Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease.

Wilson disease (WD) is a rare hereditary condition that is caused by a genetic defect in the copper-transporting ATPase ATP7B that results in hepatic copper accumulation and lethal liver failure. The present study focuses on the structural mitochondrial alterations that precede clinical symptoms in the livers of rats lacking Atp7b, an animal model for WD. Liver mitochondria from these Atp7b­/­ rats contained enlarged cristae and widened intermembrane spaces, which coincided with a massive mitochondrial accumulation of copper. These changes, however, preceded detectable deficits in oxidative phosphorylation and biochemical signs of oxidative damage, suggesting that the ultrastructural modifications were not the result of oxidative stress imposed by copper- dependent Fenton chemistry. In a cell-free system containing a reducing dithiol agent, isolated mitochondria exposed to copper underwent modifications that were closely related to those observed in vivo. In this cell-free system, copper induced thiol modifications of three abundant mitochondrial membrane proteins, and this correlated with reversible intramitochondrial membrane crosslinking, which was also observed in liver mitochondria from Atp7b­/­ rats. In vivo, copper-chelating agents reversed mitochondrial accumulation of copper, as well as signs of intra-mitochondrial membrane crosslinking, thereby preserving the functional and structural integrity of mitochondria. Together, these findings suggest that the mitochondrion constitutes a pivotal target of copper in WD.

The biogenic methanobactin is an effective chelator for copper in a rat model for Wilson disease.

Copper is an essential redox-active metal ion which in excess becomes toxic due to the formation of reactive oxygen species. In Wilson disease the elevated copper level in liver leads to chronic oxidative stress and subsequent hepatitis. This study was designed to evaluate the copper chelating efficiency of the bacterial methanobactin (MB) in a rat model for Wilson disease. Methanobactin is a small peptide produced by the methanotrophic bacterium Methylosinus trichosporium OB3b and has an extremely high affinity for copper. Methanobactin treatment of the rats was started at high liver copper and serum aspartate aminotransferase (AST) levels. Two dosing schedules with either 6 or 13 intraperitoneal doses of 200mg methanobactin per kg body weight were applied. Methanobactin treatment led to a return of serum AST values to basal levels and a normalization of liver histopathology. Concomitantly, copper levels declined to 45% and 24% of untreated animals after 6 and 13 doses, respectively. Intravenous application of methanobactin led to a prompt release of copper from liver into bile and the copper was shown to be associated with methanobactin. In vitro experiments with liver cytosol high in copper metallothionein demonstrated that methanobactin removes copper from metallothionein confirming the potent copper chelating activity of methanobactin.

Tracing copper-thiomolybdate complexes in a prospective treatment for Wilson's disease.

Wilson's disease is a human genetic disorder which results in copper accumulation in liver and brain. Treatments such as copper chelation therapy or dietary supplementation with zinc can ameliorate the effects of the disease, but if left untreated, it results in hepatitis, neurological complications, and death. Tetrathiomolybdate (TTM) is a promising new treatment for Wilson's disease which has been demonstrated both in an animal model and in clinical trials. X-ray absorption spectroscopy suggests that TTM acts as a novel copper chelator, forming a complex with accumulated copper in liver. We have used X-ray absorption spectroscopy and X-ray fluorescence imaging to trace the molecular form and distribution of the complex in liver and kidney of an animal model of human Wilson's disease. Our work allows new insights into metabolism of the metal complex in the diseased state.

Mutant rat strain lacking D-amino-acid oxidase.

D-amino-acid oxidase (DAO) is known to be associated with schizophrenia. Since the expression of DAO gene had been reported to be very low in LEA rats, we examined LEA/SENDAI rats in detail. These rats did not have DAO activity, enzyme protein or mRNA encoding this enzyme. Sequencing of the 5'-upstream region of the DAO gene revealed the deletion of one triplet in the 15 TAA repeats approximately 700-bp upstream of the transcription start point. A 1.3-kb upstream fragment containing the TAA repeats and the transcription start point was inserted into a reporter vector and was transfected into COS-1, NRK-52E and CCL-PK1 cells. Although the fragments containing 15 or 14 repeats had high promoter activity, the fragment containing 13 repeats had very weak activity. Electrophoretic mobility-shift assays showed that the nuclear extracts from COS-1 and COS-7 cells had proteins that bound to the oligonucleotides containing the TAA repeats. These results suggest that the TAA repeats are important for expression of the DAO gene. The LEA/SENDAI rats lacking DAO would be a useful tool for the investigations aimed at the elucidation of the relationships between this flavoenzyme and schizophrenia.

