Vaccine effectiveness against severe COVID-19 during the Omicron wave in Germany: results from the COViK study.

PURPOSE: COViK, a prospective hospital-based multicenter case-control study in Germany, aims to assess the effectiveness of COVID-19 vaccines against severe disease. Here, we report vaccine effectiveness (VE) against COVID-19-caused hospitalization and intensive care treatment during the Omicron wave. METHODS: We analyzed data from 276 cases with COVID-19 and 494 control patients recruited in 13 hospitals from 1 December 2021 to 5 September 2022. We calculated crude and confounder-adjusted VE estimates. RESULTS: 21% of cases (57/276) were not vaccinated, compared to 5% of controls (26/494; p < 0.001). Confounder-adjusted VE against COVID-19-caused hospitalization was 55.4% (95% CI: 12-78%), 81.5% (95% CI: 68-90%) and 95.6% (95%CI: 88-99%) after two, three and four vaccine doses, respectively. VE against hospitalization due to COVID-19 remained stable up to one year after three vaccine doses. CONCLUSION: Three vaccine doses remained highly effective in preventing severe disease and this protection was sustained; a fourth dose further increased protection.

The Function of ATPase Copper Transporter ATP7B in Intestine.

BACKGROUND & AIMS: Wilson disease is a disorder of copper (Cu) misbalance caused by mutations in ATP7B. ATP7B is highly expressed in the liver-the major site of Cu accumulation in patients with Wilson disease. The intestine also expresses ATP7B, but little is known about the contribution of intestinal ATP7B to normal intestinal copper homeostasis or to Wilson disease manifestations. We characterized the role of ATP7B in mouse intestinal organoids and tissues. METHODS: We collected intestinal tissues from ATP7B-knockout (Atp7b-/-) and control mice, and established 3-dimensional enteroids. Immunohistochemistry and x-ray fluorescence were used to characterize the distribution of ATP7B and Cu in tissues. Electron microscopy, histologic analyses, and immunoblotting were used to determine the effects of ATP7B loss. Enteroids derived from control and ATP7B-knockout mice were incubated with excess Cu or with Cu-chelating reagents; effects on cell fat content and ATP7B levels and localization were determined by fluorescent confocal microscopy. RESULTS: ATP7B maintains a Cu gradient along the duodenal crypt-villus axis and buffers Cu levels in the cytosol of enterocytes. These functions are mediated by rapid Cu-dependent enlargement of ATP7B-containing vesicles and increased levels of ATP7B. Intestines of Atp7b-/- mice had reduced Cu storage pools in intestine, Cu depletion, accumulation of triglyceride-filled vesicles in enterocytes, mislocalization of apolipoprotein B, and loss of chylomicrons. In primary 3-dimensional enteroids, administration of excess Cu or Cu chelators impaired assembly of chylomicrons. CONCLUSIONS: ATP7B regulates vesicular storage of Cu in mouse intestine. ATP7B buffers Cu levels in enterocytes to maintain a range necessary for formation of chylomicrons. Misbalance of Cu and lipid in the intestine could account for gastrointestinal manifestations of Wilson disease.

Animal models of Wilson disease.

Wilson disease (WD) is caused by ATPase copper-transporting beta (ATP7B) mutations and results in copper toxicity in liver and brain. Although the defective gene was identified in 1993, the specific mechanisms underlying copper toxicity and the remarkable phenotypic diversity of the disease are still poorly understood. Animal models harboring defects in the ATP7B homolog have helped to reveal new insights into pathomechanisms of WD. Four rodent models with ATP7B gene defects have been described - the Long-Evans Cinnamon (LEC) rat, inbred mouse models (toxic milk (tx), the Jackson Laboratory toxic milk (tx-j)), and the genetically engineered ATP7B-/- (knockout) mouse - all of which develop liver disease to different extents. Copper accumulation in parts of the brain accompanied by some neurologic involvement was revealed in LEC rats and tx/tx-j mice, but the pathology is less severe than human neurologic WD. Several dogs show hepatic copper toxicity resembling WD; however, brain involvement has not been observed and the underlying genetic defect is different. These models are of great value for examination of copper distribution and metabolism, gene expression, and investigation of liver and brain pathology. The availability of disease models is essential for therapeutic interventions such as drug, gene, and cell therapy. Findings made by animal studies may facilitate the development of specific therapies to ameliorate WD progression.

