Metallothioneins (MT) are ubiquitous low molecular weight metal-binding proteins that may act as antioxidants. We examined the sensitivity of NIH 3T3 cells transfected with a plasmid containing mouse metallothionein-I gene (NIH3T3/MT) to the membrane permeant oxidant, tert-butyl hydroperoxide (tBH). NIH3T3/MT cells had a 4-fold increase in intracellular metallothionein as compared to cells transfected with a plasmid containing an inverted gene (NIH3T3/TM). Newly expressed metallothionein appeared to be localized to the cytoplasm as determined by immunofluorescence and confocal microscopy. NIH3T3/MT cells were 6 times more resistant than NIH3T3/TM cells to the cytotoxic effects of tBH. The antioxidant activity of NIH3T3/MT cells was greater than NIH3T3/TM cells, since exposure to tBH resulted in significantly less: (a) thiobarbituric acid-reactive substances and (b) fluorescence after loading cells with the oxidant-sensitive dye, 2'7'-dichlorodihydrofluorescein diacetate. Furthermore, homogenates of NIH3T3/MT cells were more capable of scavenging in vitro generated phenoxyl radicals as quantified by electron spin resonance detection. In contrast, overexpression of cytoplasmic 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 direct support for an anti- oxidant role for MT, since physiologically relevant elevations in cytoplasmic MT interfere with tBH-induced cytotoxic peroxidation.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - May 27 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology