Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity.
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 antioxidant role for MT, since physiologically relevant elevations in cytoplasmic MT interfere with tBH-induced cytotoxic peroxidation.
Schwarz
M A
MA
Department of Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Lazo
J S
JS
Yalowich
J C
JC
Reynolds
I
I
Kagan
V E
VE
Tyurin
V
V
Kim
Y M
YM
Watkins
S C
SC
Pitt
B R
BR
D016475:3T3 Cells; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage; D004578:Electron Spin Resonance Spectroscopy; D005453:Fluorescence; D008668:Metallothionein / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D051379:Mice; D010545:Peroxides / Q000037:antagonists & inhibitors / Q000633:toxicity*; D013347:Subcellular Fractions / Q000378:metabolism; D017392:Thiobarbituric Acid Reactive Substances; D020122:tert-Butylhydroperoxide
D000975:Antioxidants; D010545:Peroxides; D017392:Thiobarbituric Acid Reactive Substances; D008668:Metallothionein; D020122:tert-Butylhydroperoxide