Hohenheim consensus workshop: copper.

Copper (Cu) is an essential trace element with many physiological functions. Homeostatic mechanisms exist to allow Cu to act as a cofactor in enzymatic processes and to prevent accumulation of Cu to toxic levels. The aim of this commentary is to better understand the role of dietary Cu supply in deficiency and under physiological and pathological conditions. The essentiality of Cu can be attributed to its role as a cofactor in a number of enzymes that are involved in the defence against oxidative stress. Cu, however, has a second face, that of a toxic compound as it is observed with accumulating evidence in hepatic, neurodegenerative and cardiovascular diseases. The destructive potential of Cu can be attributed to inherent physico-chemical properties. The main property is its ability to take part in Fenton-like reactions in which the highly reactive and extremely deleterious hydroxyl radical is formed. Diseases caused by dietary Cu overload could be based on a genetic predisposition. Thus, an assessment of risk-groups, such as infants with impaired mechanisms of Cu homeostasis regarding detoxification, is of special interest, as their Cu intake with resuspended formula milk may be very high. This implies the need for reliable diagnostic markers to determine the Cu status. These topics were introduced at the workshop by the participants followed by extensive group discussion. The consensus statements were agreed on by all members. One of the conclusions is that a re-assessment of published data is necessary and future research is required.

Dissolution of copper-rich granules in hepatic lysosomes by D-penicillamine prevents the development of fulminant hepatitis in Long-Evans cinnamon rats.

BACKGROUND/AIM: The Long-Evans cinnamon rat has a mutation homologous to the human Wilson disease gene, leading to gross copper accumulation and the development of hepatitis. D-penicillamine, a copper-chelating drug widely and efficiently used in treating Wilson disease, has also been shown to prevent hepatitis in Long-Evans cinnamon rats. The objectives of this study were: i) to investigate the effectiveness of D-penicillamine when administered to the already affected animals, and ii) to elucidate the mechanism of action of the drug. METHODS: Long-Evans cinnamon rats were divided into groups according to age and treatment with D-penicillamine. The drug was administered orally before and after the onset of hepatitis. Livers were examined by light and electron microscopy. The effect of D-penicillamine on the subcellular distribution and binding of copper was investigated in more detail. Finally, the interaction between D-penicillamine and specific hepatic copper-binding proteins was studied in vitro. RESULTS: D-penicillamine when given to either healthy or diseased animals prevented or reversed hepatitis, respectively. The drug particularly inhibited the disease-specific accumulation of copper in lysosomes of hepatocytes, tissue macrophages and Kupffer cells. When administered to diseased animals, the drug sequestered copper particularly from insoluble lysosomal particles. According to results obtained in vitro, the mobilization of this copper is likely to proceed through the solubilization of these particles. In contrast and as supported by the in vitro data, D-penicillamine had only a minor effect on copper bound to metallothionein in the cytosol. CONCLUSION: Our findings on the Long-Evans cinnamon rat provide some conclusions on the mechanism of action of D-penicillamine in Wilson disease therapy. The drug prevents the formation or promotes the solubilization of copper-rich particles which occur in lysosomes of hepatocytes and Kupffer cells in the livers of patients with Wilson disease. Once chelated with D-penicillamine copper might then be excreted into urine. However, the mobilization of copper by D-penicillamine seems to be limited due to the binding of the metal to metallothionein in liver cytosol. This copper, even at relatively high concentrations, apparently may be well tolerated.

Assaying for hydroxyl radicals: hydroxylated terephthalate is a superior fluorescence marker than hydroxylated benzoate.

Generation of hydroxyl radicals in terephthalate (benzene-1,4-dicarboxylic acid) solution yields fluorescent 2-hydroxy-terephthalate. The reaction product is stable for hours and can readily be assessed using standard fluorimeters. The efficiency, i.e. the relative increase of fluorescence per *OH radical, is about three times higher than that of the formation of salicylate (2-hydroxy-benzoate) from benzoic acid and approximately hundred-fold higher than that of the hydroxylation of phenylalanine. As the terephthalate molecule is symmetric with respect to ring-hydroxylation, only one isomer is formed; hence, mechanistic interpretation of the hydroxylation reaction is facilitated. The scavenging rate constant of terephthalate for *OH yielding the hydroxycyclohexadienyl adduct as first intermediate is close to the diffusion controlled limit (k = 3.3 x 10(9) M(-1) s(-1)). Therefore, competition of the detector molecule with biomolecules being present under physiological conditions is expected to be efficient. The assay can be used to detect 'free' *OH radicals produced by the radiolysis of water as well as 'hydroxyl analogous species' that have been suggested to arise from the interaction of complex-bound reduced metal with either oxygen or hydrogen peroxide, e.g. from Fenton reactions. Based on calibration with radiolytically generated hydroxyl radicals the detection limit of the method is estimated to be around 50 nmol/dm3. Terephthalate is classified non-toxic and hence may also prove useful for microdialysis and continuous flow experiments as observation of fluorescence is 'non-destructive' and the reporter substance does not necessarily have to be subjected to HPLC.

Metallothionein and heavy metals in brown trout (Salmo trutta) and European eel (Anguilla anguilla): a comparative study.

The levels and the cellular distribution of heavy metals, and the extent by which the metals binds to metallothionein (MT) in brown trout (Salmo trutta) and European eel (Anguilla anguilla), were analyzed in order to assess the natural conditions of MT and heavy metals in these two fish species. There were no differences in heavy metals and MT concentrations between males and females of brown trout in a nonreproductive status and between adult brown trout individuals. Brown trout presented higher Cu content than European eel. The cellular distribution of Cu was also different between the two fish species; while in brown trout most of the Cu was in the noncytosolic fraction, Cu was mainly located in the cytosol in European eel. However, the cellular distribution of Zn, Cd, and Pb was similar in the two fish species. There was also an important difference in the metal content of MT between both species. Whereas, in brown trout, Cu-binding MT represented 75% of total metal-binding MT, this value was 25% in European eel. The between-species differences found in this study are intrinsic characteristics not associated with environmental factors. These results establish the basis to use MT as a bioindicator.

Different pathomorphologic patterns in exogenic infantile copper intoxication of the liver.

Pathomorphology of the liver has been reviewed in 12 German infants with chronic exogenic copper intoxication. In 8 cases severe liver damage with diffuse accumulation of Mallory bodies and liver cell necrosis mimicking florid Indian childhood cirrhosis (ICC) was found. Seven of these children died because of liver failure. One child received liver transplantation at the age of 9 months. In contrast, 4 children with a stable clinical course had a complete micronodular cirrhosis in liver biopsy. The characteristic morphological features of ICC, especially ballooning of liver cells and accumulation of Mallory bodies, were only slightly expressed or even lacking. There was no correlation between the copper content of the liver and the severity of liver damage. The copper concentration varied between 541 micrograms/g dry weight (norm < 50 micrograms/g) and 2.154 micrograms/g dry weight in fatal cases. In surviving infants even higher concentrations of up to 698 micrograms/g fresh weight (norm < 5 micrograms/g), were found. The amount of free cytosolic copper varied between 900-4,900 ng/mg protein (13-70 times of normal). In conclusion, a spectrum of pathomorphological alterations exists in exogenic infantile copper disease which correlates with the clinical outcome in contrast to the copper content of the liver. Copper intoxication of the liver should be of diagnostic concern in any case of unclear micronodular cirrhosis in early infancy.

Association of copper to metallothionein in hepatic lysosomes of Long-Evans cinnamon (LEC) rats during the development of hepatitis [se e comments].

