Subcellular Distribution Of Copper Research Articles

Predicting Copper Toxicity with Its Intracellular or Subcellular Concentration and the Thiol Synthesis in a Marine Diatom

The accumulation and subcellular distribution of copper (Cu) in a marine diatom Thalassiosira weissflogii were examined under different nutrient conditions [nitrogen- (-N) and phosphorus-starved (-P) conditions as well as nutrient-enriched conditions with nitrate (+NO3-) and ammonium (+NH4+) as the nitrogen source]. Cu accumulation was induced in the NO3--exposed (+N03- and -P) cells, suggesting that Cu may be directly or indirectly required for intracellular NO3- reduction. The relationships between the response of the cell-specific growth rate or photosynthetic system II maximum quantum yield ((DM) to Cu exposure in different nutrient-conditioned cells and the free copper ion concentration,the intracellular Cu concentration (intra-Cu), and the distribution of Cu in different subcellular compartments were also examined. We found that the intra-Cu was the best Cu toxicity predictor, as it accounted for most of the Cu-induced PhiM response between different nutrient-conditioned cells. The synthesis of intracellular cysteine and five low molecular weight (LMW) thiols was not notably induced at high Cu levels possibly because of the existence of some other Cu detoxification mechanisms. This was further demonstrated by the much lower cysteine and LMW thiol contents in the -N cells with a similar Cu tolerance to the nutrient-enriched cells. Overall, our results suggest that Cu toxicity could be better predicted with the intra-Cu and its subcellular distribution as compared with the currently used free ion activity model and biotic ligand model. However, the LMW thiols had unexpectedly little contribution to Cu detoxification in T. weissflogii.

Hepatic metallothioneins in two neotenic salamanders, Proteus anguinus and Necturus maculosus (Amphibia, Caudata)

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12 000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.

Bioaccumulation, Subcellular Distribution, and Trophic Transfer of Copper in a Coastal Marine Diatom

Cellular uptake and subcellular distribution of copper were measured in the coastal marine diatom Thalassiosira weissflogii grown with free Cu ion concentrations (10-14.79−10-9.79 M) that are typical of coastal waters. Intracellular Cu quotas increased 2−3-fold and 4−5-fold per decade increase in free Cu ion concentration over the pCu (pCu = −log [Cu2+]) ranges of 14.79−11.79 and 11.79−9.79, respectively. The trophically available cytoplasmic pool of Cu increased by 2 orders of magnitude in T. weissflogii cells (0.53 × 10-17−75 × 10-17 mol Cu cell-1) grown with 10-14.8−10-9.8 M free Cu. A 2-fold increase in intracellular and cytoplasmic Cu concentrations was observed in response to lowering the free concentration of the potentially antagonistic metal Zn by a factor of 10. However, the percent of intracellular Cu in the cytoplasmic fraction of T. weissflogii (≅ 40%) was relatively constant over the range of cupric ion activities and [Cu2+]:[Zn2+] ratios applied. The assimilation efficiency (AE) of copper in marine copepods fed T. weissflogii, determined using the inert-tracer ratio method, was 40.3 ± 6.3% for copepods fed diatoms grown at pCu = 12.79 and 50.3 ± 9.7% for copepods fed diatoms grown at pCu = 10.79. Our results suggest that, above free Cu concentrations of 10-11.8 M, the availability of Cu for trophic transfer in coastal waters increases as a result of rapidly increasing concentrations of Cu in the assimilable, cytoplasmic fraction of diatoms and potentially higher Cu AEs at the base of the pelagic food web.

Alteration of hepatic DNA synthesis in rats treated with N-nitrosodimethylamine during early stages of copper deficiency.

The main aim of this research was to clarify that nutritional dietary copper may participate in the protective action against hepatocarcinogenesis in rats by N-nitrosodimethylamine (NDMA). The copper concentrations in serum and liver from 2 to 8d after rats were first fed a copper deficient diet (copper, 0.6 ppm) decreased significantly compared to those of pair-fed rats (copper in a control diet, 7 ppm). The subcellular distribution of copper in the liver at 5d after feeding of a copper deficient diet began was measured and the copper concentrations in soluble and nuclear fractions decreased at a similar rate in copper deficient rats treated with or without NDMA, compared to those of pair-fed rats. The incorporation of [3H]thymidine into rat liver DNA at 48 h after treatment with NDMA markedly increased under the experimental conditions used. By giving rats a copper deficient diet for a few days the increased incorporation of [3H]thymidine into liver DNA of rats treated with NDMA was enhanced compared to that of pair-fed rats treated with NDMA. The activity of thymidine kinase in liver of copper deficient rats treated with NDMA was also found to increase significantly compared to that of pair-fed rats treated with NDMA.

Postnatal changes in subcellular distribution of copper, zinc and metallothionein in the liver of bank vole ( Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferation

1. Dramatic interdependent changes in the intracellular concentrations of copper (Cu), zinc (Zn) and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were observed. 2. The post-mitochondrial Cu, Zn and MT (ZnMT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (CuMT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex CuMT was translocated to the nuclei. 3. The nuclear Cu concentration reached the highest level (62–71% of the total tissue Cu) in the period from day 3 to day 20 post-partum, just prior to and during a rapidly growing liver. 4. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.

Copper metabolism in hypercupremic human livers: Studies of its subcellular distribution, association with binding proteins and expression of mRNAs

In the present study we have used differential centrifugation, size exclusion chromatography, Western and Northern blotting to investigate the subcellular distribution of hepatic copper, the association of the metal with hepatic copper binding proteins and the expression of specific mRNAs for copper binding proteins in liver tissue from two patients with Wilson's disease, two patients with chronic liver disease and two patients with normal hepatic copper levels. Unlike previous studies the present results fail to show any gross differences in subcellular distribution of copper between the livers, with most of the copper being found in the soluble supernatant where it is associated with metallothionein. Caeruloplasmin mRNA levels were reduced in the two patients with Wilson's disease and also in a patient with fulminant hepatic failure. It remains to be confirmed if the reduction of caeruloplasmin mRNA is specific for Wilson's disease. Levels of mRNAs for copper zinc superoxide dismutase and metallothionein were variable and not related to liver copper.

Subcellular distribution of copper in the liver of fetal deer in the last month of gestation

Fetal deer, in the last month of gestation, accumulate high concentrations of copper in the liver. Livers from fetal deer in late gestation were homogenised and fractionated by continuous sucrose density gradient centrifugation. The distribution of copper closely followed that of DNA; approximately two thirds of the metal was localised to the nuclear fractions with the remaining third in the cytosolic fractions. The fractionation procedure was repeated with digitonin, a lysosomal perturbant: lysosomes were disrupted and the marker enzyme, N-acetyl-(3-glucosaminidase, shifted from the particulate fractions to the cytosolic fractions; the distribution of copper was unaffected. Differential centrifugation of homogenate confirmed that approximately two thirds of the copper was associated with the nuclear fraction. Further confirmation of a nuclear localisation of copper was provided by X-ray microanalysis of purified nuclei.

Subcellular distribution of copper in the kidneys of normal, copper-poisoned, and thiomolybdate-treated sheep

Twenty-seven sheep given either copper (Cu) and/or tetrathiomolybdate (TM) were used to study the subcellular distribution of Cu within the kidney and to monitor the location of lysosomes within the subcellular fractions using acid phosphatase (AP) as a marker enzyme. Copper dosing alone increased the Cu content in the liver and the kidneys. The administration of intravenous TM prevented the development of chronic copper poisoning (CCP) in sheep, reduced the rate of accumulation of Cu in the liver of Cu-dosed animals, but increased the Cu content of kidneys in both the control and Cu-dosed sheep. The total amount of Cu that accumulated in the kidneys of sheep given TM appears to depend on several factors: a) liver Cu concentration, b) Cu intake, and c) dosage of TM. Thus, the highest Cu concentration was found in the kidneys of sheep that continued to receive Cu orally at the same time as they were given TM. The intracellular distribution of Cu and AP in thfe kidneys showed that in the control sheep given neither Cu or TM, the highest proportion of Cu was in the cytosol fraction, and the highest specific activity of AP was in the light mitochondrial (lysosomal) fraction. Dosing with Cu markedly increased the Cu concentration and greatly elevated the total activity of AP in the heavier fractions, i.e., the nuclear (N) and heavy mitochondrial (MH). Thus, the increase in Cu observed in the N and MH fractions was not caused by an accumulation of Cu by nuclei and mitochondria, but was due to an accumulation of Cu by lysosomes that sedimented with the heavier fractions. The intracellular distribution of Cu in the kidneys of TM-treated sheep was similar to that seen in Cu-loaded sheep. Although Cu accumulated readily in the kidneys of animals receiving TM, kidney function tests showed neither glomerular nor tubular functional impairment.

