Effects Of Cu Exposure Research Articles

Metabolic changes induced by heavy metal copper exposure in human ovarian granulosa cells

Copper (Cu) is a common heavy metal and a hazardous environmental pollutant. Emerging epidemiological evidence suggests that Cu exposure is associated with female infertility, especially ovarian dysfunction. However, the mechanisms underlying ovarian toxicity remain poorly understood. Granulosa cells play crucial roles in follicle development and are the main target cells of environmental pollutants for ovarian toxicity. In this study, we investigated the effects of Cu exposure on human granulosa (KGN) cells by using cell biology and metabolomics methods, and explored the molecular mechanisms of Cu-induced cytotoxicity. We found that Cu reduced cell viability in a dose- and time-dependent manner. Then, metabolomic analyses led to the identification of 279, 368 and 466 differentially expressed metabolites (DEMs) in KGN cells exposed to 10, 60 and 240 μM Cu, respectively. Pathway enrichment analysis revealed that high Cu led to disturbances of glutathione metabolism, nucleotide metabolism, glycerophospholipid and ether lipid metabolism. Using cell biological assays, we found that exposure to high Cu significantly decreased the GSH/GSSG ratio and altered the activities of the antioxidant enzymes SOD and CAT. Exposure to high Cu significantly increased the level of mitochondrial ROS. These findings further supported the results revealed by metabolomic analysis and provided clues for elucidating the mechanism by which Cu interferes with the development of ovarian follicles.

Mitochondrial DNA release mediated by TFAM deficiency promotes copper-induced mitochondrial innate immune response via cGAS-STING signalling in chicken hepatocytes

Copper (Cu) is pollution metal that is a global concern due to its toxic effects. A recent study found that the release of mitochondrial DNA (mtDNA) into the cytoplasm can activate the innate immune response, but the exact mechanisms underlying the effect of Cu exposure remains unknown. In this study, we identified that the reduction in transcription Factor A (TFAM) led to mtDNA leakage into the cytoplasm under Cu exposure in hepatocytes, accompanied by the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway-mediated innate immunity (increased expression of cGAS, STING, TANK-binding kinase-1 (TBK1), and interferon regulatory factor-3 (IRF3)) genes and proteins, and enhanced phosphorylation levels of TBK1 and IRF3). Subsequently, silencing TFAM (siTFAM) significantly aggravated mtDNA release and the innate immune response under Cu treatment. Mitochondrial DNA depletion alleviated Cu-induced innate immunity in hepatocytes, while mtDNA transfection further enhanced the innate immune response. Notably, the inhibition of STING effectively alleviated the phosphorylation levels of the TBK1 and IRF3 proteins induced by Cu, while the upregulation of STING aggravated the Cu-induced innate immunity. Furthermore, EtBr and H-151(a STING inhibitor) treatment dramatically reversed the effect of TFAM depletion on the sharpened innate immune response induced by Cu via the cGAS-STING pathway. In general, these findings demonstrated the TFAM deficiency promotes innate immunity by activating the mtDNA-cGAS-STING signalling pathway under Cu exposure in hepatocytes, providing new insight into Cu toxicology.

Copper induced intestinal inflammation response through oxidative stress induced endoplasmic reticulum stress in Takifugu fasciatus

Copper (Cu) pollution in aquaculture water has seriously threatened the healthy and sustainable development of the aquaculture industry. Recently, many researchers have studied the toxic effects of Cu exposure on fish. However, the relationship between endoplasmic reticulum stress (ERS) and the inflammatory response, as well as its possible mechanisms, remain unclear. Particularly, information related to fish intestines must be expanded. Our study initially investigated the mechanisms underlying intestinal toxicity and inflammation resulting from Cu-induced ERS in vivo and in vitro in Takifugu fasciatus. In vivo study, T. fasciatus were treated with different concentrations (control, 20, and 100 µg/L) of Cu exposure for 28 days, causing intestinal oxidative stress, ERS, inflammatory responses, and histopathological and ultrastructural damage. Transcriptomic data further showed that Cu exposure caused ERS, as well as inflammatory responses, in the intestinal tracts of T. fasciatus. In vitro experiments on the intestinal cells of T. fasciatus showed that Cu exposure treatment (7.5 µg/mL) for 24 h induced ERS and increased mitochondrial numbers and inflammatory responses. In contrast, the addition of 4-phenylbutyric acid (4-PBA) alleviated ERS and inflammatory response in the Cu-exposed group. Furthermore, the reactive oxygen species (ROS) inhibitor, N-Acetyl-l-cysteine (NAC), effectively alleviated Cu-induced ERS. In conclusion, our in vivo and in vitro studies have confirmed that oxidative stress triggers the ERS pathway, which is involved in the intestinal inflammatory response. Our study provides new insights into the relationship among Cu-induced oxidative stress, ERS, and inflammatory responses in fish, as well as for the healthy culture of fish in aqueous environments.

