Effects Of Chronic Copper Exposure Research Articles

Effects of copper on growth, metamorphosis and endocrine disruption of Bufo gargarizans larvae

Chinese toad (Bufo gargarizans) tadpoles were exposed to copper (1, 6.4, 32 and 64μgL−1 copper) from the beginning of larval period through completion of metamorphosis. We examined the effects of chronic copper exposure on mortality, growth, time to metamorphosis, tail resorption time, body size at the metamorphic climax (Gs 42) and completion of metamorphosis (Gs 46) and thyroid gland histology. In addition, type 2 and 3 iodothyronine deiodinase (Dio2 and Dio3), thyroid hormone receptors (TRα and TRβ) mRNA levels were also measured to assess disruption of TH synthesis. Our result showed that 6.4–64μgL−1 copper concentration increased the mortality and inhibited the growth of B. gargarizans tadpoles. In addition, significant reduction in size at Gs 42 and a time delay to Gs 42 were observed at 6.4–64μgL−1 copper treatments. Moreover, histological examinations have clearly revealed that 64μgL−1 copper caused follicular cell hyperplasia in thyroid gland. According to real-time PCR results, exposure to 32 and 64μgL−1 copper significantly up-regulated mRNA expression of Dio3, but down-regulated mRNA expression of TRα and TRβ mRNA level. We concluded that copper delayed amphibian metamorphosis through changing mRNA expression of Dio3, TRα and TRβ, which suggests that copper might have the endocrine-disrupting effect.

Identification of the key molecules involved in chronic copper exposure-aggravated memory impairment in transgenic mice of Alzheimer's disease using proteomic analysis.

Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by a progressive impairment of cognitive functions including spatial learning and memory. Excess copper exposure accelerates the development of AD; however, the potential mechanisms by which copper exacerbates the symptoms of AD remain unknown. In this study, we explored the effects of chronic copper exposure on cognitive function by treating 6 month-old triple AD transgenic (3xTg-AD) mice with 250 ppm copper sulfate in drinking water for 6 months, and identified several potential key molecules involved in the effects of chronic copper exposure on memory by proteomic analysis. The behavioral test showed that chronic copper exposure aggravated memory impairment of 3xTg-AD mice. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry revealed a total of 44 differentially expressed proteins (18 upregulated and 26 down-regulated) in hippocampus between the wild-type (WT) mice and non-exposed 3xTg-AD mice. A total of 40 differentially expressed proteins were revealed (20 upregulated and 20 down-regulated) in hippocampus between copper exposed and non-exposed 3xTg-AD mice. Among these differentially expressed proteins, complexin-1 and complexin-2, two memory associated proteins, were significantly decreased in hippocampus of 3xTg-AD mice compared with the WT mice. Furthermore, the expression of these two proteins was further down-regulated in 3xTg-AD mice when exposed to copper. The abnormal expression of complexin-1 and complexin-2 identified by proteomic analysis was verified by western blot analysis. Taken together, our data showed that chronic copper exposure accelerated memory impairment and altered the expression of proteins in hippocampus in 3xTg-AD mice. The functional analysis on the differentially expressed proteins suggested that complexin-1 and complexin-2 may be the key molecules involved in chronic copper exposure-aggravated memory impairment in AD.

CHRONIC COPPER EXPOSURE CAUSES SPATIAL MEMORY IMPAIRMENT, SELECTIVE LOSS OF HIPPOCAMPAL SYNAPTIC PROTEINS, AND ACTIVATION OF PKR/EIF2 α PATHWAY IN MICE

Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. The effects of chronic copper exposure on spatial memory and the possible mechanisms remain unclear. Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the concentrations of copper in the serum and hippocampus of mice. Morris water maze test was performed to evaluate spatial learning and memory. Immunohistochemical staining and Western-blot analysis were performed to measure the changes of oxidative stress markers, synaptic proteins. TUNEL staining was performed to measure the change of apoptosis. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was up-regulated and hippocampal neuronal apopotosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway. This work was supported by NSFC (81102154), Medical Scientific Research Foundation of Guangdong Province (B2012322, A2013598) and Shenzhen Scheme of Science and Technology (Medicine and Health) (201202086, 201302148).

