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The plant decapeptide OSIP108 prevents copper-induced toxicity in various models for Wilson disease
Toxicology and Applied Pharmacology 280 (2014) 345–351
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The plant decapeptide OSIP108 prevents copper-induced toxicity in various models for Wilson disease Pieter Spincemaille a,1, Duc-Hung Pham b,1, Gursimran Chandhok c, Jef Verbeek d, Andree Zibert c, Louis Libbrecht d,e, Hartmut Schmidt c, Camila V. Esguerra b, Peter A.M. de Witte b, Bruno P.A. Cammue a,f,⁎, David Cassiman d, Karin Thevissen a a
Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium Laboratory for Molecular Biodiscovery, KU Leuven, Campus Gasthuisberg, Herestraat 49, O&N2, 3000 Leuven, Belgium c Clinic for Transplantation Medicine, Münster University Hospital, Albert-Schweitzer-Campus 1, Building A14, D-48149 Münster, Germany d Department of Hepatology and Metabolic Center, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium e Department of Pathology, University Hospital Ghent, De Pintelaan 185, 9000 Ghent, Belgium f Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Ghent, Belgium b
a r t i c l e
i n f o
Article history: Received 5 May 2014 Revised 30 July 2014 Accepted 5 August 2014 Available online 16 August 2014 Keywords: Wilson disease Copper OSIP108 Zebrafish Hepatotoxicity
a b s t r a c t Background: Wilson disease (WD) is caused by accumulation of excess copper (Cu) due to a mutation in the gene encoding the liver Cu transporter ATP7B, and is characterized by acute liver failure or cirrhosis and neuronal cell death. We investigated the effect of OSIP108, a plant derived decapeptide that prevents Cu-induced apoptosis in yeast and human cells, on Cu-induced toxicity in various mammalian in vitro models relevant for WD and in a Cutoxicity zebrafish larvae model applicable to WD. Methods: The effect of OSIP108 was evaluated on viability of various cell lines in the presence of excess Cu, on liver morphology of a Cu-treated zebrafish larvae strain that expresses a fluorescent reporter in hepatocytes, and on oxidative stress levels in wild type AB zebrafish larvae. Results: OSIP108 increased not only viability of Cu-treated CHO cells transgenically expressing ATP7B and the common WD-causing mutant ATP7BH1069Q, but also viability of Cu-treated human glioblastoma U87 cells. Aberrancies in liver morphology of Cu-treated zebrafish larvae were observed, which were further confirmed as Cuinduced hepatotoxicity by liver histology. Injections of OSIP108 into Cu-treated zebrafish larvae significantly increased the amount of larvae with normal liver morphology and decreased Cu-induced production of reactive oxygen species. Conclusions: OSIP108 prevents Cu-induced toxicity in in vitro models and in a Cu-toxicity zebrafish larvae model applicable to WD. General significance: All the above data indicate the potential of OSIP108 as a drug lead for further development as a novel WD treatment. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Wilson disease (WD), a rare (1/30.000) autosomal recessive disorder of copper (Cu) balance (Loudianos and Gitlin, 2000; Huster, 2010), is caused by mutations in the ATP7B gene. ATP7B encodes a Cutransporting protein ATPase (Bull et al., 1993) and is predominantly expressed in hepatocytes in which it localizes to the trans-Golgi network (Lutsenko et al., 2002). ATP7B plays an essential role in Cu excretion from hepatocytes into bile and for mobilization of ceruloplasmin-bound Cu from hepatocytes into the serum (Huster, 2010). ATP7B mutations in ⁎ Corresponding author at: Centre for Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium. Fax: +32 16321966. E-mail address: [email protected] (B.P.A. Cammue). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.taap.2014.08.005 0041-008X/© 2014 Elsevier Inc. All rights reserved.
WD patients cause accumulation of Cu in the liver (Ferenci, 2006) that can result in acute liver failure (ALF) or cirrhosis (Ala et al., 2007). Furthermore, elevated intracellular Cu levels cause degeneration of neuronal cells, which is also a WD-characteristic (Loudianos and Gitlin, 2000; Huster, 2010). Cu toxicity in hepatocytes of WD patients and WD animal models arises from a direct effect of Cu on the mitochondria, ultimately amounting to tissue damage in the liver (Rosencrantz and Schilsky, 2011; Zischka and Lichtmannegger, 2014). Cu-induced mitochondrial dysfunction and damage have been ascribed to a Cu-induced (i) deficiency in the mitochondrial respiratory chain, at the level of the Cu-dependent complex IV (Roberts et al., 2008); (ii) cross-linking of mitochondrial membranous proteins and subsequent contraction of the membrane as observed ultrastructurally in the livers of WD patients and rodent models (Zischka et al., 2011); (iii) oxidative stress (Seth
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et al., 2004; Sokol et al., 1994; Gaetke and Chow, 2003) and (iv) apoptosis by inducing acid sphingomyelinase (aSMase) activity, thereby altering the lipid composition of membranes and increasing the production of the apoptosis inducer ceramide (Lang et al., 2007). Current treatment options for WD are aimed at decreasing gastrointestinal Cu uptake by administration of zinc or by increasing Cu excretion via chelating agents such as D-penicillamine, trientine or tetrathiomolybdate (Smolarek and Stremmel, 1999). In the long run, these treatments are helpful in the majority of patients (Lowette et al., 2010), but none of the available drugs constitute causal treatment. Moreover, side effects such as neurological deterioration, nephrotoxicity and generation of resistance have been reported (Rodriguez et al., 2012; Czlonkowska et al., 1996). These limitations all implicate a remaining window of opportunity for novel compounds directly targeting Cu toxicity in WD. Using Tiling Array technology, we previously identified a decapeptide in the plant Arabidopsis thaliana upon treatment with the herbicide paraquat (PQ) (De Coninck et al., 2013). PQ is known to induce reactive oxygen species (ROS) (Farrington et al., 1973). This peptide, termed OSIP108 (MLCVLQGLRE), can increase tolerance of plant and yeast cells to oxidative stress agents like PQ and H2O2 respectively. Overexpression of the OSIP108-encoding sequence in the yeast Saccharomyces cerevisiae resulted in a significantly increased tolerance to H2O2, while exogenous application of OSIP108 to H2O2-treated yeast also resulted in significantly increased survival (De Coninck et al., 2013). By assessing specific markers for oxidative stress and apoptosis, we very recently demonstrated that OSIP108 indeed prevented Cu-induced oxidative stress and apoptosis in yeast and human cells (Spincemaille et al., 2014). In the present study, we investigated the effect of OSIP108 on Cu toxicity in in vitro mammalian cell lines and in an in vivo zebrafish model relevant for WD. All data point to the potential of OSIP108 as a lead for development of novel treatment options for WD. 2. Materials and methods 2.1. Ethics. All zebrafish experiments carried out were approved by the Ethics Committee of the University of Leuven (approval number P05090) and by the Belgian Federal Department of Public Health, Food Safety & Environment (approval number LA1210199). 2.2. Materials and cell lines. Chinese hamster ovary (CHO) cells and human glioblastoma U87 cells were obtained from ATCC (Rockville, MD, USA) and grown in DMEM F12 (Lonza) and DMEM High Glucose (GE Healthcare) supplemented with 10% fetal calf serum, 2 mM Lglutamine, 100 U/ml penicillin and 100 μg/ml streptomycin respectively. CHO cells were transduced by retroviral vectors expressing the coding region of human ATP7B or mutant p.H1069Q (Sauer et al., 2010). OSIP108 (MLCVLQGLRE, 1161 g/mol) was purchased from Thermo Fisher Scientific (Ulm, Germany) and dissolved in DMSO. Copper sulfate (CuSO4) and copper chloride (CuCl2) (Cu) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.3. Cellular Cu toxicity experiments. Transgenic CHO or U87 cells were seeded in triplicate at 104 cells/well in a 96-well plate. Following attachment, cells were treated with 0.75 mM Cu in the presence of control (2% DMSO) or 100 μM OSIP108. After 48 h incubation, cell viability was determined by MTT staining as described previously (Siaj et al., 2012). 2.4. Zebrafish Cu toxicity experiments. The transgenic zebrafish (Danio rerio) line zTgfabp10a-DsRed:nacre was generated using the liverspecific zebrafish fabp10a promoter (Her et al., 2003), driving the fluorescent reporter gene DsRed. Zebrafish husbandry, embryo collection, and embryo and larval maintenance were performed as described (Westerfield, 1994; Nusslein-Volhard CaD, 2002). For all experiments, 4 days post-fertilization (dpf) larvae that showed consistent fluorescent