Copper exposure effects on antibiotic degradation in swine manure vary between mesophilic and thermophilic conditions

Antibiotic and heavy metal commonly coexist in manure. This study investigated the effect of Cu exposure on antibiotic dissipation in swine manure under two typical temperature (mesophilic and thermophilic) conditions in composting, focusing on biodegradation behaviors. The results showed that Cu promoted the dissipation of norfloxacin and sulfamethazine (SMZ) in solid swine manure under mesophilic conditions at initial concentrations ranging from 407.8 to 1353.0 mg·kg−1 but insignificantly influenced or even inhibited their dissipation under thermophilic conditions. A liquid manure suspension culture experiment was designed to elucidate the response of SMZ biodegradation to Cu. In this manure suspension, biodegradation was the major mechanism for SMZ removal, but SMZ biodegradation was decreased from 23.2 % to 5.5 % when the Cu concentration increased from 0 to 10 mg L−1. Mesophilic and heat-resistant SMZ-degrading bacterial inoculants were subsequently prepared using 21 SMZ-degrading bacteria that were isolated and identified from manure suspension cultures. Inoculating both mesophilic and heat-resistant SMZ-degrading bacterial inoculants enhanced SMZ degradation in sterilized manure suspensions without Cu addition, however only mesophilic SMZ-degrading inoculum improved SMZ degradation after Cu addition. In the presence of Cu, the heat-resistant SMZ-degrading inoculum failed to enhance SMZ removal in manure suspensions. Our findings can help to answer why Cu has varied effects on antibiotic degradation during manure composting.

Exacerbation of copper pollution toxicity from ocean acidification: A comparative analysis of two bivalve species with distinct sensitivities

In estuarine ecosystems, bivalves experience large pH fluctuations caused by the anthropogenic elevation of atmospheric CO2 and Cu pollution. This study investigates whether Cu toxicity increases indiscriminately in two bivalve species from different estuarine habitats as a result of elevated Cu bioaccumulation in acidified seawater. This was carried out by evaluating the effects of Cu exposure on two bivalve species (clams and scallops) for 28 d, at a series of gradient pH levels (pH 8.1, 7.8, and 7.6). The results demonstrated an increase in the Cu content in the soft tissues of clams and scallops in acidified seawater. Cu toxicity increased under acidified seawater by affecting the molecular pathways, physiological function, biochemical responses, and health status of clams and scallops. An iTRAQ-based quantitative proteomic analysis showed increased protein turnover, disturbed cytoskeleton and signal transduction pathways, apoptosis, and suppressed energy metabolism pathways in the clams and scallops under joint exposure to ocean acidification and Cu. The integrated biomarker response results suggested that scallops were more sensitive to Cu toxicity and/or ocean acidification than clams. The proteomic results suggested that the increased energy metabolism and suppressed protein turnover rates may contribute to a higher resistivity to ocean acidification in clams than scallops. Overall, this study provides molecular insights into the distinct sensitivities between two bivalve species from different habitats under exposure to ocean acidification and/or Cu. The findings emphasize the aggravating impact of ocean acidification on Cu toxicity in clams and scallops. The results show that ocean acidification and copper pollution may reduce the long-term viability of clams and scallops, and lead to the degradation of estuarine ecosystems.

