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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

ATP7A Transgenic and Nontransgenic Mice Are Resistant to High Copper Exposure^1–3,

Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Burwood VIC 3125, Australia

^* To whom correspondence should be addressed. E-mail: jmercer{at}deakin.edu.au.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
The protein affected in Menkes disease, ATP7A, is a copper (Cu)-transporting P-type ATPase that plays an important role in Cu homeostasis, but the full extent of this role has not been defined at a systemic level. Transgenic mice that overexpress the human ATP7A from the chicken β-actin composite promoter (CAG) were used to further investigate the physiological function of ATP7A. Overexpression of ATP7A in the mice caused disturbances in Cu homeostasis, with depletion of Cu in some tissues, especially the heart. To investigate the effect of overexpression of ATP7A when dietary Cu intake was markedly increased, normal and transgenic mice were exposed to drinking water containing 300 mg/L of Cu as Cu acetate for 3 mo. Cu exposure resulted in partial restoration of heart Cu concentrations in male transgenic mice. Despite the extended period of Cu exposure, Cu concentrations in the liver remained relatively unaffected, with a significant increase in male nontransgenic mice. Liver pathology was unremarkable except for small areas of fibrosis that were detected only in livers of the Cu-exposed transgenic mice. Intracellular localization of ATP7A in various tissues was not affected by Cu exposure. Plasma Cu concentration and ceruloplasmin oxidase activity were reduced in both Cu-exposed transgenic and nontransgenic mice. The expression levels of other candidate Cu homeostatic proteins, endogenous Atp7b, ceruloplasmin, Ctr1, and transgenic ATP7A were not altered significantly by Cu exposure. Overall, mice are remarkably resistant to high Cu loads and the overexpression of ATP7A has only moderate effects on the response to Cu exposure.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

The identification of the genes affected in Menkes disease (ATP7A or MNK) (2–4) and Wilson disease (ATP7B or WND) (5–7) provided important insights into the molecular basis of Cu homeostasis. Both genes encode Cu-ATPases (ATP7A and ATP7B, respectively) that have distinct but not fully clarified physiological roles. The Menkes gene is expressed in many tissues except the liver (4,8). ATP7A is essential for efficient dietary Cu uptake in the small intestine, delivery of Cu to the brain across the blood-brain barrier, and Cu efflux in extrahepatic tissues (9,10).

ATP7B is expressed predominantly in the liver and the massive accumulation of Cu in this organ in patients with Wilson disease suggested that ATP7B is essential for the excretion of Cu from the liver. Biliary excretion of Cu is the central process required to maintain physiological Cu balance and the rate of Cu excretion in the bile increases as the Cu concentration rises in hepatocytes, thus effectively removing excess Cu from the body (11). Mammalian species differ widely in their ability to dispose of excess Cu. Sheep in particular are extremely sensitive to even a minor increase in Cu intake, because this species has very inefficient biliary Cu excretion mechanisms. Cu-loaded sheep accumulate massive amounts of Cu in the liver with subsequent liver damage and death (12). Rodents on the other hand are resistant to high Cu intakes. Rats require months of exposure to 1000–1500 mg Cu/kg body weight before liver damage is evident (13). Mice are possibly even more resistant to high Cu levels, although data are more limited. Allen et al. (14) showed that in mice loaded to water containing 300 mg/L Cu as Cu acetate, the Cu levels in the liver, kidney, and brain did not increase. The effect of Cu loading was also studied in the toxic milk mouse, a close model of Wilson disease with a defective Atp7b. The Cu-loaded toxic milk mouse showed a more rapid increase in hepatic Cu than the unloaded toxic milk mouse, but the liver pathology was not worsened (14). The molecular basis behind the different Cu sensitivities of various species is not understood, but presumably involves differences in the rate of Cu absorption, biliary excretion, and ability to safely store Cu in hepatocytes. These differences in part involve ATP7A and ATP7B.