BACKGROUND: The Long-Evans cinnamon (LEC) rat has a mutation homologous to the human Wilson's disease gene, leading to copper-induced hepatotoxicity. The mechanism of how excess copper damages the liver or what chemical form of copper is toxic is still unclear. RESULTS: In liver cytosol, copper levels were highest just before the onset of hepatitis and declined thereafter. In cytosol, total copper was bound to metallothionein (MT). Considerable amounts of both copper and iron accumulated in lysosomes with increasing age and development of liver damage. Lysosomal levels of presumably reactive non-MT-bound copper were increased. In severely affected livers, large amounts of copper were associated with insoluble material of high density which, upon ultrastructural information, was found to be derived from the lysosomes of Kupffer cells. This copper-rich material is considered to consist of polymeric degradation products of copper-MT. CONCLUSION: We suggest that chronic copper toxicity in LEC rats involves the uptake of copper-loaded MT into lysosomes, where it is incompletely degraded and polymerizes to an insoluble material containing reactive copper. This copper, together with iron, initiates lysosomal lipid peroxidation, leading to hepatocyte necrosis. Subsequent to phagocytosis by Kupffer cells, the reactive copper may amplify liver damage either directly or through stimulation of these cells.

Physiologic function of the Wilson disease gene product, ATP7B.

The genes responsible for Wilson disease and Menkes syndrome have been cloned and identified as copper ATPases. These enzymes form part of a large family of transporters, the P-type ATPases. Although copper ATPases share strong structural similarities with these other pumps, comparatively little is known about their physiologic function. In this review, we examine data relating to the Wilson disease protein, ATP7B, in the liver. We present evidence suggesting that ATP7B is located intracellularly, together with data suggesting that, at least in part, ATP7B may also be found on the canalicular membrane. We also examine the form of copper that the transporter recognizes. We then review data on the Long-Evans Cinnamon rat, a model for Wilson disease, and discuss what effect the Wilson disease mutation has on copper transport. Finally, we conclude that, although we have made major advances in our understanding of copper metabolism in the liver, there are still many questions awaiting answers.

Binding of Cu to metallothionein in tissues of the LEC rat with inherited abnormal copper accumulation.

Long-Evans Cinnamon (LEC) rats aged 16 +/- 4 weeks with histopathological alterations of liver and kidney, exhibited elevated Cu levels in liver, kidney and spleen which were 52, 27 and 5 times higher than those of the respective tissues of age-matched Wistar rats. About 61% of hepatic and about 38% of renal Cu was recovered in the cytosolic fraction. Metallothionein (MT) levels were found to correlate with the cytosolic Cu concentrations in liver and kidney. According to differential MT analysis, about 68 and 82% of hepatic and renal MT was loaded with Cu. The portion of MT which binds Cu was negatively correlated with the ratio of cytosolic Zn/Cu in all organs investigated. Despite high MT levels and the high percentage of Cu binding to MT, particularly in liver and kidney, considerable amounts of Cu remained unbound to MT. This non-MT bound Cu showed good correlation with the total cytosolic Cu content, and might play a crucial role in the pathogenesis of Cu toxicosis.

2-Chloroacetophenone is an effective glutathione depletor in isolated rat hepatocytes.

The glutathione (GSH) depleting effect of 2-chloroacetophenone (CN) was studied in freshly isolated rat hepatocytes. CN proved to be more effective in depleting GSH than diethylmaleate, phorone or styrene oxide. The reaction between GSH and CN followed a 1:1 stoichiometry, allowing adjustment of cellular GSH concentrations at distinct levels. After incubating cells (8 mg protein/ml) with 200 mumol CN/l for 5 min, GSH depletion was almost complete without signs of cytotoxicity. At 300 mumol/l CN, GSH depletion persisted, and cytotoxicity occurred after 30 min. Activities of cytochrome P450 dependent enzymes, even at concentrations up to 500 mumol CN/l, were only marginally affected. Therefore, CN is of particular value for in vitro studies at decreased availability of GSH.

Determination of Cu-containing metallothionein: comparison of Ag saturation assay, thiomolybdate assay, and enzyme-linked immunosorbent assay.

Three methods for the quantification of Cu-containing metallothionein (MT) (Ag saturation assay, thiomolybdate assay, and enzyme-linked immunosorbent assay (ELISA)) were compared for their ability to recover in vitro prepared standard Cu-MT both in absence and presence of rat liver cytosol. Uniform molar calibration of the assays was achieved using the nitrogen content of the standard Cu-MT measured by the Kjeldahl procedure. With all three methods Cu-MT reliably could be quantified. The Ag saturation assay and the thiomolybdate assay, dependent on the amount of Cu-MT and the presence of hepatic cytosol, showed a tendency to over- or underestimate the theoretical expectation. The ELISA generally performed best and moreover was three orders of magnitude more sensitive than the other two assays. With all three methods corresponding MT levels were found in the Cu-rich liver of a Long-Evans Cinnamon rat.

Quantification of oxidized metallothionein in biological material by a Cd saturation method.

A rapid and easy to perform method for determining oxidized metallothionein (MT) is described. The main features of the procedure are that oxidized MT is converted into native MT with 2-mercaptoethanol as reducing agent and Zn2+ as metal donor, and MT is subsequently quantified via Cd saturation. The procedure was effective in recovering MT oxidized either by Cu2+ or by neutralization of apothionein at pH 3, independent of the amount of MT, the origin of sample, degree of oxidation, and oxidation method. As demonstrated by HPLC analysis, the method is highly specific for MT. The described procedure provides information on both the total concentration of MT independent of its thiol redox state and the metal binding capacity of the protein; thus, it is of particular interest in studying the role of MT in metal metabolism and toxicity.

Metallothionein, copper and zinc in fetal and neonatal human liver: changes during development.

Total and cytosolic zinc (Zn) and copper (Cu), cytosolic metallothionein (MT) and the Cu-load of MT were investigated in fetal (22, 24 and 32 gestational weeks) and neonatal (2-15 months) human liver. Whereas the fraction of cytosolic Zn remained constant at 66% of the total independent of the stage of development, the fraction of cytosolic Cu increased from 26% in preterm liver to about 100% within 12 months postnatally. The MT content was higher in fetal than in neonatal liver. There was a linear correlation (r = 0.996) between cytosolic MT and Zn in both fetal and neonatal liver but not between MT and Cu. In contrast to fetal liver, the Cu-load of MT in neonatal liver seems to be determined by the Zn/Cu ratio in the cytosol. The results suggest that MT is involved in the regulation of Cu and Zn metabolism during fetal and neonatal development.

Transfer and accumulation of cadmium, and the level of metallothionein in perfused human placentae.

The purpose of the present study was to investigate the effect of perfusion with a medium containing 12 or 24 micrograms Cadmium (as CdCl2) per ml on this metal's accumulation, transfer rate and metallothionein (MT) level. The experiments were performed with an isolated lobule of a dually-perfused human term placenta. Placental cell integrity and viability were characterised by their morphology and metabolic function, manifested in the tissue's electron microscopic structure and glucose and oxygen (O2) consumption, respectively. Perfusion with 24 micrograms Cd/ml medium for 5 h resulted in significant elevation in MT. The transfer rate of Cd to the fetal side of the placenta was very slow, and not until 40 min after the addition of Cd into the maternal side was a significant increase in the metal's level observed in the fetal perfusate. Thereafter, the level of the metal increased gradually and reached a steady state about 1 h later, at a level which was less than 1/20th of its concentration in the maternal perfusate. There was a 60-fold increase in Cd level in the cytosolic fraction obtained from the Cd-treated samples. At 12 micrograms Cd/ml no significant changes were noted in morphology, metabolic function and MT content. None of the Cd levels caused a significant change in O2 and glucose consumption, in spite of the fact that with the higher Cd dose the microstructure of the tissue showed some pathological changes. The observed elevation in MT may provide the fetus some protection against the harmful effects of the metal.

Quantitation of Cu-containing metallothionein by a Cd-saturation method.