Histological alterations, oxidative stress, and inflammatory response in the liver of swamp eel (Monopterus albus) acutely exposed to copper.

Copper (Cu) is widely used as an essential trace element in diets as well as a therapeutic chemical. However, excessive Cu has deleterious effects on organisms, including teleosts. Although numerous toxic effects of Cu have been reported, the effects of Cu exposure on the swamp eel (Monopterus albus) as well as the underlying mechanisms have not yet been elucidated. In this study, swamp eels were acutely exposed to 100, 200, and 400μg/L of Cu for 96h to evaluate liver histopathology, oxidative stress, and inflammation. Dissolution of hepatocyte membrane, vacuolar degeneration, and inflammatory cell infiltration were detected in the livers of the Cu-treated swamp eels, especially in the 400μg Cu/L group. Cu-induced hepatic dysfunction was further verified by the elevated activities of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) and transcript levels of GOT and GPT genes. In addition, Cu exposure decreased the activities of total superoxide dismutase T-SOD and catalase (CAT) and the contents of glutathione (GSH) and total antioxidant capacity (T-AOC) and increased the levels of malondialdehyde (MDA). Cu exposure also significantly decreased the transcript levels of glutathione synthetase (GSS) and increased the transcript levels of SOD1, SOD2, CAT, and heme oxygenase-1 (HO-1) genes. Furthermore, pro-inflammatory genes such as interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-8 were significantly upregulated. These results indicate that Cu induces oxidative stress and inflammatory response and causes pathological changes in the liver of the swamp eel.

Copper-induced sublethal effects in Bufo gargarizans tadpoles: growth, intestinal histology and microbial alternations.

Copper (Cu) is one of the environmental contaminations which can pose significant risks for organisms. The current study explores the effects of Cu exposure on the growth, intestinal histology and microbial ecology in Bufo gargarizans. The results revealed that 0.5-1 μM Cu exposure induced growth retardation (including reduction of total body length and wet weight) and intestinal histological injury (including disordered enterocyte, changes in the villi and vacuoles) of tadpoles. Also, high-throughput sequencing analysis showed that Cu exposure caused changes in richness, diversity and structure of intestinal microbiota. Moreover, the composition of intestinal microbiota was altered in tadpoles exposed to different concentrations of Cu. At the phylum level, we observed the abundance of proteobacteria was increased, while the abundance of fusobacteria was decreased in the intestinal microbiota of tadpoles exposed to 1 μM Cu. At the genus level, a reduced abundance of kluyvera and aeromonas was observed in the intestinal microbiota of tadpoles under the exposure of 0-0.5 μM Cu. Finally, functional predictions revealed that tadpoles exposed to copper may be at a higher risk of developing metabolic disorders or diseases. Above all, our results will develop a comprehensive view of the Cu exposure in amphibians and will yield a new consideration for sublethal effects of Cu on aquatic organisms.

Toxic effects of dietary copper and EGCG on bioaccumulation, antioxidant enzyme and immune response of Korean bullhead, Pseudobagrus fulvidraco

There are few reports of dietary Cu (copper) toxicity to Korean bullhead, Pseudobagrus fulvidraco, and little is known about recovery from dietary Cu exposure. In this study, P. fulvidraco (mean length 16.9 ± 1.38 cm, and mean weight 53.2 ± 1.22 g) were exposed for 4 weeks to dietary Cu concentration of 0 (control), 700, 900, and 1100 mg Cu kg−1 dry feed to establish maximum tolerable levels of dietary Cu. All fish were then fed the dietary EGCG (Epigallocatechin gallate) concentration of 100 and 500 mg EGCG kg−1 dry feed for a further 2 weeks to assess recovery. We were measured bioaccumulation (in the intestine, liver, and gill tissue), antioxidant enzymes (SOD and CAT) and immune responses (lysozyme and phagocytosis). The Cu exposure induced a significant accumulation in the intestine, liver, and gill tissues and the highest accumulation was observed in intestinal tissues (17–34 fold), but dietary EGCG exposure decreased (about 0.8-fold) Cu concentration in each tissue (ANOVA, P < 0.05). In antioxidant enzymes, SOD and CAT significantly increased by approximately 1.6-fold by dietary Cu exposure in the liver and gill tissue, respectively, but dietary EGCG exposure decreased SOD and CAT by about 1.1-fold, respectively (ANOVA, P < 0.05). For immune responses, lysozyme and phagocytosis in the blood significantly were decreased by approximately 1.5-fold, respectively, by dietary Cu exposure, but dietary EGCG exposure increased lysozyme and phagocytosis by about 1.1-fold, respectively (ANOVA, P < 0.05). During recovery period, bioaccumulation, antioxidant enzymes (SOD and CAT activity), and immune response (lysozyme and phagocytosis activity) tended to alleviate the significant changes by Cu exposure, and the tendency to return normal state was observed in high level of EGCG. The result of this study indicate that Cu exposure to P. fulvidraco affects bioaccumulation, antioxidant enzymes, and immune responses, and high level of EGCG were effective to alleviate the toxic effects of Cu exposure.

