Metallothionein expression in canine and feline mammary and melanotic tumours.

Moderate to strong immunohistochemical metallothionein (MT) positivity (MT expression) is associated with a poor prognosis in some human tumours. The aim of this study was to determine MT expression in mammary tumours and cutaneous melanomas in dogs and cats. Canine (67) and feline (47) mammary tumours, and cutaneous melanomas (canine 40, feline 26) were immunolabelled with MT monoclonal antibody E9. The overall incidence of MT expression of these tumours was similar to that observed in various human neoplasms. However, a striking interspecies difference was detected. In dogs, MT expression occurred in 100% of benign and 57% of malignant mammary tumours. In cats, however, 30% of malignant mammary tumours expressed MT but benign mammary tumours and cases of fibroadenomatous hyperplasia did not. Moderate to strong MT immunoreactivity was detected in 30% of benign and 25% of malignant cutaneous melanomas in dogs, and in 6% of malignant melanomas in cats. The findings in feline mammary tumours resembled findings reported in human breast cancer, but the cause of tumour-associated MT expression is unknown. Studies are in progress to determine whether the MT state (apo [metal-free] or holo [metal-bound]) accounts for the paradoxical association of MT expression with individual types of tumours and the animal species in which they arise.

The failure of selenium supplementation to prevent copper-induced liver damage in Fischer 344 rats.

This study evaluates the ability of selenium (Se) supplementation to prevent experimental copper (Cu)-induced hepatocellular damage. Weanling male Fischer 344 rats were randomly assigned to groups of 15, 3 groups (A,B,C) were fed Cu-loaded diets (containing 2000 microg/g copper, added as CuSO4) and different levels of Se (added as Na2SeO3 x 5H2O) as follows: A) Cu-loaded/Se adequate diet (0.4 microg/g Se, fed basis); B) Cu-loaded/Se-supplemented diet (2 microg/g Se); and C) Cu-loaded/Se-deficient diet (< 0.2 microg/g). Three additional groups (D,E,F) were fed diets containing adequate levels of Cu (14 microg/g Cu, fed basis) and different levels of Se as follows: D) Cu-adequate/Se-adequate diet; E) Cu-adequate/Se-supplemented diet (2 microg/g Se); and F) Cu-adequate/Se-deficient (< 0.2 microg/g) diet. After 4, 8, and 12 weeks on the experimental diets, liver samples were processed for histology, histochemistry, metal analysis, glutathione peroxidase (GSH-Px) measurement, and quantification of malondialdehyde (MDA). Morphologic changes characteristic of Cu-associated hepatitis, without an increase in hepatic MDA levels, were seen in all Cu-loaded rats in each sampling. Similar changes occurred in rats fed Se-adequate, Se-supplemented and Se-deficient diets. This study demonstrates that Fischer 344 rats fed 2000 microg/g Cu develop morphologic changes due to Cu toxicity without evidence of lipid peroxidation. Furthermore, Se supplementation does not result in protection against Cu-induced liver injury.

Morphological and biochemical assessment of the liver response to excess dietary copper in Fischer 344 rats.

The aim of this study was to determine the amount of excess dietary copper (Cu) necessary to experimentally induce liver lesions characteristic of Cu-associated disease in Fischer 344 rats. Male weanling Fischer 344 rats of uniform age were divided into 6 groups (n = 5) and fed a rodent diet containing 18 (control), 750, 1000, 1250, 1500, and 2000 microg/g Cu added as CuSO4. Rats were euthanized after 3 months on the experimental diets and their livers processed for histology, histochemistry, Cu analysis (by atomic absorption spectrophotometry), and quantification of malondialdehyde (MDA) by the thiobarbituric acid reaction. Hepatic Cu levels were significantly higher (P < 0.01) in rats receiving over 1000 microg/g Cu compared to the controls (means for each diet: control = 4.8 microg/g, 750 microg/g Cu = 39.6 microg/g, 1000 microg/g Cu = 111.2 microg/g, 1250 microg/g Cu = 389 microg/g, 1500 microg/g Cu = 509.4 microg/g, and 2000 microg/g Cu = 766 microg/g). Histological lesions increased gradually according to the level of dietary Cu. Significant morphologic changes (necrosis, portal inflammation, hyaline remnants) and reduced growth rate occurred in rats receiving over 1250 microg/g Cu. However, no significant differences were found for MDA levels between groups. The present study demonstrates that compared to other species, very high levels of excess dietary Cu are needed to induce significant liver injury in Fischer 344 rats. Increased MDA content was not detected in rats with morphologic evidence of liver damage, suggesting that lipid peroxidation may not play a major role in this model of Cu toxicity.

Copper toxicosis in the Bedlington terrier: a diagnostic dilemma.

Diagnosis of copper toxicosis (CT) in Bedlington terriers by the quantitative and qualitative assessment of copper (Cu) in, and pathology of, biopsies has been largely superseded by a DNA-based assay which uses a microsatellite marker (C04107) linked to the CT disease allele. A retrospective study was conducted comprising 154 liver biopsies from Bedlington terriers with 22 matched DNA markers to compare the two methods in the diagnosis of CT. For the biopsy method, three categories (phenotypes) were identified based on analytical and morphological criteria: 'unaffected' in 83 samples (54 per cent), where Cu was much less than 400 microg/g, and there was an absence of visual Cu or liver damage; 'intermediate' in 18 samples (12 per cent), where Cu was less than 400 microg/g, and there was limited histochemical Cu and no/equivocal damage; and 'affected' in 53 samples (34 per cent), where Cu was greater than 400 microg/g, there was histochemical Cu and liver damage was poorly related to Cu content. In the DNA assay, which was used alone on unrelated individuals, the microsatellite marker failed to identify the CT status of any of the groups. Liver biopsy remains a reliable indicator of Cu accumulation and progressive liver disease in individual dogs. The microsatellite marker C04107 has a predictive value only when supported by a pedigree.

Pathogenesis of liver lesions caused by experimental infection with Piscirickettsia salmonis in juvenile Atlantic salmon, Salmo salar L.

Piscirickettsia salmonis, the etiologic agent of salmonid rickettsial septicemia (SRS), or piscirickettsiosis, causes substantial economic losses to the salmon industry. The pathogenesis of the disease has not been fully characterized. The aim of this study is to describe the hepatic lesions associated with experimental P. salmonis infection in Atlantic salmon juveniles. Fish were maintained in fresh water and inoculated intraperitoneally (IP), orally, or on the gill surface with P. salmonis. A group of uninfected fish was kept as control. Liver samples from 5 fish in each inoculated group and 3 controls were collected weekly and processed for histological and immunohistochemical examination. Thickening of the liver capsule by inflammatory cells was a characteristic histologic feature of IP inoculated fish. Three weeks post-IP inoculation, 8 fish had died and 2 fish were sampled. Histological changes at this time consisted of vasculitis, presence of fibrin thrombi, vacuolated hepatocytes and focal areas of necrosis. Leukocytes containing intracytoplasmic basophilic microorganisms were seen within hepatic sinusoids. Vasculitis and intracytoplasmic vacuoles were prominent features in fish inoculated orally and on the gill surface. The presence of P. salmonis within hepatocellular vacuoles, endothelial cells, and leucocytes was confirmed by immunohistochemistry. The intracellular location of P. salmonis and the vascular damage seen in infected fish are characteristic of rickettsial infections. Histological lesions induced by experimental infection with P. salmonis using the oral and gill surface routes were similar to those observed in natural outbreaks of piscirickettsiosis. The tropism of P. salmonis for endothelial cells explains the vascular lesions observed in SRS, whereas hepatic lesions are due to ischemic necrosis and direct injury by intracytoplasmic organisms.

Eccrine adenocarcinoma of the footpads in 2 cats.

Adenocarcinoma of sweat glands of the footpads was diagnosed in 2 cats. Clinical signs included lameness and swelling of multiple digits. Pulmonary metastasis was detected in one case. Diagnosis was based on histopathological and immunohistochemical findings. Eccrine adenocarcinoma should be included in the differential diagnosis of footpads lesions in aged cats.

The Long Evans Cinnamon (LEC) rat develops hepatocellular damage in the absence of antimicrosomal antibodies.

