Effects of low-level copper exposure on Ca2+-ATPase and carbonic anhydrase in the freshwater bivalve Anodonta anatina

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Sources of emissions and environmental levels

Natural inputs in the order of decreasing importance are volcanic eruptions, plant decomposition, forest fires, and marine aerosols (INERIS, 2010). Water and soil surrounding sites of agricultural and industrial activities are most heavily exposed to copper and, to a lesser extent, the air contaminated by road and rail traffic. In the European Union, some countries represent a large part of copper emission such as France with 43% in water, 15% in soil, and 13% in air. According to IFEN (2011), anthropogenic inputs of copper originating from industrial activities are mostly into waters and soils, while urban and agricultural activities as well as road traffic emit mostly into the air. In 2007, the emissions of copper in-to the environment in Europe were estimated as 371,000 kg year-1 to water, 146,000 kg year-1 to air, and 139,000 kg year-1 to soil (INERIS, 2010).
Air emissions are mainly non-industrial. The road transport sector is a major issue representing 51 % of emissions in France in 2004. These are mainly caused by wear of brake pads containing copper. The railway transport sector represents 32 % of total air emissions of copper which is caused by wear of overhead lines (ADEME-SOGREAH, 2007). Emissions of copper-related road transport are increasing with the development of this sector. Industrial discharges of copper are mainly from the synthesis of organic chemicals, in the production of nonferrous metals from ore, ferrous metal smelting, and the production of iron and steel. In the atmosphere, copper metal oxidizes slowly to Cu2O which covers the metal with a protective layer against corrosion. Copper is released into the atmosphere as particulate oxide, sulphate, or carbonate as particulate matter. Releases to the aquatic environment are mainly due to corrosion of equipment made from copper or brass, and urban waste is another important source of release. One of the most significant non-industrial discharges of copper is the treatment of urban wastewater. This sector represents 28 % (15617 kg year-1) of the total French emissions of copper and its derivatives to the aquatic environment (INERIS, 2010). Copper is one of the compounds always detected in the input and output of secondary sewage treatment plants (but not always in sewage treatment plants with tertiary treatment). Copper is one of the highest concentrated compounds in sewage treatment plant inflow with concentrations generally greater than 10 µg L-1. At the outlet it is usually found at concentrations between 1 and 10 µg L-1. In Europe in 2007, the main emitters of industrial origin are the United Kingdom, France, Germany, and Romania. They represent respectively 35 % (129000 kg), 15 % (55700 kg), 11 % (39800 kg) and 7 % (24500 kg) of the European industrial emissions to water (INERIS, 2010). In the European Union, the most significant sectors are thermal power plants and other combustion plants. In France, another important sector is the production of nonferrous metals from ore from concentrates and from secondary materials.
Speciation and behaviour of copper in the environment will directly influence its bioavailability. In aquatic environments, the fate of copper is influenced by many processes and factors such as chelation by organic ligands (especially on NH2 and SH groups, to a lesser extent on the OH group). Adsorption phenomena can also occur on metal oxides, clays, or particulate organic matter, bioaccumulation, presence of competing cations (Ca2+, Fe2+, Mg2+) or of anions (OH, S2-, PO43-, CO32-) plays also a role in copper behaviour (INERIS, 2010). The great portion of copper released into the water is in the particulate form and tends to settle, to precipitate, or adsorb to organic matter, hydrous iron, manganese oxides, or clay particles (ATSDR, 1990; INERIS, 2005). In hard water (carbonate concentration up to 1 mg L-1), the largest fraction of copper is precipitated as insoluble compounds. Cuprous oxide Cu2O is insoluble in water. Except in the presence of a stabilizing ligand such as sulfides, cyanide, or fluoride the oxidation state Cu(I) is easily oxidized to Cu(II) as CuSO4, Cu(OH)2 and CuCl2 more soluble in water. The Cu2+ ion forms many stable complexes with inorganic ligands such as chloride or ammonium, or organic ligands. When copper enters the aquatic environment, the chemical equilibrium of oxidation states and of soluble and insoluble species is usually reached within 24 hours (INERIS, 2005). In aquatic environments, copper is mainly absorbed on particles, and suspended solids are often heavily loaded. In Europe, according to the forum of the European geological surveys (FOREGS, 2010), copper concentrations up to 3 µg L-1 are found in continental water. In regions with intensive human activities (agricultural, urban), copper concentrations of 40 µg L-1 and even 100 µg L-1 can be found seasonally (Neal and Robson, 2000; Falfushynska et al., 2009). Trace metals as copper are mainly transported to the marine environment by rivers through estuaries. The magnitude of metal input to the marine environment depends on the levels in the river waters and on the physico-chemical processes that take place in the estuaries (Waeles et al., 2004). In sea water, copper is found in concentrations ranging from 0.1 to 4 µg L-1 (Waeles et al., 2005; Levet et al., 2009).