To more fully investigate the physiological role of ATP7A, we have produced transgenic mice overexpressing normal human ATP7A (15). The transgene is controlled by a constitutive, ubiquitously expressed promoter derived from a modified chicken β-actin (CAG) promoter (16). The expression pattern of the transgenic ATP7A is similar to the endogenous protein and it localized in a position consistent with the trans-Golgi network (TGN) in which endogenous Atp7a is found in cultured cells under basal Cu conditions (17). The overexpression of ATP7A in mice caused alterations in Cu homeostasis, with an overall depletion of Cu in most tissues (15). We demonstrated that ATP7A in the intestinal enterocytes responded to changes in Cu concentrations. As Cu was increased in a perfused loop of the small intestine, the ATP7A was induced to traffic from a tight perinuclear location to the vicinity of the basolateral membrane (18). This result suggested that ATP7A was involved in the uptake of dietary Cu but not in the downregulation of Cu uptake when excessive amounts are consumed. In this study, we investigated the effect of overexpression of ATP7A in mice exposed to very high Cu intakes for an extended period of time.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Mouse husbandry. All mice used in this study were maintained on 12-h-light/12-h-dark conditions and all experiments were approved by Deakin University Animal Welfare Committee regulations (approval no. A19/2003). The mice consumed deionized water and nonpurified diet (Supplemental Table 1; Barastoc, Ridley Agriproducts) ad libitum. The transgenic mice were produced in the C57BL/6J background. Homozygous ATP7A-transgenic mice were used rather than heterozygotes because of their higher expression of ATP7A (our unpublished data).

    Cu exposure. Oral Cu exposure was performed as described by Allen et al. (14). Cu was provided in drinking water to nontransgenic (C57BL/6J) and age-matched transgenic mice. Mice were maintained on deionized water and used as nonexposed controls. Mice were gradually Cu exposed, starting from 6 wk of age. The mice were adapted to 300 mg/L Cu by placing them on 100 mg/L Cu for 1 wk, 200 mg/L Cu for 1 wk, and then on 300 mg/L Cu and maintained at this level for 3 mo. All mice remained healthy throughout the loading period.

    Atomic absorption spectrophotometry. Tissues were analyzed for Cu by atomic absorption spectrophotometry as previously described (15). Results were expressed as µmol/g dry weight. For plasma Cu analysis, blood was collected from the heart through a 25-gauge needle, transferred to a 1-mL tube containing heparin (Interpath), and centrifuged at 2000 g (Biofuge Pico, Heraeus Instruments) for 5 min. Plasma was obtained from the supernatant. Plasma was diluted in 1:50 in 0.1% nitric acid and Cu levels were measured using the Graphite Tube Atomizer. Results were expressed as µmol/L.

    Immunolocalization of ATP7A. Due to the strong background staining in the small intestine with immunoperoxidase staining, immunofluorescence staining was used to localize the expression of ATP7A in this organ as previously described (17). ATP7A was stained in the other organs with immunohistochemistry according to the protocol from Vector ABC kit (Vector Laboratories). The expression of ATP7A was detected with an affinity-purified ATP7A antibody, R17-BX (1: 2000 and 1:1000 for liver section) as previously described (15).

    Liver histology and liver enzymes. Liver paraffin sections were stained with hematoxylin and eosin for histological assessment. Three mice were examined from each group. Liver histology was examined by Gribbles Pathology. In addition, a complete panel of liver enzymes was performed by Gribbles Pathology on plasma from transgenic and nontransgenic mice given normal or Cu water. The following liver proteins and enzymes were analyzed in 80 µL of plasma: albumin, bilirubin, alkaline phosphatase, alanine aminotransferase, aspartame aminotransferase, and glutamate dehydrogenase.

    Plasma ceruloplasmin assay. Plasma ceruloplasmin oxidase activity was determined through the reaction with o-dianisidine dihydrochloride by volume scale-down of sample and reagents from the published method of Schosinsky et al. (19). Human serum was used as a positive control for the assay and samples were analyzed in duplicate.