Long Evans Cinnamon (LEC) rats are an inbred strain with a mutation affecting a copper transporter. As a result, hepatic copper levels rise and the rats spontaneously develop hepatitis that is fatal in about 40% of the rats. The rats that die have been reported to develop anti-microsomal antibodies, most frequently against protein disulfide isomerase (PDI), prior to the onset of liver damage. The association between the presence of antibodies and death of the LEC rats, along with the detection of antibodies prior to the detection of liver damage suggested that the antibodies may have a role in the pathogenesis of liver damage. The objective of this study was to more clearly delineate the temporal relationship between antibody production and the onset of liver damage and copper accumulation. Serum was screened for the presence of anti-microsomal antibodies by immunoblotting. Liver damage was assessed by serum biochemistry and histological examination on rats between 6 and 12 weeks of age (four per group). Copper accumulation in the liver was determined by biochemistry and histological examination. Evidence of liver damage was detectable by serum biochemistry and histopathology by 11.5 weeks. Copper was rarely detected in hepatocytes, although it was detected in macrophages. Sera from only one of seven rats with evidence of liver damage had detectable anti-microsomal protein antibodies. The protein recognized was not PDI. The development of anti-microsomal autoantibodies did not precede the development of significant liver damage, suggesting that they play only a secondary role, if any, in the pathogenesis of hepatitis in this rat strain.

Effect of age and sex on liver damage due to excess dietary copper in Fischer 344 rats.

OBJECTIVE: The aim of this study was to investigate the morphologic and biochemical effects of excess dietary copper in young and adult rats of different sex. METHODS: Adult Fischer 344 male and female rats were given a diet containing 1500 ppm copper for 18 weeks. Young male and female rats were fed a similar copper-loaded diet from birth until 16 weeks of age. Age- and sex-matched control rats were fed a normal rodent diet (<10 ppm copper). Serum liver enzyme activity was determined in all rats. Livers were sampled for histology, histochemistry (rhodanine), immunohistochemical detection of metallothionein and copper analysis by atomic absorption spectrophotometry. Hepatic metallothionein and zinc concentrations were measured in adult rats. RESULTS: Excess dietary copper caused substantial liver injury, as evidenced by morphologic changes and increased activity of serum ALT, GGT, and SDH. All copper-loaded rats had significantly (p< 0.05) increased hepatic copper concentrations compared to controls. However, young copper-loaded rats accumulated more hepatic copper, had more severe liver changes, and had higher serum liver enzyme activities than adult rats. Histologic changes in copper-loaded rats consisted of multifocal hepatitis and widespread single-cell necrosis. Cytoplasmic copper was detected histochemically in centroacinar zone 1 (portal) and mid-zone in copper-loaded rats. Immunoreactivity for metallothionein was prominent in necrotic hepatocytes and within inflammatory foci in copper-loaded rats. However, differences in hepatic metallothionein concentrations were not detected between adult copper-loaded and control rats. CONCLUSIONS: Young Fischer 344 rats are more susceptible than adults to copper-induced liver injury.

Purification and partial characterization of a cadmium-binding protein from the liver of rainbow trout (Onchorynchus mykiss).

This study describes the isolation and partial characterization of a low molecular weight (approximately 14 kDa), cadmium-binding protein from rainbow trout (Onchorynchus mykiss) liver. Rainbow trout were injected intraperitoneally with 3.5 mg/kg cadmium chloride (total body dose) twice weekly for 3 wk. Livers were removed and a cadmium-binding protein was isolated. Monoclonal antibodies produced against this protein were used in the affinity purification process. Amino acid analysis showed the protein contained 3.8 mol% cysteine, 3.5 mol% phenylalanine, 2.2 mol% tyrosine and 1.9 mol% histidine. The low cysteine content suggests that it was distinct from metallothionein. The monoclonal antibodies were also used to identify the protein in liver homogenates from both cadmium-exposed and control fish and in the testes of cadmium-exposed mice lacking the gene for both metallothionein-1 and metallothionein-II. The compound identified in this study represents a non-metallothionein cadmium-binding protein that appears to be highly conserved.

Immunohistochemical demonstration of metallothionein in benign and malignant canine mammary tumours.