Copper metabolism and its physiological role in animals (generalities)

Copper is an essential trace element in microorganisms, plants, and animals. It plays a basic role being in the active centre of enzymes involved in connective tissue formation with lysyl oxidase, in respiration with cytochrome C oxidase (López de Romana, 2011), in photosynthesis with plastocyanin (Grotz and Guerinot, 2006), and in controlling the level of oxygen radicals with Cu / Zn-superoxide dismutase (Table 1). It allows also the transport of oxygen in the haemolymph of many invertebrates. Copper is used in several cell compartments, and the intracellular distribution of copper is regulated in response to metabolic demands and changes in the cell environment (Tapiero et al., 2003). The same properties that make transition metal ions indispensable for life at low exposure level are also the ones that are responsible for toxicity when present in excess. Copper metabolism must be tightly regulated, ensuring a sufficient supply without toxic accumulation. Copper homeostasis involves a balance between absorption, distribution, use, storage, and detoxification.

Toxicology of copper (general)

In living organisms, cupric ion (Cu2+) is fairly soluble whereas cuprous (Cu+) solubility is in the sub-micromolar range. Cu is present mainly as Cu2+ since in the presence of oxygen or other electron acceptors, Cu+ is readily oxidized. Strong reductants such as ascorbate or reduced glutathione can reduce Cu2+ back to Cu+ (Arredondo and Núñez, 2005). As in the case of iron through the Haber-Weiss and Fenton reactions, free copper ions can catalyse the production of hydroxyl radicals (HO•). Copper toxicity results also from nonspecific binding which can inactivate important regulatory enzymes by displacing other essential metal ions from catalytic sites, by binding to catalytic Cys groups or by allosterically altering the functional conformation of proteins (Mason and Jenkins, 1996).
Thus, the mechanisms of toxicity are associated both with oxidative stress and direct interactions with cellular compounds.
Free copper ions have high affinity to sulfur-, nitrogen-, and oxygen-containing functional groups in biological molecules which can inactivate and damage them. Cytotoxicity observed in copper poisoning results from inhibition of the pyruvate oxidase system by competing for the protein’s sulfhydryl groups. Glucose-6-phospho-dehydrogenase and GR are also inhibited (competitive inhibition) proportionally to the concentration of intracellular copper (Barceloux, 1999). The same applies to some transporters as the ATPase(s) which are also inhibited by copper, causing disruption of homeostasis of the respective transported entities. Toxic effects of copper can also result from its affinity to DNA (Agarwal et al., 1989; Bremner, 1998; Sagripanti et al., 1991). Another mechanism of toxicity of excessive concentrations of copper is the modification of the zinc finger structures of transcriptional factors which cannot any longer bind to DNA (Pena et al., 1999). Copper in excess can also promote apoptosis (Kozlowski et al., 2009) wile copper deficiency may be the cause of many diseases due to cuproprotein and copper dependent reaction inhibition.
In mammals, copper homeostasis is primordial. Wilson’s and Menkes diseases are caused by genetic mutations in copper transporter proteins. The former results from accumulation of copper in several organs and tissues. There are different varieties, the most common being liver disease and anaemia (Hejl et al., 2009). The accumulation of copper arises from a defect in the P-type Cu-protein ATP7B (called Wilson protein), a specific transporter of copper. The gene which encodes this protein is located on autosome number 13 in humans. It also allows the incorporation of copper in cuproproteins and excretion of copper into the bile. Accumulation of copper leads to liver cirrhosis and neurodegeneration.
Menkes disease is a neurodegenerative disease. Copper, after ingestion, accumulates in the intestine and absorption by other organs and tissues (blood, liver, brain) is defective. Menkes syndrome is caused by a mutation in the ATP7A gene located on chromosome X which encodes a protein ATP7A Cu-type P. This membrane protein is the first specific transporter of copper found in eukaryotes.
Copper (Cu), besides cadmium, is one of the major metals causing environmental problems in fresh water ecosystems. Since it is highly toxic to fish, it is also used as piscicide (Manzl et al., 2004).


