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

The mechanisms associated with metallothionein (MT) gene regulation are com plex and poorly understood. Only a modest increase in brain MT expression l evels 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 introd uced as a zero background model of MT expression. MT protein levels were de termined by western blot analysis. MT proteins in MT-I and MT-II null astro cytes were undetectable. Transient MT-I gene transfection increased the lev els 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 e fflux and D-[2,3-H-3]aspartate uptake were studied as indices of acute meth ylmercury (MeHg) (5 mu M) cytotoxicity. In MT-I and MT-II knockout astrocyt es 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) decr eased the effect of MeHg on (Na2CrO4)-Cr-51 efflux in MT null, as well as w ild-type astrocytes. MT-I gene transfection in MT-I and MT-II null astrocyt es reversed the inhibitory effect of MeHg on D-[2,3-3H]aspartate uptake, su ch that initial rates of uptake in MT-I transfected cells in the presence a nd absence of MeHg (5 mu M) were indistinguishable. These results demonstra te that. (1) astrocytes lacking MTs are more sensitive to MeHg than those w ith 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) tha t foreign MT expression endows astrocytes with increased resistance to MeHg . (C) 2000 Elsevier Science B.V. All rights reserved.