Effects of marginal iron overload on iron homeostasis and immune function in alveolar macrophages isolated from pregnant and normal rats.

The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx and egress have been evaluated. Macrophages isolated from iron deficient rats, or pregnant rats at day 21 of gestation, either supplemented with a single dose of iron dextran, 10 mg, at the commencement of pregnancy, or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic Hamp mRNA expression. IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy +/- a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplementation, alone or combined with pregnancy, increased the activities of both NADPH oxidase and nuclear factor kappa B (NFkappaB). In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimuli. Increasing iron status, if only marginally, will reduce the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms.

Isolation of highly pure rat liver mitochondria with the aid of zone-electrophoresis in a free flow device (ZE-FFE).

This protocol describes the purification of mitochondria from rat liver with the aid of zone electrophoresis in a free flow device (ZE-FFE). Starting from liver homogenate, cell debris and nuclei are removed by low speed centrifugation. A crude mitochondrial fraction is obtained by medium speed centrifugation and is further purified by washing followed by a Nycodenz gradient centrifugation. Lysosomes and microsomes are located at the upper parts of the gradient, whereas mitochondria are found in the medium part of the gradient. A subsequent purification step with ZE-FFE efficiently removes remaining lysosomes and microsomes and, importantly, damaged mitochondrial structures. The resulting purified mitochondria can be concentrated by centrifugation and used for further experiments. Finally, possible modifications of this protocol with respect to the isolation of pure lysosomes are discussed.

D-amino-acid oxidase is involved in D-serine-induced nephrotoxicity.

D-serine is nephrotoxic in rats. Based on circumstantial evidence, it has been suspected that D-amino-acid oxidase is involved in this nephrotoxicity. Since we found that LEA/SENDAI rats lacked D-amino-acid oxidase, we examined whether this enzyme was associated with D-serine-induced nephrotoxicity using the LEA/SENDAI rats and control F344 rats. When d-propargylglycine, which is known to have a nephrotoxic effect through its metabolism by D-amino-acid oxidase, was injected intraperitoneally into the F344 rats, it caused glucosuria and polyuria. However, injection of d-propargylglycine into LEA/SENDAI rats did not cause any glucosuria or polyuria, indicating that D-amino-acid oxidase is definitely not functional in these rats. D-serine was then injected into the F344 and LEA/SENDAI rats. It caused glucosuria and polyuria in the F344 rats but not in the LEA/SENDAI rats. These results indicate clearly that D-amino-acid oxidase is responsible for the D-serine-induced nephrotoxicity.

Phenomenon of hepatic overload of copper in Mugil cephalus: role of metallothionein and patterns of copper cellular distribution.

In this work we describe a phenomenon of accumulation of copper (Cu) in livers of a teleost fish commonly known as mullet, Mugil cephalus. High levels of Cu, up to 1936 microg/g wet weight were found. The high Cu levels seem not to be associated with environmental Cu contamination, since the fish were collected from widely separated regions with low Cu concentrations. Other fish species sharing the same environment did not show high levels of Cu. The accumulation of Cu in mullet was seen in liver and most of the hepatic Cu was located in the non-cytosolic fraction. The intrahepatic distribution of Cu in mullet seems to depend on the total Cu content in the liver; as the total liver burden of Cu rose, Cu was increasingly recovered from the non-cytosolic fraction. Metallothionein in hepatic cytosols from mullet contained the most Cu. However, the Cu concentration not bound to metallothionein rose when total cytosolic Cu increased; which show that metallothionein, particularly at higher Cu levels, is not the major hepatic Cu-binding protein in cytosols of mullet. This report shows mullet as a very useful model to study the accumulation of Cu in the liver, which may lead to a better understanding of cellular mechanisms which control Cu homeostasis.

Whole animal copper flux assessed by positron emission tomography in the Long-Evans cinnamon rat--a feasibility study.