Immunocytochemical demonstration of metallothionein (MT) has been reported as a useful prognostic tool in human breast cancer. The aim of this study was to determine the immunohistochemical location of MT in canine mammary tumours and its possible correlation with the morphologic characteristics of these tumours. Surgical specimens from spontaneous malignant (n = 20) and benign mammary neoplasms (n = 20) were processed for routine histological examination and immunohistochemical study. An indirect immunoperoxidase technique, using monoclonal antibody E9 against horse MT was employed. Intensity of the stain, the percentage of immunoreactive tumour cells and immunohistochemical overexpression of MT was estimated for each case. Metallothionein over-expression, defined as those cases with more than 10% immunopositive cells, was detected in both benign and malignant mammary tumours. However, strong immunostaining intensity was seen in benign tumours, whereas in malignant tumours immunopositive cells stained weakly. Positive MT immunostaining occurred in neoplastic epithelial cells, and some chondrocytes present in mixed mammary tumours. However, staining intensity was variable in immunopositive cells. Differences in staining intensity between the primary malignant mammary tumour, tumour emboli and metastatic cells within a lymph node were also noted. Myoepithelial cells and connective tissue did not stain for MT. We concluded that metallothionein immunostaining cannot be used as a diagnostic or prognostic tool in canine mammary neoplasms. However, results of this study support the hypothesis that MT has a role in tumour proliferation and tumour progression.

Immunohistochemical detection of metallothionein in liver, duodenum and kidney after dietary copper-overload in rats.

Metallothionein (MT) has been used in immunohistochemical techniques to indicate presence and distribution of heavy metals within biological tissues. This study describes a comparison of the pattern of MT-immunostaining in the liver, duodenum and kidney during dietary copper overload in rats. Sixteen male 10-week-old Wistar rats were randomly allocated into groups of four. Two groups were fed a pelleted diet containing 1,500 mg/kg copper and two control groups received a rodent diet containing 10 mg/kg copper. After 6 weeks samples of liver, kidney and duodenum were collected for immunohistochemistry and histology. An indirect immunoperoxidase technique, using monoclonal antibody E9 against horse MT and polyclonal sera against rabbit MT, was employed. Copper-loaded rats had marked MT-immunoreactivity within the nucleus and cytoplasm of many periportal hepatocytes, renal proximal convoluted tubule epithelial cells, intestinal columnar epithelial cells and Paneth cells. Immunohistochemical staining was similar using either mouse anti-MT polyclonal serum, or monoclonal antibody E9. Hepatocytes surrounding inflammatory foci were positive for MT, supporting the proposed role of this protein in free radical scavenging. The presence of MT in the kidney appears to be associated with renal excretion of copper-metallothionein (Cu-MT) in copper-loaded rats. Paneth cells were easily detected using MT-immunostaining. MT may play a part in absorption of copper from intestinal contents and possible storage as Cu-MT in Paneth cells. The function of Paneth cells remains unknown but the presence of marked MT-immunoreactivity in these cells, observed in copper-loaded rats, suggests their involvement in homeostasis and metabolism of copper.

Pathobiology of copper-induced injury in Bedlington terriers: ultrastructural and microanalytical studies.

The pathogenesis of copper (Cu)-induced liver injury has been investigated in Bedlington terriers with familial Cu toxicosis using ultrastructural and microanalytical techniques. Livers from 3 affected and 1 non-affected Bedlington terrier were fixed in 4% paraformaldehyde and 2% glutaraldehyde for transmission electron microscopy and X-ray electron probe microanalysis. Cu analysis was performed on formalin fixed liver by atomic absorption spectrophotometry. In the dog with liver Cu concentration < 2000 micrograms/g, Cu was located only in electron dense lysosomes with minimal cytoplasmic change. With increasing concentrations of liver Cu, the metal became apparent in the nucleus with early signs of nuclear disturbance. In the dog with highest liver Cu content > 7000 micrograms/g X-ray microanalysis identified Cu peaks in lysosomes, nucleus and cytoplasm in descending order with profound cellular changes. The hepatocytes were shrunken with compacted electron dense organelles and the nuclei were contracted, misshapen with chromatin condensation and fragmentation. Apoptotic bodies were identified in sinusoids. It was concluded that excess Cu is initially sequestered in lysosomes but following increasing saturation of this compartment nuclear accumulation of Cu occurs with DNA damage. Apoptosis follows probably by induction of p53 protein.

The role of the liver, kidney and duodenum in tolerance in the copper-loaded rat.

