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

Measurement of hydroxyl radical (√OH) 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 generation of √OH is believed to be one of the major triggers of radiation injury. In this study, √OH generation was monitored by spin trapping the secondary methyl radical formed by the reaction of √OH with dimethyl sulfoxide (DMSO). Rats were injected intraperitoneally with a DMSO solution of α-phenyl-N-tert-butylnitrone (PBN). X-irradiation of the rats remarkedly increased the six-line EPR signal in the bile. The strengthened signal was detectable above 40 Gy. Use of 13C-substituted DMSO revealed that the signal included the methyl radical adduct of PBN as a major component. The EPR signal of the PBN–methyl radical adduct was completely suppressed by preadministration of methyl gallate, a scavenger of √OH 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 √OH generated in vivo by x-irradiation. This is the first report of the in vivo monitoring of √OH generation at a radiation dose close to what people might receive in the case of radiological accident or radiation therapy.