Cytotoxicity of nitric oxide is alleviated by zinc-mediated expression of antioxidant genes

Metallothioneins (MTs) are small, cysteine-rich zinc binding proteins that are powerful antioxidants. In this study, we investigated the interaction between zinc, MTs, and other components of the antioxidant defense system in HepG2 cells. Cells were preincubated with zinc and then exposed to sodium nitroprusside (SNP), a nitric oxide (NO) donor. Both zinc pretreatment and SNP exposure separately induced transcription of MT genes (MT1A, MT2A, MT1E, MT1X), as measured using real time-polymerase chain reaction (PCR) after reverse transcription (RT). Pretreatment of HepG2 cells with zinc sulfate (ZnSO ₄) followed by SNP exposure caused MT and glucose-6-phosphate dehydrogenase (G6PD) mRNA levels to increase more than in cells only exposed to SNP. However, when cells were incubated with N,N,N′,N′-tetrakis(2- pyridylmethyl)ethylenediamine (TPEN), a membrane-permeant Zn²⁺ chelator, the stimulation of MT transcription by SNP was blocked, suggesting that SNP-induced upregulation of these genes is zinc-dependent. Human glutathione-S-transferase (hGSTA1) and G6PD mRNA levels in the cells treated with 5 μM TPEN decreased. Additionally, the induction of MT by SNP after zinc pretreatment appears to be mediated by metal-activated transcription factor-1 (MTF-1), which is induced by labile zinc in the cytosol. SNP cytotoxicity was inhibited by preincubation with zinc. Taken together, these results suggest that NO plays an important role in regulation of cellular zinc homeostasis and that NO-mediated release of protein-bound Zn²⁺ may be an important signal in antioxidant defense.