CYTOPLASMIC METALLOTHIONEIN OVEREXPRESSION PROTECTS NIH 3T3 CELLS FROM TERT-BUTYL HYDROPEROXIDE TOXICITY

Metallothioneins (MT) are ubiquitous low molecular weight metal-bindin g proteins that may act as antioxidants. We examined the sensitivity o f NIH 3T3 cells transfected with a plasmid containing mouse metallothi onein-I gene (NIH3T3/MT) to the membrane permeant oxidant, tert-butyl hydroperoxide (tBH). NIH3T3/MT cells had a 4-fold increase in intracel lular metallothionein as compared to cells transfected with a plasmid containing an inverted gene (NIH3T3/TM). Newly expressed metallothione in appeared to be localized to the cytoplasm as determined by immunofl uorescence and confocal microscopy. NIH3T3/MT cells were 6 times more resistant than NIH3T3/TM cells to the cytotoxic effects of tBH. The an tioxidant activity of NIH3T3/MT cells was greater than NIH3T3/TM cells , since exposure to tBH resulted in significantly less: (a) thiobarbit uric acid-reactive substances and (b) fluorescence after loading cells with the oxidant-sensitive dye, 2'7'-dichlorodihydrofluorescein diace tate. Furthermore, homogenates of NIH3T3/MT cells were more capable of scavenging in vitro generated phenoxyl radicals as quantified by elec tron spin resonance detection. In contrast, overexpression of cytoplas mic MT did not protect against tBH-induced DNA damage, suggesting that subcellular location of MT is important for its function and that DNA damage is not a key determinant of cytotoxicity. These data provide d irect support for an antioxidant role for MT, since physiologically re levant elevations in cytoplasmic MT interfere with tBH-induced cytotox ic peroxidation.