The purpose of this study was to help characterize the pathway of copper in the liver, kidney and duodenum during copper loading and unloading in the rat. Male Wistar rats were allocated randomly into four groups: Group A (control) was composed of 16 animals fed a normal rodent diet. Group B had 16 animals fed a high copper diet (1500 ppm copper). Four rats from each group were killed at 1, 5, 10 and 15 weeks. Group C had 4 animals fed the high copper diet for 5 weeks and normal diet for 5 weeks. Group D consisted of 4 animals fed the high copper diet for 5 weeks, normal diet for 5 weeks, followed by 5 weeks of high copper diet. At termination of each experimental period liver, kidney and duodenum were collected for histochemistry and copper analysis by atomic absorption spectrophotometry. Hepatic copper concentration in Group B rose to 726 +/- 170 micrograms/g after 5 weeks; renal and duodenal copper levels were 285 +/- 14 micrograms/g and 134 +/- 49 micrograms/g, respectively. A significant (P < 0.005) decrease in copper concentration was observed after 15 weeks in all three organs. Duodenal copper concentration in group C was similar to control rats. Changes in copper tissue distribution and efficient unloading were demonstrated in all copper-loaded groups in the three organs studied.

Mechanisms of tolerance in the copper-loaded rat liver.

The aim of this study was to contribute to the understanding of the pathogenesis of copper-induced damage and subsequent recovery and tolerance to copper in the copper-loaded rat liver. Male Wistar rats were allocated randomly into groups of four, fed a pelleted diet containing 1500 mg/g copper, and killed at 1, 5, 6, 10, and 15 weeks. Two additional groups were treated as follows: (a) 5 weeks copper loading followed by 5 weeks with normal rodent diet (group 5-0), (b) 5 weeks copper loading followed by 5 weeks normal diet and 5 weeks of copper reloading (group 5-0-5). Control rats were fed a normal rodent diet that contained 18 mg/kg of copper. Tissues were collected for histology, histochemistry, immunocytochemistry, and copper analysis by atomic absorption spectrophotometry and X-ray microanalysis. In the rats continuously fed the high copper diet, copper concentration rose to 444 +/- 32 micrograms/g of liver (wet weight) by Week 1 and to 726 +/- 170 micrograms/g at Week 5, decreasing to 417 +/- 9 micrograms/g at Week 15. X-ray microanalysis and dot mapping microanalysis demonstrated copper within the nucleus, nucleolus, and lysosomes of these continuously loaded rats. Recovery with unloading of liver copper was demonstrated by both qualitative and quantitative methods in group 5-0 (41.32 +/- 19 micrograms/g). Recovery and tolerance were associated with a reduction in nucleolar copper. Copper tolerance was demonstrated in group 5-0-5 and in continuously copper-loaded rats by Weeks 10 and 15. Copper tolerance was reflected by a change in intracellular levels and distribution of copper and an efficient copper unloading mechanism.

Hepatic lesions in rabbits infected with Encephalitozoon cuniculi administered per rectum.

Microsporidia have been recognized recently as opportunistic pathogens in acquired immunodeficiency syndrome patients. In an attempt to develop an animal model of enteric microsporidiosis, adult (5 to 6 months old) male Flemish Giant rabbits from a closed New York colony were administered 5 x 10(3), 5 x 10(5), and 5 x 10(7) Encephalitozoon cuniculi per rectum. Rabbits given 5 x 10(5) and 5 x 10(7) E. cuniculi had moderate granulomatous periportal infiltrates, characterized by the presence of numerous macrophages, epithelioid cells and a few multinucleated giant cells, lymphocytes, and plasma cells. Inflammatory cells also were seen infiltrating the tunica adventitia and tunica media of hepatic portal veins and branches of the hepatic artery. This study demonstrates that administration of E. cuniculi per rectum to rabbits results in infection that is characterized by high frequency and severity of hepatic lesions.

Amebic meningoencephalitis in a sheep.

A 1.5-year-old Suffolk ewe with acute onset of incoordination and blindness unresponsive to antibiotic treatment was examined at necropsy. The meninges were congested, opaque, and thick. Microscopically, focal areas of hypercellularity in the left cortical gray matter and the meninges were observed. The inflammatory response consisted of gliosis and perivascular cuffing (lymphocytes, plasma cells, and variable numbers of eosinophils). An amebic organism in 2 life stages was found in the cerebral parenchyma. Numerous large (15 to 35 microns in diameter) organisms, interpreted as trophozoites, were characterized by vacuolated cytoplasm and small nuclei with a prominent eosinophilic nucleolus (karyosome). The smaller (10 to 17 microns in diameter) encysted stage was surrounded by a capsule-like membrane, and contained a large central body sometimes surrounded by a clear halo. Immunofluorescence studies for amebic antigens were strongly positive for an ameba recently isolated in human beings and baboons (Leptomyxid sp).