levels in the liver were selected. OSIP108 (2.5 mg/kg–5 mg/kg) was co-injected with Rhodamine B Dextran as an injection marker (10% g/v) into the bloodstream via the Duct of Cuvier (Isogai et al., 2001) prior to incubation of the zebrafish larvae in Cu-containing medium (10 μM–100 μM) for three days. As control treatment, larvae were injected with 10% Rhodamine B Dextran. At 7 dpf, larvae liver phenotypes were scored using fluorescence stereomicroscopy (Leica MZ 10F). Liver morphology was not affected by injection. 2.5. Zebrafish histology. Zebrafish larvae were incubated or not (control) with Cu-containing medium (50 μM). Ten larvae within each group were fixed in 10% neutral formalin buffer before aligning two arrays of five parallel larvae in an agarose block. These blocks were processed manually through a series of 70% ethanol, 100% xylene and paraffin as described (Sabaliauskas et al., 2006). Next, blocks were cut sagittally into slides of 7 μM thickness by a microtome (Microm HM 360, Thermo Scientific, Belgium) and adhered onto glass slides. Glass slides with larvae were stained with hematoxylin and eosin (H&E) and imaged by microscopy. A certified pathologist performed histopathological assessment of zebrafish larval livers. All zebrafish larvae were alive when processed for the evaluation of liver pathology. 2.6. Detection of oxidative stress markers in zebrafish larvae. As the transgenic line zTgfabp10a-DsRed:nacre exhibits high fluorescence levels in the liver, we used the wild-type AB zebrafish line to determine Cuinduced ROS production by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining. Wild type AB zebrafish larvae (4 dpf) were injected with 10% Rhodamine B Dextran (control) or 5 mg/kg OSIP108 prior to incubation in Cu-containing growth media (50 μM). After 4 h of incubation, H2DCFDA (1 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA) was added to the growth medium. Subsequently, 5 dpf larvae were washed carefully with embryo medium (1.5 mM HEPES, pH 7.6, 17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO4, and 0.18 mM Ca(NO3)2) and immobilized in 3% methylcellulose. Images of live samples were captured by fluorescence stereomicroscopy (Leica MZ 10F). Fluorescence levels in the liver region were quantified using ImageJ software. Correction for fluorescence levels was made by subtracting auto-fluorescence values of non-H2DCFDAexposed control larvae. 2.7. Statistical analysis. All values are presented as mean with standard error (SEM). Data were analyzed by GraphPad Prism 6 software. P b 0.05 was considered as statistically significant. 3. Results 3.1. OSIP108 increases Cu tolerance of CHO cells expressing H1069Q In the present study we investigated the effect of the anti-apoptotic plant peptide OSIP108 (MLCVLQGLRE) (De Coninck et al., 2013; Spincemaille et al., 2014), in in vitro models and in a zebrafish model relevant for WD. In the first step, we tested the effect of OSIP108 on Cu tolerance of a Chinese hamster ovary (CHO) cell line, which lacks intrinsic expression of the WD disease gene ATP7B (Forbes and Cox, 2000). This cell line is highly sensitive to Cu and was previously used as a cell model to study ATP7B mutants (La Fontaine et al., 2001). The ATP7B mutation H1069Q is the predominant mutation found in WD patients of various regions in Europe (Ferenci, 2006). We first evaluated the Cu sensitivity of CHO cell lines and found that the minimal Cu dose that induces at least 50% cell death was 0.75 mM Cu. Likewise, we previously studied the effect of different OSIP108 doses on HepG2 viability in the presence of toxic Cu, and found that 100 μM OSIP108 proved maximally efficient (Spincemaille et al., 2014). Hence, to investigate the effect of OSIP108 on acute Cu-induced toxicity of CHO cell lines, we chose to use a Cu dose of 0.75 mM and OSIP108 dose of 100 μM. Addition of 100 μM OSIP108 to CHO cell lines expressing either ATP7B
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or ATP7BH1069Q cDNA significantly increased cell viability upon coincubation with 0.75 mM Cu (Fig. 1): viability of CHO cells expressing ATP7B or ATP7BH1069Q cDNA in the presence of 0.75 mM Cu was 18% and 16% respectively, and increased to 38% and 28%, respectively upon coincubation with 100 μM OSIP108. Note that in the absence of the peptide, toxicity of 0.75 mM Cu was similar in CHO cell lines expressing wild type ATP7B or the relative mild mutant ATP7B H1069Q cDNA (Gromadzka et al., 2006), while the rescuing capacity of OSIP108 was higher in CHO cells expressing wild type ATP7B cDNA. These data indicate that OSIP108 can significantly increase the Cu tolerance of CHO cell lines in both wild type and mutant ATP7B expressing lines albeit that the absolute level of recovery by the peptide may be higher for wild type ATP7B. In addition, 100 μM OSIP108 was also effective at a broader range of Cu (0.25 mM– 0.75 mM) in CHO cells expressing wild type ATP7B cDNA (data not shown). 3.2. OSIP108 prevents Cu-induced cell death in human U87 glial cells Next, we evaluated the effect of OSIP108 on Cu tolerance of the human U87 glioblastoma cell line. In addition to Cu-induced cirrhosis in WD, progressive degeneration of basal ganglia in the brain is also a prominent clinical feature of WD (Loudianos and Gitlin, 2000; Huster, 2010). U87 cells have been studied in the context of Cu-induced toxicity in glioblastoma cells (Li et al., 2013) as well as in Cu-induced astrocyte apoptosis during the progression of WD (Merker et al., 2005). Incubation of U87 cells with 0.75 mM Cu decreased cell viability to 10% while OSIP108 treatment (100 μM) significantly increased cell viability to 39% (Fig. 2). These data indicate that OSIP108 can also prevent Cuinduced cell death of human glioblastoma cells. 3.3. Cu induces hepatotoxicity in zebrafish larvae In an effort to translate our in vitro data to an in vivo model relevant for WD, we developed a zebrafish larvae model for Cu-induced liver damage. To this end, a zebrafish transgenic line was generated that specifically expresses the gene encoding the fluorescent DsRed reporter in hepatocytes. This allowed us to observe liver morphology of zebrafish larvae by fluorescence stereomicroscopy. Upon incubation with Cu, we observed a range of aberrant liver phenotypes including reduced and enlarged livers (Fig. 3a), both denoted from hereon as ‘aberrant’. We found that the amount of larvae with aberrant liver phenotypes significantly increased by addition of 50 μM Cu (up to 80%) and 100 μM Cu (up to 63%) as compared to control larvae (approx. 20% of the population) (Fig. 3b). Vice versa, the amount of larvae with normal liver
Fig. 2. OSIP108 reduces Cu sensitivity of human U87 cells. U87 cells were incubated with 0.75 mM Cu in the absence or presence of 100 μM OSIP108. Cell viability was determined by MTT assay as compared to cells receiving no Cu. Mean and SEM of six experiments are shown. (***P b 0.001; Student t-test.)