Changes to Cuticle Surface Ultrastructure and Some Biological Functions in the Nematode Caenorhabditis Elegans Exposed to Excessive Copper

Copper is an essential metal, but its toxic effects are pronounced when organisms are exposed to it in excessive amounts. However, information about the effects of chronic copper exposure on the cuticle ultrastructure of organisms is insufficient. Studies of the model organism, Caenorhabditis elegans, could further our understanding of the effect of chronic excessive copper exposure on human health. In this study, the cuticle surface ultrastructure of C. elegans was observed using scanning electron microscopy after excessive copper exposure. In addition to this, some biological functions, such as chemotaxis, reproduction, and development, were also analyzed. After chronic excessive copper exposure, the worms' body surface from vulva to tail was extensively wrinkled and folded along with the annulus. The worm's vulva size was significantly decreased, and the middle ridge of the alae was disrupted. Furthermore, some of the biological functions of nematodes were also affected: the chemotaxis index was partially changed, bags-of-worms were induced, development was delayed, and egg-laying number was decreased by copper treatment. The results of the present study shed new light on the effects of copper on C. elegans cuticle as well as some biological functions.

Effects of chronic copper exposure on development and survival in the southern leopard frog (Lithobates [Rana] sphenocephalus)

Exposure to environmental contaminants contributes to the global decline of amphibian populations. The impacts of organic contaminants on amphibians are well documented. However, substantially less is known concerning the potential effects of metals on amphibian populations. Copper (Cu) is an essential element, but it can be toxic at concentrations only slightly higher than the normal physiological range. The present study examines the effects of chronic Cu exposure on embryos and larvae of southern leopard frogs, Lithobates (Rana) sphenocephalus. Groups of eggs from multiple clutches were collected from two wetlands and exposed to a range of Cu concentrations (0-150 µg/L) until they reached the free-swimming stage, and then individual larvae were reared to metamorphosis. Higher Cu concentrations significantly reduced embryo survival to the free-swimming stage but did not further reduce survival to metamorphosis. Larval period was affected by Cu treatment, but the clutch from which larvae originated (i.e., parentage) explained a higher proportion of the variation. Embryo survival to hatching varied significantly among clutches, ranging from 42.9 to 79.2%. Measurable levels of Cu were found in larvae with body burdens up to 595 µg Cu/g dry mass in the 100 µg/L treatment, and larval Cu body burdens were higher than in metamorphs. The present study also demonstrated that higher initial egg density ameliorated embryo mortality at higher Cu levels and should be accounted for in future studies.

The effects of chronic copper exposure on the amyloid protein metabolisim associated genes’ expression in chronic cerebral hypoperfused rats

The pathogens of Alzheimer's disease (AD) are still unclear, while accumulating evidences have indicated that both genetic and environmental factors are involved in the pathogenesis of AD. Recent studies suggest that AD is primarily a vascular disorder and copper (Cu) may play an important role in AD pathology. However, the consequences of chronic Cu exposure at the presence of other AD risk factors remain to be clarified. To investigate the effects of chronic Cu intake on cerebral hypoperfusion-induced AD pathology, Sprague-Dawley rats suffered bilateral common carotid artery occlusion (2VO) were administrated with 250ppm copper-containing water or not. Morris water maze test showed that Cu exposure for 3 months exacerbated cognitive impairment induced by 2VO. Elevated amyloid precursor protein (APP) and beta-site APP-cleaving enzyme 1 (BACE1) expression in mRNA and protein levels were also observed in brain of Cu-exposed rats suffered 2VO. In contrast, these Cu-exacerbated changes were ameliorated after Cu was withdrawn from drinking water. In summary, our findings demonstrate that chronic Cu exposure might exacerbate AD pathology in 2VO rats.

Effects of chronic copper exposure on fluvial systems: Linking structural and physiological changes of fluvial biofilms with the in-stream copper retention

Long-term metal exposure is known to be responsible for a large variety of structural and functional changes in periphyton communities which allow these communities to adapt to metal-polluted conditions. This study aimed to link the changes that chronic copper (Cu) exposure causes on the structure and physiology of fluvial biofilms with the efficiency of the river systems in retaining phosphate and Cu. The effects of a chronic Cu exposure on the structure, physiology and induction of Cu tolerance of the community were evaluated by comparing this community with a non-exposed one. Results showed that periphyton chronically exposed to Cu had lower algal biomass, higher proportion of green algae, lower proportion of brown algae, and higher EPS content per unit of biomass than the un-exposed community. In addition, the chronically-exposed community showed a Cu content (both total and intracellular Cu content) ten times higher than the un-exposed community. While in-stream phosphate retention was not markedly influenced by chronic Cu exposure; Cu retention was clearly reduced, as was shown by a reduction in Cu retention efficiency (Cu– S w) and demand (Cu–Vf). The chronically-exposed periphyton, in spite of having high intracellular Cu concentration, showed similar photosynthetic efficiency than the un-exposed community and showed a higher Cu tolerance. It indicated that this community was acclimatized to Cu exposure and that this acclimatization was probably linked to the ability to detoxify and immobilize metals. These observations suggest that the fate of Cu in fluvial ecosystems will be influenced by the exposure history of the system. The results from this study indicate that metals will travel longer distances in metal-polluted streams compared to pristine systems having effects on water quality farther downstream.

