Hepatic Copper Concentrations Research Articles

Effects of D-galactosamine hydrochloride and partial hepatectomy on spontaneous hepatic injury and hepatocarcinogenesis in Long-Evans Cinnamon rats.

To examine the effect of nongenotoxic chemicals on hepatocarcinogenesis in Long‐Evans Cinnamon (LEC) rats, we gave 6‐week‐old male and female LEC rats (n=18) weekly subcutaneous injections of d‐galactosamine hydrochloride (GalN, 300 mg/kg) in 0.9% NaCl or only 0.9% NaCl for 50 weeks, and killed them in week 62. GalN‐treated male rats unexpectedly showed no lethal necrotizing hepatitis. GalN treatment increased the incidence of cholangiofibrosis in males and its severity in females, but did not cause significant increases of hepatocellular tumors in either sex. GalN treatment increased the 5‐bromo‐2′‐deoxyuridine (BrdU)‐labeling index of hepatocytes and plasma hepatocyte growth factor, and accelerated megalocytic alterations without reduction of the hepatic copper concentration. Next, male and female LEC rats were subjected to two‐thirds partial hepatectomy (PH) or sham hepatectomy in week 8 (n=12) or in week 14 (n=9), and killed in week 62. PH in week 14 inhibited lethal hepatitis, but PH in week 8 was less effective. PH reduced the hepatic copper concentration to half that of controls. The present data suggest that induction of hepatocyte regeneration by repeated injections of GalN, or by PH just before the onset of jaundice has a significant effect in prevention of hepatic injury of LEC rats, but not enhancement of spontaneous hepatocarcinogenesis.

Folate and homocysteine metabolism in copper-deficient rats

To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague–Dawley rats were fed semi-purified diets containing either 0.1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group ( P<0.05). Copper deficiency resulted in a 21% reduction in hepatic methionine synthase activity as compared to the control group ( P<0.01). This change most likely caused the increased hepatic 5-methyltetrahydrofolate and plasma homocysteine concentrations in the copper-deficient group. Our results indicate that hepatic methionine synthase may be a cuproenzyme, and plasma homocysteine concentrations are influenced by copper nutriture in rats. These data support the concept that copper deficiency can be a risk factor for cardiovascular disease.

Hepatocyte-specific localization and copper-dependent trafficking of the Wilson’s disease protein in the liver

Wilson's disease is an inherited disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration. In this current study, a polyclonal antiserum specific for the Wilson's disease ATPase was used to examine the hepatic expression of this protein. Immunoblot analysis of lysates from human and rat liver detected a single 165-kDa protein, which by immunofluorescence was present only in hepatocytes and localized predominantly to the trans-Golgi network and exclusively in this compartment under low hepatic copper concentrations. Although hepatic copper concentration had no effect on the steady-state levels of the Wilson's disease protein, copper administration in vivo resulted in redistribution of this protein to a cytoplasmic vesicular compartment localized toward the hepatocyte canalicular membrane. The relative abundance of the Wilson's disease protein in the liver was found to be greatest in the fetus before the onset of biliary copper excretion. Taken together, these studies reveal a novel posttranslational mechanism of copper homeostasis in vivo consistent with the proposed function of the Wilson's disease protein in holoceruloplasmin biosynthesis and biliary copper excretion and of relevance to the broad clinical heterogeneity observed in this disease.

Wilson disease; analysis of 34 Turkish patients

The aim of this study was to determine clinical and laboratory findings of 34 Turkish children with Wilson disease (WD) at diagnosis before treatment. Presenting symptom was chronic liver disease in 27 patients. Five patients also had neurological symptoms. Seven patients were detected by family screening (siblings) and two were asymptomatic. Evaluation included neurological and ophthalmic examination, routine laboratory tests and parameters of copper metabolism, including liver copper content in 20 liver biopsy specimens. In the whole group, serum ceruloplasmin level was 20 mg/dl in 76.5%, urinary copper excretion was increased in 81.4% and liver copper content was elevated in 45% at diagnosis. Kayser–Fleischer rings were detected in 47% of patients. We concluded that, in patients presenting with liver disease and/or neurologic abnormalities of unknown origin, even normal ceruloplasmin and hepatic copper concentration, WD should always be kept in mind. On the other hand, family screening must be done in order to find siblings who suffer from WD in the early stages.

The effects of copper on blood and biochemical parameters of rainbow trout (Oncorhynchus mykiss).