Cu-induced mitochondrial dysfunction is mediated by abnormal mitochondrial fission through oxidative stress in primary chicken embryo hepatocytes

BackgroundExcess copper (Cu) is an oxidative stress factor which associates with a variety of diseases. The aim of this study was to evaluate the effect of Cu in primary chicken embryo hepatocytes (CEHs). MethodsCEHs were isolated from 13 days old chicken embryos and followed by different concentration Cu (0, 10, 100, 200 μM) and/or ALC treatment (0.3 mg/mL) for 12 or 24 h. The effects of Cu exposure in CEHs were determined by detecting reactive oxygen species (ROS), malondialdehyde (MDA), mitochondrial membrane potential (MMP), and ATP levels. The expression of mitochondrial dynamics-related genes and proteins were also detected. ResultsResults showed that Cu treatment (100 or 200 μM) significantly decreased CEHs viability, MMP and ATP levels, increased ROS and MDA levels in 12 or 24 h. The up-regulated mitochondrial fission genes and protein in 100 and 200 μM Cu groups suggested Cu promoted mitochondrial division but not fusion. However, the co-treatment of ALC and Cu alleviated those changes compared with the 100 or 200 μM Cu groups. ConclusionIn conclusion, we speculated that Cu increased the oxidative stress and induced mitochondria dysfunction via disturbing mitochondrial dynamic balance in CEHs, and this process was not completely reversible.

Act now before its too late: Copper exposure drives chemo-ecology of predator-prey dynamics of freshwater common spiny loach, Lepidocephalichthys thermalis (Valenciennes, 1846)

Due to the extensive use of copper (Cu) in various commercial products, its existence in aquatic bodies (freshwater and marine) is not unusual. Cu is well known for its effect on the olfactory physiology of fish. However, there are limited studies on the effect of Cu on important ecological functions in fish (predator-prey dynamics) that are primarily influenced by olfaction. In a series of experiments, we studied the effect of Cu exposure on the chemoreceptive behavior of the prey fish, Lepidocephalichthys thermalis. Prey fishes were exposed to an environmentally relevant concentration (5 μg/L) of Cu for 3 h and the anti-predator responses against native (Channa gachua) and alien predatory fish (tilapia) were quantified using an ethological assay. Cu exposed prey fishes did not recognize the native predator and had a lower survival rate than control (unexposed) fishes in predation trials. Cu exposed prey fishes have failed to learn associatively to detect a non-native predator resulting in higher mortality in prey population in direct encounters with tilapia. However, such a lack of predator recognition was found to be short-term and the treated prey fishes recovered anti-predator responses within 72 h. In addition, Cu inactivated the alarm cue which acts as a signal for the presence of predators and ensures associative learning and therefore it was considered to be an ‘info-disruptor’ in the present study. These outcomes together demonstrate that even at low concentration, Cu influences ecological decisions and survival against predators. Owing to the ubiquitous occurrence of Cu in water bodies, the present investigation will contribute to the knowledge of how environmental stressors alter the crucial ecological decisions of prey individuals in aquatic ecosystems. In addition, we suggest that freshwater reservoirs containing high levels of Cu could be unsuitable for the long-term survival of prey fishes and freshwater biodiversity.