Adverse effects of chronic copper exposure in larval northern leopard frogs (Rana pipiens)

Northern leopard frogs (Rana pipiens) were exposed to environmentally relevant concentrations of copper in water (control and 5, 25, and 100 microg/L, as CuSO4) in a static renewal system for 154 d from posthatch stage to metamorphosis. After 10 d of copper exposure (from Gosner stages 19-25), survival of tadpoles was not significantly different between treatments (averages range from 96.7 to 99.3%), but a significantly higher incidence of deformities (p < 0.05) was observed in the 100-microg/L treatment. Tadpole growth rate was decreased in the 25- and 100-microg/L treatments relative to controls. Tadpole survival, swimming performance, percent metamorphosis, time to metamorphosis, and survival during and time required for tail resorption were also negatively affected in the 100-microg/L treatment. Copper concentrations in tadpole tissues ranged from 46.5 to 769.5 mg/ kg dry mass and were positively related to copper concentrations in the water. In conclusion, copper was shown to affect development, growth, survival, and behavior in larval northern leopard frogs at ecologically relevant concentrations. The existing U.S. Environmental Protection Agency freshwater-quality chronic criterion for copper appears to be protective for larval Rana pipiens.

Early Histological and Functional Effects of Chronic Copper Exposure in Rat Liver

Cu is an essential trace element capable of producing toxic effects in animals and man when ingested acutely or chronically in excess. Although chronic Cu exposure is increasingly recognized as a public health issue, its early effects remain largely unknown. We approached the significance of a moderate chronic Cu load in young rats to correlate early hepatic histopathological changes with functional alterations of liver cells. For this purpose, supplementation with 1,200 ppm of Cu in rat food for 16 weeks was chosen. In these conditions, Cu load elicited a significant decrease in growth curves. There were mild light microscopy alterations in Cu-treated rats, although increasing intracellular Cu storage was correlated with longer Cu exposure both by histological and biochemical measurements. Ultrastructural alterations included lysosomal inclusions as well as mitochondrial and nuclear changes. Liver perfusion studies revealed higher rates of basal O(2) consumption and colloidal carbon-induced O(2) uptake in Cu-treated rats, with enhanced carbon-induced O(2)/carbon uptake ratios and NF-kappaB DNA binding activity. These changes were time-dependent and returned to control values after 12 or 16 weeks. It is concluded that subchronic Cu loading in young rats induces early hepatic morphological changes, with enhancement in Küpffer cell-dependent respiratory burst activity and NF-kappaB DNA binding, cellular responses that may prevent or alleviate the hepatotoxicity of the metal.

Effects of chronic copper exposure during early life in rhesus monkeys

Whether infants regulate copper absorption and the potential effects of excess copper in early life remain poorly defined. The objective of the study was to assess copper retention, liver copper content, and liver function in infant rhesus monkeys fed infant formula containing 6.6 mg Cu/L. From birth to 5 mo of age, infant rhesus monkeys were fed formula that was supplemented with copper (0.6 mg Cu/L; n = 5) or not supplemented (n = 4). In all animals, weight and crown-rump length (by anthropometry), hemoglobin, hematocrit, plasma ceruloplasmin activity, and zinc and copper concentrations were measured monthly (birth to 6 mo) and at 8 and 12 mo. When the animals were 1, 5, and 8 mo old, liver copper and metallothionein concentrations, liver histology (by light and electron microscopy), and the number of Kupffer cells were assessed, and 67Cu retention was measured. Liver function was assessed by measurement of plasma alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, and alkaline phosphatase activities and protein, albumin, bilirubin, and blood urea nitrogen concentrations. 67Cu retention was 19.2% and 10.9% after 1 and 5 mo of copper treatment, respectively, compared with approximately 75% in controls at age 2 mo. At age 8 mo, 67Cu retention was 22.9% in copper-treated animals and 31.5% in controls. Liver histology remained normal by light microscopy, with mild ultrastructural signs of cell damage at 5 mo. Liver copper concentration was 4711, 1139, and 498 microg/g dry tissue at 1, 5, and 8 mo, respectively, in copper-treated animals and 250 microg/g at 2 mo in controls. Measurements could not be completed in all animals. No clinical evidence of copper toxicity was observed. Copper absorption was down-regulated; increases in liver copper content at ages 1 and 5 mo did not result in histologic damage. Ultrastructural changes at age 5 mo could signal early cellular damage.