Effect of dietary copper on the hepatic concentration and subcellular distribution of copper and zinc in the rainbow trout ( Salmo gairdneri)

Feeding trials were conducted with six groups of rainbow trout (mean initial weight of 35 g) using purified diets with an estimated gross energy of 1442 kJ 100 g and copper contents ranging from 3.5 to 1000 mg Cu/kg diet. With the exception of groups 1 and 2 (given 102 and 194 mg Cu/kg), which showed an atypical growth response, the weight gain and feed conversion were reduced with increasing levels of Cu in the diet. No gross pathologies and very low mortalities were noted even in the group receiving the highest dietary copper level. Copper and zinc were determined in freeze-dried samples and in subcellular fractions from individual trout livers using atomic absorption spectrophotometry. A linear relationship was observed between liver copper concentration and copper provided in the diet. Copper intake had no effect on hepatic zinc concentration. Increased dietary copper also gave increased copper levels in whole fish but the concentration factor was much lower than in the liver. The relative retention of copper given at levels above 100 mg Cu/kg dry diet was low and was estimated to be 1.3%. Subcellular fractionation studies demonstrated that 43–67% and 36–49%, respectively, of hepatic copper and zinc were sedimented with the fractions nuclei, mitochondria and lysosomes. Increased dietary levels of copper had apparently no effect on the subcellular distribution of zinc except at the highest level (group 6) where an increase in zinc recovered in the nuclear fraction was observed.

LIVER DISTRIBUTION OF COPPER AND 99Mo IN SHEEP FOLLOWING INTRAVENOUS ADMINISTRATION OF COPPER SULFATE AND [99Mo]-TETRATHIOMOLYBDATE

The effect of a single intravenous dose of 0, 50 or 100 g molybdenum (Mo) as [99Mo] labelled sodium tetrathiomolybdate (Na2MoS4) on the subcellular distribution of copper (Cu) in the liver was studied in three groups of four sheep. The sheep received a 35-mg intravenous dose of Cu 20 h prior, and were killed 24 h after the dosing with Mo. Subcellular fractionation of liver homogenates revealed that [Formula: see text] may react with Cu in the nuclear (lysosomal) fraction. Key words: Sheep, copper, molybdenum, tetrathiomolybdate, liver fractions

Copper tolerance in Silene cucubalus

The uptake, translocation and subcellular distribution of copper as well as its effect on chloroplasts and plastocyanin synthesis were studied in a copper-sensitive and a copper-tolerant population of Silene cucubalus (L.) Wib. As a function of time, the copper concentration in roots of tolerant plants increased more slowly than that in roots of sensitive ones. Translocation to the shoot occurred more rapidly in tolerant plants than in sensitive ones. Although it was accumulated in leaf cells, copper was not accumulated in the chloroplasts of either sensitive or tolerant plants. Chlorophyll content was not affected by copper in tolerant plants, whereas sensitive plants became chlorotic. Plastocyanin synthesis was not enhanced as a result of high copper concentrations and no difference in plastocyanin content between tolerant and sensitive plants was detected. Measurements of copper in purified cell walls revealed that storage of the metal in cell-wall material does not play an important role in tolerance mechanism. Uptake characteristics, distribution and cytoplasmic detoxification of copper are discussed.

Regulation and subcellular distribution of copper in the freshwater molluscs Anodonta cygnea (L.) and Unio elongatulus (Pf.)

