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 Na(2)51CrO(4) efflux and D-[2,3-3H]aspartate uptake were studied as indices of acute methylmercury (MeHg) (5 microM) cytotoxicity. In MT-I and MT-II knockout astrocytes MeHg led to significant (p<0.01) increase in Na(2)51CrO(4) efflux and a significant (p<0.05) decrease in the initial rate (1 min) of D-[2, 3-3H]aspartate uptake compared to MT-I and MT-II knockout controls. Transfection of the MT-I gene in MT-I and MT-II null mice significantly (p<0.01) decreased the effect of MeHg on Na(2)51CrO(4) 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 microM) 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.
Yao
C P
CP
Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
Allen
J W
JW
Mutkus
L A
LA
Xu
S B
SB
Tan
K H
KH
Aschner
M
M
D000818:Animals; D001224:Aspartic Acid / Q000493:pharmacokinetics; D001253:Astrocytes / Q000201:enzymology*; D001692:Biological Transport / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002840:Chromates; D002860:Chromium Radioisotopes; D003603:Cytotoxins / Q000494:pharmacology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D008297:Male; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism; D008767:Methylmercury Compounds / Q000633:toxicity*; D051379:Mice; D018345:Mice, Knockout; D010957:Plasmids; D017670:Sodium Compounds; D014162:Transfection
D002840:Chromates; D002860:Chromium Radioisotopes; D003603:Cytotoxins; D008767:Methylmercury Compounds; D017670:Sodium Compounds; D001224:Aspartic Acid; C028982:sodium chromate(VI); D008668:Metallothionein