The effect of diazinon on Na+/K+-ATPase activity

Abstract

Beside the main toxic action of diazinon to inhibit acetylcholineesterase activity, it is established that some organophosphates inhibit different ATPases, the group of enzymes playing an important role in biochemical processes. The aim of the work was to investigate influence of diazinon on Na+/K+-ATPase activity using different model systems: commercial purified Na+/K+-ATPase from porcine cerebral cortex, cultivated human blood cells (lymphocytes and erythrocytes) and skin fibroblasts. Both blood cells and fibroblasts were cultivated in the presence of increasing doses of diazinon (2 × 10−8–2 × 10−4 M) during 72 h, while commercial Na+/K+-ATPase was in vitro exposed to the same concentrations. Specific activity of Na+/K+-ATPase in the absence (control) and presence of diazinon was determined in a standard incubation medium containing (in mM): 50 Tris–HCl (pH 7.4), 100 NaCl, 20 KCl, 5 MgCl2, 2 ATP, via released inorganic orthophosphate (Pi) using spectrophotometric method. Results show that activity of commercially purified Na+/K+-ATPase was not affected by diazinon concentrations lower than 1 × 10−4 M. On the contrary, dose-dependent Na+/K+-ATPase inhibition was obtained for all investigated cells. The concentrations of diazinon with capability to inhibit 50% of the enzyme after given exposure time (IC50 values (72 h)) (M) were 3.4 × 10−5, 6.6 × 10−5, and 4.6 × 10−5 for fibroblast, erythrocyte and lymphocyte Na+/K+-ATPase, respectively. In addition, in all cell types diazinon dose-dependent increase of malondialdehyde content (as a product of lipid peroxidation) was observed within the same diazinon concentration range as found for cells Na+/K+-ATPase inhibition. Since purified Na+/K+-ATPase is insensitive to diazinon, it could be concluded that oxidative membrane injury by the investigated organophosphate is probably responsible for the obtained Na+/K+-ATPase inhibition in cells.