Metals are released into aquatic systems from many sources, often at sublethal concentrations. The effects of sublethal concentrations of metals on fish are not entirely understood. The objective of this study was to determine the hematological and biochemical effects of a range of copper concentrations (6.4, 16.0, 26.9 microg Cu/L) on rainbow trout (Oncorhynchus mykiss) over a prolonged period of time. Trout were exposed to copper, and, at intervals of 3, 7, 14, and 21 days, selected parameters were evaluated. Hemoglobin, hematocrit, plasma glucose, and plasma cortisol levels were elevated in trout exposed to 26.9 microg Cu/L at day 3 and then returned to levels comparable to control fish. Plasma protein and lactate levels were not significantly altered in trout from any copper treatment. Hepatic copper concentration and hepatic metallothionein mRNA expression were consistently elevated in trout exposed to 26.9 microg Cu/L. Both of these parameters stabilized by day 3, with only hepatic copper concentration showing a further increase at day 21. Hepatic copper concentration and hepatic metallothionein mRNA expression appear to be robust indicators of copper exposure. Most blood-based parameters evaluated appear to be associated with a transitory, nonspecific stress response. The return of elevated hematological and biochemical parameters to control levels after 3 days and the stabilization of hepatic metallothionein mRNA expression and copper concentration over a similar time period suggested acclimation to dissolved copper at 26.9 microg/L. Further analysis of the data on blood-based parameters indicated that certain parameters (hemoglobin, hematocrit, plasma glucose, plasma cortisol) may be useful in field monitoring.

A critical evaluation of copper metabolism in Indian Wilson's disease children with special reference to their phenotypes and relatives.

Wilson's disease is an autosomal recessive disorder of copper accumulation in various organs, with most common clinical manifestations such as hepatic, neurological, and renal dysfunctions. Serum copper and ceruloplasmin in Wilson's disease were significantly lower as compared to normals, controls, and relatives of Wilson's disease patients, whereas marked hypercupriuria (145+/-7 microg/24 h) was observed in Wilson's children only. A good correlation (r=0.92) was found between non-ceruloplasmin-bound copper and 24-h urinary copper excretion in Wilson's disease patients. Further, copper studies among the different phenotypes of Wilson's disease revealed substantially low serum ceruloplasmin and a marked hypercupriuria in Wilson's disease children associated with renal tubular acidosis as compared to the patients with either hepatological or neurological manifestations. Serum ceruloplasmin levels in 14 patients of Wilson's disease were between 14 and 20 mg/dL. These patients of Wilson's disease were confirmed by measuring liver biopsy copper, which was about nine times higher than normal hepatic copper content. During the family screening by copper studies, four asymptomatic siblings were diagnosed for Wilson's disease. These subjects were then started on D-penicillamine therapy because presymptomatic treatment prevents progression of the disease complications.

Effects of copper on immune system parameters of rainbow trout (Oncorhynchus mykiss)

AbstractAricultural, urban, industrial, and mining sources release metals into waterways. The effects of sublethal concentrations of metals on integrated physiological processes in fish, such as immunocompetency, are not well understood. The objective of this study was to determine the physiological effects of a range of sublethal copper concentrations (6.4, 16.0, and 26.9 μg Cu/L) on Shasta‐strain rainbow trout (Oncorhynchus mykiss) exposed in soft water. Trout were sampled after 3, 7, 14, and 21 d of exposure to copper. The percentage of monocytes was consistently elevated at 26.9 μg Cu/L, and the percentage of lymphocytes was decreased. A consistent increase in the percentage of neutrophils occurred at 26.9 and 6.4 μg Cu/L. Respiratory burst activity was decreased for all concentrations at all sampling days, but a significant reduction occurred only at 14 and 21 d of exposure to copper. B‐like cell proliferation was decreased by exposure to the higher copper concentrations. Proliferation results, however, had high variability. T‐like cell proliferation and phagocytosis were not altered. Hepatic copper concentration was consistently elevated in trout exposed to 26.9 μg Cu/L; no correlation was found between hepatic copper concentration and the immune system responses investigated. Consistent alterations in immunological parameters suggest that these parameters could serve as indicators of chronic metal toxicity in natural systems.

Effect of different degrees of moderate iron deficiency on the activities of tricarboxylic acid cycle enzymes, and the cytochrome oxidase, and the iron, copper, and zinc concentrations in rat tissues.

