Antioxidant activity is believed to be an i
mportant intracellular function of
metallothioneins(MT), yet the specific
mechanis
ms of their antioxidant action are not known. Under conditionswhen cells are challenged with elevated concentrations of free copper as a result of
metabolicdi
sturbances or environ
mental and occupational exposures, MTs
may be ideally suited forantioxidant function as effective copper chelators. In the
study presented here, we te
sted thishypothesis using a recently e
stablished
model of copper nitrilotriacetate-induced oxidative
stressin HL-60 cells. Since copper-induced oxidative
stress triggers apoptosis, we further inve
stigatedantiapoptotic function of MTs in HL-60 cells. Using a Sephadex G-75 chro
matographic partialpurification of MTs fro
m cell ho
mogenates with subsequent i
mmuno-dot-blot assay, we showedthat zinc pretreat
ment yielded a pronounced induction of MTs in HL-60 cells. We report thatzinc-induced MTs were able to (i) co
mpletely bind intracellular copper, (ii) co
mpletely quenchredox-cycling activity of copper, (iii) significantly inhibit copper-dependent oxidative
stress in
me
mbrane phospholipids, and (iv) prevent copper-dependent apoptosis and its characteri
sticbioche
mical features (cytochro
me
c release fro
m mitochondria into cytosol, caspase-3 activation,and externalization of phosphatidylserine in plas
ma
me
mbranes). In separate experi
ments,we used lung fibrobla
sts derived fro
m MT1, MT2 knockout
mice (MT
-/-) and MT wild-type(MT
+/+)
mice. ZnCl
2 pretreat
ment resulted in a
more than 10-fold induction of MTs in MT
+/+cells, whereas the MT content in MT
-/- cells re
mained low, at levels
mages/entities/ap.gif">100-fold lower than intheir MT wild-type counterparts. MT
-/- cells were very sensitive to Cu-NTA and,
mo
sti
mportantly, showed no response to ZnCl
2 pretreat
ment. In contra
st, MT
+/+ cells were relatively
more resi
stant to Cu-NTA, and this resi
stance was re
markably enhanced by ZnCl
2 pretreat
ment. Co
mbined, our results de
mon
strate that
metallothioneins function as effective antioxidants and an antiapoptotic
mechanis
m in copper-challenged HL-60 cells.