shape and size was significantly reduced when larvae were treated with 50 μM or 100 μM Cu compared to control treatment (Fig. 3b). At 100 μM Cu we observed that approx. 23% of larvae died, but this effect was not statistically significant. However, treatment with 150 μM and 200 μM Cu significantly increased death of the population to approx. 80% and 100% of the larvae population, respectively (data not shown). These results indicate that Cu affects liver morphology of zebrafish larvae as observed by fluorescence stereomicroscopic analysis. In order to validate our larval model for Cu-induced hepatotoxicity, we performed histological analysis of larvae treated with or without Cu. We chose the least toxic Cu concentration (50 μM) that induced significant differences between control treated larvae and Cu-treated larvae. Larval zebrafish phenotypic assays to detect hepatotoxicants starting from 3 days post-fertilization (dpf) have been described (He et al., 2013; McGrath and Li, 2008). Since the left liver lobe is present and functioning at 4 dpf (Chng et al., 2012; Korzh et al., 2008), and we were able to locate fluorescent livers with a consistent fluorescent signal at 4 dpf, the latter time point was chosen to initiate Cu exposure. Zebrafish livers not exposed to Cu showed hepatocytes with homogenous eosinophilic cytoplasm and recognizable nuclei of non-parenchymal liver cells (Figs. 4a,c). The livers of Cu-exposed zebrafish larvae consistently showed larger hepatocytes, which was evident from the decreased nucleocytoplasmatic index (i.e. the ratio between nuclear size and hepatocyte size) (Figs. 4b,d). The cytoplasm of Cu-exposed zebrafish hepatocytes showed uniform eosinophilic clumping. There was a clear decrease in the number of nuclei from non-parenchymal liver cells in the Cu-exposed zebrafish larvae. These results show that Cu indeed affects the hepatocytes of zebrafish and that the observed liver fluorescence of zebrafish larvae (Figs. 3a,b) correlates with the histological analyses of the liver (Figs. 4a–d).
3.4. OSIP108 prevents Cu-induced hepatotoxicity in zebrafish
Fig. 1. OSIP108 reduces Cu sensitivity of CHO cells. CHO cells expressing ATP7B wild type (‘ATP7B’) or mutation p.H1069Q (‘ATP7BH1069Q’) were incubated with 0.75 mM Cu in the absence (black bars) or presence of 100 μM OSIP108 (gray bars). Cell viability was determined by MTT assay and calculated as compared to cells receiving no Cu. Mean and SEM of three experiments are shown. (**P b 0.01; ***P b 0.001; Student t-test.)
As the above described zebrafish model can be used to determine liver aberrancies, we evaluated the effect of OSIP108 injections on Cu-induced aberrancies in liver morphology of zebrafish larvae. We found that injection of untreated control larvae with different doses of OSIP108 (2.5 mg/kg–5 mg/kg) did not affect normal liver morphology (Fig. 5), while OSIP108 (5 mg/kg) injection in the bloodstream of 50 μM Cu-treated larvae significantly increased the number of larvae with normal livers (Fig. 5). Interestingly, 2.5 mg/kg OSIP108 did not result in such effect, indicating that a minimal OSIP108 dose is required to observe this protective effect. These data indicate that OSIP108 can
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protect against Cu-induced changes in liver morphology in zebrafish larvae, thereby preventing Cu-induced hepatotoxicity.
3.5. OSIP108 prevents Cu-induced hepatotoxicity in zebrafish larvae by reducing oxidative stress
Fig. 3. Cu induces liver damage in zebrafish larvae. (a) Fluorescent visualization of liver morphology. Each treatment condition was conducted with 10–20 larvae. (b) Zebrafish larvae were treated with control (distilled water, C.) or different Cu doses (10 μM– 100 μM). Liver morphology was assessed by fabp10a.DsRed expression in the liver. Normal (black bars) or aberrant (comprises enlarged and reduced liver phenotypes (as defined in (a), gray bars)) liver phenotypes were determined. Mean and SEM of at least 3 biological repeats are shown. (**P b 0.01; ***P b 0.001; ANOVA test using Tukey corrections.)
To gain insights into the underlying mechanistic events characteristic for Cu-induced toxicity and changes in liver morphology in zebrafish larvae, as well as in the protective effect of OSIP108, we evaluated Cuinduced ROS production in zebrafish larvae by H2DCFDA staining in the absence (control) or presence of OSIP108 and Cu. In all larvae, including the non-Cu-treated controls, we observed fluorescence signals in larvae within the gut and intestine (Fig. 6a). This background signal might reflect natural oxidative stress processes (Kim et al., 2014). Larvae treated with Cu (50 μM), however, showed much higher levels and area of fluorescence. More specifically, the fluorescence was localized in specific regions such as the liver, heart, spinal cord and brain (Fig. 6a), indicating that Cu-induced ROS production is widespread in zebrafish larvae. In addition, by assessing fluorescence levels in the liver region with ImageJ software, we observed that Cu-treated larvae exhibited significantly increased fluorescence levels as compared to control treated larvae (fluorescence units 5.21 and 2.83, respectively), indicating Cu-induced ROS production in the livers of zebrafish larvae. In contrast, injection of larvae with OSIP108 (5 mg/kg) in the presence of Cu (50 μM) abrogated this effect as the fluorescence intensity at different areas, including the liver region, returned to the level of the controls (Fig. 6a). Quantification of this effect confirmed that OSIP108 significantly decreased fluorescence levels, expressed as the ratio of fluorescence levels of OSIP108-injected larvae to non-Cu-treated control (Fig. 6b), as compared to larvae injected with control, and thus indicates decreased Cu-induced ROS production. Taken together, these results point to the ability of OSIP108 to prevent Cu-induced ROS production in zebrafish larvae, including the liver.
Fig. 4. Cu induces histological changes in zebrafish larvae livers. Liver histology of zebrafish larvae treated without Cu (a, c) or 50 μM Cu (b, d). Representative H&E-stained images of 10 larvae per condition. Magnification (zebrafish larvae 7 dpf) is 4 ×, inset (liver) is 40×; panels c & d are at maximum magnification, scale bar = 100 μm.
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Fig. 5. OSIP108 protects zebrafish against Cu-induced liver damage. Total amount of larvae with normal liver upon treatment without (0 mg/kg) or with OSIP108 (2.5 mg/kg–5 mg/kg) in the absence or presence of 50 μM Cu. For each condition, 10–20 larvae were evaluated. Mean and SEM of at least 3 biological repeats. (**P b 0.01; ***P b 0.001; ANOVA test using Tukey corrections.)