A rapid and sensitive method for determining Cu-containing metallothionein (MT) is described. The main features of this Cd-saturation assay are: high-molecular-weight Cd-binding compounds are denatured with acetonitrile (50% final concentration), Cu bound to MT is removed with ammonium tetrathiomolybdate, excessive tetrathiomolybdate and its Cu complexes are removed with DEAE-Sephacel, apothionein is saturated with Cd, and excessive Cd is bound to Chelex 100. The thiomolybdate assay is capable of reliably detecting 14 ng MT and thus is particularly suitable for measuring MT in small tissue samples (e.g., biopsies), in extrahepatic tissues, and in cultured cells. Moreover, the combination of the thiomolybdate assay with the recently developed Cd-Chelex assay also makes it possible to determine the portion of MT which binds Cu (Cu load of MT), provided that the amount of non-Cu-thionein exceeds 100 ng, the detection limit of the Cd-Chelex assay.

Characterization of Cat-2t, a radiation-induced dominant cataract mutation in mice.

A dominant cataract mutation was detected recently among the offspring of x-ray-irradiated male mice. The mutation, which causes total lens opacity, has provisionally been designated by the gene symbol Cat-2t. In the lenses of heterozygous and homozygous Cat-2t mutants, the epithelial and fiber cells were swollen and the lens capsule was ruptured. The histologic analysis demonstrated a complete destruction of the cellular organization of the lens, which might be caused by its altered developmental processes. The data derived from biochemical investigations indicate that biochemistry of the cataractous Cat-2t lenses is affected: the osmotic state as indicated by the increased water content and increased Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity; the energy state as indicated by the decreased adenosine triphosphate (ATP) concentration; and the redox state as indicated by the enhanced content of oxidized glutathione. Additionally, the lenticular protein composition is altered because of the presence of vimentin in the water-soluble fraction. This cannot be explained by the enhanced crosslinking activity of transglutaminase. The changes of the osmotic, energy, and redox states are considered to be secondary in relation to the altered lenticular development. In contrast, the variations concerning vimentin and transglutaminase might be a biochemical indication of the changed development. Possible similarities to other dominantly expressed murine cataract mutants are discussed.

The Cd-Chelex assay: a new sensitive method to determine metallothionein containing zinc and cadmium.

A rapid and sensitive one-vial procedure to determine metallothionein (MT) containing zinc (Zn) and cadmium (Cd) is described. New features of this Cd-saturation method are: high molecular weight Cd-binding proteins are denatured by treatment with acetonitrile (50% final concentration), and excess of Cd is bound to a cation exchange resin (Chelex-100). With this method, MT has been measured, e.g. in liver of control and zinc- or cadmium-treated rats, in human liver and in cultured human fibroblasts down to absolute amounts of 0.1 microgram. The Cd-Chelex assay is 10 times more sensitive than the established Cd-heme assay (Dieter et al. 1986) and therefore is particularly suitable to quantify MT in small tissue samples (e.g., liver biopsies of a few milligrams) and in extrahepatic tissues or cell cultures with low MT concentrations.

Metallothionein induction by nonsteroidal antiinflammatory drugs.

In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).

Mutagenicity and cytotoxicity of 2-chlorobenzylidene malonitrile (CS) and metabolites in V79 Chinese hamster cells.

The genotoxicity of the sensory irritant 2-chlorobenzylidene malonitrile (CS) to V79 Chinese hamster cells was investigated using the induction of gene mutations, micronuclei and DNA repair synthesis as biological endpoints. CS efficiently induced micronuclei and mutants resistant to 6-thioguanine in these cells, but it did not elicit DNA repair synthesis. Induction of micronuclei and mutants showed very similar courses of concentration dependence, suggesting that both events were caused by the same mechanism. The hydrolysis products of CS, o-chlorobenzaldehyde and malononitrile dit not induce micronuclei and were much less cytotoxic than CS. The observation of heritable genetic changes in cells exposed to CS in the absence of detectable DNA damage suggests that the genetic effects of CS are not caused by an interaction of the compound or its hydrolysis products with DNA. It appears more likely that the mutagenic activity is the consequence of effects of CS on the mitotic apparatus of the cells causing chromosomal aneuploidy.