Effects of chronic copper exposure on the nutritional composition of Hyalella azteca

The toxicity of dietborne metals currently is a topic of high interest in aquatic toxicology. We hypothesized that, in addition to causing direct physiological effects when ingested by organisms, dietborne metals might also cause indirect foodweb effects. One such indirect effect might be alteration of the nutritional content of prey, leading to decreased growth and (or) survival of consumers. Because dietborne and waterborne metal exposures usually occur concurrently in aquatic ecosystems, we exposed two generations of a representative grazer species—the freshwater amphipod Hyalella azteca—to dietborne copper (Cu) concurrent with waterborne Cu in the laboratory for a total of 27 days (first generation) and 45–57 days (second generation). The aqueous Cu concentrations were 0.0099 (control), 0.054 and 0.11 μM (0.6, 3.4 and 7.0 μg Cu per l, respectively) at 24 °C, pH 7.5–8.3, alkalinity of ∼3 mEq/l and hardness of ∼4 mEq/l; whereas the dietborne Cu exposures were via biofilm growing on cotton gauze in the same exposure aquaria. In a preliminary test, we demonstrated that the highest Cu concentrations were at the threshold for adversely affecting survival and reproduction. In the two-generation test, first- and second-generation amphipods exposed to the intermediate and high aqueous Cu concentrations accumulated significantly higher amounts of Cu than control amphipods. However, none of the traditional nutritional composition categories (protein, total lipid, ash and ‘other’) differed among the treatments. The only significant difference in nutritional composition was an elevated concentration of linolenic acid (18:3 n−3) in the second-generation amphipods exposed to 0.054 μM Cu.

EFFECT OF COPPER ON ALGAL COMMUNITIES FROM OLIGOTROPHIC CALCAREOUS STREAMS1

Two sets of experiments were done to quantify the effects of chronic copper exposure on natural peri‐ phyton in a nonpolluted calcareous river. The results of short‐term (up to 6 h exposure) experiments corroborated the significance of pH on copper toxicity. Copper toxicity increased when pH was reduced from 8.6 to 7.7, and this was related to the effect of pH on copper speciation (free copper concentration increased from 0.2% to 2.3% of total copper). Longer term experiments demonstrated that periphyton communities exposed to copper under pH variation (8.2–8.6) were already affected at 10 μg·L−1 (20–80 ng·L−1 Cu2+) after 12 days of exposure. Copper exposure caused stronger effects on structural (algal biomass and community structure) than on functional (photosynthetic efficiency) parameters of peri‐ phyton. Changes in community composition included the enhancement of some taxa (Gomphonema gracile), the inhibition of others (Fragilaria capucina and Phormidium sp.), and the appearance of filament malformations (Mougeotia sp.). The results of our study demonstrated that several weeks of exposure to copper (10–20 μg·L−1) were sufficient to cause chronic changes in the periphyton of oligotrophic calcareous rivers. This degree of copper pollution can be commonly found in the Mediterranean region as a result of agricultural practices and farming activities.

Effects of chronic copper exposure on the green mussel Perna viridis

The effects of chronic copper exposure on growth and physiological responses of the green mussel Perna viridis were investigated by exposing the mussels to 50 μg l−1 Cu for 3 mo at 17 and 25 °C. These temperatures represent, respectively, the winter and summer seawater temperatures in Hong Kong. Differences in the level of response between mussels exposed for 3 mo to 50 μg Cu l−1 generally increased with duration of exposure. The tissue concentration of copper had increased by 280 and 450% after 3 mo exposure at 17 and 25 °C, and growth performances were reduced, with the 25 °C sets suffering from larger negative impact of copper in most responses. The inhibitory effects of copper on production of the various body components generally followed the order linear shell growth (greatest) > tissue production > byssus production > shell production. There were also decreases in the condition index (43 and 35% reductions at 17 and 25 °C), clearance rates (10.3 and 18.5%), faeces production (11 and 16.3%), assimilation efficiency (6.8 and 9.2%) and oxygen consumption rate (12.8 and 24.8%). In contrast, the organic content of the faeces (9.2 and 13.2% increases at 17 and 25 °C) and rate of ammonia excretion (21 and 28.6%), increased upon chronic copper exposure. Many of the responses (e.g. changes in tissue copper content, body dry wt, shell organic content, clearance rate and oxygen consumption rate) exhibited fluctuating levels of impact during prolonged copper exposure, while others (e.g. faecal production rate, assimilation efficiency, tissue organic content) demonstrated steady decreasing trends with increasing exposure time.

