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2008 Annual Meeting Abstracts


Hydrogen Sulfide: A Novel Cytoprotectant Against Radiation Induced Cellular Injury
Esther H. Teo, BS1, Sunil P. Singh, BA2, Daniel J. Kadouch, BA3, Gabriella A. Wernicke, MD1, Jason A. Spector, MD1.
1Weill Cornell Medical College, New York, NY, USA, 2New York University School of Medicine, New York, NY, USA, 3Vrije Universiteit Medisch Centrum, Amsterdam, Netherlands.

Background
Despite improved delivery protocols, radiation damage to the skin is a common sequela to standard radiation therapy. Cytotoxicity resulting from radiation treatment produces significant morbidity as evidenced by the development of fibrosis in the treatment area. Radiation damaged skin is more prone to injury and less capable of repair. Radiation-induced cellular damage is caused, in part, by formation of free oxygen radicals when tissue is exposed to ionizing radiation. Recent evidence from our lab has demonstrated that hydrogen sulfide significantly ameliorates cellular damage resulting from ischemia-reperfusion injury, possibly through its ability to reversibly inhibit cellular metabolism and scavenge free radicals. We hypothesized that hydrogen sulfide might provide a similar cytoprotective effect to ameliorate radiation-induced cellular injury.
Methods
NIH 3T3 embryonic mouse fibroblasts were plated in 6-well plates and allowed to grow to confluency. Sodium hydrogen sulfide was added to the culture media to a concentration of 500 µM one hour prior to radiation exposure. Untreated controls received an equivalent volume of phosphate-buffered saline. Cells were then exposed to a single dose of 20 Gy of ionizing radiation. Non-irradiated controls were placed on the radiation field without turning on the radiation source. Cytospin preparations were made at equal cell densities for each condition at 2 and 24 hours post-radiation exposure. Slides were stained with TUNEL and visualized under florescence microscopy. Apoptosis was quantified by counting the number of TUNEL-positive nuclei in 5 random high-powered fields per slide.
Results
At two hours post-radiation with a 20 Gy dose, 2.6 ± 1.3 TUNEL-positive nuclei were present per high-power field for cells treated with saline. Cells treated with 500 µM sodium hydrogen sulfide had 0.8 ± 0.8 TUNEL-positive nuclei per high-power field. This is a 3-fold reduction in the number of TUNEL-positive nuclei for cells treated with hydrogen sulfide compared to saline alone (p=0.030). At twenty-four hours post-radiation, no apoptosis was visualized in either the control or untreated groups. No overt evidence of toxicity resulting from hydrogen sulfide treatment was seen as evidenced by lack of TUNEL staining in non-irradiated controls.
Conclusion
These data demonstrate that hydrogen sulfide treatment prior to radiation exposure results in a significant decrease in radiation-induced apoptosis in 3T3 fibroblast cells. This cytoprotective effect of hydrogen sulfide against ionizing radiation has significant clinical implications for the development of treatments designed to protect patients from the side effects of radiation therapy. Further studies are in progress to determine if this cytoprotective effect is seen in other cellular components of the skin including keratinocytes and endothelial cells.