Basis To Understand Toxic Effects Associated With Chronic Exposure To Copper

In mammals, adaptation to high and low copper exposure depends on general mechanisms of cellular copper management that control plasmatic levels of this metal. In different tissues, biochemical and molecular studies have led to a better understanding of how different organs adapt to copper excess and deficit. These studies provide evidence on the molecular components that participate in copper metabolism. In this section, we will discuss the molecular mechanisms involved in three important steps of copper intracellular metabolism of intestinal and hepatic tissues: (1) transport of copper into the cells; (2) intracellular storage of copper; (3) export of copper from the cells. These mechanisms will be discussed considering whether their alteration may introduce toxic effects to the organism.

4.1. Chronic Effects at Intestinal Tissues Level

The process of copper transport and absorption must supply adequate amounts of systemic copper to fulfill the requirement of a variety of copper-dependent enzymes (31); at the same time, intestinal mechanisms must exist to prevent copper excess in the organism. As mentioned in Section 2., copper absorption in humans occurs in the small intestine, after processing of food in the stomach, duodenum, and jejunum (13,32). In general, evidence indicates that intestinal tissue is able to modulate the rate of copper absorption; however, the cellular and molecular mechanisms involved in this function have not been identified, leaving the precise limits of intestinal copper adaptation undetermined.

In eukariotic cells, the redox state of copper is important to the process of copper uptake and may be a first point of regulation in the absorption pathway. In the intestinal lumen, copper is mainly bound to proteins, and during the uptake process, there is equilibrium between cuprous [Cu(I)] and cupric forms [Cu(II)]. Dietary components such as phytates, amino acids (mainly histidine and cys-teine), ascorbate, and other reducing agents modulate the predominance of one or the other copper ions, determining the bioavailability of copper (33). Protein(s) with storage or reductase activity such as cellular prion (PrPc) or amyloid precursor protein (APP), respectively, have been identified in the apical epithelial membrane (34-36). However, their physiological potential capacity to modulate the concentration and uptake of the cuprous form in enterocytes has been not evaluated. Several reports indicate that structural alterations of APP and PrPc affect neuronal copper homeostasis, suggesting the participation of this metal in pathologies such as Alzheimer's disease, Parkinson's disease, and amyotropic lateral sclerosis (37-41). Two types of copper transporter have been identified in the epithelial plasmatic membrane: DCT1 (42,43) and human copper transporter 1 (hCTRl) and 2 (hCTR2) (44). Although there is no evidence of mutations affecting their function, any alteration of them (sorting to membrane, interaction with other plasmatic proteins, etc.) could lead to defective copper homeostasis.

Distribution and storage of copper in epithelial cells can be envisioned as a highly regulated process. Once copper is in the cytoplasm of the intestinal cells, it is rapidly transferred to the tripeptide glutathion (gamma-L-Glu-L-Cys-Gly, GSH) and/or to the protein metallothionein (MT) (45). In intestinal cells, close to 80% of the newly absorbed Cu is found in the cytoplasm, essentially bound to MT and GSH (32). GSH and MTs are important intracellular reducing agents for Cu(II) and com-plexation agents for Cu(I). The high thermodynamic stability of Cu(I)-S bonds in both Cu(I)-GSH and Cu(I)-MT complexes, coupled with their kinetic fragility, provide efficient and specific pathways for intracellular copper storage and transport (45,46). To investigate the role of MT in copper homeostasis, mice bearing these targeted deletions were crossed with the mottled-brindled-J (Mo-brJ) strain of mice, which lack a functional copy of the Menkes protein (47). The Mo-brJ mice died from copper deficiency because of the inability to export copper out of the intestine, into circulation. Mutant mice accumulate copper in the intestine, which induce the expression of MT-I and MT-II genes, leading to an increase of copper-MT complexes. Considering that MT binds copper with high affinity, it was proposed that elimination of MT from the intestine might enhance copper efflux into the circulation. However, the work of Kelly and Palmiter (48) showed that inactivation of MT-I and MT-II genes in the MT-/-, Mo-brJ mutant exacerbated the phenotype of Mo-brJ mice. These results support the idea that the elevated MT levels observed in Menkes disease are mitigating copper toxic-ity rather than exacerbating the disease. In addition, they showed that in the absence of a functional mechanism of copper export, MT is required for a normal embryonic development.

Recently, a new group of proteins named "chaperones" have been associated with intracellular copper sorting (49,50). These proteins bind copper and are involved in copper transfer from the cell surface to specific intracellular copper proteins. In humans, copper chaperone HAH1 (or ATOX1) has been shown to deliver copper to the Wilson's disease protein ATP7B, in the Golgi apparatus (51). COX17 is involved in transferring copper to cytochrome oxidase in the mitochondria (52,53) and CCS is involved in delivering copper to SOD1 (54). Thus, regulation of intracellular copper levels corresponds to the summation of relative contributions of each of these components that may be modified as a function of epithelial cell requirements.