Physiological effects of chronic copper exposure to rainbow trout (Oncorhynchus mykiss) in hard and soft water: Evaluation of chronic indicators

AbstractEffects of chronic copper exposure on a suite of indicators were examined: acute toxicity, acclimation, growth, sprint performance, whole‐body electrolytes, tissue residues, and gill copper binding characteristics. Juvenile rainbow trout were exposed for 30 d to waterborne copper in hard water (hardness = 120 mg/L as CaCO3, pH = 8.0, Cu = 20 and 60 μg/L) and soft water (hardness = 20 mg/L as CaCO3, pH = 7.2, Cu = 1 and 2 μg/L). Significant acclimation to the metal occurred only in fish exposed to 60 μg/L, as seen by an approx. twofold increase in 96‐h LC50 (153 vs 91 μg Cu/L). Chronic copper exposure had little or no effect on survival, growth, or swimming performance in either water hardness, nor was there any initial whole‐body electrolyte loss (Na+ and Cl−). The present data suggest that the availability of food (3% wet body weight/day, distributed as three 1% meals) prevented growth inhibition and initial ion losses that usually result from Cu exposure. Elevated metal burdens in the gills and livers of exposed fish were measures of chronic copper exposure but not of effect. Initial gill binding experiments revealed the necessity of using radiolabeled Cu (64Cu) to detect newly accumulated Cu against gill background levels. Using this method, we verified the presence of saturable Cu‐binding sites in the gills of juvenile rainbow trout and were able to make estimates of copper‐binding affinity (log Kgill=Cu) and capacity (Bmax). Furthermore, we showed that both chronic exposure to Cu and to low water calcium had important effects on the Cu‐binding characteristics of the gills.

Effects of chronic copper exposure on the macrophage chemiluminescent response and gill histology in goldfish (Carassius auratusL.)

The effects of chronic copper exposure on the macrophage chemiluminescent response and gill histology in goldfish (Carassius auratusL.) were determined. Fish were exposed to sublethal concentrations (30, 65, 100, and 175ppb) of copper sulphate for 96h. Pronephric (head kidney) cells were collected and were comprised on lymphocytes, thrombocytes, monocytes/macrophages neutrophils and eosinophils/basophils (cell types grouped due to difficulty in identification). There were no significant differences in population profiles of cells used for chemiluminescence studies after exposure to 100ppb copper. Density gradient-separated cells were stimulated with zymosan, the luminol-enhanced chemiluminescence (CL) response was measured and the results were compared to non-copper exposed controls. There was a significant correlation between copper concentration and reduction in the time at which the peak CL response [time to peak (TTP) CL] occurred. In addition, there was a significant parabolic dose response curve for peak CL response, which suggests increased production of reactive oxygen intermediates by phagocytes from fish exposed to concentrations up to 100ppb, followed by inhibition at 175ppb. These results indicate that copper affects phagocyte function in a complex manner, stimulating or inhibiting production of reactive oxygen intermediates depending on copper concentration. Exposure to concentrations of copper also resulted in varying degrees of gill hyperplasia. The relative median severity score (a score assigned to each gill based upon the amount of hyperplasia seen in a histological section) correlated significantly with the copper concentration. In addition, there was a very high correlation between the degree of hyperplasia and the TTP CL response. Levels of copper in the head kidney of goldfish exposed to 100ppb were similar to controls as measured by atomic absorption spectroscopy. Thus, changes in the CL response of head kidney phagocytes of fish exposed to copper may be caused indirectly, via effects on other immunoregulatory cells or other physiological mechanisms. We conclude that addition of heavy metals such as copper to water can modify the CL responses of phagocytes and alter the histopathology of the gills inC. auratus.

Filtration and phototactic behavior as indices of chronic copper stress inDaphnia magna straus

Filtration rate and negative phototactic behavior ofDaphnia magna were evaluated as potential predictors of the chronic no-effect copper concentration. The effects of copper on filtration and phototactic behavior were compared to the effects of chronic copper exposure on survivorship, number of juvenile molts, age at reproductive maturity, and neonate body length. Animals exposed to copper concentrations ⩾20μg/L exhibited reductions in filtration rate, negative phototaxis, body length of neonates and survival time. Animals exposed to 10μg/L exhibited a reduction in filtration rate, negative phototaxis and body length of neonates, but not in survival time. Number of juvenile molts, age at reproductive maturity, and mean brood size each responded erratically to copper exposure and are poor indices of copper stress. Since phototactic behavior, filtration rate, and neonate body length were all reduced at copper concentrations which did not reduce longevity or reproduction, bioassays in which only the latter are examined may underestimate the toxicity of copper and other chemicals. This may occur if bioassays are conducted for the frequently chosen 21-day interval.