4. Discussion We show here that the plant-derived peptide OSIP108 (De Coninck et al., 2013), which was previously shown to prevent Cu-induced apoptosis in yeast and human cells (Spincemaille et al., 2014), protects against Cu-induced toxicity in various in vitro mammalian cell models and in a newly developed zebrafish model for Cu-induced hepatotoxicity. In the recent past, several assays have been developed to detect drug-induced toxicity in zebrafish and previous studies also reported a perfect match between visual observation of zebrafish livers and hepatotoxicity based on quantitative image analysis and liver histology of zebrafish treated with known mammalian hepatotoxicants (He et al., 2013; McGrath and Li, 2008; Berghmans et al., 2008). Besides, combining high-content screening by using fluorescent markers in human hepatocytes or HepG2 cells, and zebrafish larvae to detect hepatotoxicants in early stages of drug development has been suggested to be a powerful combination (Hill et al., 2012). In addition, the ability to identify toxic metabolites, potential drug–drug interactions via liver enzyme induction or inhibition and liver pathologies allows
zebrafish to serve as a valuable model to detect hepatotoxicity (McGrath and Li, 2008; Hill et al., 2012; Alderton et al., 2010; Embry et al., 2010). Our zebrafish larvae model for Cu-induced hepatotoxicity, based on fluorescent observation of liver morphology, was indeed confirmed by liver histology. Noteworthy is that changes in liver histology specific for advanced states of WD (interface hepatitis, Mallory bodies, hepatocytes with glycogenated nuclei (Ala et al., 2007; Honma et al., 2011; Hayashi et al., 2012; Pilloni et al., 1998, 2004) were absent in zebrafish larvae livers. However, biopsies of early stage WD patient livers do not show such changes, suggesting that our model rather reflects the early stages in WD pathogenesis. In contrast to rodent models for WD and our zebrafish model for Cuinduced hepatotoxicity, a genetic zebrafish model for WD is to our knowledge not yet available. However, a zebrafish model for Menkes disease (MD), a copper-deficiency linked disorder (Tumer and Moller, 2010) due to mutations in the ATP7A gene encoding a Cu translocating ATPase (Vulpe et al., 1993), termed calamity, is available. This MD zebrafish model is characterized by splicing defects in the zebrafish
Fig. 6. OSIP108 prevents Cu-induced ROS production in zebrafish larvae. In vivo ROS production in zebrafish larvae was assessed by H2DCFDA staining and fluorescence stereomicroscopy. (a) Left panels are brightfield images and right panels are corresponding fluorescence photomicrographs as observed upon H2DCFDA excitation by the GFP channel. White arrow heads indicate background fluorescence observed in all evaluated zebrafish larvae. (b) Fluorescence in the liver region, as indicated by circles in the right panel of a, was quantified by ImageJ software and is represented as the ratio of mean fluorescence as compared to the mean fluorescence of non-Cu-treated control larvae. For every condition, 12 larvae in total were evaluated on two biological repeats. Brightness and contrast of all fluorescence images were processed in a similar way. (*P b 0.05; Student t-test.)
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orthologue of the ATP7A gene with embryonic zebrafish mutants showing a similar phenotype as MD patients (Mendelsohn et al., 2006). In terms of Cu toxicity, reports in literature describe that Cu accumulates in the gills and liver of adult zebrafish and zebrafish larvae even upon incubation with low Cu concentrations (6.75 μM) (Chen et al., 2011; Leung et al., 2014; Craig et al., 2009). Zebrafish exposed to Cu display an increased mRNA expression of genes encoding Cu efflux pumps and cytoplasmic Cu-capturing proteins such as ATP7A, ATP7B, ATOX1 and metallothionein 2 (MT2) in the gills, intestine, kidney and liver while that of Cu importer protein CTR1 remains stable (Hill et al., 2012; Chen et al., 2011). Similarly, an additional report also showed an increased gene expression pattern of those Cu transporter proteins in the zebrafish hepatocyte cell line ZFL and zebrafish larvae at 3 dpf (Berghmans et al., 2008). In contrast to zebrafish, CTR1 expression increased when ZFL or larvae were exposed to Cu. These results implicate that Cu overload in zebrafish gills and liver could be explained by the elevated levels of ATOX1, while the expression of CTR1 was unaffected or increased. Given that ATOX1 is a cytosolic chaperone that binds Cu and transports it to ATP7A and ATP7B in the trans-Golgi network, increased levels ATOX1 are required to capture the accumulated Cu. While the expression of the importer protein CTR1 was not down-regulated, increased levels of Cu are expected to enter tissues and accumulate. Consequently, although the expression of genes encoding MT2, ATP7A and ATP7B is upregulated (Hill et al., 2012; Chen et al., 2011), excessive Cu subsequently gives rise to Cu toxicity. Furthermore, in zebrafish larvae exposed to excess Cu, the expression of the gene encoding Cu/Zn superoxide dismutase increases, which implies that the mechanism related to Cu-induced hepatotoxicity is via the generation of reactive oxygen species (Berghmans et al., 2008). Likewise, Cu was shown to induce oxidative stress and alter mitochondrial properties in zebrafish livers (Craig et al., 2007). Indeed, our results indicate that Cu induces the production of ROS in zebrafish larvae, specifically also the liver, and that this effect is abrogated upon injection with OSIP108. As OSIP108 prevents Cu-induced apoptosis, decreases Cuinduced oxidative stress, preserves mitochondrial ultrastructure upon Cu overload, and affects sphingolipid homeostasis (Spincemaille et al., 2014), this suggests that OSIP108, at least regarding oxidative stress, directly affects known Cu-induced events associated with WD (Rosencrantz and Schilsky, 2011; Zischka and Lichtmannegger, 2014; Zischka et al., 2011; Seth et al., 2004; Sokol et al., 1994; Gaetke and Chow, 2003; Lang et al., 2007; Spincemaille et al., 2014) in zebrafish larvae. Given the efficacy of OSIP108 in our zebrafish model for Cu-induced hepatotoxicity, this peptide is a promising lead molecule for further development as a treatment for human diseases involving Cu toxicity such as WD. In addition to neurodegeneration in the context of WD, the finding that OSIP108 increases the tolerance of glioblastoma cells to Cu is of interest since Cu dysregulation is recognized as a key player in additional neurodegenerative diseases such as Alzheimer disease and Parkinson disease (Scheiber et al., 2013). Hence, a putative application of OSIP108 in the context of Cu-induced toxicity is not restricted to WD. In conclusion, in the present study we report on the protective effect of OSIP108 on Cu-induced toxicity in mammalian cell models of WD. Moreover, these data could be translated to a newly established in vivo zebrafish model for Cu intoxication. Our results show that OSIP108 could be a new candidate for treatment of WD. For practical applications in e.g. WD, the bioavailability and efficacy of the OSIP108 peptide have to be determined in different organs, including the liver and brain, for instance by oral and intravenous administration of OSIP108 in rodent models for WD (Mori et al., 1994; Theophilos et al., 1996).