Activation of liver X receptor/retinoid X receptor pathway ameliorates liver disease in Atp7B(-/-) (Wilson disease) mice.

UNLABELLED: Wilson disease (WD) is a hepatoneurological disorder caused by mutations in the copper-transporter, ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurological symptoms. We demonstrate that in Atp7b(-/-) mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b(-/-) mice identified dysregulation of nuclear receptors (NRs), especially the liver X receptor (LXR)/retinoid X receptor heterodimer, as an important event in WD pathogenesis. Treating Atp7b(-/-) mice with the LXR agonist, T0901317, ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized, and liver function and histology were improved. CONCLUSIONS: The results demonstrate the major role of an altered NR function in the pathogenesis of WD and suggest that modulation of NR activity should be explored as a supplementary approach to improving liver function in WD. (Hepatology 2016;63:1828-1841).

Hepatobiliary malignancies in Wilson disease.

BACKGROUNDS & AIMS: Reports of hepatobiliary malignancies in Wilson disease are sparse. The aim of this study was to evaluate hepatobiliary malignancies in Wilson disease patients concerning the clinical course of tumour disease and pathological analysis of tumour tissue. METHODS: Multicenter cohort study of patients with confirmed diagnosis of Wilson disease treated at the Second Department of Neurology, Institute of Psychiatry and Neurology, Warsaw, Poland, the university hospitals Heidelberg, Duesseldorf and Dresden, Germany, and the Department of Hepatology, University Leuven, Belgium. Occurrence, treatment and outcome of hepatobiliary tumours were analysed retrospectively. RESULTS: Of a total of 1186 patients, fourteen developed hepatobiliary malignancies. Eight were hepatocellular carcinomas (HCC) and six were intrahepatic cholangiocellular carcinomas (ICC). The prevalence of hepatobiliary malignancies in the cohort was 1.2% and the incidence was 0.28 per 1000 person years. Pathological analysis of tumour material showed no abnormal copper concentration. CONCLUSIONS: The rate of hepatobiliary malignancies in Wilson disease is very low, even in cirrhotic patients. As a result of the relevant number of ICC in addition to HCC histological analysis through surgical resection or biopsy should be mandatory when a suspect liver lesion is detected. The influence of copper depletion from Wilson disease-specific medical treatment on tumour activity remains to be elucidated.

Structural and metabolic changes in Atp7b-/- mouse liver and potential for new interventions in Wilson's disease.

Wilson's disease (WD) is caused by ATP7B mutations and results in copper accumulation and toxicity in liver and brain tissues. The specific mechanisms underlying copper toxicity are still poorly understood. Mouse models have revealed new insights into pathomechanisms of hepatic WD. Mitochondrial damage is observed in livers of WD patients and in mouse models; copper induces fragmentation of mitochondrial membrane lipids, particularly cardiolipin, with deleterious effects on both mitochondrial integrity and function. Copper accumulation also induces chronic inflammation in WD livers, which is followed by regeneration in parts of the liver and occasionally neoplastic proliferation. Gene expression studies using microarrays have aided our understanding of the molecular basis of these changes. Copper overload alters cholesterol biosynthesis in hepatocytes resulting in reduced liver and serum cholesterol. Experiments are currently underway to elucidate the link between copper and cholesterol metabolism. These findings may facilitate the development of specific therapies to ameliorate WD progression.

Non-invasive evaluation of hepatic manifestation in Wilson disease with transient elastography, ARFI, and different fibrosis scores.