Long-term effects of copper exposure to agricultural soil function and microbial community structure at a controlled and experimental field site

The long-term effect of heavy metals on soil microbial communities and their function is relatively unknown and little work has been done in field settings. To address this gap, we revisited a field-based experiment, 12 years after the application of copper (Cu) to agricultural soils, with treatment concentrations ranging from 0 to 3310 mg Cu kg−1 soil. We measured the long-term effects of Cu exposure to soils using multiple functionality assessments and environmental DNA-based community analyses. The assessment results revealed that soils that received moderate to high Cu doses had still not recovered functionality 12-years post exposure. However, plots that received doses of 200 mg kg−1 Cu or less appeared to have a functionality index not dissimilar to control plots. Environmental DNA analyses of the microbial communities revealed a high level of beta diversity in low Cu treatment plots, whereas communities within high Cu treatment plots had similar community structures to one another (low beta diversity), indicating that specific Cu-tolerant or dormant taxa are selected for in high-Cu environments. Interestingly, high Cu plots had higher within-sample taxa counts (alpha diversity) compared with controls and low Cu plots. We hypothesise that taxa in high Cu plots activated dormancy mechanisms, such that their genetic signal remained present, whilst the functionality of the soil was reduced. Many species identified in high Cu plots are known to have associated dormancy mechanisms and survive in high stress environments. Understanding how these mechanisms collectively contribute to contaminant outcomes is of great importance for the goals of predicting and managing microbial communities and their function. As we found that Cu concentrations above 200 mg kg−1 can cause significant functionality loss and a selective pressure on microbial communities, it is recommended that Cu concentrations above 200 mg kg−1are avoided in agricultural soils.

Copper exposure alters the metabolism of the blue crab Callinectes sapidus submitted to osmotic shock

Copper (Cu) is an essential metal capable to alter many metabolic and physiological processes in animal species, depending on the environmental concentration and salinity. The present study evaluated the effects of Cu exposure on the metabolism of the blue crab Callinectes sapidus under different osmotic situations. Crabs were acclimated at two different salinities conditions (30 and 2). Subsequently, they were exposed to Cu during 96 h at each salinity and under hypo-osmotic shock. Results demonstrated that Cu exposure increased whole-body oxygen consumption. In addition, the activity of LDH decreased while citrate synthase increased in anterior gills from animals submitted to hypo-osmotic shock. This scenario indicates extra stress caused by sudden environmental osmotic changes, as commonly observed in estuarine environments, when combined with copper exposure. Therefore, the activity of LDH and citrate synthase enzymes might be sensitive indicators for aquatic toxicology studies approaching Cu contamination in estuarine environments.

Effects of copper exposure and increased temperatures on Collembola in western Washington, USA

In the city of Seattle and the surrounding Puget Sound region, increased human population and the resulting increased domestic and commercial transportation has led to higher inputs of heavy metal pollutants in the region, many of which have been shown to increase in toxicity for various organisms at higher temperatures. One pollutant of concern in the region is copper (Cu) from brake and tire attrition. The extent to which Cu can affect other organisms that are part of the Puget Sound ecosystem is largely unknown. We studied the effect of Cu exposure on two species of Collembola, one of which is a native species (Choreutinula americana Palacios-Vargas, Castaño-Meneses & Gao) inhabiting the region's epiphytic mosses and lichen while the other is a model species commonly used in ecotoxicology studies (Folsomia candida Willem). We then extended this work by examining the effect of Cu exposure when Collembola are exposed to increased temperatures as predicted by the B1 and A2 climate change scenarios. We found that the native Collembolan was significantly more sensitive to Cu than the model species when maintained under a constant 20 °C, suggesting that ecotoxicology studies using the model species F. candida might underestimate the effect of toxicants to native Collembola. We also observed that increased temperatures significantly lowered the tolerance of both species to Cu. The results of this study highlight the joint effect of warming temperatures and pollutants on a representative species of Collembola in the Pacific Northwest.

Behavioral effects of copper on larval white sturgeon.

