Thimerosal-induced cytosolic Ca 2+ elevation and subsequent cell death in human osteosarcoma cells

Abstract

The effect of the oxidizing agent thimerosal on cytosolic free Ca 2+ concentration ([Ca 2+] i) and proliferation has not been explored in human osteoblast-like cells. This study examined whether thimerosal alters Ca 2+ levels and causes cell death in MG63 human osteosarcoma cells. [Ca 2+] i and cell death were measured using the fluorescent dyes fura-2 and WST-1, respectively. Thimerosal at concentrations above 5 μM increased [Ca 2+] i in a concentration-dependent manner. The Ca 2+ signal was reduced by 80% by removing extracellular Ca 2+. The thimerosal-induced Ca 2+ influx was sensitive to blockade of La 3+, and dithiothreitol (50 μM) but was insensitive to nickel and several l-type Ca 2+ channel blockers. After pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor), thimerosal failed to induce [Ca 2+] i rises. Inhibition of phospholipase C with 2 μM U73122 did not change thimerosal-induced [Ca 2+] i rises. At concentrations of 5, 10 and 20 μM thimerosal killed 33, 55 and 100% cells, respectively. The cytotoxic effect of 5 μM thimerosal was reversed by 54% by prechelating cytosolic Ca 2+ with BAPTA. Collectively, in MG63 cells, thimerosal induced a [Ca 2+] i rise by causing Ca 2+ release from endoplasmic reticulum stores and Ca 2+ influx from extracellular space. Furthermore, thimerosal can cause Ca 2+-related cytotoxicity in a concentration-dependent manner.