Severe iron deficiency results in complex systemic disorders e.g., including metabolism of energy and minerals. To investigate whether also moderate iron depletion may alter the activities of citric cycle enzymes and the cytochrome oxidase, the trace element status, and serum enzymes indicative of cell damage, this experiment was carried out with rats supplied with sub-optimal iron (9, 13 and 18 mg iron per kg diet) over a total of 5 weeks. The study included 3 pair-fed groups and an ad libitum group, fed with 50 mg iron/kg diet. All iron-restricted rats were classified as iron-deficient on the basis of reduced iron concentrations in body and iron-depending blood parameters. Body weight gain and catalase activity in kidney were lowered in rats receiving the lowest dietary iron level, exclusively. Rats fed 9 and 13 mg iron per kg diet had nearly 6- and 3-fold, respectively higher platelet counts in blood than their corresponding pair-fed controls. The activities of transaminases ASAT and ALAT, alkaline phosphatase, glutamate dehydrogenase and lactate dehydrogenase in serum which are indicative of cell damage were also markedly influenced by moderate dietary iron restriction, in which the enzyme levels in serum increased with intensifying iron depletion. Although, moderate iron restriction to young male rats was associated with marked alterations in iron status and serum enzymes, the activities of tricarboxylic acid cycle enzymes including malic dehydrogenase, fumarase, and isocitric dehydrogenase as well as cytochrome oxidase in liver remained largely unaffected. Only hepatic aconitase showed a somewhat reduction with iron depletion. Moreover, iron restriction was also accompanied with an accumulation of copper in liver which was significant for rats fed 9 and 13 mg iron per kg diet, whereas zinc status remained completely unaffected by moderate iron deficiency. It can be concluded, that a short-term moderate iron deficiency with ranging hemoglobin concentrations from 66 and 121 g/L, was accompanied with altered platelet counts, serum enzyme activities indicative of cell damage, and hepatic copper concentrations, but the activities of the tricarboxylic acid cycle enzymes and cytochrome oxidase in liver remained largely unaffected.

Histomorphological and immunohistochemical studies of chronic active hepatitis in Doberman Pinschers.

Liver tissue samples were reviewed from 35 Doberman Pinschers with chronic active hepatitis in the precirrhotic stage. Thirty dogs had elevated hepatic copper concentrations, and five had normal liver copper concentrations. The earliest changes were inflammation and scar tissue deposition around the small hepatic vein branches. There was also apoptosis of scattered hepatocytes in zone 3. Inflammation consisted of macrophages, lymphocytes, and plasma cells. As the disease progressed, collagen deposition increased around the hepatic veins; in some liver specimens, thin scar tissue septa radiated from the hepatic vein branches, and inflammation spread to include the portal tracts. The sinusoids adjacent to the scar tissue were converted to endothelial-lined, thin-walled vessels. Chronic active hepatitis (commonly referred to as Doberman hepatitis or chronic active hepatitis of Dobermans) is a progressive fibrosis, inflammation and hepatocyte loss beginning among zone 3 hepatocytes around the terminal hepatic vein branches. The histomorphologic changes were the same among those Dobermans with elevated hepatic copper and those with normal hepatic copper. The cause was not determined, but these morphologic studies support the idea of immune-mediated disease.

Value of histochemical stains for copper in the diagnosis of Wilson's disease.

The histochemical demonstration of hepatic copper is important in the diagnosis of Wilson's disease (WD). Conflicting results have been published with regard to the ability of different histochemical methods to demonstrate copper storage in the liver. Therefore, we evaluated the diagnostic value of three available histochemical methods in a large series of patients affected by WD. Seventy-nine consecutive liver needle biopsies, from 74 patients, 39 males and 35 females, aged 4-60 years (mean age 28.5 years) were stained with orcein, rhodanine and using Timm's method. On the basis of the histological picture, liver biopsies were subdivided into three groups: group A, steatosis; group B, interface hepatitis; group C, chronic hepatitis with bridging fibrosis and/or cirrhosis. In group A, 30.4% of the cases were positive using Timm's method, vs 13.2% using the rhodanine and 17.5% using the orcein method. In group B, Timm's method was positive in 40.1% while rhodanine and orcein showed positivity in 26.7%. In group C, the Timm's method stained 58.6%, rhodanine 36.6% and orcein 29.3% positively. Our data show that: (1) Timm's silver stain is the most sensitive method for the demonstration of copper in all cases of WD; (2) rhodanine and orcein have minor value in the diagnosis of WD, especially in the early stages of the disease; (3) to increase the diagnostic value of histochemistry for copper multiple histochemical stains in serial sections are required; and (4) although hepatic copper concentration is highest in the early stages of WD, the histochemical demonstration fails in a large number of cases.