OBJECTIVE: Noninvasive investigation of liver fibrosis with ultrasound-based elastography and laboratory-based fibrosis indices have been established in various chronic liver diseases within the last years. We aimed to evaluate feasibility and diagnostic value of transient elastography (TE), acoustic radiation force impulse imaging (ARFI), and different serologic fibrosis indices in Wilson's disease (WD). MATERIALS AND METHODS: TE and ARFI were performed in 50 Wilson patients. In addition, AST/Platelet Ratio Index (APRI), FIB-4, and Forns score were calculated. Hepatic fibrosis was classified by a clinical score. RESULTS: Of the 50 Wilson patients 41 had hepatic manifestation of WD. TE results were significantly increased in advanced hepatic fibrosis (7.0 ± 2.2 kPa; p < 0.05) and cirrhosis (10.1 ± 6.73 kPa; p < 0.05) compared to individuals without hepatic manifestation (5.0 ± 1.4 kPa). Right liver lobe ARFI (R-ARFI) values were only increased in cirrhotic patients (1.43 ± 0.28 vs. 1.19 ± 0.14 m/s; p < 0.05). The cutoff values to best discriminate cirrhosis were 6.1 kPa for TE and 1.29 m/s for R-ARFI. Left lobe ARFI failed to provide additional diagnostic benefit. Elastography methods displayed a significant correlation with APRI, FIB-4, and Forns indices (Pearson's rho > 0.33; p < 0.03). CONCLUSIONS: TE displayed a gradual increase between different stages of hepatic manifestation in WD and could significantly discriminate cirrhosis. The TE cutoff for cirrhosis may be clinically more relevant than the R-ARFI value.

Diverse functional properties of Wilson disease ATP7B variants.

BACKGROUND & AIMS: Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS: We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper ((64)Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS: Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS: Variants in ATP7B associated with Wilson disease disrupt the protein's transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype-phenotype correlation and mechanisms of disease pathogenesis.

Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b-/- mouse model of Wilson's disease.

Cellular copper overload as found in Wilson's disease may disturb mitochondrial function and integrity. Atp7b(-/-) mice accumulate copper in the liver and serve as an animal model for this inherited disease. The molecular mechanism of copper toxicity in hepatocytes is poorly understood. Total mitochondrial lipids from liver of wild-type mice were subjected to oxidative stress by the Cu(2+)/H(2)O(2)/ascorbate system. Phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were detected as cardiolipin fragmentation products by thin-layer chromatography combined with MALDI-TOF mass spectrometry in oxidized samples, but not in unperturbed ones. The formation of PA and PHA in copper-treated model membrane correlated well with the decrease of cardiolipin. Mitochondrial lipids from Atp7b(-/-) mice of different age were analyzed for the presence of PA. While 32-weeks old wild-type (control) and Atp7b(-/-) mice did not show any PA, there was a steady increase in the amount of this lipid in Atp7b(-/-) mice in contrast to control with increasing age. Hepatocytes from elder Atp7b(-/-)mice contained morphologically changed mitochondria unlike cells from wild-type animals of the same age. We concluded that free-radical fragmentation of cardiolipin with the formation of PA is a likely mechanism that damages mitochondria under conditions of oxidative stress due to copper overload. Our findings are relevant for better understanding of molecular mechanisms for liver damage found in Wilson's disease.

Wilson disease.

Wilson disease is an inherited autosomal recessive disorder of copper balance leading to hepatic damage and neurological disturbance of variable degree. The defective gene, ATP7B, encodes a hepatic copper-transporting protein, which plays a key role in human copper metabolism. Our knowledge of the genetic basis of Wilson disease has increased dramatically; however, understanding of genotype-phenotype correlation and multifarious effects of copper toxicity as basis for targeted and individualised therapy strategies is still insufficient. Clinical manifestations are related to copper accumulation predominantly in the liver and brain and include hepatic disease ranging from mild hepatitis to acute liver failure or cirrhosis and/or neurological symptoms such as dystonia, tremor, dysarthria, psychiatric disturbances. Mixed presentations occur frequently. Early recognition by means of clinical, biochemical or genetic examination and initiation of therapy with copper chelators, zinc salts or even liver transplantation in cases of acute and chronic liver failure are essential for favourable outcome.

Wilson disease at a single cell level: intracellular copper trafficking activates compartment-specific responses in hepatocytes.