1. The effects of copper exposure have been studied in two freshwater bivalves, Anodonta cygnea and Unio elongatulus. 2. Copper exposure induces a small degree of copper accumulation in both organisms leading to the hypothesis of a regulatory mechanism for copper intake. 3. Copper distribution in the particulate fractions is affected by copper treatments, with an increase in the lysosome-enriched fraction. 4. Copper exposure does not affect zinc levels and distribution. 5. The percentage distribution of copper and zinc among the eytosol fractions obtained from Sephadex G-75 varies with organism, tissue and copper exposure suggesting that high molecular weight chelating compounds are actually present in the two organisms.

Copper toxicosis and tolerance in the rat: III. Intracellular localization of copper in the liver and kidney

Male rats fed a 3 g/kg copper diet were killed sequentially up to 14 weeks. Copper in liver and kidney cortex was identified histochemically and assayed in homogenates and gradient fractions following analytical subcellular fractionation on reorientating sucrose density gradients. Copper accumulated fastest in the liver postnuclear (PNS) supernatant fraction which became saturated at 2 weeks; there was a distinct localization of copper in hepatic lysosomes which displayed progressively enhanced fragility. Rapid accumulation of copper then occurred in liver and kidney nuclear (N) fractions, peak copper concentrations coinciding with hepatic and renal tubular necrosis. Copper accumulated slowly up to 4 weeks and was maintained to 14 weeks in the cytosol of the proximal renal tubules from which it appeared to be excreted. Subsequently liver copper declined in both liver fractions and kidney (N) fractions; hepatic lysosomes regained stability, regeneration of liver and kidney occurred, and the rats were tolerant to continued copper administration. These findings do not support a primary role for copper-loaded lysosomes in the genesis of cell injury, but suggest that nuclear saturation may be the destabilizing event. Recovery is associated with changes in the subcellular distribution of copper within liver and kidney and renal excretion of excess.

Influence of tansy ragwort ( Senecio jacobaea), dietary copper, and feed restriction on the subcellular distribution of minerals in rat liver

Young male rats were pair-fed diets containing 0 or 5% tansy ragwort (TR), with each diet containing 0 or 50 μg/g copper. Other animals given TR-free diets were also maintained as ad libitum controls. After 5 weeks of feeding these diets, the rats were killed for the determination of the trace metal contents of liver fractions. TR with no dietary copper supplement increased the calcium content of all hepatic fractions except nuclei and debris, but had no effect on the subcellular concentrations of copper, zinc, or iron. When 50 μg/g copper was included in the TR diet, the subcellular distribution of copper, iron, and calcium was altered. This alteration was shown by an increase (as compared to pair-fed controls given copper-supplemented diet with no TR) in the copper content of all fractions except cytosol, the iron content of all fractions, and the calcium content of mitochondria, microsomes, lysosomes, and cytosol. Feed restriction (pair-fed versus ad libitum controls) using diets with no additional copper produced an increase in the copper and calcium contents of mitochondria; the copper, zinc, and calcium contents of cytosol; and the calcium content of lysosomes. Addition of 50 μg/g copper to the diets abolished these effects of feed restriction. These findings suggest that the subcellular distribution of copper, zinc, iron, and calcium in rat liver is subject to perturbation by pyrrolizidine alkaloid-containing diet, dietary copper, and feed restriction.

Sub-cellular distribution of copper and caeruloplasmin in chemically induced tumour tissue

Copper and caeruloplasmin concentrations were determined in the subcellular fraction of tumour tissue from 15 Strain A mice bearing an induced fibrosarcoma. It was observed that copper concentration was higher in the mitochondrial fraction of tumour tissue when compared to that of tissues of similar site from normal controls. However, the caeruloplasmin concentration was lower in the various fractions of tumour tissue when compared to that of normal control tissue.

Copper accumulation in the soluble and particulate fractions of renal cortex in the streptozotocin-diabetic rat

