Protective Role of Glutathione against Peroxynitrite-Mediated DNA Damage During Acute Inflammation

Abstract

Inflammation is an immune response to protect against various types of infections. When unchecked, acute inflammation can be life-threatening, as seen with the current coronavirus pandemic. Strong oxidants, such as peroxynitrite produced by immune cells, are major mediators of the inflammation-associated pathogenesis. Cellular thiols play important roles in mitigating inflammation-associated macromolecular damage including DNA. Herein, we have demonstrated a role of glutathione (GSH) and other thiols in neutralizing the effect of peroxynitrite-mediated DNA damage through stable GSH-DNA adduct formation. Our observation supports the use of thiol supplements as a potential therapeutic strategy against severe COVID-19 cases and a Phase II (NCT04374461) open-label clinical trial launched in early May 2020 by the Memorial Sloan Kettering Cancer Center.

Figure 1.

Protective effect of GSH on PN-mediated DNA damage. (A) Ampicillin-resistant colonies transformed with ampicillin-resistant pUC19 plasmid (20 μg) treated with PN (50 μM) or PN followed by GSH (5 mM). (B) Green fluorescence protein-expressing HEK-293T cells transiently transfected with green fluorescence plasmid pEGFPN1 (50 μg) treated with PN (5 μM) or PN followed by GSH (5 mM).

Figure 2.

DNA cleavage assay. (A) Agarose gel images of ctDNA or plasmid following various treatments. ctDNA (1 mg) or pUC19 (1 μg) were incubated with PN (0.5 mM for ctDNA and 1 mM for pUC19) for 10 min at 25 °C (ctDNA) or on ice (pUC19) followed by the addition of GSH (5 mM, 37 °C for 2 h). (B) pUC19 (1 μg) DNA cleavage by PN in the presence and absence of thiols (5 mM). (C) pUC19 (1 μg) DNA cleavage by PN (50 μM) in the presence of varying concentrations of thiols.

Figure 3.

LC-MS extracted ion chromatogram and LC-MS/MS spectrum of m/z 573 peak eluting at 5.8 min (GS-dG adduct).

Figure 4.

Detection of GS-DNA adduct. (a) UV–vis and fluorescence spectra of DNA after various treatments; (b) LC-MS and LC-MSMS chromatogram showing m/z 573.17 and 573 → 328 peak, respectively.

Figure 5.

Ames mutagenicity test with PN and PN + GSH or PN + Cys. The figure shows pictures of colonies and its quantitation. Sodium azide (NaN₃, 15 μM) and degraded PN were used as positive and negative controls, respectively. The concentration of PN was 10 μM and that of GSH/Cys was 5 mM.

Scheme 1.

Cellular Fate of 8-Nitroguanine Formed in DNA