    Western blot analysis. The 150-d-old mice were killed by CO₂ asphyxiation. Tissues were prepared as previously described (15). Proteins (50 µg) were boiled in reducing buffer (final concentrations: 16.7 mmol/L Tris-HCl, pH6.8, 2% SDS, 0.83% β-mercaptoethanol, 3.3% glycerol, and 0.016% bromophenol blue), electrophoresed in a 10% SDS-PAGE gel (120V) and transferred to Hybond-C nitrocellulose membrane (Amersham) using 1.5 mA/cm² for 2 h. Membranes were microwaved for 5 min to expose the epitope, blocked with 5% skim milk at 4°C overnight, and incubated with the sheep anti-ATP7B antibody, NC36 (1:500) (20), rabbit anti-ceruloplasmin antibody (1:1000, Dako), rabbit anti-hCTR1 antibody (1:500) (21), sheep anti-ATP7A antibody, R17-BX (1:2000) (15), or rabbit anti-calnexin polyclonal antibody (1:500, Santa Cruz Biotechnology) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature or ATP7B antibody at 37°C for 2 h. Membranes were then washed with TBST and incubated for 1 h at room temperature with peroxidase-conjugated anti-goat IgG (1:4000, Sigma) or anti-rabbit IgG (1:4000, Chemicon) in TBST. Proteins were detected by chemiluminescence (Lumi-Light Western Blotting Substrate, Roche Diagnostics). Densitometry was used to evaluate immunolabeled protein intensity. Pixel intensities were quantified using Multi Gauge software V2.2 (Fujifilm) and their levels were normalized against Calnexin controls.

    Data analysis. Data for tissue, plasma Cu concentrations, and plasma ceruloplasmin activity were analyzed by 3-way ANOVA with factors of gender (male and female), Cu treatment (with and without Cu exposure), and mouse type (nontransgenic and transgenic mice) using the SPSS (version 12.0.1) program. When a significant interaction of Cu treatment and mouse type was observed, we performed post hoc analysis using Welch test and Dunnett's T3 test between the following groups: nontransgenic nonexposed (N–Cu)⁴ vs. nontransgenic Cu-exposed (N+Cu); transgenic nonexposed (T–Cu) vs. transgenic Cu-exposed (T+Cu), N–Cu vs. T–Cu, and N+Cu vs. T+Cu, as the data have unequal variance. Differences were considered significant at P < 0.05. All data are presented as means ± SD.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Tissue Cu concentrations. For analyses of the changes in tissue Cu concentrations, a 3-way ANOVA was carried out with factors of gender, Cu exposure, and mouse type for each tissue (Table 1). In the heart, main effects were found in Cu exposure and mouse type. There was an interaction between Cu exposure and mouse type (P = 0.028) but no gender effects. Data from male and female were pooled and analyzed with Welch test and Dunnett's T3 test. Cu exposure increased heart Cu concentrations in T+Cu mice (P = 0.036) but not in N+Cu mice. The expression of the transgene reduced heart Cu levels in both nonexposed (P < 0.001) and exposed (P = 0.026) mice compared with nontransgenic mice. In the liver, main effects were observed in gender and Cu exposure. Overall, the males had higher Cu concentrations than females (P < 0.001). The interaction of Cu exposure and mouse type differed (P = 0.034). Cu levels increased in N+Cu males (P = 0.035). In the small intestine, males and females differed (P = 0.002), which is due to the higher Cu concentrations in Cu-exposed females than in Cu-exposed males. The interaction of gender and Cu exposure was significant (P = 0.003), further suggesting that the difference between males and females was due to the Cu treatment rather than the overexpression of transgene. Cu levels increased in the Cu-treated group (P < 0.001). Cu concentrations in the kidney and spleen were not altered by Cu exposure or overexpression of transgene. In the brain, Cu exposure increased Cu concentrations in the treated mice (7% elevation in pooled Cu-exposed groups) (P = 0.018). Males and females did not differ. The overexpression of transgene did not seem to affect the response of mice to Cu exposure.

    Immunolocalization of ATP7A. Immunofluorescence and immunohistochemical analyses were performed to investigate if chronic Cu exposure affected the localization of ATP7A. We used immunofluorescence staining to localize ATP7A in the small intestine and this showed the protein was localized to the apical side of the enterocytes in nonexposed mice as seen by Monty et al. (Supplemental Fig. 2A) (18). When the mice were exposed to chronic Cu intake, the majority of ATP7A was still localized on the apical side of the nuclei but with a slight relocalization toward the basolateral side (Supplemental Fig. 2B). In the kidney, Cu exposure did not affect the localization of ATP7A in the tubules (Supplemental Fig. 2C,D). Similar to the kidney, the localization of ATP7A did not change in the Cu-exposed brain compared with nonexposed control, with staining observed in the Purkinje cells of the cerebellum (Supplemental Fig. 2E,F) and CA2 region of the hippocampus (Supplemental Fig. 2G,H). In the nonexposed mice, ATP7A was localized in patches in the plasma membrane of hepatocytes (Supplemental Fig. 2I) and Cu exposure did not affect the localization of ATP7A in hepatocytes (Supplemental Fig. 2J).