Conflicts of interest The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by grants from FWO-Vlaanderen (G.A062.10N and G.0414.09) and ‘Bijzonder Onderzoeksfonds KU Leuven’ (GOA/2008/11). P.S. is supported by IWT-Vlaanderen (IWT 101449); D-H.P. by a doctoral scholarship of the KU Leuven (DBOF); G.C. by FP7-PEOPLE (grant 247506); D.C. by FWO-Vlaanderen as a fundamental-clinical researcher (grant 1830412N); K.T. and C.V.E. hold an Industrial Research Fund (IOF) mandate of the KU Leuven. References Ala, A., Walker, A.P., Ashkan, K., Dooley, J.S., Schilsky, M.L., 2007. Wilson's disease. Lancet 369 (9559), 397–408. http://dx.doi.org/10.1016/S0140-6736(07)60196-2. Alderton, W., Berghmans, S., Butler, P., Chassaing, H., Fleming, A., Golder, Z., Richards, F., Gardner, I., 2010. Accumulation and metabolism of drugs and CYP probe substrates in zebrafish larvae. Xenobiotica 40 (8), 547–557. http://dx.doi.org/10.3109/ 00498254.2010.493960. 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The plant decapeptide OSIP108 prevents copper-induced toxicity in various models for Wilson disease Pieter Spincemaille a,1, Duc-Hung Pham b,1, Gursimran Chandhok c, Jef Verbeek d, Andree Zibert c, Louis Libbrecht d,e, Hartmut Schmidt c, Camila V. Esguerra b, Peter A.M. de Witte b, Bruno P.A. Cammue a,f,⁎, David Cassiman d, Karin Thevissen a a
Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium Laboratory for Molecular Biodiscovery, KU Leuven, Campus Gasthuisberg, Herestraat 49, O&N2, 3000 Leuven, Belgium c Clinic for Transplantation Medicine, Münster University Hospital, Albert-Schweitzer-Campus 1, Building A14, D-48149 Münster, Germany d Department of Hepatology and Metabolic Center, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium e Department of Pathology, University Hospital Ghent, De Pintelaan 185, 9000 Ghent, Belgium f Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Ghent, Belgium b
a r t i c l e
i n f o
Article history: Received 5 May 2014 Revised 30 July 2014 Accepted 5 August 2014 Available online 16 August 2014 Keywords: Wilson disease Copper OSIP108 Zebrafish Hepatotoxicity
a b s t r a c t Background: Wilson disease (WD) is caused by accumulation of excess copper (Cu) due to a mutation in the gene encoding the liver Cu transporter ATP7B, and is characterized by acute liver failure or cirrhosis and neuronal cell death. We investigated the effect of OSIP108, a plant derived decapeptide that prevents Cu-induced apoptosis in yeast and human cells, on Cu-induced toxicity in various mammalian in vitro models relevant for WD and in a Cutoxicity zebrafish larvae model applicable to WD. Methods: The effect of OSIP108 was evaluated on viability of various cell lines in the presence of excess Cu, on liver morphology of a Cu-treated zebrafish larvae strain that expresses a fluorescent reporter in hepatocytes, and on oxidative stress levels in wild type AB zebrafish larvae. Results: OSIP108 increased not only viability of Cu-treated CHO cells transgenically expressing ATP7B and the common WD-causing mutant ATP7BH1069Q, but also viability of Cu-treated human glioblastoma U87 cells. Aberrancies in liver morphology of Cu-treated zebrafish larvae were observed, which were further confirmed as Cuinduced hepatotoxicity by liver histology. Injections of OSIP108 into Cu-treated zebrafish larvae significantly increased the amount of larvae with normal liver morphology and decreased Cu-induced production of reactive oxygen species. Conclusions: OSIP108 prevents Cu-induced toxicity in in vitro models and in a Cu-toxicity zebrafish larvae model applicable to WD. General significance: All the above data indicate the potential of OSIP108 as a drug lead for further development as a novel WD treatment. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Wilson disease (WD), a rare (1/30.000) autosomal recessive disorder of copper (Cu) balance (Loudianos and Gitlin, 2000; Huster, 2010), is caused by mutations in the ATP7B gene. ATP7B encodes a Cutransporting protein ATPase (Bull et al., 1993) and is predominantly expressed in hepatocytes in which it localizes to the trans-Golgi network (Lutsenko et al., 2002). ATP7B plays an essential role in Cu excretion from hepatocytes into bile and for mobilization of ceruloplasmin-bound Cu from hepatocytes into the serum (Huster, 2010). ATP7B mutations in ⁎ Corresponding author at: Centre for Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium. Fax: +32 16321966. E-mail address: [email protected] (B.P.A. Cammue). 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.taap.2014.08.005 0041-008X/© 2014 Elsevier Inc. All rights reserved.
WD patients cause accumulation of Cu in the liver (Ferenci, 2006) that can result in acute liver failure (ALF) or cirrhosis (Ala et al., 2007). Furthermore, elevated intracellular Cu levels cause degeneration of neuronal cells, which is also a WD-characteristic (Loudianos and Gitlin, 2000; Huster, 2010). Cu toxicity in hepatocytes of WD patients and WD animal models arises from a direct effect of Cu on the mitochondria, ultimately amounting to tissue damage in the liver (Rosencrantz and Schilsky, 2011; Zischka and Lichtmannegger, 2014). Cu-induced mitochondrial dysfunction and damage have been ascribed to a Cu-induced (i) deficiency in the mitochondrial respiratory chain, at the level of the Cu-dependent complex IV (Roberts et al., 2008); (ii) cross-linking of mitochondrial membranous proteins and subsequent contraction of the membrane as observed ultrastructurally in the livers of WD patients and rodent models (Zischka et al., 2011); (iii) oxidative stress (Seth
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et al., 2004; Sokol et al., 1994; Gaetke and Chow, 2003) and (iv) apoptosis by inducing acid sphingomyelinase (aSMase) activity, thereby altering the lipid composition of membranes and increasing the production of the apoptosis inducer ceramide (Lang et al., 2007). Current treatment options for WD are aimed at decreasing gastrointestinal Cu uptake by administration of zinc or by increasing Cu excretion via chelating agents such as D-penicillamine, trientine or tetrathiomolybdate (Smolarek and Stremmel, 1999). In the long run, these treatments are helpful in the majority of patients (Lowette et al., 2010), but none of the available drugs constitute causal treatment. Moreover, side effects such as neurological deterioration, nephrotoxicity and generation of resistance have been reported (Rodriguez et al., 2012; Czlonkowska et al., 1996). These limitations all implicate a remaining window of opportunity for novel compounds directly targeting Cu toxicity in WD. Using Tiling Array technology, we previously identified a decapeptide in the plant Arabidopsis thaliana upon treatment with the herbicide paraquat (PQ) (De Coninck et al., 2013). PQ is known to induce reactive oxygen species (ROS) (Farrington et al., 1973). This peptide, termed OSIP108 (MLCVLQGLRE), can increase tolerance of plant and yeast cells to oxidative stress agents like PQ and H2O2 respectively. Overexpression of the OSIP108-encoding sequence in the yeast Saccharomyces cerevisiae resulted in a significantly increased tolerance to H2O2, while exogenous application of OSIP108 to H2O2-treated yeast also resulted in significantly increased survival (De Coninck et al., 2013). By assessing specific markers for oxidative stress and apoptosis, we very recently demonstrated that OSIP108 indeed prevented Cu-induced oxidative stress and apoptosis in yeast and human cells (Spincemaille et al., 2014). In the present study, we investigated the effect of OSIP108 on Cu toxicity in in vitro mammalian cell lines and in an in vivo zebrafish model relevant for WD. All data point to the potential of OSIP108 as a lead for development of novel treatment options for WD. 2. Materials and methods 2.1. Ethics. All zebrafish experiments carried out were approved by the Ethics Committee of the University of Leuven (approval number P05090) and by the Belgian Federal Department of Public Health, Food Safety & Environment (approval number LA1210199). 