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Fig. 4. (A) Electron micrograph of the high-density fraction (fraction no. 1 of the density gradient) of crude lysosomes from a jaundiced LEC rat (original magnification 52,400x). (B) Energy-dispersive X-ray microanalysis of the lysosomal electron-dense matrix. Aluminum (Al), nickel (Ni), and platinum (Pt) peaks are derived from the stub, the grid, and the surface coating of the specimen, respectively. [From ref. 18, with permission.]

these polymers relates to the uptake of highly copper-loaded MT from the cytosol into the lysosomes. Generally, the fate of MT in the lysosome is mainly determined by the metal composition of the protein: Because of the acidic pH of the lysosomal matrix, zinc-containing MT will easily lose the metal, and the apoprotein, consequently, will be degraded (27). Because metal removal from copper-MT requires a lower pH (28), the lysosomal pH of typically 4.7 is likely not low enough to remove significant amounts of copper from highly copper-loaded MT, thus rendering the protein fairly stable

Fig. 5. HPLC gel chromatography of cytosol, solubilized lysosomes (density gradient fraction no. 9) and solubilized dense bodies (gradient fraction no. 1) from the liver of a jaundiced rat. The inset shows the relative elution volumes of chicken ovalbumin (molecular weight [MW] 44,000), equine myoglobin (MW 17,000), aprotinin (MW 6500), P-endorphin (MW 3465), and angiotensin (MW 1297). The bracket indicates the relative elution volume of native MT from rat liver. (From ref. 18, with permission.)

Fig. 5. HPLC gel chromatography of cytosol, solubilized lysosomes (density gradient fraction no. 9) and solubilized dense bodies (gradient fraction no. 1) from the liver of a jaundiced rat. The inset shows the relative elution volumes of chicken ovalbumin (molecular weight [MW] 44,000), equine myoglobin (MW 17,000), aprotinin (MW 6500), P-endorphin (MW 3465), and angiotensin (MW 1297). The bracket indicates the relative elution volume of native MT from rat liver. (From ref. 18, with permission.)

against proteolysis. Consistently, copper-containing MT in vitro was reported to be fairly resistant against hydrolysis by lysosomal enzymes (29). Furthermore, in vivo, the intralysosomal pH has been found to be increased after copper overload (25). Our results suggest that the highly copper-loaded MT in lysosomes is only partly degraded. The resulting proteolysis-resistant product presumably represent the P-domain of MT, which in copper-containing MT (30,31) is more stable than the a-domain. The P-domain then might polymerize and form an oxidized, insoluble material that apparently cannot be excreted into bile. The polymerization process likely proceeds through a radical-mediated mechanism involving iron which has been shown to be massively elevated in the hepatocyte lysosomes of LEC rats (18). The degradation products of copper-containing MT obviously are polymerized through disulfide bridges because the solubilization of the polymer by guanidinium thiocy-anate required the presence of 2-mercaptoethanol. Therefore, it is reasonable to assume that copper associated with the polymer is more loosely bound and therefore more reactive than copper bound to native MT. Supporting this assumption, copper in the copper-rich granules is histochemically stain-able with rhodanine, whereas copper bound to native MT is not (32,33). The reactive polymer-associated copper may initiate lipid peroxidation of lysosomal membranes, resulting in lysosomal rupture and release of hydrolytic enzymes into the cytoplasm.

According to our ultrastructural findings, copper is massively located in Kupffer cells in the liver of jaundiced LEC rats. This is most likely the result of phagocytotic copper uptake from necrotic or apoptotic hepatocytes. Once within the macrophages, this material seems to further aggregate to copper-rich granules. The accumulation of high amounts of presumably reactive copper in Kupffer cells may amplify the liver damage either directly or through stimulation of these cells. A similar

Fig. 6. Copper elution profile of gel chromatographically separated liver cytosol (circles) from a LEC rat aged 77 d and copper-rich polymers (squares) from a jaundiced LEC rat before (open symbols) and after (closed symbols) incubation with D-penicillamine (DPA, 185 mjtf, 5 min, mean ± SD, n = 3). The brackets indicate the elution volume of native metallothionein (MT) from rat liver and a mixture of copper:DPA of 1:1.250 mol/mol, respectively. The inset shows the relative elution volumes of protein standards (see Fig. 5). [From Klein, D., et al., J. Hepatol. 32, 193-201 (2000), with permission.]

Fig. 6. Copper elution profile of gel chromatographically separated liver cytosol (circles) from a LEC rat aged 77 d and copper-rich polymers (squares) from a jaundiced LEC rat before (open symbols) and after (closed symbols) incubation with D-penicillamine (DPA, 185 mjtf, 5 min, mean ± SD, n = 3). The brackets indicate the elution volume of native metallothionein (MT) from rat liver and a mixture of copper:DPA of 1:1.250 mol/mol, respectively. The inset shows the relative elution volumes of protein standards (see Fig. 5). [From Klein, D., et al., J. Hepatol. 32, 193-201 (2000), with permission.]

stimulatory role of Kupffer cells has been also reported in chemical- and metal-induced hepatotoxic-ity (34,35).

The therapeutic principle of DPA in treating Wilson's disease is thought to involve the depletion of excess copper in the liver through chelation or to facilitate copper detoxification through induction of MT. Accordingly, in Wilson's disease, DPA treatment resulted in diminished levels of hepatic copper (36), although a negative copper balance is probably not maintained after the first year of treatment (33). Scheinberg et al. (37) reported that the removal of copper from the liver is incomplete and inconsistent, and the hepatic copper levels may remain elevated even after years of therapy. These findings led the authors to suggest that DPA detoxifies copper by induction of MT rather than removes the metal from the liver (33). Induction of MT, however, has been described only in rats and mice after single intraperitoneal administration of the drug (38,39) and in our LEC rats there was no evidence of MT induction after DPA treatment (results not shown).

Consistent with earlier findings (10, 11), DPA administered to nondiseased LEC animals prevented the onset of fulminant hepatitis. The drug particularly inhibited the disease-specific accumulation of copper in the noncytosolic fraction. In diseased LEC rats, DPA treatment resulted in the reversal of liver damage. In these animals DPA sequestered copper particularly from the lysosomes (i.e., it mobilized the metal likely from the MT polymers). In agreement, upon ultrastructural examination, the electron-dense particles were absent and, simultaneously, the copper content of the polymer-containing fractions declined. The mobilization of copper from the polymers by DPA could also be demonstrated in vitro and obviously involves their solubilization and the formation of copper-DPA complexes. DPA is known to form stable high-molecular-weight complexes with copper (20,40)

and is able to undergo disulfide-exchange reactions (40). Thus, the solubilization is likely to proceed through the reduction of the disulfide bridges of the polymers.

According to the in vitro findings and in agreement with findings of others (42,43), DPA did not remove copper that was bound to MT in liver cytosol. Also in our LEC rats, most of the cytosolic MT-bound copper remained in this compartment after DPA treatment. The poor availability of MT-bound copper for binding to DPA may explain the observation that copper concentrations in normal livers of LEC rats treated with DPA for 3 mo were half of those observed in untreated animals but still 30 times greater than those of control animals (10). This, again, supports the assumption that elevated levels of cytosolic copper bound to MT is of minor importance for the hepatotoxicity of the metal. Assuming our findings on LEC rats are applicable to Wilson's disease, DPA particularly mobilizes copper, which is not bound to cytosolic MT. This fits well to the observation (37) that patients with Wilson's disease may not be truly decoppered by DPA.

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