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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Molecular mechanism on cadmium-induced activity changes of catalase and superoxide dismutase
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Jing Wang a , Hao Zhang a , Tong Zhang a , Rui Zhang a , Rutao Liu a,∗ , Yadong Chen b a Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment & Health, 27# Shanda South Road, Jinan 250100, Shandong Province, PR China b Laboratory of Molecular Design and Drug Discovery, School of Basic Science, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
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Article history: Received 1 June 2014 Received in revised form 23 August 2014 Accepted 23 February 2015 Available online xxx
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Keywords: Cadmium Oxidative stress Catalase and superoxide dismutase
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1. Introduction
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Cadmium contributes to adverse effects of organisms probably because of its ability to induce oxidative stress via alterations in activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), but their molecular mechanisms remain unclear. We investigated the molecular mechanism of CAT and SOD response under Cd-induced oxidative stress in the liver of zebrafish. The enzyme activity changes observed in vitro were consistent with those seen in vivo, indicating the direct interaction of CAT and SOD with Cd contributes to their activity change in vivo. Further experiments utilizing multiple spectroscopic methods, isothermal titration calorimetry and a molecular docking study were performed to explore the mechanism of molecular interaction of CAT and SOD with Cd. Different interaction patterns were found that resulted in misfolding and changed the enzyme activities. Taken together, we suggest the misfolding of CAT and SOD contributes to their activity change under Cd-induced oxidative stress in vivo. © 2015 Elsevier B.V. All rights reserved.
Cadmium (Cd) is a known environmental contaminant [1]. Prolonged and various application of Cd has resulted in large releases into the environment. Natural levels of Cd found in water bodies range from 0.1 to 10 nM; while Cd level is 0.2–15 g/g in the larvae of Chaoborus punctipennis [2]. Food and tobacco smoking are the main sources of Cd uptake for the non-occupationally exposed population. For the age of 40–50 group, it is estimated that Cd concentration is 1.4–2 g/g and 26–70 g/g in the liver and kidney cortex of human subjects [3]. And human exposure to Cd is predicted to continue to increase in the next decades. Significant attention has been paid to the multiple toxic effects of Cd on human health due to its common presence in the environment. Cd has been officially listed as a potential carcinogenic agent by the International Agency for Research and banned by the European Union’s Restriction on Hazardous Substances directive [4]. Growing evidence indicates that the accumulation of Cd has a significant negative impact on organisms via the induction of oxidative stress mediated by the excessive generation of reactive oxygen species (ROS). Previous investigations have suggested multifactorial mechanisms responsible for Cd-induced oxidative stress.
∗ Corresponding author. Tel.: +86 531 88364868; fax: +86 531 88364868. E-mail address: [email protected] (R. Liu).
Generally, as a redox-inactive metal, Cd depletes the cell’s major antioxidants and causes damage to antioxidant enzymes, resulting in ROS accumulation [5]. Furthermore, excessive ROS can overwhelm cell’s intrinsic antioxidant defenses and attack biomolecules including lipids, proteins and DNA, causing lipid peroxidation, protein oxidation and DNA damage [5,6]. The role of antioxidant enzymes has been recognized as an important factor in Cd-induced oxidative stress. Catalase (CAT) and superoxide dismutase (SOD) are essential for maintaining the level of ROS in organisms and are used as biomarkers to indicate ROS production [7,8]. Inactivation of CAT and SOD will allow the generation of excess ROS and then induce oxidative stress in organisms [9]. Conflicting results have been reported on the activity changes of CAT and SOD involved in Cd-induced oxidative stress in vivo and in vitro. López et al. found that in in vitro studies on rat cortical neuron cells SOD activity displayed a dose-dependent increase but CAT activity was not affected by Cd exposure [10]. Shi et al. examined CAT and SOD activity in the liver of Carassius auratus, and their results indicated that Cd inhibited CAT activity and increased SOD activity [11]. Studies by Hussain et al. and Casalino et al. showed that the activity of SOD was inhibited by Cd in the liver of rats both in vivo and in vitro due to direct interaction of Cd with the enzyme molecule [12,13]. Previous research has demonstrated that Cd induce oxidative stress by altering the activity of CAT and SOD both in vitro and in vivo. The modulation of the related genes and the direct
