Influence of calcium and EDTA on copper ion bioavailability in copper nanoparticle toxicity tests improves understanding of nano-specific effects.

Metal-based nanoparticles (NPs) can release metal ions that are toxic to aquatic organisms; however, whether the toxicity is from metal ions rather than unique "nano-scale" effects of the NPs is unresolved. The present study aimed to compare the toxicity of Cu2+ and Cu-NPs in larval zebrafish (Danio rerio) to clarify whether toxic effects are attributable to release of Cu ions and to determine the effect of the chelating agent ethylenediaminetetraacetic acid (EDTA) and calcium hardness (as CaCO3) on the Cu toxicity. First, the acute toxicity (96-h lethality) of Cu-NPs was determined in comparison to aqueous Cu in larvae exposed to CuSO4, and subsequently, sublethal tests with Cu-NPs and CuSO4 were conducted with additions of EDTA or calcium ions to evaluate alterations in expression of metallothionein-2 (MT2) gene transcripts (quantitative real-time polymerase chain reaction). Acute toxicity of Cu in larvae exposed to CuSO4 was greater (LC50 = 226 µg Cu/L) than for larvae exposed to Cu-NPs (LC50 = 648 µg Cu/L). The expression of MT2 increased with Cu concentration (p < 0.05), and the slope of the linear regression was significantly greater in fish exposed to CuSO4 (slope = 0.090) compared to Cu-NPs (slope = 0.011). Cu2+ was 2.9-fold more toxic than Cu-NPs. The presence of 5 mg/L EDTA and 220 mg/L CaCO3 significantly reduced the expression of MT2 (1.8-fold for EDTA, 2.3-fold for CaCO3) in larvae exposed to CuSO4. For larvae exposed to Cu-NPs, the presence of EDTA reduced the expression of MT2 (1.7-fold) relative to Cu-NP concentration. While Cu-NPs induced MT2 expression, the differences in concentration relationships of MT2 expression between Cu-NPs and CuSO4 indicated that factors other than release of Cu ions from Cu-NPs influenced acute toxicity of Cu-NPs. The conclusion drawn from this ecotoxicological risk assessment was that EDTA and calcium significantly decreased Cu toxicity in freshwater fish.

The effect of feed supplementation with a copper-glycine chelate and copper sulphate on selected humoral and cell-mediated immune parameters, plasma superoxide dismutase activity, ceruloplasmin and cytokine concentration in broiler chickens.

The varied bioavailability and different effects of organic forms of copper on the immune system of poultry have prompted the search for new feed additives based on copper compounds containing modified chelate complexes. The aim of the study was to determine the effect of inorganic and organic forms of copper on selected parameters of the cellular and humoral immune response in broiler chickens by determining the percentages of CD3+ CD4+ , CD3+ CD8+ and CD25+ lymphocytes, cells with MHC Class II expression, and BU-1+ cells, as well as the concentrations of SOD, IL-2, IL-10 and TNF-α in the peripheral blood. The experiments were conducted using 500 one-day-old Ross 308 roosters divided into five groups. Cu was added in inorganic form (CuSO4 ), in inorganic form with the addition of phytase (CuSO4 + F), in organic form in combination with glycine (Cu-Gly) and in organic form in combination with glycine and a phytase supplement (Cu-Gly+F). The results of the study indicate an increase in the percentage of CD3+ CD4+ and CD3+ CD8+ T lymphocytes, CD25+ T cells, and cells expressing MHC class II molecules, and in the concentration of ceruloplasmin, activity of superoxide dismutase and the concentration of IL-2 in the groups that received copper, particularly copper-glycine chelates. Based on the study, we can conclude that supplementation of poultry feed with copper chelates activates mainly the Th1 cellular immune response and the response of peripheral blood T lymphocytes. Furthermore, it promotes secretion of cytokines, which are involved in potentiation and regulation of the immune response in birds.

Histopathological changes in snail, Pomacea canaliculata, exposed to sub-lethal copper sulfate concentrations.

The acute toxicity test of Cu including range-finding and definitive test, was performed on golden apple snails, Pomacea canaliculata. The median lethal concentrations (LC50) of Cu at exposure times of 24, 48, 72 and 96 h were 330, 223, 177 and 146 µg/L, respectively. P. canaliculata were exposed to Cu at 146 µg/L for 96 h to study bioaccumulation and histopathological alterations in various organs. Snails accumulated elevated levels of Cu in gill, and lesser amounts in the digestive tract, muscle, and digestive gland. Histopathological investigation revealed several alterations in the epithelia of gill, digestive tract (esophagus, intestine, rectum), and digestive gland. The most striking changes were observed in the epithelium of the gill in which there was loss of cilia, an increase in number of mucus cells, and degeneration of columnar cells. Similar changes occurred in digestive tract epithelium. The digestive gland showed moderate alterations, vacuolization and degeneration of cells and an increase in the number of basophilic cells. We concluded that, P. canaliculata has a great potential as a bioindicator for Cu, and a biomarker for monitoring Cu contamination in aquatic environment.

[Metabolic memory enhances hormesis effect to the copper ions in age-depended manner].

The ability of young and old rats to manifest the hormesis effect to lethal doses of copper sulphate and the ability to save the induced "adaptive" pattern of redistribution of copper ions after the transfer of animals in the standard conditions is the mechanism of metabolic memory. It was found that pretreatment of animals with low-dose (1 mg per 100 g body mass, i.e. 33% of the lethal dose) of copper sulfate induced the formation of their resistance to lethal doses (3 mg per 100 g), so the hormesis effect was manifested. Hormesis effect depended on the number of pre injections of small doses of copper sulphate in an S-shaped manner. The protective effect increased after 1 to 3 of preliminary injections of copper sulfate, and after four or more injections the hormesis effect decreased. It is shown that the cardinal role in intracellular pattern of copper ion redistribution play heat-stable copper binding proteins 12 kDa cytosolic proteins. The formed "adaptive" pattern of intracellular distribution of the copper ions may be reproduced, after at least, one month. The prolonged hormesis effect can be attributed to the forming metabolic memory. The intracellular distribution pattern of the copper ions was age-dependent. Age-related differences were found in hormesis effect induced by copper ions, which results in increased binding capacity of copper binding proteins in old animals, with a higher content of copper ions in the mitochondria and microsomes as compared to young animals.

Proteomic analysis of leaves and roots of common wheat (Triticum aestivum L.) under copper-stress conditions.

Proteomic studies were performed to identify the protein species involved in copper (Cu) stress responses in common wheat. Two-week-old wheat seedlings were exposed to 100 µM CuSO4 treatment for 3 days. Growth of shoots and roots was markedly inhibited and lipid peroxidation was greatly increased. Cu was readily absorbed by wheat seedlings, with greater Cu contents in roots than in leaves. Using 2-DE method, 98 protein spots showed significantly enhanced or reduced abundance, of which 93 were successfully identified. Of these identified protein species, 49 and 44 were found in roots and leaves, respectively. Abundance of most of identified protein species, which function in signal transduction, stress defense, and energy production, was significantly enhanced, while that of many protein species involved in carbohydrate metabolism, protein metabolism, and photosynthesis was severely reduced. The Cu-responsive protein interaction network revealed 36 key proteins, most of which may be regulated by abscisic acid (ABA), ethylene, jasmonic acid (JA), and so on. Exogenous JA application showed a protective effect against Cu stress and significantly increased transcripts of the glutathione S-transferase (GST) gene. This study provides insight into the molecular mechanisms of Cu responses in higher plants.

Relative bioavailability of organic and hydroxy copper sources in growing steers fed a high antagonist diet1.

To assess relative bioavailability (RBV) of hydroxy and organic Cu sources compared with CuSO4 in steers fed a high Cu antagonist diet, 84 steers were stratified by BW to pens randomly assigned to dietary treatments for 90 d. Steers received a common corn silage-based diet supplemented with Cu antagonists (diet analyzed 0.25% S; 6.8 mg Mo/kg DM). Supplemental (SUPP) Cu treatments included: control (CON; no SUPP Cu), low or high inorganic (ING5 or ING10; 5 or 10 mg Cu/kg DM from CuSO4; Old Bridge Chemical Inc., Old Bridge, NJ, USA), low or high organic (ORG5 or ORG10; 5 or 10 mg Cu/kg DM from Cu lysine; CuPlex 100, ZinPro Corp., Eden Prairie, MN), and low or high hydroxy (HYD5 or HYD10; 5 or 10 mg Cu/kg DM; IntelliBond CII, Micronutrients USA LLC, Indianapolis, IN). Body weights were recorded on days -7, -6, 28, 56, 84, and 85, and plasma samples collected on days -7, 28, 56, and 85. Liver samples were collected to start and end the trial. Data were analyzed using the mixed procedure of SAS and the model included treatment with initial liver Cu values used as a covariate in analysis of final liver Cu. Contrast statements were used to separate treatment means: 0 vs. 5 mg SUPP Cu/kg DM, 0 vs. 10 mg SUPP Cu/kg DM, 5 vs. 10 mg SUPP Cu/kg DM, HYD vs. ORG, HYD vs. ING, and ORG vs. ING. Initial liver Cu concentrations were similar across all treatment comparisons (P ≥ 0.22). Final liver Cu concentrations were lesser in CON compared with either 5 or 10 mg Cu/kg DM (P ≤ 0.001). Final liver concentrations were lesser in ORG compared with HYD and ING (P ≤ 0.009), but HYD was similar to ING (P = 0.14). There was a treatment × time interaction (P ≤ 0.001) for plasma Cu concentrations where CON exhibited a rapid decline in plasma Cu, steers receiving 5 mg SUPP Cu/kg DM were decreased to a greater extent in ORG, and steers supplemented with 10 mg Cu/kg DM did not differ at the end of the trial. Assessment of RBV was conducted for liver and plasma Cu concentrations using a slope-ratio assay in the GLM procedure. The RBV of Cu tended (P = 0.07) to be increased in HYD (112%) compared with ING (100%) for liver Cu values, but RBV was similar for all other source comparisons based on liver and plasma Cu values (P ≥ 0.22). These data suggest in steers fed high antagonist diets hydroxy Cu may be more available. Based on plasma and liver Cu concentrations, supplementation of 10 mg Cu/kg DM is needed to maintain Cu status in cattle fed diets high in S and Mo.

Role of Na+, K+-ATPase alpha1 subunit in the intracellular accumulation of cisplatin.

The present study was undertaken to identify what regulates intracellular cisplatin (CDDP) accumulation and what changes in membrane fraction of CDDDP-resistant cell line. The CDDP-resistant rat hepatoma cell line, H4-II-E/CDDP, shows a significant decrease in intracellular platinum accumulation compared with parental H4-II-E cells, although there was no difference in the efflux of CDDP between these two cell lines. In this study, we examined the contribution of functional change in active transport to the CDDP resistance of H4-II-E/CDDP cells. Compared with the resistant cells, platinum accumulation in the parental cells was clearly decreased by low temperature or ATP depletion. In addition, the Na+, K+-ATPase inhibitor ouabain and the K+ channel inhibitor tetraethylammonium decreased platinum accumulation in parental cells but did not change the accumulation in resistant cells. Amphotericin B, an antifungal agent, increased the intracellular platinum accumulation in resistant cells to the same level as in parent cells. Western blot analysis demonstrated that the Na+, K+-ATPase alpha1 subunit was reduced in resistant cells compared with the parental cells, although there was no difference in the expression of the beta1 subunit between the two cell lines. Furthermore, the Na+, K+-ATPase alpha1 subunit of H4-II-E was decreased following a 24-h exposure to CDDP. These results suggest that Na+, K+-ATPase-dependent active transport of CDDP does not occur in resistant cells, and, furthermore, our findings provide the first evidence that the Na+, K+-ATPase alpha1 subunit plays an important role in the transport of CDDP.

Acquisition of resistance to cisplatin is accompanied by changes in the cellular pharmacology of copper.

Impaired uptake of cisplatin (DDP) consistently accompanies the acquisition of resistance to the platinum drugs. The pathways by which DDP enters or exits from cells remain poorly defined. Using three pairs of human ovarian carcinoma cell lines, each consisting of a sensitive parental line and a stably DDP-resistant subline derived by in vitro selection, resistance to DDP was found to be accompanied by cross-resistance to Cu. Accumulation of DDP in the resistant sublines ranged from 38 to 67% of that in the parental line at 1 h, and DNA adduct formation varied from 10 to 38% of that in the sensitive cells. The DDP-resistant cells had 22-56% lower basal levels of copper, and the copper levels were only 27-46% of those observed in the sensitive parental lines after a 24-h exposure to medium supplemented with copper. The initial influx rate for DDP in the three resistant cell lines ranged from 23 to 55% of that in the sensitive cells of each pair; the initial influx rate for copper in the resistant cells varied from 56 to 75% of control. Studies performed using one pair of cell lines demonstrated that for both copper and DDP the initial efflux rate was lower, whereas the terminal efflux rate was higher in the resistant cells. On Western blot analysis all three resistant lines exhibited increased expression of one or the other of the two copper export pumps (ATP7A or ATP7B) with no change in the HAH1 chaperone. We conclude that the acquisition of DDP resistance in ovarian carcinoma is accompanied by alterations in the cellular pharmacology of DDP that are paralleled by similar changes in the uptake and efflux of copper. These results are consistent with the concept that DDP enters and exits from the cell via transporters that normally mediate copper homeostasis.

Suppressive Effects of Copper Sulfate Accumulation on the Spermatogenesis of Rats.

This study investigated the effect of copper sulfate (CuSO4) in the rat spermatogenesis. Forty male rats, weighing 70-80 g, were randomly divided into four groups: control group (CG, 0 mg/kg BW), low-dose group (LG, 100 mg/kg BW), mid-dose group (MG, 200 mg/kg BW), and high-dose group (HG, 400 mg/kg BW). Rats were administered CuSO4 by gavage for 30 days. A variety of measurements were taken including the testis coefficients, the sperm count, the abnormal malformation rate, testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations in the serum. In addition, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, and malondialdehyde (MDA) concentration in the testis were determined. The results showed that in the CuSO4-treated groups, the testis coefficients, sperm count, T, LH, and FSH concentrations, and GSH-Px and SOD activities decreased, while the abnormal malformation rate and MDA concentration increased, compared with the CG. It indicates that CuSO4 exposure impairs the sperm quality and inhibits secretion of sex hormone and gonadotropin, and testis anti-oxidative function, suppressing the rat spermatogenesis.

Relative bioavailability of copper in tribasic copper chloride to copper in copper sulfate for laying hens based on egg yolk and feather copper concentrations.

This experiment was conducted to determine the relative bioavailability (RBV) of Cu in tribasic copper chloride (TBCC) to Cu in copper sulfate (monohydrate form; CuSO4·H2O) for layer diets based on egg yolk and feather Cu concentrations. A total of 252, 72-wk-old Hy-Line Brown laying hens were allotted to 1 of 7 treatments with 6 replicates consisting of 6 hens per replicate in a completely randomized design. Hens were fed corn-soybean meal-based basal diets supplemented with 0 (basal), 100, 200, or 300 mg/kg Cu from CuSO4 or TBCC for 4 wk. Results indicated that egg production, egg weight, and egg mass were not affected by dietary treatments. However, increasing inclusion levels of Cu in diets from CuSO4 decreased (P < 0.05) feed conversion ratio (FCR), whereas increasing inclusion levels of Cu in diets from TBCC did not affect FCR, indicating significant interaction (P < 0.05). Increasing inclusion levels of Cu from TBCC or CuSO4 increased (P < 0.05) Cu concentrations of egg yolk and feathers. Feather Cu concentrations were greater (P < 0.01) for hens fed diets containing CuSO4 than for hens fed diets containing TBCC. The values for the RBV of Cu in TBCC to Cu in CuSO4 based on log10 transformed egg yolk and feather Cu concentrations were 107.4% and 69.5%, respectively. These values for the RBV of Cu in TBCC did not differ from Cu in CuSO4 (100%). The RBV measured in egg yolk did not differ from the RBV measured in feather. In conclusion, the RBV of Cu in TBCC to Cu in CuSO4 can be determined using Cu concentrations of egg yolk and feathers although the values depend largely on target tissues of laying hens. For a practical application, however, the RBV value of Cu in TBCC to Cu in CuSO4 could be 88.5% when the RBV values determined using egg yolk and feather Cu concentrations were averaged.

New insights in the acute toxic/genotoxic effects of CuO nanoparticles in the in vivo Drosophila model.

