reserved.