Early-life stage white sturgeon are sensitive to copper (Cu), with adverse behavioral responses observed during previous studies. The objectives of the present study were to quantify the effects of Cu exposure on white sturgeon swimming and feeding behaviors and determine their time to response. Larval sturgeon (1-2, 28, or 35 d posthatch [dph]) were exposed to Cu (0.5-8 μg/L) for 4 to 14 d. Abnormal behavioral changes were observed within the first few days of exposure including loss of equilibrium and immobilization. Digital video tracking software revealed decreased swimming activity with increasing Cu concentration. Significant changes in behavior and mortality occurred at concentrations of Cu between 1 and 8 μg/L. Juvenile white sturgeon, 58 dph, exposed to 12 μg/L Cu consumed 37 to 60% less food than controls after 3 d of exposure. The present results indicate that behavioral endpoints were more sensitive than some standard toxicity test endpoints and can effectively expand the sensitivity of standard toxicity tests for white sturgeon. Swimming behavior was impaired to the extent that survival in the field would likely be jeopardized. Such data would provide managers a useful metric for characterizing the risks of Cu contamination to white sturgeon. Environ Toxicol Chem 2019;38:132-144. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Excess copper promotes photoinhibition and modulates the expression of antioxidant-related genes in Zostera muelleri

Copper (Cu) is an essential micronutrient for plants and as such is vital to many metabolic processes. Nevertheless, when present at elevated concentrations, Cu can exert toxic effects on plants by disrupting protein functions and promoting oxidative stress. Due to their proximity to the urbanised estuaries, seagrasses are vulnerable to chemical contamination via industrial runoff, waste discharges and leachates. Zostera muelleri is a common seagrass species that forms habitats in the intertidal areas along the temperate coast of Australia. Previous studies have shown the detrimental effects of Cu exposure on photosynthetic efficiency of Z. muelleri. The present study focuses on the impacts of sublethal Cu exposure on the physiological and molecular responses. By means of a single addition, plants were exposed to 250 and 500 μg Cu L−1 (corresponding to 3.9 and 7.8 μM, respectively) as well as uncontaminated artificial seawater (control) for 7 days. Chlorophyll fluorescence parameters, measured as the effective quantum yield (ϕPSII), the maximum quantum yield (Fv/Fm) and non-photochemical quenching (NPQ) were assessed daily, while Cu accumulation in leaf tissue, total reactive oxygen species (ROS) and the expression of genes involved in antioxidant activities and trace metal binding were determined after 1, 3 and 7 days of exposure. Z. muelleri accumulated Cu in the leaf tissue in a concentration-dependent manner and the bioaccumulation was saturated by day 3. Cu exposure resulted in an acute suppression of ϕPSII and Fv/Fm. These two parameters also showed a concentration- and time-dependent decline. NPQ increased sharply during the first few days before subsequently decreasing towards the end of the experiment. Cu accumulation induced oxidative stress in Z. muelleri as an elevated level of ROS was detected on day 7. Lower Cu concentration promoted an up-regulation of genes encoding Cu/Zn-superoxide dismutase (Cu/Zn-sod), ascorbate peroxidase (apx), catalase (cat) and glutathione peroxidase (gpx), whereas no significant change was detected with higher Cu concentration. Exposure to Cu at any concentration failed to induce regulation in the expression level of genes encoding metallothionein type 2 (mt2), metallothionein type 3 (mt3) and cytochrome c oxidase copper chaperone (cox17). It is concluded that chlorophyll fluorescence parameters provide timely probe of the status of photosynthetic machinery under Cu stress. In addition, when exposed to a moderate level of Cu, Z. muelleri mitigates any induced oxidative stress by up-regulating transcripts coding for antioxidant enzymes.