The newly absorbed Cu bound to MT, to chaperones, and to GSH must be continuously released from them to regenerate the capacity of these molecules to form the Cu complex. The process as a whole induces an apical-to-basolateral cellular flux of the metal (see Fig. 2), which permits transfer-

Fig. 2. Schematic representation of the effect of copper preloading on cellular copper homeostasis. The figure illustrates the absorption process of copper at cellular levels and the different components involved in the interaction with this metal.

ring copper from the intestinal lumen to the portal circulation (32,45). Our results indicate that in Caco-2 cells grown in a low copper medium, 64% of the cellular-to-basolateral flux corresponds to newly incorporated copper (64Cu). This suggests that in these conditions, copper uptake is strongly associated to efflux pathway(s) (Fig. 2). In contrast, only a minor fraction (4%) of the 64Cu was delivered to the basolateral domain, when copper concentration was increased in the medium. In these latter conditions, the intracellular copper was elevated and the copper efflux was enhanced (14). When copper offered to the cell is high, copper uptake by the cell may be higher than the concentration of the copper-chelating agents, making it possible that intracellular copper becomes toxic unless the cells can induce protective mechanisms. Induction of MT is one protective mechanism (55). Another mechanism is given by the facilitation of copper efflux during excess, by increasing the expression, and by a copper-dependent redistribution of the copper transporter Menkes P-type ATPase (MNK) (56-60). These evidences along with our studies on copper transport in Caco-2 led us to propose the model schematized in Fig. 2. In monolayer intestinal cells (Caco-2) chronically exposed to high extracellular copper concentration, the trans-cellular traffic of copper is shifted from a direct (uptake/efflux) pathway to an indirect (copper storage/efflux) pathway. Thus, the overall process results in an increase on both storage and cellular-to-basolateral cellular flux of copper.

4.2. Chronic Effects at Hepatic Tissues Level

Once copper enters portal circulation, the metal is transported bound to albumin, transcuprein, amino acids (histidine, threonine, cysteine), or peptides containing these amino acids (61-63). Hepatic tissue removes copper from circulation by rapidly trapping it into chelating copper proteins that transfer copper to cuproenzymes or ceruloplasmin. Copper returns to the extrahepatic circulation mainly bound to ceruloplasmin, and copper excess is excreted into the bile (32,45,64).

Whatever the mechanism involved in copper uptake, the metal is preferentially transported as Cu(I). Because vitamin C reduces Cu(II) to Cu(I), it stimulates Cu uptake into liver plasma membranes and isolated hepatocytes (65,66); a reductase system that uses NADH to reduce Cu has been described in liver cells (65).

The processes of storing and exporting copper in the liver are critical to maintaining the physiological level of this element in plasma. Hepatic cells possess protective mechanisms that allow them to act as a buffering system against extracellular copper fluctuations. Measurement of intracellular copper concentration in cells growing in a culture medium containing 0.44 pmol/L of Cu revealed that hepatic cell lines (HepG2) contained significantly more copper than HeLa (from adenocarcinoma of cervix), NIH 3T3 (fibroblasts of embryo), N2A (neuroblastoma), and B12 (glioblastoma) cell lines. In these four cell lines, analyzed the order of relative abundance for trace metals was Cu < Fe < Zn (67,68). Analysis of the subcellular distribution of copper indicated that Cu was mainly in the cytosol fraction (81.7%) (69), whereas only about 30% of the newly incorporated 64Cu was recovered in this fraction (70). Considering that only about 10% of total proteins is present in the cytoplasmatic fraction, copper concentration was highly enriched in this fraction (13.1+1.2 nmol/mg protein) as compared to the corresponding homogenate (1.35 ± 0.24 nmol/mg protein) (69). In this context, it has been reported that free ions in the intracellular [Cu] pool are limited to less than one free-copper ion per cell, suggesting that a pool of free-copper ions is not used in physiological activation of metalloenzymes (71). These studies also suggest that in hepatic cells, copper is handled by mechanisms that control intracellular levels of the metal and its return to the extrahepatic system by bile excretion (32,45,64). One of these mechanisms consists of the increase in the number of intracellular copper-chelator units (such as MT or GSH) that can sequester copper excess (45,55,72-77).

