Identification of superoxide dismutase as a potential urinary marker of carbon tetrachloride-induced hepatic toxicity

The aim of this study was the identification of a novel protein marker of hepatotoxicity in rat urine. Rats were dosed by gavage with carbon tetrachloride (CCl4) to induce acute liver injury. Surface enhanced laser desorption/ionisation (SELDI) ProteinChip technology revealed the appearance of a 15.7 kDa protein in the CCl4-treated rat urine. One-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis (SDS–PAGE) identified an 18.4 kDa protein in the CCl4-treated rat urine. The appearance of either protein was coincident over a time course during which they first appeared at 12 h post-dosing, peaked at 36 h and had disappeared again within 3 days post-dosing. The protein was identified by in-gel digestion and nano-electrospray (nano-ES)-tandem mass spectrometry as Cu/Zn superoxide dismutase (SOD-1). SOD activity was found to be increased by 61.4-fold in CCl4-treated rat urine. Western blots of tissue homogenates from the rats revealed a time-dependent loss of SOD-1 from the livers of CCl4-treated rats matching the time course of SOD-1 appearance in urine. SOD-1 is not specifically located in liver; however, its appearance in urine in response to acute CCl4-induced hepatotoxicity is a novel finding; this coupled with loss from the liver following injury suggests urinary SOD-1 may be a potential marker of hepatotoxicity.