Metal oxide nanoparticles are highly reactive from the biological point of view and, for this reason, it exists important reservations in regard human health impact. We used Drosophila as a promising in vivo model to diagnose the biological effects of copper oxide nanoparticles (CuO-NPs). Due to the potential role of ions release the effects of CuO-NPs were compared with those induced by copper sulfate, CuSO4. A wide battery of approaches has been used including toxicity, cell and body internalization, induction of reactive oxygen species (ROS) as well as changes in gene expression, related to both general stress and alterations in the intestinal barrier, and genotoxicity. The obtained results show that CuO-NPs have the ability to be distributed inside midgut cells and translocate to the general body compartment (internal hemolymph) interacting with hemocytes. Its exposure leads to reduced larval growth, decreased flies viability, delaying their emergency periods, especially at higher doses (2 and 10 mM). Moreover, deregulation of stress genes including antioxidant genes, and genes involved in wound healing were also observed. In this point it should be emphasized the novelty of using genes such as Duox, Upd3, PPO2, and Hml to determine injury on the intestinal barrier. On the other hand, CuO-NPs had non-genotoxic potential, in agreement with their inability to increase ROS production. In general dissolved copper produced higher toxic/genotoxic effects than those induced by CuO-NPs which would indicate that copper ions alone are more important in inducing harmful effects than copper nanoparticles itself.

Effects of dietary supplementation with copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability, and oxidation stability of vitamin E in feed.

An experiment was conducted using a total of 420, 1-d-old, Arbor Acres commercial male chicks to compare copper sulfate and tribasic copper chloride (TBCC) as sources of supplemental copper for broilers. Chicks were randomly allotted to 1 of 7 treatments for 6 replicates of 10 birds each and were fed a basal corn-soybean meal diet (11.45 mg/kg copper) supplemented with 0, 150, 300, or 450 mg/kg copper from copper sulfate or TBCC for 21 d. Chicks fed 450 mg/kg copper as copper sulfate had lower (P < 0.01) average daily feed intake and average daily gain than those consuming other diets. Feeding supplemental copper increased linearly (P < 0.0001) liver copper concentrations regardless of copper source. The slopes of regressions of log10 liver copper on different independent variables used in regressions differ (P < 0.05) between the 2 copper sources. Linear regression over nonzero dietary levels of log10 transformed liver copper concentration on added copper intake resulted in a slope ratio estimate of 109.0 +/- 3.4% (with a 95% confidence interval from 102.2 to 115.8) for bioavailability of copper from TBCC compared with 100 for that in copper sulfate. When the feeds were stored at room temperature for 10 or 21 d, the vitamin E content in the feed fortified with 300 mg/kg copper as TBCC was higher (P < 0.01) than that in the feed added with 300 mg/kg copper as CuSO4. The vitamin E contents in liver and plasma of broilers given TBCC were also higher (P < 0.01) than those of birds fed copper sulfate. The results from this study indicate that TBCC is a safer product and more available to broilers than copper sulfate, and it is chemically less active than copper sulfate in promoting the oxidation of vitamin E in feed.

Assessment of the Polychlorinated Biphenyl (PCB) Occurrence in Copper Sulfates and the Influential Role of PCB Levels on Grapes.

Copper sulfates (CuSO4) are widely used as the primary component of fungicides in the grape industry. The agricultural-grade CuSO4 that we collected from Chinese nationwide markets were found to be contaminated by polychlorinated dibenzo-p-dioxins and dibenzofurans and high levels of polychlorinated biphenyls (Σ19PCBs: 0.32~9.51 ng/g). In the following research, we studied the impact of CuSO4 application on PCB levels in grape products through a field experiment, and conducted a national survey to speculate the role that CuSO4 played on the occurrence of PCB in grapes. In the field experiment, an obvious increase of PCBs in grape leaves (from 174 to 250 pg/g fw) was observed after Bordeaux mixture (the main component of which is CuSO4) application. As to the main PCB congener in CuSO4, the most toxic CB 126 (toxic equivalency factor = 0.1) also increased in grape peels (from 1.66 to 2.93 pg/g fw) after pesticide spray. Both the correlation study and the principal component analysis indicated that environmental factors were dominant PCB contributors to grapes, and grapes from e-waste dismantling area containing the highest PCBs also proved the notion. It is worth noting that this report describes the first research examining PCBs in CuSO4 and its influence on agricultural products to date.

Copper bioavailability, blood parameters, and nutrient balance in mink.

