Mg2+Antagonism on Ni2+-Induced Changes in Microtubule Assembly and Cellular Thiol Homeostasis

Abstract

As an essential metal for cell metabolism, Mg2+is known to exert antagonism on Ni2+genotoxic and other effects. This study examined the influence of Mg2+on Ni2+-induced changes in microtubule (MT) assemblyin vitro,cytoplasmic MT organization, cellular glutathione (GSH), and cytoskeletal and cytosolic protein sulfhydryls (PSH). As determined by a turbidity assay at 27°C, Ni2+enhanced thein vitroMT assembly in a Pipes buffer by shortening the initial lag (nucleation phase) and increasing the rate of polymerization with a higher final plateau. However, presence of 1 mMexogenous MgCl2abolished the Ni2+enhancing effect. Exposure of 3T3 cells to 2 mMNiCl2for 20 hr resulted in perinuclear bundling of MTs and decreases in cytoskeletal and cytosolic PSH and cellular GSH levels. However, coincubation of cells with MgCl2(1.25–20 mM) added to the culture medium markedly diminished the Ni2+injury to MT organization. Under these conditions the Ni2+interference with PSH was blocked such that both the cytoskeletal and cytosolic PSH levels returned to the range of control cells without metal treatment. Treatment of cells with Mg2+(1.25–5 mM) for 20 hr slightly increased, while with higher Mg2+doses (>10 mM) decreased, cellular GSH content. Importantly, in Ni2+-treated cultures, addition of Mg2+(1.25–10 mM) elevated GSH levels to ≥200% of that in cells treated with Ni2+alone. Furthermore, these Ni2+and Mg2+(1.25–10 mM) treated cells actually maintained GSH levels which were essentially unchanged from the basal level of control cells with no metal treatment. Although Mg2+replacement of Ni2+bound to MT proteins could be an important mechanism, cellular GSH may also be a critical factor in Mg2+antagonism on Ni2+-enhanced MT assembly in view of the essential role of tubulin PSH in modulating MT assembly and, in turn, the GSH modulation of PSH.