Measurement of radical-scavenging ability in hepatic metallothionein of rat using in vivo electron spin resonance spectroscopy

In this study, the ability of metallothionein (MT) to scavenge free radicals was determined by in vivo electron spin resonance (ESR) spectroscopy using a carbamoyl-PROXYL, nitroxyl radical, as a spin probe. Production of metallothionein was induced in the liver of rats with ZnSO4 (0.2 mol/kg, ip) and the intensity of the carbamoyl-PROXYL ESR signal was measured at the upper abdominal level which is a position of the liver. After the injection of carbamoyl-PROXYL, the peak of ESR signal gradually decreased and showed a linear decay curve. The rate of decay of carbamoyl-PROXYL, the spin clearance rate, was determined over the first 3 min. The spin clearance rate did not differ significantly between ZnSO4-treated and control rats. When rats were fasted for 24 h, hepatic glutathione (GSH) concentrations decreased significantly and the spin clearance rate was significantly lower than non-fasted rats. However, the spin clearance rate of the fasted rats treated with Zn returned to the control level. To reduce GSH concentrations in the liver, buthionine sulfoximine (BSO, 2 mmol/kg, ip) was injected into the rats. The spin clearance rate of rats treated with BSO was significantly decreased as compared with that of control rats without BSO treatment. In rats treated with Zn, the decay rate of carbamoyl-PROXYL increased significantly in spite of the depletion of the hepatic GSH caused by BSO treatment, and returned to the control level. These results indicate that when the hepatic GSH concentration was significantly decreased by fasting and the administration of BSO, hepatic MT acted as a scavenger of free radicals. We suggest that GSH and MT act cooperatively as antioxidants to scavenge free radicals produced in response to various forms of stress, and MT serves as a second rather than the first line of defense.