The LEC Rat Possesses Reduced Hepatic Selenium, Contributing to the Severity of Spontaneous Hepatitis and Sensitivity to Carcinogenesis

The hepatic concentrations of copper, zinc, magnesium, calcium, and selenium were measured in LEC rats, which develop a spontaneous form of hepatitis at 3-4 months of age, and compared to trace metal concentrations in the LEA rat, its asymptomatic congenic strain. Consistent with results found by other groups, copper was found to accumulate within the liver of LEC rats to levels more than 50 times those measured in LEA rats. In addition, liver selenium concentration in LEC rats was found to be around 50% of that in LEA rats. The enzyme activity, and RNA for the selenium dependant enzyme, glutathione peroxidase, was also found to be reduced in LEC rat liver. These results indicate that hepatic selenium in the LEC rat is depleted and that, as a result of this, the capacity to protect cells from copper-induced free-radical damage is reduced.

Determination of copper, manganese and zinc in human liver.

Autopsied liver tissue samples collected from 42 males and 31 females were analyzed for copper, manganese and zinc using atomic absorption spectrometry (AAS). With the exception of two liver samples for which the copper levels were determined to be 74.8 and 104.0 micrograms/g (dry weight), hepatic copper concentrations were found to range from 1.7 to 32.4 micrograms/g with a mean concentration of 14.2 micrograms/g and standard deviation of 7.0 micrograms/g. Manganese concentrations (with the exception of one sample having 12.9 micrograms/g) ranged from 0.22 to 4.6 micrograms/g with a mean of 2.26 +/- 1.00 micrograms/g. Hepatic zinc levels averaged 118.3 +/- 44.4 micrograms/g and ranged from 38.5 to 231.3 micrograms/g. There were no apparent trends for the levels of any metals versus age nor were there any differences in average hepatic metal concentrations for males and females.

Bioavailability and interactions with other micronutrients of three dietary iron sources in Atlantic salmon, Salmo salar , smolts

Atlantic salmon, Salmo salar L., with mean initial weight of 60 g were fed a diet based on cod muscle meal supplemented with elemental iron, iron sulphate or haem-bound iron in concentrations of 0, 25, 50, 100, 500 and 1500 mg iron kg ‐1 for 8 weeks. No significant differences in growth or mortality were found, except in fish fed 1500 mg haem iron kg ‐1 , which showed reduced growth. In fish fed diets supplemented with elemental iron below 1500 mg iron kg ‐1 , blood haemoglobin and hepatic iron concentration decreased compared with fish fed the unsupplemented diet. Fish fed diets supplemented with iron sulphate showed increased blood haemoglobin and hepatic iron concentrations between 25 and 100 mg iron kg ‐1 . Fish fed diets supplemented with haem-bound iron showed increased hepatic iron at all dietary iron levels, while blood haemoglobin concentration decreased in the group fed 1500 mg haem iron kg ‐1 . The bioavailability of haem iron relative to sulphate iron was calculated by the slope ratio method to be 239% and 148% using blood haemoglobin and hepatic iron, respectively. Relative bioavailability of elemental iron was zero when dietary supplementation levels were between 25 and 500 mg iron kg ‐1 , while a small part was utilized when 1500 mg elemental iron kg ‐1 was supplemented. Additions of 500 and 1500 mg haem-bound iron kg ‐1 resulted in a complete loss of ascorbic acid in these diets. When these groups were discounted, no significant relationship between hepatic iron and hepatic ascorbic acid was found. There was no significant effect of dietary iron on whole-body manganese concentration and only a weak effect on whole-body zinc concentration. No significant correlations between dietary iron and hepatic copper concentration were found in any of the dietary treatments. This study also showed that the level of inorganic iron supplementation may be reduced by inclusion of 20 g blood meal kg ‐1 in the diet.