Copper is an essential trace element. However, excess copper can lead to oxidation of biomolecules and cell damage and copper levels must be carefully controlled. While copper homeostasis has been studied extensively at the cellular level, short-term body copper fluxes are poorly understood. Here, we assessed for the first time the feasibility of measuring whole body copper flux by positron emission tomography, using 64Cu. A comparative approach comparing the Long-Evans cinnamon (LEC) rat to the wild type was chosen. LEC rats are an accepted model for Wilson disease, an inherited disorder of copper excretion in humans. In LEC rats as well as in Wilson patients, the copper transporting ATPase, ATP7B, is defective. This ATPase is primarily expressed in the liver and serves in copper secretion via the bile. Dysfunction of ATP7B leads to accumulation of copper in the liver. A control and an LEC rat were transgastrically injected with 10 microg of 64Cu and the copper flux followed for three hours by whole animal PET and concomitant collection of bile, as well as the analysis of tissue following tomography. As seen by PET, the administered copper was largely trapped in the stomach and the proximal intestine, and without a significant difference between control and LEC rat. Due to an insufficient dynamic range of the PET technology, copper which was systemically absorbed and primarily transported to the liver could only be followed by sampling and by beta-counting. Biliary copper excretion ensued after 15 min in the control rat, but was absent in the LEC rat. Biliary excretion reached saturation one hour after copper administration. The trapping of orally administered copper in the gastrointestinal tract may be an important mechanism to prevent copper toxicity under conditions of a sudden, excessive copper load, which cannot be alleviated by increased biliary secretion. This trapping does however limit the utility of PET to measure whole animal copper flux.

Tetrathiomolybdate in the treatment of acute hepatitis in an animal model for Wilson disease.

BACKGROUND/AIMS: Tetrathiomolybdate (TTM) is a potent copper-chelating agent that has been shown to be effective in Wilson disease patients with neurological symptoms. Here, we investigate the potential use of TTM in treating the acute hepatic copper toxicosis in Long-Evans Cinnamon (LEC) rats, an authentic model for Wilson disease. METHODS: After the onset of acute hepatitis, LEC rats were treated once with 10 mg TTM/kg. After 1 and 4 days, parameters of liver toxicity and the subcellular distribution and binding of copper and iron were studied. RESULTS: In 11 out of 12 rats TTM rapidly improved acute hepatitis. Hepatic copper decreased through removal from cytosolic metallothionein and lysosomal metallothionein polymers. The remaining lysosomal copper forms a metallothionein-copper-TTM complex. In an almost moribund rat, however, TTM caused severe hepatotoxicity with fatal outcome. CONCLUSIONS: TTM is effective in treating acute hepatitis in LEC rats when applied before the animals become moribund. TTM appears to act by removing the presumable reactive copper associated to lysosomal metallothionein polymers. The remaining lysosomal copper seems to be inactivated by forming a complex with TTM. Moreover, TTM removes copper from cytosolic copper-containing metallothionein. As a consequence, metallothionein is degraded and the uptake of copper-metallothionein into the lysosomes and the formation of the metallothionein polymer associated copper is reduced.

Gene expression in the liver of Long-Evans cinnamon rats during the development of hepatitis.

The Long-Evans cinnamon (LEC) rat, an authentic model for Wilson disease, is characterized by a mutation in the Atp7b gene leading to a defective copper excretion and, as a consequence, to an accumulation of the metal in the liver and copper-associated hepatotoxicity. In the present communication expression profiles of genes in the liver from wild-type Long-Evans agouti (LEA) and LEC rats at different stages of copper accumulation and liver disease were investigated. Disease states were defined according to serum aspartate aminotransferase activity and bilirubin levels in serum and from histopathology of the liver. Gene expression was determined with the Affymetrix RTU34 oligonucleotide array covering 1031 genes. Compared to the LEA rat, the nondiseased LEC rat with already increased hepatic copper level showed an enhanced expression of genes, particularly related to oxidative stress and DNA damage. During the progression of the liver disease, in particular genes related to oxidative stress, DNA damage, apoptosis and inflammation with acute-phase reaction were upregulated.

Tetrathiomolybdate causes formation of hepatic copper-molybdenum clusters in an animal model of Wilson's disease.

Wilson's disease is an autosomal recessive human illness in which large quantities of copper accumulate in various organs, including the brain and the liver. If left untreated, it results in hepatitis, neurological complications, and death. Long-Evans Cinnamon (LEC) rats have a homologous mutation to Wilson's disease and thus provide an animal model. Liver lysosomes from tetrathiomolybdate-treated LEC rats were isolated and analyzed by Cu and Mo K-edge X-ray absorption spectroscopy. The lysosomes contained a Cu-Mo-S cluster in which the Mo is coordinated by four sulfurs at 2.24 A with approximately three copper neighbors at 2.70 A. Each Cu is coordinated to 3-4 sulfurs at 2.28 A with approximately one Mo neighbor at 2.70 A. These results indicate the formation of a biologically novel molybdenum-copper-sulfur cluster.