Antioxidant and antiapoptotic function of metallothioneins in HL-60 cells challenged with copper nitrilotriacetate

Antioxidant activity is believed to be an important intracellular function of metallothioneins (MT), yet the specific mechanisms of their antioxidant action are not known. Under conditions when cells are challenged with eleva ted concentrations of free copper as a result of metabolic disturbances or environmental and occupational exposures, MTs may be ideally suited for ant ioxidant function as effective copper chelators. In the study presented her e, we tested this hypothesis using a recently established model of copper n itrilotriacetate-induced oxidative stress in HL-60 cells. Since copper-indu ced oxidative stress triggers apoptosis, we further investigated antiapopto tic function of MTs in HL-60 cells. Using a Sephadex G-75 chromatographic p artial purification of MTs from cell homogenates with subsequent immune-dot -blot assay, we showed that zinc pretreatment yielded a pronounced inductio n of MTs in HL-60 cells. We report that zinc-induced MTs were able to (i) c ompletely bind intracellular copper, (ii) completely quench redox-cycling a ctivity of copper, (iii) significantly inhibit copper-dependent oxidative s tress in membrane phospholipids, and (iv) prevent copper-dependent apoptosi s and its characteristic biochemical features (cytochrome c release from mi tochondria into cytosol, caspase-3 activation, and externalization of phosp hatidylserine in plasma membranes). In separate experiments, we used lung f ibroblasts derived from MT1, MT2 knockout mice (MT-/-) and MT wild-type (MT +/+) mice. ZnCl2 pretreatment resulted in a more than 10-fold induction of MTs in MT+/+ cells, whereas the MT content in MT-/- cells remained low, at levels approximate to 100-fold lower than in their MT wild-type counterpart s. MT-/- cells were very sensitive to Cu-NTA and, most importantly showed n o response to ZnCl2 pretreatment. In contrast, MT+/+ cells were relatively more resistant to Cu-NTA, and this resistance was remarkably enhanced by Zn Cl2 pretreatment. Combined, our results demonstrate that metallothioneins f unction as effective antioxidants and an antiapoptotic mechanism in copper- challenged HL-60 cells.