METALLOTHIONEIN AND APOPTOSIS IN THE TOXIC MILK MUTANT MOUSE

Toxic milk mutant (tx) mice accumulate excess copper (Cu) in liver wit h age and develop symptoms similar to those seen in human Wilson disea se. Because metallothionein (MT) is the major Cu-binding protein in tu mouse liver and Cu-MT can enhance lipid peroxidation initiated by an organic hydroperoxide, the potential genotoxicity of Cu-MT in tx mice was assessed in male tx mice (11 to 12 months old) and in age-and sex- matched control wild-type (DL) mice. Toxic milk mutant mice, but not c ontrol DL mice, developed regenerative liver nodules (tx-N) with norma l histologic appearance. Residual, non-nodular tu mouse liver (tx-R) w as microscopically abnormal with large, atypical hepatocytes. The leve ls of Cu, zinc (Zn), and MT, and the numbers of apoptotic cells (APC) in tx-N, tx-R, and DL livers were measured by atomic absorption spectr ophotometry, (109)cadmium-heme assay, and the TUNEL method, respective ly. Significantly higher levels of MT, Cu, and Zn, as well as increase d numbers of APC were found in both tx-N and tx-R compared with DL mou se livers. Intense nuclear and cytoplasmic immunohistochemical stainin g for MT was observed in both normal and atypical hepatocytes of the t x mouse, whereas only cytoplasmic staining for MT was detected in DL m ouse liver tissue. Accumulated Cu could be detected in tu-R and tx-N l iver by rhodanine staining but was not detected in other tu mouse orga ns, or in mouse liver or other organs of DL. The number of APC and lev el of MT were significantly higher in tx-R liver compared with both tx -N and DL liver. These results suggest that: (a) aged tx mouse accumul ate excess Cu in liver accompanied by striking morphologic changes, an d (b) although MT binds to Cu in tx mouse liver, the presence of high Cu-MT and Cu in the nucleus can be genotoxic and may lead to enhanced apoptosis.