In vivo monitoring of hydroxyl radical generation caused by x-ray irradiation of rats using the spin trapping/EPR technique

The measurement of hydroxyl radical in living animals irradiated with ionizing radiation should be required to clarify the mechanisms of radiation injury and the in vivo assessment of radiation protectors, because the generation of hydroxyl radical is believed to be one of the major triggers of radiation injury. In this study, hydroxyl radical generation was monitored by spin trapping the secondary methyl radical formed by the reaction of the hydroxyl radical with dimethyl sulfoxide (DMSO). Rats were injected intraperitoneally with a DMSO solution of a-phenyl-N-tert-butylnitrone (PBN). X-ray irradiation of the rats remarkably increased the six-line EPR signal in the bile. The strengthened signal was detectable above 40 Gy. The use of 13C-substituted DMSO revealed that the signal included methyl radical adduct of PBN as a major component. The EPR signal of PBN-methyl radical adduct was completely suppressed by pre-administration of methyl gallate, a scavenger of hydroxyl radical but not of methyl radical. Methyl gallate did not reduce the spin adducts to EPR-silent forms. These observations indicate that what we were measuring was the hydroxyl radical generated in vivo by x-ray irradiation. This is the first report of the in vivo monitoring ofhydroxyl radical generation at a radiation dose close to what people might receive in the case of radiological accident or radiation therapy