S-Nitrosylation in Organs of Mice Exposed to Low or High Doses of γ-Rays: The Modulating Effect of Iodine Contrast Agent at a Low Radiation Dose

Abstract

The covalent addition of nitric oxide (NO•) onto cysteine thiols, or S-nitrosylation, modulates the activity of key signaling proteins. The dysregulation of normal S-nitrosylation contributes to degenerative conditions and to cancer. To gain insight into the biochemical changes induced by low-dose ionizing radiation, we determined global S-nitrosylation by the “biotin switch” assay coupled with mass spectrometry analyses in organs of C57BL/6J mice exposed to acute 0.1 Gy of 137Cs γ-rays. The dose of radiation was delivered to the whole body in the presence or absence of iopamidol, an iodinated contrast agent used during radiological examinations. To investigate whether similar or distinct nitrosylation patterns are induced following high-dose irradiation, mice were exposed in parallel to acute 4 Gy of 137Cs γ rays. Analysis of modulated S-nitrosothiols (SNO-proteins) in freshly-harvested organs of animals sacrificed 13 days after irradiation revealed radiation dose- and contrast agent-dependent changes. The major results were as follows: (i) iopamidol alone had significant effects on S-nitrosylation in brain, lung and liver; (ii) relative to the control, exposure to 0.1 Gy without iopamidol resulted in statistically-significant SNO changes in proteins that differ in molecular weight in liver, lung, brain and blood plasma; (iii) iopamidol enhanced the decrease in S-nitrosylation induced by 0.1 Gy in brain; (iv) whereas a decrease in S-nitrosylation occurred at 0.1 Gy for proteins of ~50 kDa in brain and for proteins of ~37 kDa in liver, an increase was detected at 4 Gy in both organs; (v) mass spectrometry analyses of nitrosylated proteins in brain revealed differential modulation of SNO proteins (e.g., sodium/potassium-transporting ATPase subunit beta-1; beta tubulins; ADP-ribosylation factor 5) by low- and high-dose irradiation; and (vi) ingenuity pathway analysis identified major signaling networks to be modulated, in particular the neuronal nitric oxide synthase signaling pathway was differentially modulated by low- and high-dose γ-irradiation.

Ionizing radiation biomarkers in epidemiological studies – An update

“…[205,206]. A study of cognitive defects in irradiated mice showed a decrease of total malondialdehyde-modified protein content in the hippocampus at 1 Gy [178] and the analysis of Snitrosothiols, a post-translational modification of proteins, by mass spectrometry in brain, liver and plasma showed a decreased level after exposure to 100 mGy [207] suggesting that markers of redox imbalance might be associated indicative of cognitive defects. Persistent (40 week) increases in protein carbonylation in the cardiovascular system after a single heart dose to C57BL/6 mice were detected after exposure to 2 Gy but not after 0.2 Gy [176].…”

Direct Measurement of S-Nitrosothiols with an Orbitrap Fusion Mass Spectrometer: S-Nitrosoglutathione Reductase as a Model Protein

“…Subsequent biotin or tandem mass tag conjugation to the ascorbate-reduced cysteines facilitates immunoprecipitation and proteomics for identification of SNO-modified proteins [6,7]. Site-specific cysteine SNOs have been inventoried in Arabidopsis thaliana cell suspensions and rosette leaves [8,9] and mouse liver and brain [10].…”