A 3 × 3 + 1 factorial experiment was conducted based on a completely randomized design to evaluate the effects of different sources of copper on plasma metabolites, nutrient digestibility, relative copper bioavailability, and retention of some minerals in male mink. Animals in the control group were fed a basal diet, which mainly consisted of corn, fish meal, meat and bone meal, and soybean oil, with no copper supplementation. Mink in the other 9 treatments were fed the basal diet supplemented with Cu from reagent-grade copper sulfate (CuSO4), tribasic copper chloride (TBCC), or copper methionine (CuMet). Copper concentrations of the experimental diets were 50, 100, and 150 mg Cu/kg DM. Blood samples were collected via the toe clip at the end of study (d 42) to determine blood hematology and blood metabolites. A metabolism trial of 4 d was conducted during the last week of experimental feeding. There was a linear (P < 0.01) effect of dose of Cu on plasma Cu concentrations, ceruloplasmin concentration, and Cu-Zn superoxide dismutase activity. A linear response to Cu dose was noted for fat (P < 0.05) digestibility. Supplemental dose of Cu linearly increased (P < 0.05) liver Cu and decreased (P < 0.05) liver Zn level but did not alter liver Fe. The concentration of liver Cu of the mink fed with TBCC and CuMet diets was greater (P < 0.05) than that fed CuSO4. Compared with CuSO4 (100%), relative bioavailability values of TBCC were 104 and 104%, based on serum ceruloplasmin and liver copper, respectively, and relative bioavailability values of CuMet were 130 and 111%. CuMet and TBCC are more bioavailable than CuSO4. In conclusion, the relative bioavailability of CuMet obtained in this study was greater than that of CuSO4 and TBCC. Dose of Cu had an important effect on the regulating ceruloplasmin concentration, Cu-Zn superoxide dismutase activity, and the digestion of dietary fat in mink.

Copper status of ewes fed increasing amounts of copper from copper sulfate or copper proteinate.

The Cu status of mature, crossbred ewes fed two sources (CuSO4 vs. Cu proteinate) and three levels (10, 20, or 30 mg/kg) of dietary Cu was determined in a 73-d feeding trial. Ewes (n = 30) were fed a basal diet containing rice meal feed, cottonseed hulls, cottonseed meal, meat and bone meal, cracked corn, and vitamin-mineral supplements at 2.5% of BW to meet NRC requirements for protein, energy, macrominerals, and microminerals, excluding Cu. The basal diet contained 5 mg/kg Cu, 113 mg/kg Fe, .1 mg/kg Mo, and .17% S. Copper sulfate or Cu proteinate was added to the basal diet to supply 10, 20, or 30 mg/kg of dietary copper in a 2x3 factorial arrangement of treatments. Ewes were housed in 3.7- x 9.1-m pens in an open-sided barn. Blood samples were collected on d 28 and 73. Ewes were slaughtered on d 74, and liver and other tissues were collected to determine Cu concentrations. An interaction (P = .08) occurred between source and level for liver Cu. The interaction existed due to an increase in liver Cu concentrations when ewes were fed increasing dietary Cu from CuSO4 but not when fed Cu proteinate diets. There was no source x level interaction (P>.10) for the blood constituents measured. On d 73, plasma ceruloplasmin activity was greater (P<.05) in ewes fed Cu proteinate than in those fed CuSO4 (33.1 vs. 26.8 microM x min(-1) x L(-1)). Increasing the concentration of dietary Cu did not affect (P>.10) plasma ceruloplasmin. Packed cell volume (PCV), red blood cell count (RBC), white blood cell count, whole blood hemoglobin (wHb), plasma hemoglobin, and plasma Cu were similar between sources of Cu. Ewes fed 20 mg/kg Cu had lower (P<.05) PCV, RBC, and wHb than those fed 10 or 30 mg/kg Cu diets. Feeding up to 30 mg/kg Cu from these sources did not cause an observable Cu toxicity during the 73-d period.

Chemical characteristics and relative bioavailability of supplemental organic copper sources for poultry.