Wilson disease (WD) is a severe hepato-neurologic disorder that affects primarily children and young adults. WD is caused by mutations in ATP7B and subsequent copper overload. However, copper levels alone do not predict severity of the disease. We demonstrate that temporal and spatial distribution of copper in hepatocytes may play an important role in WD pathology. High resolution synchrotron-based x-ray fluorescence imaging in situ indicates that copper does not continuously accumulate in Atp7b(-/-) hepatocytes, but reaches a limit at 90-300 fmol. The lack of further accumulation is associated with the loss of copper transporter Ctr1 from the plasma membrane and the appearance of copper-loaded lymphocytes and extracellular copper deposits. The WD progression is characterized by changes in subcellular copper localization and transcriptome remodeling. The synchrotron-based x-ray fluorescence imaging and mRNA profiling both point to the key role of nucleus in the initial response to copper overload and suggest time-dependent sequestration of copper in deposits as a protective mechanism. The metabolic pathways, up-regulated in response to copper, show compartmentalization that parallels changes in subcellular copper concentration. In contrast, significant down-regulation of lipid metabolism is observed at all stages of WD irrespective of copper distribution. These observations suggest new stage-specific as well as general biomarkers for WD. The model for the dynamic role of copper in WD is proposed.

Genetic regulation of serum phytosterol levels and risk of coronary artery disease.

BACKGROUND: Phytosterols are plant-derived sterols that are taken up from food and can serve as biomarkers of cholesterol uptake. Serum levels are under tight genetic control. We used a genomic approach to study the molecular regulation of serum phytosterol levels and potential links to coronary artery disease (CAD). METHODS AND RESULTS: A genome-wide association study for serum phytosterols (campesterol, sitosterol, brassicasterol) was conducted in a population-based sample from KORA (Cooperative Research in the Region of Augsburg) (n=1495) with subsequent replication in 2 additional samples (n=1157 and n=1760). Replicated single-nucleotide polymorphisms (SNPs) were tested for association with premature CAD in a metaanalysis of 11 different samples comprising 13 764 CAD cases and 13 630 healthy controls. Genetic variants in the ATP-binding hemitransporter ABCG8 and at the blood group ABO locus were significantly associated with serum phytosterols. Effects in ABCG8 were independently related to SNPs rs4245791 and rs41360247 (combined P=1.6 x 10(-50) and 6.2 x 10(-25), respectively; n=4412). Serum campesterol was elevated 12% for each rs4245791 T-allele. The same allele was associated with 40% decreased hepatic ABCG8 mRNA expression (P=0.009). Effects at the ABO locus were related to SNP rs657152 (combined P=9.4x10(-13)). Alleles of ABCG8 and ABO associated with elevated phytosterol levels displayed significant associations with increased CAD risk (rs4245791 odds ratio, 1.10; 95% CI, 1.06 to 1.14; P=2.2 x 10(-6); rs657152 odds ratio, 1.13; 95% CI, 1.07 to 1.19; P=9.4 x 10(-6)), whereas alleles at ABCG8 associated with reduced phytosterol levels were associated with reduced CAD risk (rs41360247 odds ratio, 0.84; 95% CI, 0.78 to 0.91; P=1.3 x 10(-5)). CONCLUSION: Common variants in ABCG8 and ABO are strongly associated with serum phytosterol levels and show concordant and previously unknown associations with CAD.

Hepatocyte GP73 expression in Wilson disease.

BACKGROUND/AIMS: Wilson disease (WD) is a disorder of copper transport caused by mutations within the ATP7B gene. WD is phenotypically variable and can present with predominantly hepatic or neurologic manifestations. The mechanisms responsible for this variability are unknown. GP73, a Golgi membrane protein, is expressed in hepatocytes in response to acute and chronic liver disease. METHODS: Hepatocyte GP73 expression was examined in the livers of WD patients by semiquantitative immunohistochemistry. GP73 mRNA levels were measured in mice with a deletion of the WD gene (Atp7b(-/-)) by real-time PCR, and these values were compared to the concomitant histological abnormalities and previously reported copper levels. RESULTS: Hepatocyte GP73 expression was more frequently observed in patients with hepatic versus neurologic presentation (79% vs. 30%, p<0.05). Furthermore, GP73 expression was significantly higher (44.7+/-14.0 vs. 2.0+/-0.81, p<0.05) in patients with hepatic phenotype. In Atp7b(-/-) mice, GP73 mRNA was significantly elevated at 20-46 weeks of age, coincident with extensive hepatic inflammation and fibrosis, but not at 6 weeks, when hepatic histology was normal despite significant copper overload. GP73 mRNA levels normalized concomitantly with the resolution of hepatic injury at 60-weeks. However, in tumor-like nodules GP73 was strikingly elevated. CONCLUSION: Increased hepatocyte GP73 expression is more commonly a feature of hepatic than neurologic WD, and is triggered in response to inflammation, fibrosis, and dysplasia, rather than copper overload.