    Plasma Cu concentrations and ceruloplasmin activity. For analyses of the changes in plasma Cu concentrations and ceruloplasmin activity, a 3-way ANOVA was performed with factors of gender, Cu exposure, and mouse type (Table 2). Main effects were found in gender, Cu exposure, and mouse type for both plasma Cu levels and ceruloplasmin activity. Overall, males had lower plasma Cu concentrations (P < 0.001) and ceruloplasmin activity (P < 0.001) than females. Cu exposure decreased both plasma Cu concentrations (P < 0.001) and ceruloplasmin activity (P < 0.001). Transgenic mice had lower plasma Cu levels (P < 0.001) and ceruloplasmin activity (P < 0.001) than nontransgenic mice.

View larger version (45K): [in this window] [in a new window]   FIGURE 1  Western blot analysis of Cu-related proteins in the livers of nontransgenic and transgenic mice that were or were not exposed to Cu for 12 wk. Cp, Ceruloplasmin. Calnexin antibody was used as indication of protein loading. Four mice were examined in each group.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

A possible explanation for the remarkable resistance of mice to the high Cu exposure is that Cu uptake from the small intestine was greatly reduced in response to Cu exposure. Our data indicate that the localization of ATP7A in the enterocyte was similar in both unexposed and exposed mice (Supplemental Fig. 2A,B). This result differs from Monty et al. (18) who showed that ATP7A traffics to the basolateral membrane in response to short term (1 h) perfusion of the small intestine with Cu solutions (18). This observation suggests that the Cu-induced trafficking of ATP7A does not occur in the chronically Cu-exposed mice despite the high Cu concentration in the small intestine. The overexpression of ATP7A in the small intestine would be expected to increase the rate of transfer of Cu from the enterocyte into the circulation; however, the concentration of Cu in the small intestine was just as high or even higher in T+Cu mice. Furthermore, liver concentrations were also not elevated in transgenic mice, which would have been expected if there was a higher rate of intestinal absorption. This result may suggest that long-term Cu exposure results in a change in the uptake mechanisms in the small intestine. As the levels remain high in the Cu-exposed mice, it may be that the Cu is sequestered by induced metallothioneins in the enterocyte, preventing uptake, as has been suggested with animals exposed to high levels of zinc (24).

Heart Cu levels were much lower in the T+Cu mice than N+Cu mice and this is presumably due to enhanced Cu efflux capacity as a result of overexpression of ATP7A (Table 1). Cu in the brains of Cu-exposed mice was elevated by 7%, but surprisingly there was no difference in brain Cu levels in the transgenic mice. Previously, ATP7A heterozygous transgenic mice showed a 21–30% reduction of brain Cu (15). This discrepancy is difficult to explain. Age may provide a possible explanation for these differences; 150-d-old homozygous mice were used in this study, whereas 60-d or 300-d-old heterozygous mice used in the previous study (15). Further work will be needed to clarify if there are distinct changes in brain Cu in the 150-d-old group.