2.2. Materials and cell lines. Chinese hamster ovary (CHO) cells and human glioblastoma U87 cells were obtained from ATCC (Rockville, MD, USA) and grown in DMEM F12 (Lonza) and DMEM High Glucose (GE Healthcare) supplemented with 10% fetal calf serum, 2 mM Lglutamine, 100 U/ml penicillin and 100 μg/ml streptomycin respectively. CHO cells were transduced by retroviral vectors expressing the coding region of human ATP7B or mutant p.H1069Q (Sauer et al., 2010). OSIP108 (MLCVLQGLRE, 1161 g/mol) was purchased from Thermo Fisher Scientific (Ulm, Germany) and dissolved in DMSO. Copper sulfate (CuSO4) and copper chloride (CuCl2) (Cu) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.3. Cellular Cu toxicity experiments. Transgenic CHO or U87 cells were seeded in triplicate at 104 cells/well in a 96-well plate. Following attachment, cells were treated with 0.75 mM Cu in the presence of control (2% DMSO) or 100 μM OSIP108. After 48 h incubation, cell viability was determined by MTT staining as described previously (Siaj et al., 2012). 2.4. Zebrafish Cu toxicity experiments. The transgenic zebrafish (Danio rerio) line zTgfabp10a-DsRed:nacre was generated using the liverspecific zebrafish fabp10a promoter (Her et al., 2003), driving the fluorescent reporter gene DsRed. Zebrafish husbandry, embryo collection, and embryo and larval maintenance were performed as described (Westerfield, 1994; Nusslein-Volhard CaD, 2002). For all experiments, 4 days post-fertilization (dpf) larvae that showed consistent fluorescent
levels in the liver were selected. OSIP108 (2.5 mg/kg–5 mg/kg) was co-injected with Rhodamine B Dextran as an injection marker (10% g/v) into the bloodstream via the Duct of Cuvier (Isogai et al., 2001) prior to incubation of the zebrafish larvae in Cu-containing medium (10 μM–100 μM) for three days. As control treatment, larvae were injected with 10% Rhodamine B Dextran. At 7 dpf, larvae liver phenotypes were scored using fluorescence stereomicroscopy (Leica MZ 10F). Liver morphology was not affected by injection. 2.5. Zebrafish histology. Zebrafish larvae were incubated or not (control) with Cu-containing medium (50 μM). Ten larvae within each group were fixed in 10% neutral formalin buffer before aligning two arrays of five parallel larvae in an agarose block. These blocks were processed manually through a series of 70% ethanol, 100% xylene and paraffin as described (Sabaliauskas et al., 2006). Next, blocks were cut sagittally into slides of 7 μM thickness by a microtome (Microm HM 360, Thermo Scientific, Belgium) and adhered onto glass slides. Glass slides with larvae were stained with hematoxylin and eosin (H&E) and imaged by microscopy. A certified pathologist performed histopathological assessment of zebrafish larval livers. All zebrafish larvae were alive when processed for the evaluation of liver pathology. 2.6. Detection of oxidative stress markers in zebrafish larvae. As the transgenic line zTgfabp10a-DsRed:nacre exhibits high fluorescence levels in the liver, we used the wild-type AB zebrafish line to determine Cuinduced ROS production by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining. Wild type AB zebrafish larvae (4 dpf) were injected with 10% Rhodamine B Dextran (control) or 5 mg/kg OSIP108 prior to incubation in Cu-containing growth media (50 μM). After 4 h of incubation, H2DCFDA (1 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA) was added to the growth medium. Subsequently, 5 dpf larvae were washed carefully with embryo medium (1.5 mM HEPES, pH 7.6, 17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO4, and 0.18 mM Ca(NO3)2) and immobilized in 3% methylcellulose. Images of live samples were captured by fluorescence stereomicroscopy (Leica MZ 10F). Fluorescence levels in the liver region were quantified using ImageJ software. Correction for fluorescence levels was made by subtracting auto-fluorescence values of non-H2DCFDAexposed control larvae. 2.7. Statistical analysis. All values are presented as mean with standard error (SEM). Data were analyzed by GraphPad Prism 6 software. P b 0.05 was considered as statistically significant. 3. Results 3.1. OSIP108 increases Cu tolerance of CHO cells expressing H1069Q In the present study we investigated the effect of the anti-apoptotic plant peptide OSIP108 (MLCVLQGLRE) (De Coninck et al., 2013; Spincemaille et al., 2014), in in vitro models and in a zebrafish model relevant for WD. In the first step, we tested the effect of OSIP108 on Cu tolerance of a Chinese hamster ovary (CHO) cell line, which lacks intrinsic expression of the WD disease gene ATP7B (Forbes and Cox, 2000). This cell line is highly sensitive to Cu and was previously used as a cell model to study ATP7B mutants (La Fontaine et al., 2001). The ATP7B mutation H1069Q is the predominant mutation found in WD patients of various regions in Europe (Ferenci, 2006). We first evaluated the Cu sensitivity of CHO cell lines and found that the minimal Cu dose that induces at least 50% cell death was 0.75 mM Cu. Likewise, we previously studied the effect of different OSIP108 doses on HepG2 viability in the presence of toxic Cu, and found that 100 μM OSIP108 proved maximally efficient (Spincemaille et al., 2014). Hence, to investigate the effect of OSIP108 on acute Cu-induced toxicity of CHO cell lines, we chose to use a Cu dose of 0.75 mM and OSIP108 dose of 100 μM. Addition of 100 μM OSIP108 to CHO cell lines expressing either ATP7B
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or ATP7BH1069Q cDNA significantly increased cell viability upon coincubation with 0.75 mM Cu (Fig. 1): viability of CHO cells expressing ATP7B or ATP7BH1069Q cDNA in the presence of 0.75 mM Cu was 18% and 16% respectively, and increased to 38% and 28%, respectively upon coincubation with 100 μM OSIP108. Note that in the absence of the peptide, toxicity of 0.75 mM Cu was similar in CHO cell lines expressing wild type ATP7B or the relative mild mutant ATP7B H1069Q cDNA (Gromadzka et al., 2006), while the rescuing capacity of OSIP108 was higher in CHO cells expressing wild type ATP7B cDNA. These data indicate that OSIP108 can significantly increase the Cu tolerance of CHO cell lines in both wild type and mutant ATP7B expressing lines albeit that the absolute level of recovery by the peptide may be higher for wild type ATP7B. In addition, 100 μM OSIP108 was also effective at a broader range of Cu (0.25 mM– 0.75 mM) in CHO cells expressing wild type ATP7B cDNA (data not shown). 3.2. OSIP108 prevents Cu-induced cell death in human U87 glial cells Next, we evaluated the effect of OSIP108 on Cu tolerance of the human U87 glioblastoma cell line. In addition to Cu-induced cirrhosis in WD, progressive degeneration of basal ganglia in the brain is also a prominent clinical feature of WD (Loudianos and Gitlin, 2000; Huster, 2010). U87 cells have been studied in the context of Cu-induced toxicity in glioblastoma cells (Li et al., 2013) as well as in Cu-induced astrocyte apoptosis during the progression of WD (Merker et al., 2005). Incubation of U87 cells with 0.75 mM Cu decreased cell viability to 10% while OSIP108 treatment (100 μM) significantly increased cell viability to 39% (Fig. 2). These data indicate that OSIP108 can also prevent Cuinduced cell death of human glioblastoma cells. 3.3. Cu induces hepatotoxicity in zebrafish larvae In an effort to translate our in vitro data to an in vivo model relevant for WD, we developed a zebrafish larvae model for Cu-induced liver damage. To this end, a zebrafish transgenic line was generated that specifically expresses the gene encoding the fluorescent DsRed reporter in hepatocytes. This allowed us to observe liver morphology of zebrafish larvae by fluorescence stereomicroscopy. Upon incubation with Cu, we observed a range of aberrant liver phenotypes including reduced and enlarged livers (Fig. 3a), both denoted from hereon as ‘aberrant’. We found that the amount of larvae with aberrant liver phenotypes significantly increased by addition of 50 μM Cu (up to 80%) and 100 μM Cu (up to 63%) as compared to control larvae (approx. 20% of the population) (Fig. 3b). Vice versa, the amount of larvae with normal liver
Fig. 2. OSIP108 reduces Cu sensitivity of human U87 cells. U87 cells were incubated with 0.75 mM Cu in the absence or presence of 100 μM OSIP108. Cell viability was determined by MTT assay as compared to cells receiving no Cu. Mean and SEM of six experiments are shown. (***P b 0.001; Student t-test.)
