Foreign metallothionein-I expression by transient transfection in MT-I and MT-II null astrocytes confers increased protection against acute methylmercury cytotoxicity

The mechanisms associated with metallothionein (MT) gene regulation are complex and poorly understood. Only a modest increase in brain MT expression levels is attained by exposure to metals, MT gene transfection, and MT gene knock-in techniques. Accordingly, in the present study, MT null astrocytes isolated from transgenic mice deficient in MT-I and MT-II genes were introduced as a zero background model of MT expression. MT protein levels were determined by western blot analysis. MT proteins in MT-I and MT-II null astrocytes were undetectable. Transient MT-I gene transfection increased the levels of foreign MT expression in MT-I and MT-II null astrocytes by 2.3-fold above basal levels in wild-type astrocytes. Intracellular (Na2CrO4)-Cr-51 efflux and D-[2,3-H-3]aspartate uptake were studied as indices of acute methylmercury (MeHg) (5 mu M) cytotoxicity. In MT-I and MT-II knockout astrocytes MeHg led to significant (p < 0.01) increase in Na-2(5)'CrO4 efflux and a significant (p < 0.05) decrease in the initial rate (1 min) of D-[2,3-H-3]aspartate uptake compared to MT-I and MT-II knockout controls. Transfection of the MH-I gene in MT-I and MT-II null mice significantly (p < 0.01) decreased the effect of MeHg on (Na2CrO4)-Cr-51 efflux in MT null, as well as wild-type astrocytes. MT-I gene transfection in MT-I and MT-II null astrocytes reversed the inhibitory effect of MeHg on D-[2,3-3H]aspartate uptake, such that initial rates of uptake in MT-I transfected cells in the presence and absence of MeHg (5 mu M) were indistinguishable. These results demonstrate that. (1) astrocytes lacking MTs are more sensitive to MeHg than those with basal MT protein levels, (2) the MT-I gene can be overexpressed in MT-I and MT-II null astrocytes by transient MT-I gene transfection, and (3) that foreign MT expression endows astrocytes with increased resistance to MeHg. (C) 2000 Elsevier Science B.V. All rights reserved.