Metallothionein and apoptosis in the toxic milk mutant mouse

Toxic milk mutant (tx) mice accumulate excess copper (Cu) in liver with age and develop symptoms similar to those seen in human Wilson disease. Because metallothionein (MT) is the major Cu-binding protein in tx 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 control DL mice, developed regenerative liver nodules (tx-N) with normal histologic appearance. Residual, non-nodular tx mouse liver (tx-R) was microscopically abnormal with large, atypical hepatocytes. The levels 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 spectrophotometry, 109cadmium-heme assay, and the TUNEL method, respectively. Significantly higher levels of MT, Cu, and Zn, as well as increased numbers of APC were found in both tx-N and tx-R compared with DL mouse livers. Intense nuclear and cytoplasmic immunohistochemical staining for MT was observed in both normal and atypical hepatocytes of the tx mouse, whereas only cytoplasmic staining for MT was detected in DL mouse liver tissue. Accumulated Cu could be detected in tx-R and tx-N liver by rhodanine staining but was not detected in other tx mouse organs, or in mouse liver or other organs of DL. The number of APC and level 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 accumulate excess Cu in liver accompanied by striking morphologic changes, and (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.