shape and size was significantly reduced when larvae were treated with 50 μM or 100 μM Cu compared to control treatment (Fig. 3b). At 100 μM Cu we observed that approx. 23% of larvae died, but this effect was not statistically significant. However, treatment with 150 μM and 200 μM Cu significantly increased death of the population to approx. 80% and 100% of the larvae population, respectively (data not shown). These results indicate that Cu affects liver morphology of zebrafish larvae as observed by fluorescence stereomicroscopic analysis. In order to validate our larval model for Cu-induced hepatotoxicity, we performed histological analysis of larvae treated with or without Cu. We chose the least toxic Cu concentration (50 μM) that induced significant differences between control treated larvae and Cu-treated larvae. Larval zebrafish phenotypic assays to detect hepatotoxicants starting from 3 days post-fertilization (dpf) have been described (He et al., 2013; McGrath and Li, 2008). Since the left liver lobe is present and functioning at 4 dpf (Chng et al., 2012; Korzh et al., 2008), and we were able to locate fluorescent livers with a consistent fluorescent signal at 4 dpf, the latter time point was chosen to initiate Cu exposure. Zebrafish livers not exposed to Cu showed hepatocytes with homogenous eosinophilic cytoplasm and recognizable nuclei of non-parenchymal liver cells (Figs. 4a,c). The livers of Cu-exposed zebrafish larvae consistently showed larger hepatocytes, which was evident from the decreased nucleocytoplasmatic index (i.e. the ratio between nuclear size and hepatocyte size) (Figs. 4b,d). The cytoplasm of Cu-exposed zebrafish hepatocytes showed uniform eosinophilic clumping. There was a clear decrease in the number of nuclei from non-parenchymal liver cells in the Cu-exposed zebrafish larvae. These results show that Cu indeed affects the hepatocytes of zebrafish and that the observed liver fluorescence of zebrafish larvae (Figs. 3a,b) correlates with the histological analyses of the liver (Figs. 4a–d).
3.4. OSIP108 prevents Cu-induced hepatotoxicity in zebrafish
Fig. 1. OSIP108 reduces Cu sensitivity of CHO cells. CHO cells expressing ATP7B wild type (‘ATP7B’) or mutation p.H1069Q (‘ATP7BH1069Q’) were incubated with 0.75 mM Cu in the absence (black bars) or presence of 100 μM OSIP108 (gray bars). Cell viability was determined by MTT assay and calculated as compared to cells receiving no Cu. Mean and SEM of three experiments are shown. (**P b 0.01; ***P b 0.001; Student t-test.)
As the above described zebrafish model can be used to determine liver aberrancies, we evaluated the effect of OSIP108 injections on Cu-induced aberrancies in liver morphology of zebrafish larvae. We found that injection of untreated control larvae with different doses of OSIP108 (2.5 mg/kg–5 mg/kg) did not affect normal liver morphology (Fig. 5), while OSIP108 (5 mg/kg) injection in the bloodstream of 50 μM Cu-treated larvae significantly increased the number of larvae with normal livers (Fig. 5). Interestingly, 2.5 mg/kg OSIP108 did not result in such effect, indicating that a minimal OSIP108 dose is required to observe this protective effect. These data indicate that OSIP108 can
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protect against Cu-induced changes in liver morphology in zebrafish larvae, thereby preventing Cu-induced hepatotoxicity.
3.5. OSIP108 prevents Cu-induced hepatotoxicity in zebrafish larvae by reducing oxidative stress
Fig. 3. Cu induces liver damage in zebrafish larvae. (a) Fluorescent visualization of liver morphology. Each treatment condition was conducted with 10–20 larvae. (b) Zebrafish larvae were treated with control (distilled water, C.) or different Cu doses (10 μM– 100 μM). Liver morphology was assessed by fabp10a.DsRed expression in the liver. Normal (black bars) or aberrant (comprises enlarged and reduced liver phenotypes (as defined in (a), gray bars)) liver phenotypes were determined. Mean and SEM of at least 3 biological repeats are shown. (**P b 0.01; ***P b 0.001; ANOVA test using Tukey corrections.)
To gain insights into the underlying mechanistic events characteristic for Cu-induced toxicity and changes in liver morphology in zebrafish larvae, as well as in the protective effect of OSIP108, we evaluated Cuinduced ROS production in zebrafish larvae by H2DCFDA staining in the absence (control) or presence of OSIP108 and Cu. In all larvae, including the non-Cu-treated controls, we observed fluorescence signals in larvae within the gut and intestine (Fig. 6a). This background signal might reflect natural oxidative stress processes (Kim et al., 2014). Larvae treated with Cu (50 μM), however, showed much higher levels and area of fluorescence. More specifically, the fluorescence was localized in specific regions such as the liver, heart, spinal cord and brain (Fig. 6a), indicating that Cu-induced ROS production is widespread in zebrafish larvae. In addition, by assessing fluorescence levels in the liver region with ImageJ software, we observed that Cu-treated larvae exhibited significantly increased fluorescence levels as compared to control treated larvae (fluorescence units 5.21 and 2.83, respectively), indicating Cu-induced ROS production in the livers of zebrafish larvae. In contrast, injection of larvae with OSIP108 (5 mg/kg) in the presence of Cu (50 μM) abrogated this effect as the fluorescence intensity at different areas, including the liver region, returned to the level of the controls (Fig. 6a). Quantification of this effect confirmed that OSIP108 significantly decreased fluorescence levels, expressed as the ratio of fluorescence levels of OSIP108-injected larvae to non-Cu-treated control (Fig. 6b), as compared to larvae injected with control, and thus indicates decreased Cu-induced ROS production. Taken together, these results point to the ability of OSIP108 to prevent Cu-induced ROS production in zebrafish larvae, including the liver.
Fig. 4. Cu induces histological changes in zebrafish larvae livers. Liver histology of zebrafish larvae treated without Cu (a, c) or 50 μM Cu (b, d). Representative H&E-stained images of 10 larvae per condition. Magnification (zebrafish larvae 7 dpf) is 4 ×, inset (liver) is 40×; panels c & d are at maximum magnification, scale bar = 100 μm.
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Fig. 5. OSIP108 protects zebrafish against Cu-induced liver damage. Total amount of larvae with normal liver upon treatment without (0 mg/kg) or with OSIP108 (2.5 mg/kg–5 mg/kg) in the absence or presence of 50 μM Cu. For each condition, 10–20 larvae were evaluated. Mean and SEM of at least 3 biological repeats. (**P b 0.01; ***P b 0.001; ANOVA test using Tukey corrections.)