Five commercially available organic Cu products and reagent-grade CuSO4 x 5H2O (Cu Sulf) were evaluated by polarographic analysis and solubility in 0.1 M K2HPO4-KH2PO4 buffer (pH 5), 0.2 M HCl-KCl buffer (pH 2), or deionized water. Fractions from these solubility tests were evaluated by gel filtration chromatography for structural integrity. The organic sources were Cu lysine complex (Cu Lys), Cu amino acid chelate (Cu AA), Cu proteinate A (Cu ProA), Cu proteinate B (Cu ProB), and Cu proteinate C (Cu ProC). Separation of peaks in the chromatograms for the soluble Cu fraction from deionized water indicated that 77, 31, 69, 94, and 16% of the Cu remained chelated for the above sources, respectively. Two experiments were conducted to estimate the relative bioavailability of Cu from the organic Cu supplements for chicks when added at high dietary concentrations to practical corn-soybean meal diets. Liver Cu concentration increased (P < 0.0001) as dietary Cu increased in both experiments. When Cu Sulf was assigned a value of 100% as the standard, linear regression slope ratios of log10 liver Cu concentration regressed on added dietary Cu concentration gave estimated relative bioavailability values of 124 +/- 5.1, 122 +/- 5.3, and 111 +/- 6.0 for Cu Lys, Cu AA, and Cu ProC, respectively, in Exp. 1. The bioavailability estimates for Cu Lys and Cu AA were greater (P < 0.05) than that for Cu Sulf. Values in Exp. 2 were 111 +/- 7.6, 109 +/- 8.4, and 105 +/- 7.5 for Cu Lys, Cu ProA, and Cu ProB, respectively, and all sources were similar in value for chicks. Solubility of Cu in pH 2 buffer provided the best prediction of bioavailability (r2 = 0.924). Other indicators of chelation integrity and solubility had little value as predictors of bioavailability (r2 < or = 0.445).

Copper proteinate in weanling pig diets for enhancing growth performance and reducing fecal copper excretion compared with copper sulfate.

Two 28-d experiments were conducted to evaluate the efficacy of low dietary concentrations of Cu as Cu-proteinate compared with 250 ppm Cu as CuSO4 with growth performance, plasma Cu concentrations, and Cu balance of weanling swine as the criteria. In the production study (Exp. 1), 240 crossbred pigs that averaged 19.8 d of age and 6.31 kg BW initially were group-fed (two or three pigs per pen) the basal diets (Phase 1: d 0 to 14 and Phase 2: d 14 to 28) supplemented with 0 (control), 25, 50, 100, or 200 ppm Cu as Cu-proteinate, or 250 ppm Cu as CuSO4 (as-fed basis). The basal diets contained 16.5 ppm Cu supplied as CuSO4 before supplementation with Cu-proteinate or 250 ppm Cu as CuSO4. There were quadratic responses (P < or = 0.05) in ADFI and ADG for wk 1, Phases 1 and 2, and overall because ADFI was higher for pigs fed 25 or 50 ppm Cu as Cu-proteinate, and ADG increased with increasing Cu-proteinate up to 50 ppm Cu. The Cu-proteinate treatment groups combined had a higher (P < or = 0.05) Phase 2 and overall ADFI and ADG than the CuSO4 group. In the mineral balance study (Exp. 2), 20 crossbred barrows that averaged 35 d of age and 11.2 kg/BW initially were placed in individual metabolism pens with total urine and fecal grab sample collections on d 22 to 26. Treatments were the basal Phase 2 diet supplemented with 0, 50, or 100 ppm Cu as Cu-proteinate, or 250 ppm Cu as CuSO4 (as-fed basis). Treatments did not differ in growth performance criteria. There were linear increases (P < 0.001) in Cu absorption, retention, and excretion (milligrams per day) with increasing Cu-proteinate. Pigs fed 100 ppm Cu as Cu-proteinate absorbed and retained more Cu and excreted less Cu (mg/d, P < or = 0.003) than pigs fed 250 ppm Cu as CuSO4. Plasma Cu concentrations increased linearly (P = 0.06) with increasing Cu-proteinate. In conclusion, weanling pig growth performance was increased by 50 or 100 ppm Cu as Cu-proteinate in our production Exp. 1, but not in our balance Exp. 2, compared with 250 ppm Cu as CuSO4. However, 50 or 100 ppm Cu as Cu-proteinate increased Cu absorption and retention, and decreased Cu excretion 77 and 61%, respectively, compared with 250 ppm Cu as CuSO4.

The effect of dietary supplementation with copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability, and dietary prooxidant activity.

