Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity

Margaret Schwarz, John S. Lazo, Jack C. Yalowich, Ian Reynolds, Valerian E. Kagan, Vladimir Tyurin, Young Myeong Kim, Simon C. Watkins, Bruce R. Pitt

Research output: Contribution to journalArticle

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Abstract

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 languageEnglish (US)
Pages (from-to)15238-15243
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number21
StatePublished - May 27 1994
Externally publishedYes

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tert-Butylhydroperoxide
NIH 3T3 Cells
Metallothionein
Toxicity
Oxidants
DNA Damage
Plasmids
Antioxidants
Genes
Thiobarbituric Acid Reactive Substances
Confocal microscopy
DNA
Scavenging
Electron Spin Resonance Spectroscopy
Cytotoxicity
Fluorescence Microscopy
Confocal Microscopy
Paramagnetic resonance

ASJC Scopus subject areas

  • Biochemistry

Cite this

Schwarz, M., Lazo, J. S., Yalowich, J. C., Reynolds, I., Kagan, V. E., Tyurin, V., ... Pitt, B. R. (1994). Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity. Journal of Biological Chemistry, 269(21), 15238-15243.

Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity. / Schwarz, Margaret; Lazo, John S.; Yalowich, Jack C.; Reynolds, Ian; Kagan, Valerian E.; Tyurin, Vladimir; Kim, Young Myeong; Watkins, Simon C.; Pitt, Bruce R.

In: Journal of Biological Chemistry, Vol. 269, No. 21, 27.05.1994, p. 15238-15243.

Research output: Contribution to journalArticle

Schwarz, M, Lazo, JS, Yalowich, JC, Reynolds, I, Kagan, VE, Tyurin, V, Kim, YM, Watkins, SC & Pitt, BR 1994, 'Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity', Journal of Biological Chemistry, vol. 269, no. 21, pp. 15238-15243.
Schwarz M, Lazo JS, Yalowich JC, Reynolds I, Kagan VE, Tyurin V et al. Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity. Journal of Biological Chemistry. 1994 May 27;269(21):15238-15243.
Schwarz, Margaret ; Lazo, John S. ; Yalowich, Jack C. ; Reynolds, Ian ; Kagan, Valerian E. ; Tyurin, Vladimir ; Kim, Young Myeong ; Watkins, Simon C. ; Pitt, Bruce R. / Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 21. pp. 15238-15243.
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