Studies on different cell lines (including HepG2 cells) (72) exposed to graded copper concentration have shown great complexity on the MT gene expression (78,79). As an example, Sadhu and Gedamu (80) have shown that exposure of HepG2 cells to heavy metals results in a differential increase in the relative amounts of MT transcripts. To date, the functional significance of the multiple MT isoforms is unknown. Whether each isoform contributes separately to copper homeostasis or there is redundancy in their cellular roles is not known. GSH is the main nonproteinaceous component that binds intracellular copper. It has a well-defined role in hepatic copper turnover and also has the potential to carry copper into bile during the copper excretion process (81). It has been proposed that GSH functions as a first chelating agent for incoming Cu(I), binding the metal as soon as it enters the cell (74,82). The role of GSH as an intermediate in intracellular copper distribution has been evaluated in hepatic cells by using a GSH synthesis inhibitor that has no effect on MT levels (74). Results of those studies showed a significant decreased in MT-bound copper levels, supporting a model in which Cu(I) is complexed by GSH immediately after entering the cell and then transferred from GSH to MT, where is stored.

A second protective mechanism used by hepatic cells is based on transporters that facilitate copper excretion during excess (32,45,46,83,84), so that the amount of copper excreted into the bile is directly proportional to the excess of absorbed copper (85). Transport of hepatic copper across the canalicular membrane follows at least three routes:

1. Lysosomal exocytosis: Under certain conditions such as chronic copper overload, copper tends to accumulate in liver lysosomes (84). 64Cu orally administered to Wilson patients, who have gross accumulation of hepatic copper, results in a similar specific activity both in lysosomes and the bile, suggesting that lysosomes may be a source of biliary copper (86).

2. Glutathione-mediated copper excretion. Evidence indicate that depletion of hepatic GSH leads to a decrease in biliary copper, suggesting coupling between hepatic transport of GSH and copper excretion into bile; this mechanism requires that copper conjugation with GSH occurs within the hepatic cell (45). Studies with mutant Eisai hyperbilirrubinuric (EHB) rats suggested that Cu-GSH complexes are secreted via a GSH-conjugated transporting system located in the canalicular membrane. These studies indicate that the intracellular levels of GSH available to form Cu-GSH complexes and the activity of a GSH-conjugated transporting system are necessary for hepatic copper excretion (87-89).

3. P-Type ATPase facilitated copper excretion. Mutations in the ATP7B gene lead to excessive hepatic copper accumulation because of impaired biliary copper excretion (90). Biochemical and immunohistochemi-

cal studies have localized the Wilson protein mainly to the trans-Golgi reticulum and late endosomes (49,85,90). However, Dijkstra et al. (87), using liver plasma membrane vesicles, provided biochemical evidence for the presence of a vanadate-inhibited copper transport in human liver, which was mainly localized at the canalicular domain of the hepatocyte plasma membrane. The presence of inmunoreactive Wilson proteins in canalicular membrane in HePG2 cells was confirmed using antibodies against ATP7B protein after treatment with copper, suggesting that copper induces trafficking of its own transporter from the trans-Golgi network to the apical membrane, where it may facilitate biliary copper excretion (90). Thus, the ATPase may represent another pathway of biliary copper excretion that works in concert with lysosomal exocytosis and GSH-mediated excretion.

Alternatively, a redistribution of copper by chaperon units (HAH1) would promote an increase of copper flux at specific cellular compartments in which copper efflux is carried out by the ATP7B-ceruloplasmine system (49). Based on structural analysis of ATP7B, it was assumed that copper binds as Cu(I) to cysteine residues on the copper-binding site of this protein (91). Once copper is incorporated to the secretory pathway, it may be delivered to plasma, bound to ceruloplasmin (92), or it can be used to be incorporated into newly synthesized cuproproteins (51,93).

Copper is secreted from the liver to blood and then distributed to the various organs. Ceruloplas-min (CP) is a blue multicopper oxidase that contains 80-90% of the copper found in the sera of vertebrate species (85). The crystal structure of CP confirms the presence of six tightly bound copper ions, three of them forming type-1 copper centers involved in electron-transfer processes. Three other copper ions (one type 2 and two type 3) are in a single trinuclear center, which is the oxygen-activating site during the catalytic cycle of the enzyme (92). Copper in CP is not exchangeable, suggesting that CP does not function as an essential copper-transport protein (84). Normal copper metabolism in patients with aceruloplasminemia support this concept. There is 5-10% of copper bound to nonceruloplasmin components, which is exchangeable. Several studies demonstrate that copper in plasma is bound to albumin and histidine. Albumin seems to play a regulatory role in controlling the copper flow between plasma and tissues. Histidine competes with albumin for copper and a ternary complex was found to be the rate-determining step in the exchange of copper between histidine and albumin (84). Histidine has been shown to enhance copper uptake in human trophoblast cells, in the presence of serum; this has been interpreted as the result of copper release from albumin (94). The relevance of this copper-transport mechanism in plasma deserves further investigation.

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