The accumulation and subcellular distribution of copper in the kidney of streptozotocin-diabetic rats were investigated. Male Sprague-Dawley rats received streptozotocin (50 mg/kg body wt on two consecutive days) intraperitoneally and were fed either commercial or purified diet. The concentrations of copper, zinc, iron, and manganese present in intact kidney, renal cortex, and renal medulla were compared at various times. Chow-fed diabetic rats had a renal copper concentration 2.6 times greater than age-matched controls after 2 weeks. The concentration of zinc was only 30% higher in diabetic kidney than in control tissue, whereas the iron and manganese concentrations were similar for both groups. The additional complement of renal copper was localized entirely in the cortex and was significantly reduced by oral treatment with penicillamine, a copper chelating agent. When diabetic rats were fed purified diet (15-20 ppm Cu), the quantity of copper accumulated in the renal cortex increased from 2.3 to 8.7-fold higher than in control tissue from 1 to 4 weeks, respectively, after injection with streptozotocin. Copper levels in. both the soluble and particulate (165, 000g pellet) fractions of diabetic renal cortex were similarly increased at each time. Gel filtration Chromatographic analysis of the cytosol showed that all of the copper accumulated in the soluble fraction was associated with metallothionein. The distribution of excess copper in the particulate fraction was determined by differential centrifugation. The additional quantity of metal was localized in the crude nuclear fraction of renal cortex in the diabetic rat. Further analysis revealed that the lysosomal fraction from 3-weeek diabetic rats had a copper level 16-fold higher than in the controls. The possibility that accumulation of excessive levels of copper in the streptozotocin-diabetic kidney may contribute to the development of diabetic nephropathy is discussed.

The role of lysosomes in the pathogenesis of copper-induced hepatotoxicity: II. Intracellular distribution of copper in hepatocytes

The effects of altered lysosomal function on the pathogenesis of copper-induced hepatotoxicity were studied in C57B1 6 bg/bg (beige) and C57B1 6 bg/+ (conventional) mice. Copper loading was accomplished through daily ip injections of aqueous copper chloride at a dosage rate of 8 mg/kg body weight for 1, 2, and 4 weeks. The subcellular distribution of copper in copper-treated beige mice was significantly different than that in conventional mice. Beige mice had consistently higher levels of copper in the nuclear and cytosolic fractions with lower levels in the heavy and light mitochondrial fractions. The copper levels were lowest in the microsomal fractions of both groups of copper-treated mice and remained similar throughout the 4-week experiment. Levels of total hepatic copper were similar in both groups of mice receiving parenteral copper except at Day 14, when beige mice had higher levels.

Influence of sex hormones on the subcellular distribution of copper in sheep liver

At the 14th to 16th week of pregnancy the copper content in the maternal liver was low while that in the fetus was high. After ovariectomy there was a decrease in liver copper and ceruloplasmin which was prevented by treatment with oestrogens but not with progesterone. Five groups of sheep were studied: control (barren ewes), pregnant, ovariectomised and ovariectomised treated with oestrogens or progesterone. In all, with high or low liver copper, the nuclear fraction contained the most copper. This suggests a species specificity of the copper metabolism in sheep.

The subcellular distribution of copper, zinc and iron in liver and kidney. Changes during copper deficiency in the rat.

1. The subcellular distribution of copper, zinc and iron was investigated in liver and kidney homogenates from Cu-deficient and control rats. The supernatant fraction contained most Cu and Zn in control animals, but the highest proportion of Fe was in the microsomal fraction.2. Cu deficiency reduced the concentration of the metal in all fractions, but the depletion was most severe and developed most rapidly in the supernatant fraction, indicating that the soluble cytoplasm is the site of Cu storage in the cell. The Fe content of the liver increased during Cu deficiency with Fe being deposited preferentially in the mitochondria.3. All the Cu and most of the Zn in liver and kidney supernatant fractions occurred in four protein-containing fractions that were of similar molecular weights in both organs. A fraction of molecular weight 30000 was primarily concerned with Cu storage in mature rats.4. The reduction in liver Zn during Cu deficiency appeared to be the result of impaired intestinal absorption and it is suggested that a small amount of Cu facilitates the absorption of Zn.

Spectrophotometric determination of copper with N,N,N′,N′-tetraethylthiuram disulfide and an application of this method for studies on subcellular distribution of copper in rat brain

A method for spectrophotometric determination of copper with N,N,N′,N′-tetraethylthiuram disulfide is described. This method was applied for determination of copper in serum, urine, and brain subcellular fractions. Of the total copper of rat brain, 49% was in crude mitochondria fraction, 31% in the supernatant, 13% in nuclear fraction, and 7% in microsomal fraction. Of the crude mitochondrial copper, more than half was recovered in F 4 and F 5.