The Effects of Dietary Soybean Versus Skim Milk Protein on Plasma and Hepatic Concentrations of Zinc in Veal Calves

We assessed the zinc status of veal calves that were fed milk replacers containing either skim milk protein as the sole source of protein or a mixture of skim milk protein and soybean protein. After the milk replacers had been fed for 26 wk, mean body weight gain was 3kg lower for calves fed the skim milk plus soybean proteins; this decrease was not significant. Inclusion of dietary protein from soybeans versus milk protein alone reduced plasma concentrations of zinc by 43% and reduced hepatic concentrations of zinc by 81%. The impairment of zinc status that was induced by the inclusion of soybean protein was probably caused by its phytate component. The effect of soybean protein on zinc status was rather specific because plasma and hepatic concentrations of copper were unaffected. Despite the high concentration of zinc (142mg/kg of dry matter) in the milk replacer that contained milk plus soybean proteins, calves displayed a shortage of zinc because their plasma and hepatic concentrations of zinc were significantly reduced.

Screening for Wilson's disease in patients with liver diseases by serum ceruloplasmin

Background/Aims: A low serum ceruloplasmin level is considered a diagnostic test for Wilson's disease. To examine whether it is useful to detect presymptomatic patients with Wilson's disease, serum ceruloplasmin was determined by radial immunodiffusion (normal: 20–60 mg/dl) in all patients ( n=2867) admitted for evaluation of a liver disease in 1993 and 1994. Methods: Patients with levels lower than 20 mg/dl were further evaluated by determination of serum copper concentration, urine copper excretion and ophthalmological examination. If possible, a liver biopsy was performed and the hepatic copper content was determined by flame atomic absorption spectroscopy. Results: Seventeen patients had serum ceruloplasmin levels <20 mg/dl. One had asymptomatic Wilson's disease (no Kayser-Fleischer rings or neurological symptoms). In the other 16 patients Wilson's disease was excluded. Based on elevated hepatic copper concentration, three were considered as heterozygous carriers of the WD gene. The remaining patients had various liver diseases (acute viral hepatitis in three, chronic hepatitis in two, drug-induced liver disease in three, alcoholic induced liver disease in two) or malabsorption ( n=3). Conclusions: The positive predictive value of low serum ceruloplasmin was only 5.9%. Although helpful for identifying presymptomatic Wilson's disease, screening by determining of serum ceruloplasmin in unselected patients with clinical or laboratory evidence of liver disease in neither feasible nor cost effective.

Age‐related copper, zinc, and iron metabolism in Long‐Evans cinnamon rats and copper‐eliminating effects of D‐penicillamine and trienthine‐2HCl

The Long-Evans cinnamon (LEC) rat is an animal model of human Wilson's disease. The hepatic copper content of LEC rats increased in an age-dependent manner from 4–5 days of age and was maintained at a high level from 15–16 weeks of age. The renal copper content of LEC rats showed a tendency to increase from 10 weeks of age and increased rapidly from 15 weeks of age. No difference in whole-brain copper concentration was observed between LEC and Long-Evans agouti (LEA) rats. LEC rats treated with D-penicillamine and trienthine-2HCl had reduced liver dysfunction. These agents reduced the hepatic copper content by 1/2–2/3 and the renal copper content to within the normal range. They also decreased the copper content of hair and nails, and there was also a good correlation between the copper content of liver and white hair. J. Trace Elem. Exp. Med. 10:49–59, 1997. © 1997 Wiley-Liss, Inc.

Studies on Removal of Copper from LEC Rats Liver by Dithiocarbamates.

The effects of N-benzyl-D-glucaminedithiocarbamate (BGD), N-benzyl-D-galactamine dithiocarbamate (BGalD), N-p-methylbenzyl-D-glucamine dithiocarbamate (MeBGD), penicillamine (D-PEN), triethylenetetramine·2HCl (Trien), on the excretion and the distribution of copper were compared in male LEC rats (5 weeks). Rats were injected intraperitoneally with chelating agents (2 mmol/kg) every other day for 10 d. The hepatic copper content after treatment with BGD and MeBGD decreased more than that with D-PEN and Trien. Those treatment with dithiocarbamates did not redistribute copper to kidneys and brain. BGD, BGalD and MeBGD all significantly enhanced the biliary excretion of copper, zinc and iron.

Serum and hepatic copper concentrations used to define normal, marginal and deficient copper status in horses.