Ecotoxicological interest

Molluscs are common, highly visible, ecologically and commercially important on a global scale as food and as non-food resources (Rittschof and McClellan-Green, 2005). In some aquatic ecosystems (lakes, slow streams) molluscs can represent up to 80% of the total biomass of the benthic macroinvertebrates, so their impact can become major. Populations of bivalves filter large amounts of water (Unionidae 300ml/individual/h) and take an active part in sedimentation and water purification. Faeces and pseudofaeces concentrate sometimes a large fraction of planktonic microorganisms not used, making them accessible to detritivorous invertebrates such as oligochaetes, and many diptera. But they also change the quality of sediment by concentration and excretion of many substances (metals, pesticides, radionuclides). Because of their sedentary lifestyle, their filtration capacity, and their wide distribution, molluscs and bivalves are excellent sentinels for monitoring the fraction of bioavailable pollutants in their environment (Hayer and Pihan, 1996). In close contact with water-suspended particles and sediment, they are widely used for controlling the bioaccumulation and toxic effects of metallic and organic pollutants in aquatic ecosystems (Viarengo et al., 2000; Rittschof and McClellan-Green, 2005). Several Unionidae mussels, especially from the Anodonta genus have since been used as biomonitor organisms in toxicity assessment of numerous compounds released in continental water. The anatomy and physiology of these animals has been studied for a long time, allowing the study of toxic effects of compounds but also the mechanisms of detoxification. Anodonta cygnea and Anodonta anatina are biological models widely used in ecotoxicology (Falfushynska et al., 2009). They are present in large quantities, sedentary, easy to collect and to acclimatize in aquaria.

