Glutathione (GSH) and metallothioneins (MT) are believed to play important roles in protecting cells against high copper (Cu) concentrations. Little is known, however, about their specific intracellular interactions and the coordination of protective functions. We investigated contributions of GSH and MT to protection against Cu toxicity in fibroblasts derived from wild-type (MT+/+) and knockout (MT-/-) mice that were challenged with cupric nitrilotriacetate (Cu-NTA). Endogenous levels of GSH and MT were manipulated using an inhibitor of gamma-glutamylcysteine synthetase, buthionine sulfoximine (BSO, 5 microM), as GSH depletor and ZnCl(2) (100 microM) as inducer of MT expression. BSO pretreatment markedly decreased cellular GSH levels in MT+/+ and MT-/- cells, by 65% and 70%, respectively, which resulted in Cu cytotoxicity accompanied by its elevated redox-cycling activity and enhanced Cu-induced membrane phospholipid peroxidation. BSO-pretreated MT-/- cells were markedly more sensitive to Cu despite the fact that the residual levels of GSH were similar in both BSO-pretreated MT+/+ and MT-/- cells. Zn pretreatment resulted in more than 10-fold induction of MT in MT+/+ cells but not in MT-/- cells. Accordingly, Zn pretreatment afforded significant protection of MT+/+ cells against Cu cytotoxicity, likely associated with MT-dependent suppression of Cu redox-cycling activity and phospholipid peroxidation, but it exerted no protection in MT-/- cells (as compared to naive cells). To determine whether MT functions specifically in Cu regulation or rather acts as a nonspecific Cu-binding cysteine-rich nucleophile, experiments were performed using MT+/+ and MT-/- cells pretreated with both BSO and Zn. BSO pretreatment did not affect Zn-induced MT expression in MT+/+ cells. As compared with BSO pretreatment alone, exposure to Cu of MT+/+ cells after Zn/BSO pretreatment resulted in the following: (i) a significantly higher viability; (ii) attenuated Cu-dependent redox-cycling activity; and (iii) a lower level of phospholipid peroxidation. In BSO/Zn-pretreated MT-/- cells, the redox-cycling activity of Cu and the level of phospholipid peroxidation remained remarkably higher than in naive cells and were not significantly different from those in cells pretreated with BSO alone. Cu-induced toxicity was remarkably higher in BSO/Zn-pretreated MT-/- cells than in naive or Zn-pretreated cells, although slightly lower than in the MT-/- cells pretreated with BSO alone.