Equine Veterinary JournalVolume 28, Issue 6 p. 497-499 Serum and hepatic copper concentrations used to define normal, marginal and deficient copper status in horses N. F. SUTTLE, N. F. SUTTLE Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JHSearch for more papers by this authorJ. N. W. SMALL, J. N. W. SMALL Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JHSearch for more papers by this authorE. A. COLLINS, E. A. COLLINS Wellburn, York, YO6 7EWSearch for more papers by this authorD. K. MASON, D. K. MASON Milngavie, Glasgow, G62 6QZ, UKSearch for more papers by this authorK. L. WATKINS, K. L. WATKINS Royal Hong Kong Jockey Club, Veterinary Department, Sha Tin Racecourse, New Territories, Hong Kong.Search for more papers by this author N. F. SUTTLE, N. F. SUTTLE Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JHSearch for more papers by this authorJ. N. W. SMALL, J. N. W. SMALL Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JHSearch for more papers by this authorE. A. COLLINS, E. A. COLLINS Wellburn, York, YO6 7EWSearch for more papers by this authorD. K. MASON, D. K. MASON Milngavie, Glasgow, G62 6QZ, UKSearch for more papers by this authorK. L. WATKINS, K. L. WATKINS Royal Hong Kong Jockey Club, Veterinary Department, Sha Tin Racecourse, New Territories, Hong Kong.Search for more papers by this author First published: November 1996 https://doi.org/10.1111/j.2042-3306.1996.tb01624.xCitations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume28, Issue6November 1996Pages 497-499 RelatedInformation

The relationship between hepatic copper content and morphologic changes in the liver of West Highland White Terriers.

Liver tissue from 17 West Highland White Terriers (WHWTs) with cirrhosis, subacute bridging necrosis, hepatitis, or massive necrosis were examined for the presence, composition, and distribution of inflammatory foci. Copper analysis was performed on the specimens. The foci of inflammation and necrosis composed a significant part of the lesion in 15 of the samples. The foci were of two types. One, characteristic of idiopathic chronic active hepatitis, consisted of one or two apoptotic hepatocytes attended by lymphocytes and plasma cells. These foci were found primarily in the vicinity of the portal tracts, not associated with centrolobular copper-laden hepatocytes. The other type of focus was characteristic of copper toxicosis. These foci were larger and composed of debris-filled macrophages, lymphocytes, plasma cells, and scattered neutrophils, and on occasion apoptotic hepatocytes were found at the periphery. These foci were always found around the central vein among the copper-laden hepatocytes. Such foci were found only in dogs with copper concentration > 2,000 parts/million on a dry weight basis. These morphologic studies show that clinical liver disease in WHWTs is caused by more than one etiologic agent. Among 17 WHWTs with clinical liver disease, two had copper toxicosis, five had idiopathic chronic active hepatitis, and 10 had hepatic disease of undetermined type.

Retrorsine in breast milk influences copper handling in suckling rat pups

Aims: To explore the hypothesis that a second xenobiotic agent is required with excess copper to produce Indian Childhood Cirrhosis, this study investigated the effect of the pyrrolizidine alkaloid retrorsine fed to the mother during the suckling period upon the serial changes in neonatal copper status. Methods: Female Wistar rats with new-born litters were fed either a control or a retrorsine (50 mg/kg) diet. At 0, 4, 8, 11, 15, 18 and 21 days, pups from each litter were weighed, sacrificed and their livers removed for copper, DNA and metallothionein analysis. Serum samples were assayed for caeruloplasmin oxidase activity and albumin. Results: 1) Higher than adult level of hepatic copper in normal rats which rose post-natally before declining from day 11 after birth, 2) raised hepatic copper concentrations and total copper in the retrorsine group from day 15; levels were higher than adult at birth, 3) reduced serum caeruloplasmin oxidase activity and albumin levels in retrorsine group, but both groups lower than adult, 4) lower hepatic metallothionein levels in retrorsine group, but both groups higher than adult, and 5) reduced liver DNA in the retrorsine group when expressed as total DNA and per gram of tissue. These changes were not secondary to under-nutrition as a small study on under-nourished rat neonates showed that copper handling is not significantly altered when compared to well-nourished rats. Conclusions: Retrorsine passing to rat neonates via breast milk causes: 1) the accumulation of hepatic copper, 2) impairment of the rise in serum caeruloplasmin, which could indicate a decline in synthesis or failure of copper incorporation into the apo-protein, 3) a decrease in hepatic metallothionein and serum albumin levels, again suggesting diminished protein synthesis, and 4) reduced hepatic DNA indicative of decreased cell number but increased cell size. Accumulation of liver copper but reduction of copper-binding proteins could result in free copper and explain the synergistic hepatotoxicity of copper and retrorsine.