Copper effects on biomarkers associated with photosynthesis, oxidative status and calcification in the Brazilian coral Mussismilia harttii (Scleractinia, Mussidae)

Seawater contamination with metals, such as copper (Cu), is a notable local impact threatening coral reefs. Cu effects on biomarkers associated with photosynthesis, oxidative status and calcification were evaluated in the Brazilian coral Mussismilia harttii using a marine mesocosm facility. Polyps were kept under control conditions (1.9 μg L−1 Cu) or exposed to dissolved Cu (3.0, 4.8, and 6.7 μg L−1) for 12 days. Photochemical efficiency of the photosystem II of symbiotic algae (zooxanthellae) was measured and polyps were analyzed for antioxidant capacity, lipid peroxidation, DNA damage, and carbonic anhydrase Ca-ATPase, Mg-ATPase and (Ca,Mg)-ATPase activities after 12 days. Results highlighted the effects of Cu exposure, leading corals to an oxidative stress condition [increased total antioxidant capacity (TAC) and DNA damage] and a possible reduced calcification ability [decreased (Ca,Mg)-ATPase activity]. Therefore, biomarkers associated with oxidative status (TAC and DNA damage) and calcification [(Ca, Mg)-ATPase] are indicated as good predictors of corals health.

Stable isotope analysis to detect copper (Cu) accumulation in species with high endogenous Cu concentrations: linking Cu accumulation with toxic effects in the gastropod Bembicium nanum

Tissue metal concentrations are widely used as indicators of environmental contamination. High endogenous concentrations of essential metals such as copper (Cu) can mask changes in tissue metal concentrations. Experiments using enriched isotopes can provide greater insight into endogenous Cu exchange and more broadly the exposure–bioaccumulation–response relationship. The aim of the present study was to investigate the effect of Cu exposure through the dietary uptake route in the intertidal gastropod Bembicium nanum. The marine alga Tetraselmis chuii was cultured at different Cu concentrations and fed to B. nanum. The Cu concentrations in T. chuii and B. nanum were analysed. Effects were measured using the biomarkers – total antioxidant capacity, lipid peroxidation and lysosomal membrane destabilisation in B. nanum. Dietary uptake of Cu in B. nanum was investigated with a feeding experiment using a 65Cu isotopically enriched solution. Although there were no significant differences in the Cu concentrations in B. nanum, lysosomal membrane destabilisation increased with the dietary Cu concentration. The 65Cu experiment demonstrated that B. nanum accumulated Cu under the experimental conditions. The study showed that tissue Cu concentrations are not always indicative of effects in organisms and that enriched isotope analysis assists in understanding the exposure–bioaccumulation–response relationship.

Endoplasmic reticulum stress and dysregulation of calcium homeostasis mediate Cu-induced alteration in hepatic lipid metabolism of javelin goby Synechogobius hasta

The present study was conducted to investigate the effect of Cu exposure on endoplasmic reticulum (ER) stress and Ca2+ homeostasis, and also explore the underlying mechanism of the ER stress and Ca2+ homeostasis in the Cu-induced change of hepatic lipid metabolism in javelin goby Synechogobius hasta. To this end, four experiments were conducted. In experiment 1, the full-length cDNA sequences of two ER molecular chaperones [glucose-regulated protein 78 (GRP78) and calreticulin (CRT)] and three ER stress sensors [PKR-like ER kinase (PERK), inositol requiring enzyme (IRE)-1α, and activating transcription factor (ATF)-6α] cDNAs were firstly characterized from S. hasta. The predicted amino acid sequences for the S. hasta GRP78, CRT, PERK, IRE-1α and ATF-6α revealed that the proteins contained all of the structural features characteristic in other species. mRNAs of the five genes were expressed in various tissues, but their mRNA levels varied among tissues. In experiment 2, S. hasta were exposed to four waterborne Cu concentrations (control, 19μg/l, 38μg/l, and 57μg/l, respectively) for 60days. Cu exposure evoked ER stress in liver of S. hasta in a time- and concentration-course change. In experiment 3, specific inhibitors, 2-aminoethyldiphenyl borate (2-APB) and dantrolene, were used to explore whether Ca2+ release from ER was involved in the Cu-induced ER stress change. Dantrolene and 2-APB prevented Cu-induced intracellular Ca2+ elevation, which demonstrated the release of Ca2+ from the ER was mediated by both RyR and IP3R. In experiment 4, a chemical chaperone, 4-phenyl butyric acid (4-PBA), was used to demonstrate whether Cu-induced alteration in lipid metabolism was suppressed through the attenuation of ER stress. Cu exposure evoked ER stress and sterol-regulator element-binding protein-1c (SREBP-1c) activation in hepatocytes of S. hasta, resulting in dysregulation of hepatic lipid metabolism. 4-PBA attenuated the Cu-induced elevation of mRNA expression of ER stress-related genes. For the first time, our study cloned GRP78, CRT, PERK, IRE-1α and ATF-6α genes in S. hasta and demonstrated their differential expression among tissues. Moreover, the study demonstrated the molecular mechanism by which ER stress might underlie the change of lipid metabolism induced by Cu in S. hasta.