Functional interactions of Cu-ATPase ATP7B with cisplatin and the role of ATP7B in the resistance of cells to the drug.

Cisplatin is a widely used chemotherapeutic agent for treatment of ovarian, testicular, lung, and stomach cancers. The initial response to the drug is robust; however, tumor cells commonly develop resistance to cisplatin, which complicates treatment. Recently, overexpression of the Cu-ATPase ATP7B in ovary cells was linked to the increased cellular resistance to cisplatin; and the role for Cu-ATPases in the export of cisplatin from cells was proposed. Our results support functional interactions between cisplatin and ATP7B but argue against the active transport through the copper translocation pathway as a mechanism of drug resistance. In hepatocytes, we observed no correlation between the levels of endogenous ATP7B and the resistance of cells to cisplatin. Unlike copper, cisplatin does not induce trafficking of ATP7B in hepatoma cells, neither does it compete with copper in a transport assay. However, cisplatin binds to ATP7B and stimulates catalytic phosphorylation with EC(50) similar to that of copper. Mutations of the first five N-terminal copper-binding sites of ATP7B do not inhibit the cisplatin-induced phosphorylation of ATP7B. In contrast, the deletion of the first four copper-binding sites abolishes the effect of cisplatin on the ATP7B activity. Thus, cisplatin binding to ATP7B and/or general changes in cellular copper homeostasis are likely contributors to the increased resistance to the drug. The link between changes in copper homeostasis and cisplatin resistance was confirmed by treating the Huh7 cells with copper chelator and increasing their resistance to cisplatin.cisplatin.

Free radical fragmentation of cardiolipin by cytochrome c.

The effect of cytochrome c (cyt c) on degradation of cardiolipin in its polar part was investigated in cardiolipin/phosphatidylcholine (CL/PC) liposomes incubated with cyt c/H(2)O(2)/and (or) ascorbate by high-performance thin layer chromatography and MALDI-TOF mass spectrometry. It has been shown that phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were formed in the system under conditions where hydrogen peroxide favours a release of heme iron from cyt c. The formation of PA and PHA occurs via an OH-induced fragmentation taking place in the polar moiety of cardiolipin. Formation of fragmentation products correlated with the loss of CL in CL/PC liposomes incubated with cyt c/H(2)O(2)/ascorbate or with Cu(2+)/H(2)O(2)/ascorbate.

Formation of phosphatidic acid in stressed mitochondria.

Mitochondria are an important intracellular source of ROS as well as a sensitive target for oxidative damage under certain pathological conditions such as iron or copper overload. Mitochondrial membranes are rich in the tetraacyl phospholipid cardiolipin. Its integrity is important for efficient oxidative phosphorylation. Mouse liver mitochondria were subjected to oxidative stress by the Cu(2+)(Fe(2+))/H(2)O(2)/ascorbate system. Phosphatidic acid was detected in oxidized mitochondria, but not in unperturbed mitochondria. The Cu(2+)/H(2)O(2)/and (or not) ascorbate system caused the formation of phosphatidic acid and phosphatidylhydroxyacetone in cardiolipin liposomes. These products proceed via an HO*-radical induced fragmentation taking place in the polar moiety of cardiolipin. Mass spectrometry analysis of phosphatidic acid newly formed in mitochondria revealed that it has been derived from fragmentation of cardiolipin. Thus, free-radical fragmentation of cardiolipin in its polar part with the formation of phosphatidic acid is a likely mechanism that damages mitochondria under conditions of oxidative stress.