4. Discussion We show here that the plant-derived peptide OSIP108 (De Coninck et al., 2013), which was previously shown to prevent Cu-induced apoptosis in yeast and human cells (Spincemaille et al., 2014), protects against Cu-induced toxicity in various in vitro mammalian cell models and in a newly developed zebrafish model for Cu-induced hepatotoxicity. In the recent past, several assays have been developed to detect drug-induced toxicity in zebrafish and previous studies also reported a perfect match between visual observation of zebrafish livers and hepatotoxicity based on quantitative image analysis and liver histology of zebrafish treated with known mammalian hepatotoxicants (He et al., 2013; McGrath and Li, 2008; Berghmans et al., 2008). Besides, combining high-content screening by using fluorescent markers in human hepatocytes or HepG2 cells, and zebrafish larvae to detect hepatotoxicants in early stages of drug development has been suggested to be a powerful combination (Hill et al., 2012). In addition, the ability to identify toxic metabolites, potential drug–drug interactions via liver enzyme induction or inhibition and liver pathologies allows
zebrafish to serve as a valuable model to detect hepatotoxicity (McGrath and Li, 2008; Hill et al., 2012; Alderton et al., 2010; Embry et al., 2010). Our zebrafish larvae model for Cu-induced hepatotoxicity, based on fluorescent observation of liver morphology, was indeed confirmed by liver histology. Noteworthy is that changes in liver histology specific for advanced states of WD (interface hepatitis, Mallory bodies, hepatocytes with glycogenated nuclei (Ala et al., 2007; Honma et al., 2011; Hayashi et al., 2012; Pilloni et al., 1998, 2004) were absent in zebrafish larvae livers. However, biopsies of early stage WD patient livers do not show such changes, suggesting that our model rather reflects the early stages in WD pathogenesis. In contrast to rodent models for WD and our zebrafish model for Cuinduced hepatotoxicity, a genetic zebrafish model for WD is to our knowledge not yet available. However, a zebrafish model for Menkes disease (MD), a copper-deficiency linked disorder (Tumer and Moller, 2010) due to mutations in the ATP7A gene encoding a Cu translocating ATPase (Vulpe et al., 1993), termed calamity, is available. This MD zebrafish model is characterized by splicing defects in the zebrafish
Fig. 6. OSIP108 prevents Cu-induced ROS production in zebrafish larvae. In vivo ROS production in zebrafish larvae was assessed by H2DCFDA staining and fluorescence stereomicroscopy. (a) Left panels are brightfield images and right panels are corresponding fluorescence photomicrographs as observed upon H2DCFDA excitation by the GFP channel. White arrow heads indicate background fluorescence observed in all evaluated zebrafish larvae. (b) Fluorescence in the liver region, as indicated by circles in the right panel of a, was quantified by ImageJ software and is represented as the ratio of mean fluorescence as compared to the mean fluorescence of non-Cu-treated control larvae. For every condition, 12 larvae in total were evaluated on two biological repeats. Brightness and contrast of all fluorescence images were processed in a similar way. (*P b 0.05; Student t-test.)
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orthologue of the ATP7A gene with embryonic zebrafish mutants showing a similar phenotype as MD patients (Mendelsohn et al., 2006). In terms of Cu toxicity, reports in literature describe that Cu accumulates in the gills and liver of adult zebrafish and zebrafish larvae even upon incubation with low Cu concentrations (6.75 μM) (Chen et al., 2011; Leung et al., 2014; Craig et al., 2009). Zebrafish exposed to Cu display an increased mRNA expression of genes encoding Cu efflux pumps and cytoplasmic Cu-capturing proteins such as ATP7A, ATP7B, ATOX1 and metallothionein 2 (MT2) in the gills, intestine, kidney and liver while that of Cu importer protein CTR1 remains stable (Hill et al., 2012; Chen et al., 2011). Similarly, an additional report also showed an increased gene expression pattern of those Cu transporter proteins in the zebrafish hepatocyte cell line ZFL and zebrafish larvae at 3 dpf (Berghmans et al., 2008). In contrast to zebrafish, CTR1 expression increased when ZFL or larvae were exposed to Cu. These results implicate that Cu overload in zebrafish gills and liver could be explained by the elevated levels of ATOX1, while the expression of CTR1 was unaffected or increased. Given that ATOX1 is a cytosolic chaperone that binds Cu and transports it to ATP7A and ATP7B in the trans-Golgi network, increased levels ATOX1 are required to capture the accumulated Cu. While the expression of the importer protein CTR1 was not down-regulated, increased levels of Cu are expected to enter tissues and accumulate. Consequently, although the expression of genes encoding MT2, ATP7A and ATP7B is upregulated (Hill et al., 2012; Chen et al., 2011), excessive Cu subsequently gives rise to Cu toxicity. Furthermore, in zebrafish larvae exposed to excess Cu, the expression of the gene encoding Cu/Zn superoxide dismutase increases, which implies that the mechanism related to Cu-induced hepatotoxicity is via the generation of reactive oxygen species (Berghmans et al., 2008). Likewise, Cu was shown to induce oxidative stress and alter mitochondrial properties in zebrafish livers (Craig et al., 2007). Indeed, our results indicate that Cu induces the production of ROS in zebrafish larvae, specifically also the liver, and that this effect is abrogated upon injection with OSIP108. As OSIP108 prevents Cu-induced apoptosis, decreases Cuinduced oxidative stress, preserves mitochondrial ultrastructure upon Cu overload, and affects sphingolipid homeostasis (Spincemaille et al., 2014), this suggests that OSIP108, at least regarding oxidative stress, directly affects known Cu-induced events associated with WD (Rosencrantz and Schilsky, 2011; Zischka and Lichtmannegger, 2014; Zischka et al., 2011; Seth et al., 2004; Sokol et al., 1994; Gaetke and Chow, 2003; Lang et al., 2007; Spincemaille et al., 2014) in zebrafish larvae. Given the efficacy of OSIP108 in our zebrafish model for Cu-induced hepatotoxicity, this peptide is a promising lead molecule for further development as a treatment for human diseases involving Cu toxicity such as WD. In addition to neurodegeneration in the context of WD, the finding that OSIP108 increases the tolerance of glioblastoma cells to Cu is of interest since Cu dysregulation is recognized as a key player in additional neurodegenerative diseases such as Alzheimer disease and Parkinson disease (Scheiber et al., 2013). Hence, a putative application of OSIP108 in the context of Cu-induced toxicity is not restricted to WD. In conclusion, in the present study we report on the protective effect of OSIP108 on Cu-induced toxicity in mammalian cell models of WD. Moreover, these data could be translated to a newly established in vivo zebrafish model for Cu intoxication. Our results show that OSIP108 could be a new candidate for treatment of WD. For practical applications in e.g. WD, the bioavailability and efficacy of the OSIP108 peptide have to be determined in different organs, including the liver and brain, for instance by oral and intravenous administration of OSIP108 in rodent models for WD (Mori et al., 1994; Theophilos et al., 1996).
Conflicts of interest The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by grants from FWO-Vlaanderen (G.A062.10N and G.0414.09) and ‘Bijzonder Onderzoeksfonds KU Leuven’ (GOA/2008/11). P.S. is supported by IWT-Vlaanderen (IWT 101449); D-H.P. by a doctoral scholarship of the KU Leuven (DBOF); G.C. by FP7-PEOPLE (grant 247506); D.C. by FWO-Vlaanderen as a fundamental-clinical researcher (grant 1830412N); K.T. and C.V.E. hold an Industrial Research Fund (IOF) mandate of the KU Leuven. References Ala, A., Walker, A.P., Ashkan, K., Dooley, J.S., Schilsky, M.L., 2007. Wilson's disease. Lancet 369 (9559), 397–408. http://dx.doi.org/10.1016/S0140-6736(07)60196-2. Alderton, W., Berghmans, S., Butler, P., Chassaing, H., Fleming, A., Golder, Z., Richards, F., Gardner, I., 2010. Accumulation and metabolism of drugs and CYP probe substrates in zebrafish larvae. Xenobiotica 40 (8), 547–557. http://dx.doi.org/10.3109/ 00498254.2010.493960. 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