Effects of copper exposure and detoxification mechanisms

Trace elements are known to be highly accumulated by aquatic molluscs. Bivalves are in close contact with sediments which constitute a major environmental sink for metals, with an important filtering activity to satisfy respiration and nutrition, and tolerance mechanisms that involve metal sequestration rather than metal exclusion or elimination. They provide accurate and integrated information about the environmental impact and bioavailability of chemicals. They are therefore extensively applied in marine environments using mussels and oysters, but are also implemented in freshwater systems using other bivalve species such as Anodonta sp., Dreissena polymorpha, Elliptio complanata, and Asiatic clams. Among freshwater organisms, unionid molluscs are widely recognised for their capacity to accumulate a variety of environmental contaminants including metals in their tissues (Winter, 1996; Bilos et al., 1998; Kádár et al., 2001; Falfushynska et al., 2009). The widespread recent decline in the species diversity and population density of freshwater mussels (Lydeard et al., 2004) may be partly related to chronic, low-level exposure to toxic metals (Frank and Gerstmann, 2007). Freshwater mussels are exposed to metals that are dissolved in water, associated with suspended particles, and deposited in bottom sediments. Thus, freshwater mussels can bioaccumulate certain metals to concentrations that greatly exceed those dissolved in water. In adult mussels, the most common site of metal uptake is the gills, followed by the digestive gland, the mantle, and the kidneys (Pagliarani et al., 1996; Bonneris et al., 2005). Bioaccumulation of metals varies strongly according the water chemistry conditions, pH and water hardness being important parameters. Aqueous concentrations of calcium probably enhance the bioavailability and toxicity of metal cations, because the permeability of membranes is inversely related to aqueous calcium concentration; calciumions apparently compete with other metal cations for binding sites on the gill surface, decreasing the direct uptake of other cationic metals. The pH influences both the chemistry of metals and macromolecules of surface structures. A modification of membrane permeability causes an alteration in metal diffusion. Additionally, changes in membrane potential modify the transport of polar metal species (Winter, 1996). In bivalves, the biological barriers are the gill epithelium, the wall of the digestive tract, and the shell (which is often reported as a site of bioaccumulation). Metals or metalloids in solution are more easily absorbed by the surfaces in direct contact with the external environment, while those associated with the particulate phase are rather ingested and internalized after solubilization in the digestive tract, or transferred by endocytosis to then undergo lysosomal digestion (Wang and Rainbow, 2005). Once past the first barrier, the mechanisms of transfer of metals or metalloids into the cell involve intracellular diffusion (passive or facilitated), active transport, and endocytosis (phagocytosis and pinocytosis). The oxygen concentration of the water or the density of microalgae are parameters that will directly influence the ventilatory activity of the bivalve and so its exposure to metals in solution or particulate (Tran et al., 2000; Tran et al., 2004). Copper and metal bioaccumulation can change between individuals of the same species with the difference in sizes, or between different species mussels due to different physiology (breathing, eating) (Winter, 1996; Bilos et al., 1998; Gundacker, 2000; Hédouin et al., 2006; Falfushynska et al., 2009). In bivalves, the determination of trace metal concentrations in whole individuals presents little interest, since determination of bioconcentration factors in various tissues suggested that the principal accumulating organs are: the gills, the digestive gland, the kidney, and the mantle, also the shell acting as a storage matrix (Viarengo and Nott, 1993; Roméo and Gnassia-Barelli, 1995; Das and Jana, 1999; Bonneris et al., 2005). The intracellular sequestration of metals is based on a sequence of cellular events involving a cascade of different ligands with increasing metal binding strengths. Anodonta sp. and other molluscs accumulate metals to high levels in their tissues (Falfushynska et al., 2009). Metal tolerance in such accumulator organisms involves sequestration of metals in non-toxic forms. Among the best studied intracellular sites involved in the sequestration of essential and non-essential metals in aquatic invertebrates are lysosomes, granules, and soluble Cys-rich ligands as metal-binding peptide and proteins. Unionids also lay down calcium microspherule concretions, particularly in the connective tissue of the gills, in the mantle, and in the digestive gland (Pynnönen et al., 1987; Moura et al., 2000; Lopes-Lima et al., 2005). Dissimilar mechanisms for copper and metal uptake, storage, mobilisation, and excretion performed by different cell types in different organs explain the pattern of metal accumulation and tissue distribution (Soto et al., 1997).

Table of contents :

1 General introduction
2 Background
2.1 Copper
2.1.1 Origin and use
2.1.2 Sources of emissions and environmental levels
2.1.3 Copper metabolism and its physiological role in animals (generalities)
2.1.4 Toxicology of copper (general)
2.2 Biological models studied: Anodonta anatina and Anodonta cygnea
2.2.1 Taxonomy, description, and distribution
2.2.2 Anatomy and ecology
2.2.3 Ecotoxicological interest
2.3 Effects of copper exposure and detoxification mechanisms
2.3.1 Copper exposure effects Copper bioaccumulation Oxidative stress Calcium transport and perturbation of bio-mineralization
2.3.2 Detoxification mechanisms Cysteine thiol rich compounds Metal detoxification mechanisms in bivalves
3 Extended summary
3.1 Optimization of analytical protocols
3.2 Effects of copper on calcium transport in Anodonta anatina
3.3 Metal detoxification mechanism in Anodonta cygnea
3.4 Decline of the Unionidae populations
4 Conclusion
5 Article 1 (published): Effects of low-level copper exposure on Ca2+-ATPase and carbonic anhydrase in the freshwater bivalve Anodonta anatina
6 Article 2: Copper effects on Na+/K+-ATPase and H+-ATPase in the freshwater bivalve Anodonta anatina
7 Article 3: Phytochelatins in the freshwater bivalve Anodonta cygnea
8 Article 4: Phytochelatins, a group of metal-binding peptides induced by copper exposure in the bivalve Anodonta cygnea


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