Discussion

In agreement with the key role of copper for the development of hepatitis in LEC rats, the metal accumulated in the liver with age. During the progression of hepatitis, the metal shifted from the cytosolic to the noncytosolic compartment of the liver. The decrease in the concentration of hepatic copper in severely affected animals is considered to result from the leakage of cytosolic copper from necrotic hepatocytes into blood. Accordingly, in these animals, elevated copper levels were observed in the blood and kidney (21).

Here, we report that in the LEC rat, the noncytosolic copper was mainly detected in lysosomes. Also in Wilson's disease, the localization of hepatic copper varies with the stage of the disease: In the early stage, copper is diffuse in the cytoplasm of hepatocytes, whereas later, when fibrosis and cirrhosis are the predominant histopathological features, the metal is mainly located in hepatocyte lyso-somes (22).

There is considerable uncertainty on the subcellular site of hepatic injury caused by excess copper. From studies with Wilson's disease patients, it has been postulated that hepatic copper in the cytosol, which is predominant in the early stage of the disease, is toxic (22). In the liver of our LEC animals, copper levels were also fairly high in the cytosol before the onset of acute hepatitis; however, the total of copper was bound to MT. Consistent with the role of MT in detoxifying metals, these findings do not support a prominent role of cytosolic copper in initiating hepatotoxicity.

The assumption that the accumulation of copper in lysosomes represents a detoxification pathway (23) is questionable because findings in humans (1) and animals (24,25) suggest that, in particular, hepatocytes packed with copper-loaded lysosomes are the cells that undergo necrosis (26). Undoubtedly, the molecular association of copper within the lysosomes is of crucial importance with regard to its

Fig. 3. Copper (top) and MT (bottom) in density gradient fractions of crude lysosomes from livers of LEC rats (mean values, n = 3-11). Crude lysosomes were obtained by differential centrifugation as described in ref. 18. To separate lysosomes from mitochondria, crude lysosomes were layered on a Nycodenz gradient of the following composition: 1 mL 40%, 1 mL 33%, 3 mL 28%, 2 mL 27% and 2 mL 24% (w/v) Nycodenz in 10 mM Tris-HCl (pH 7.4) and centrifuged at 74,100gmax (swing-out rotor) for 3 h. After centrifugation, the gradient was fractionated in aliquots of 1 mL. The inset shows a typical distribution profile of the lysosome-specific acid phosphatase (AP) and the mitochondria-specific succinate dehydrogenase (SDH) activities. MT was determined by the thiomolybdate-method (19) after incubating the sample with guanidinium thiocyanate and 2-mercaptoethanol as described in ref. 18. [In part from Klein, D., et al., J. Hepatol. 32, 193-201 (2000), with permission.]

Fig. 3. Copper (top) and MT (bottom) in density gradient fractions of crude lysosomes from livers of LEC rats (mean values, n = 3-11). Crude lysosomes were obtained by differential centrifugation as described in ref. 18. To separate lysosomes from mitochondria, crude lysosomes were layered on a Nycodenz gradient of the following composition: 1 mL 40%, 1 mL 33%, 3 mL 28%, 2 mL 27% and 2 mL 24% (w/v) Nycodenz in 10 mM Tris-HCl (pH 7.4) and centrifuged at 74,100gmax (swing-out rotor) for 3 h. After centrifugation, the gradient was fractionated in aliquots of 1 mL. The inset shows a typical distribution profile of the lysosome-specific acid phosphatase (AP) and the mitochondria-specific succinate dehydrogenase (SDH) activities. MT was determined by the thiomolybdate-method (19) after incubating the sample with guanidinium thiocyanate and 2-mercaptoethanol as described in ref. 18. [In part from Klein, D., et al., J. Hepatol. 32, 193-201 (2000), with permission.]

toxicity. As shown here, the lysosomal copper was associated with insoluble material, which could be solubilized under reducing conditions. The solubilized product had an apparent molecular weight of 7.3 kDa and crossreacted with MT-specific antibodies. Accordingly, the insoluble material is considered to consist of polymers of degradation products of copper-containing MT. The formation of

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