Three experiments were conducted to study Cu sulfate and tribasic Cu chloride (TBCC) as sources of supplemental Cu for poultry. In Experiment 1, 252 chicks were fed the basal corn-soybean meal diet (26 ppm Cu) supplemented with either 0, 150, 300, or 450 ppm Cu from Cu sulfate or TBCC for 21 d. Chicks fed 450 ppm Cu from sulfate had lower (P < 0.05) feed intake than those consuming other diets. Feeding supplemental Cu increased (P < 0.0001) liver Cu concentration linearly with increasing dietary Cu regardless of Cu source. The slopes of regression of log10 liver Cu on dietary Cu intake did not differ between sources (P > 0.10). Linear regression over nonzero dietary levels of log10 transformed liver Cu concentration (parts per million of DM) on analyzed total Cu intake (micrograms) resulted in a slope ratio estimate of 106+/-19 for bioavailability of Cu from TBCC compared to 100 for that in Cu sulfate. In Experiment 2, a 42-d floor pen study was conducted with 1,260 broiler chicks given the basal corn-soybean meal diet supplemented with 0, 200, 400, or 600 ppm Cu from either feed-grade Cu sulfate or TBCC. Body weight and feed conversion did not differ in birds fed up to 400 ppm Cu from either source. Birds given 600 ppm Cu from either source had lower feed intake, poorer growth, and feed conversion (P < 0.0001). Liver Cu increased (P < 0.0001) linearly with increasing dietary Cu. Based on log10 liver Cu concentration, Cu in TBCC was 112% available compared to 100% for the standard Cu sulfate. In Experiment 3, Cu sources were added to broiler starter diets at concentrations of 25, 100, and 300 ppm Cu and diets were stored at an elevated temperature to examine the effect of particle size on oxidation. Diets were stored at 37 C for up to 20 d and samples were removed at 4-d intervals. At 300 ppm added Cu, oxidation in TBCC diets was lower (P < 0.0001) than oxidation in diets fortified with coarse Cu sulfate, even though TBCC modal diameter for particle size was almost seven times smaller. Oxidation promotion by Cu sulfate was much greater with fine than in coarse sized particles for all three fortification levels.

Effect of sublethal concentrations of waterborne copper on lipid peroxidation and enzymatic antioxidant response in Gambusia holbrooki.

The aim of the current research was to assess the possible influence of copper sulphate contamination on the antioxidant enzymatic defenses and lipid peroxidation (LPO) in the mosquitofish (Gambusia holbrooki). Quadruplicated lots of this fish were exposed to three increasing sub-lethal concentrations of Cu (0.10; 0.17 and 0.25mgCu/L) and a control without Cu for 20 days. Previous to laboratory acclimation, 8 fish were taken to define the initial population. At the end of the trials, 12 fish/sex/treatment were sampled for the determination of levels of copper in gills, metallothioneins (MTs) content, total lipids, fatty acids profiles and antioxidant enzymatic activity, as well as lipid peroxidation. Most of the antioxidant enzymatic defenses assayed were not activated and lipid peroxidation decreased significantly in fish exposed to any concentration of copper applied. This leads us to presume the existence of a protective mechanism against peroxidation other than the enzymatic antioxidant defense, which could be related to the observed increase of copper content in the gills.

Copper in organic proteinate or inorganic sulfate form is equally bioavailable for broiler chicks fed a conventional corn-soybean meal diet.

An experiment was conducted to investigate the bioavailability of organic copper (Cu) proteinate relative to inorganic Cu sulfate for broiler chicks fed a conventional corn-soybean meal basal diet. A total of 320 day-old Arbor Acres commercial male chicks were assigned to one of five treatments in a completely randomized design involving a 2 × 2 factorial arrangement with two levels of added Cu (125 or 250 mg Cu/kg) and two Cu sources (Cu proteinate and Cu sulfate) plus a control with no added Cu for an experimental phase of 42 days. Plasma and liver tissue samples were collected at both 21 and 42 days of age, and bile samples were also obtained at 42 days of age for Cu analyses. The Cu concentrations in liver and bile increased linearly (P < 0.001) on both days 21 and 42 as dietary Cu levels increased. No significant (P > 0.17) linear regression relationships were observed between plasma Cu concentrations on days 21 and 42 or log10 liver Cu concentration on day 21 and daily analyzed Cu intake. Therefore, based on the slope ratios from multiple linear regressions of log10 liver and bile Cu concentrations with daily analyzed Cu intake on day 42, when Cu sulfate was set as 100%, the estimated relative bioavailability values of Cu proteinate were 78.8% for log10 liver Cu concentration and 79.3% for log10 bile Cu concentration, respectively. There was no significant (P > 0.08) difference in bioavailability between Cu proteinate and Cu sulfate for broilers chicks in this experiment.