The liver is a target organ for Cu toxicity; e.g. Cu-exposed rats develop a pronounced liver pathology (13). Overall, males had higher hepatic Cu than females. In the Cu-exposed mice, liver Cu concentrations increased only in N+Cu males (by 59%). The small effect of Cu exposure on liver Cu is consistent with the observation that liver Cu did not increase even after 20 mo of excess Cu exposure (300 mg/L Cu) in normal mice (25) and indicates that mice are surprisingly resistant to high Cu exposure. The liver histology also indicated that there were no major changes in response to the Cu exposure. Surprisingly, the most pronounced difference was observed in T+Cu livers, which showed mild to moderate hypercellularity of portal areas and regions of mild fibrosis (arrow, Supplemental Fig. 1). Fibrosis was observed in 2 out of 3 T+Cu mice but not in N–Cu, N+Cu, and T–Cu mice. Thus, it is possible that some damage has occurred in discrete regions of the livers of T+Cu mice, but the reason for this occurring in only the transgenic mice is not obvious. One possibility is that the regions of damage correspond to the patches of hepatocytes that express ATP7A in the transgenic mice, because these regions are similar in extent (Supplemental Fig. 2J). But it is not clear why the expression of ATP7A would be associated with development of fibrosis. Further experiments are required to confirm the association of fibrosis and ATP7A expression.

Surprisingly, when the mice were Cu exposed, both plasma Cu concentration and ceruloplasmin activity decreased (Table 2). A possible explanation for this observation is that Atp7b in hepatocytes, which delivers Cu to ceruloplasmin in the TGN, is relocalized from the TGN to the subapical membrane in response to Cu exposure (26), thus reducing Cu incorporation into ceruloplasmin and resulting in lower ceruloplasmin activity and low Cu concentration in the plasma. Unfortunately, this hypothesis could not be tested, because the available antibody was raised to human ATP7B and could not detect Atp7b in the liver. A similar lowering of ceruloplasmin activity has been previously observed in Cu-exposed mice (25).

Several Cu-related proteins have been shown to be involved in Cu uptake and transport in cells. Little is known about how these Cu-related proteins are regulated in an animal in response to Cu exposure. In this study, we investigated the expression of Cu-related proteins in the liver, the key organ involved in Cu homeostasis. The expression of endogenous hepatic Atp7b did not differ with Cu exposure (Fig. 1). The reduction of ceruloplasmin protein in the liver was only marginal but could be contributing to the reduction of plasma ceruloplasmin oxidase activity. There was a small reduction of Ctr1 in Cu-exposed mice, possibly suggesting that Ctr1 expression may decrease in response to constant Cu exposure, thus reducing cellular Cu uptake as a protective mechanism. This result was consistent with cell studies in which the Ctr1 protein was found to internalize and degrade in elevated Cu conditions (27).

In summary, we found some differences between transgenic and nontransgenic mice in response to Cu exposure, but the effect of ATP7A overexpression was not as marked as expected. Tissue Cu levels in T+Cu mice tended to be lower (particularly in the heart) than in N+Cu mice presumably because of the enhanced Cu efflux capacity resulting from the overexpression of ATP7A. Plasma Cu levels and ceruloplasmin oxidase activity were reduced in the Cu-exposed mice. Cu exposure did not induce the relocalization of ATP7A in tissues investigated, suggesting that there was a reduced availability of Cu to ATP7A in the cells of chronically Cu-exposed mice and this effect may be an important part of the resistance of mice to chronic Cu exposure. There was no evidence of major changes in the levels of a range of possible Cu homeostatic proteins in response to chronic Cu exposure.

	ACKNOWLEDGMENTS

 
We thank Maree Mc Glynn and Erin Willams for providing technical assistance in the implementation of work presented in this manuscript and Professor Gerry Quinn for his kind help with statistical analysis.

	FOOTNOTES

 
¹ Supported by a grant from the International Cu Association.

² Author disclosures: B. X. Ke, R. M. Llanos, and J. F. B. Mercer, no conflicts of interest.

³ Supplemental Table 1 and Supplemental Figures 1 and 2 are available with the online posting of this paper at jn.nutrition.org.

⁴ Abbreviations used: N–Cu, nontransgenic mice given deionized water; N+Cu, nontransgenic mice given copper water; TBST, Tris-buffered saline containing 0.1% Tween-20; TGN, trans-Golgi network; T–Cu, transgenic mice given deionized water; T+Cu, transgenic mice given copper water.

Manuscript received 20 August 2007. Initial review completed 10 October 2007. Revision accepted 21 December 2007.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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This Article Abstract Full Text (PDF) Online Supporting Material Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ke, B.-X. Articles by Mercer, J. F. B. Search for Related Content PubMed PubMed Citation Articles by Ke, B.-X. Articles by Mercer, J. F. B.