Salinity-dependent mechanisms of copper toxicity in the galaxiid fish, Galaxias maculatus

The euryhaline galaxiid fish, inanga (Galaxias maculatus) is widely spread throughout the Southern hemisphere occupying near-coastal streams that may be elevated in trace elements such as copper (Cu). Despite this, nothing is known regarding their sensitivity to Cu contamination. The mechanisms of Cu toxicity in inanga, and the ameliorating role of salinity, were investigated by acclimating fish to freshwater (FW), 50% seawater (SW), or 100% SW and exposing them to a graded series of Cu concentrations (0–200μgL−1) for 48h. Mortality, whole body Cu accumulation, measures of ionoregulatory disturbance (whole body ions, sodium (Na) influx, sodium/potassium ATPase activity) and ammonia excretion were monitored. Toxicity of Cu was greatest in FW, with mortality likely resulting from impaired Na influx. In both FW and 100% SW, ammonia excretion was significantly elevated, an effect opposite to that observed in previous studies, suggesting fundamental differences in the effect of Cu in this species relative to other studied fish. Salinity was protective against Cu toxicity, and physiology seemed to play a more important role than water chemistry in this protection. Inanga are sensitive to waterborne Cu through a conserved impairment of Na ion homeostasis, but some effects of Cu exposure in this species are distinct. Based on effect concentrations, current regulatory tools and limits are likely protective of this species in New Zealand waters.

Sublethal effects of copper sulphate compared to copper nanoparticles in rainbow trout (Oncorhynchus mykiss) at low pH: physiology and metal accumulation

A few studies have investigated the interaction between copper toxicity and water pH in fishes, but little is known about the effects of acidic pH on the toxicity of copper nanoparticles (Cu-NPs). This study aimed to describe the sub-lethal toxic effects of Cu-NPs compared to CuSO4 at neutral and acidic water pH values in juvenile rainbow trout. Fish were exposed in triplicate (3 tanks/treatment) to control (no added Cu), or 20μgl−1 of either Cu as CuSO4 or Cu-NPs, at pH 7 and 5 in a semi-static aqueous exposure regime for up to 7 days. Acidification of the water altered the mean primary particle size (at pH 7, 60±2nm and pH 5, 55±1nm) and dialysis experiments to measure dissolution showed an increased release of dissolved Cu from Cu-NPs at pH 5 compared to pH 7. Copper accumulation was observed in the gills of trout exposed to CuSO4 and Cu-NPs at pH 7 and 5, with a greater accumulation from the CuSO4 treatment than Cu-NPs at each pH. The liver also showed Cu accumulation with both Cu treatments at pH 7 only, whereas, the spleen and kidney did not show measurable accumulation of Cu at any of the water pH values. Exposure to acid water caused changes in the ionoregulatory physiology of control fish and also altered the observed effects of Cu exposure; at pH 5, branchial Na+/K+-ATPase activity was greater than at pH 7 and the inhibition of Na+/K+-ATPase activity caused by exposure to CuSO4 at pH 7 was also not observed. There were some changes in haematology and depletion of plasma Na+ at pH 7 and 5 due to Cu exposure, but there were few material-type or pH effects. Overall, the data show that the accumulation of Cu is greater from CuSO4 than Cu-NPs; however, understanding of the effects of low pH on bioavailability of CuSO4 may not be directly transferred to Cu-NPs without further consideration of the physico-chemical behaviour of Cu-NPs in acid water.