Wilson disease: not just a copper disorder. Analysis of a Wilson disease model demonstrates the link between copper and lipid metabolism.

Copper is an essential nutrient required for normal growth and development in many organisms. In humans, the disruption of normal copper absorption and excretion is associated with two severe disorders, known as Menkes disease and Wilson disease, respectively. The consequences of insufficient copper supply that is characteristic of Menkes disease have been largely linked to the inactivation of key metabolic enzymes, although other non-enzymatic processes may also be involved. In contrast, the consequences of copper accumulation in Wilson disease have been generally ascribed to copper-induced radical-mediated damage. Recent studies suggest that the cellular response to copper overload, particularly at the early stages of copper accumulation, involves more specific mechanisms and specific pathways. Genetic and metabolic characterization of animal models of Wilson disease has provided new insights into the pre-symptomatic effects of copper that is accumulated in the liver. The studies have uncovered unexpected links between copper metabolism, cell-cycle machinery, and cholesterol biosynthesis. We discuss these new findings along with the earlier reports on dietary effects of copper. Together these experiments suggest a tight link between lipid and copper metabolism and identify several candidate proteins that may mediate the cross-talk between copper status and lipid metabolism.

High copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of Wilson disease.

Copper is essential for human physiology, but in excess it causes the severe metabolic disorder Wilson disease. Elevated copper is thought to induce pathological changes in tissues by stimulating the production of reactive oxygen species that damage multiple cell targets. To better understand the molecular basis of this disease, we performed genome-wide mRNA profiling as well as protein and metabolite analysis for Atp7b-/- mice, an animal model of Wilson disease. We found that at the presymptomatic stages of the disease, copper-induced changes are inconsistent with widespread radical-mediated damage, which is likely due to the sequestration of cytosolic copper by metallothioneins that are markedly up-regulated in Atp7b-/- livers. Instead, copper selectively up-regulates molecular machinery associated with the cell cycle and chromatin structure and down-regulates lipid metabolism, particularly cholesterol biosynthesis. Specific changes in the transcriptome are accompanied by distinct metabolic changes. Biochemical and mass spectroscopy measurements revealed a 3.6-fold decrease of very low density lipoprotein cholesterol in serum and a 33% decrease of liver cholesterol, indicative of a marked decrease in cholesterol biosynthesis. Consistent with low cholesterol levels, the amount of activated sterol regulatory-binding protein 2 (SREBP-2) is increased in Atp7b-/- nuclei. However, the SREBP-2 target genes are dysregulated suggesting that elevated copper alters SREBP-2 function rather than its processing or re-localization. Thus, in Atp7b-/- mice elevated copper affects specific cellular targets at the transcription and/or translation levels and has distinct effects on liver metabolic function, prior to appearance of histopathological changes. The identification of the network of specific copper-responsive targets facilitates further mechanistic analysis of human disorders of copper misbalance.

Consequences of copper accumulation in the livers of the Atp7b-/- (Wilson disease gene) knockout mice.

Wilson disease is a severe genetic disorder associated with intracellular copper overload. The affected gene, ATP7B, has been identified, but the molecular events leading to Wilson disease remain poorly understood. Here, we demonstrate that genetically engineered Atp7b-/- mice represent a valuable model for dissecting the disease mechanisms. These mice, like Wilson disease patients, have intracellular copper accumulation, low-serum oxidase activity, and increased copper excretion in urine. Their liver pathology developed in stages and was determined by the time of exposure to elevated copper rather than copper concentration per se. The disease progressed from mild necrosis and inflammation to extreme hepatocellular injury, nodular regeneration, and bile duct proliferation. Remarkably, all animals older than 9 months showed regeneration of large portions of the liver accompanied by the localized occurrence of cholangiocarcinoma arising from the proliferating bile ducts. The biochemical characterization of Atp7b-/- livers revealed copper accumulation in several cell compartments, particularly in the cytosol and nuclei. The increase in nuclear copper is accompanied by marked enlargement of the nuclei and enhanced DNA synthesis, with these changes occurring before pathology development. Our results suggest that the early effects of copper on cell genetic material contribute significantly to pathology associated with Atp7b inactivation.