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interaction of Cd with the enzyme might account for the activity change of the enzyme [14,15]. However, the underlying molecular mechanisms have not been fully elucidated [16]. A number of in vitro studies have utilized multiple spectroscopic methods to study the molecular mechanism of the interaction between the enzyme and xenobiotics. Yet, the molecular mechanism of the interaction of Cd with CAT and SOD has not been reported. Zebrafish (Danio rerio) is a popular model in many areas of vertebrate research instead of mammalian model because of its low cost and ease of manipulation. A number of studies have utilized zebrafish as a model system for studying Cd-induced oxidative stress both in vitro and in vivo [14,15,17] due to its capacity to sustain soft water [18], which is a basic requirement for testing biological response of Cd to avoid the interference with other cation [19]. The liver is the primary target organ for Cd accumulation; and it can be critically damaged by chronic Cd exposure from food [20]. The hepatotoxicity of Cd has been extensively studied and widely reported in occupationally and environmentally exposed populations, as well as in various experimental models. Therefore, the liver of zebrafish was selected as a model in this study. This study introduced a combined in vitro and in vivo method to clarify the molecular mechanisms for the alterations of CAT and SOD activity under Cd-induced oxidative stress in the liver of zebrafish. To assess the physiological responses under oxidative stress, we monitored the content of reduced glutathione (GSH), oxidized glutathione (GSSG) and malondiadehyde (MDA), and the activity of CAT and SOD in the liver of zebrafish after exposure to Cd. To evaluate whether the direct interactions of CAT and SOD molecules with Cd resulted in their activity change in vivo, we performed enzyme activity assays on CAT and SOD in vitro. The molecular mechanism of the interaction between Cd and the two antioxidant enzymes was further explored by multiple spectroscopic methods, isothermal titration calorimetry and a molecular docking study. Our study demonstrated that the direct interaction of Cd with CAT and SOD in vitro contributed to the enzyme activity change under Cd-induced oxidative stress in vivo. Different interaction patterns were found that resulted in misfolding and the different activity change of CAT and SOD. Taken together, the activity changes of CAT and SOD in vivo are attributed to the misfolding of the enzymes upon Cd binding. This study can help to clarify the mechanism of oxidative stress and biomacromolecule response induced by heavy metal exposure.
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Cd(NO3 )2 ·4H2 O was purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). CAT from beef liver was obtained from Sigma (USA). Cu/Zn SOD from pig blood was purchased from Biodee Biotechnology Co. (Beijing, China). Tris–HCl (NaCl) buffer (0.02 M, pH = 7.4) was used to control pH in the in vitro experiments. For the in vivo experiment, 0.01 M phosphate buffer saline (PBS, pH = 7.4) was used as the homogenization medium. All reagents were of analytical grade. Ultrapure water (18.25 M cm) was used throughout the experiments. 2.2. In vivo experiment 2.2.1. Model organism and Cd treatment The present study was conducted in accordance with the Guiding Principles outlined in the Use of Animals in Toxicology adopted by the Society of Toxicology in 1989. Adult zebrafish were purchased from the Boning Aquarium market (Jinan, China) and acclimatized in dechlorinated tap water with a natural light/dark
cycle of 12 h:12 h at 26 ± 1 ◦ C [21] in the environmental chamber before experimentation. Zebrafish were fed twice daily with brine shrimp (Artemia) and starved 24 h before exposure to avoid fecal interference in the assay. The water samples used in the current study were filtered prior to use. The pH of the water was 7.4 ± 0.2, and its electrical conductivity was 140 ± 10 S/cm. The dissolved oxygen in the water was 8.9 ± 0.6 mg/L. After adaptive feeding for 15 days, three experimental groups of adult zebrafish were exposed to Cd by adding a solution of Cd(NO3 )2 to each beaker (1 L) for 24 h. The water sample without Cd was set as the control group. The in vitro and in vivo exposure time were selected as 30 min [22,23] and 24 h [24,25] after consulting the literature, which is sufficient to induce oxidative stress and the biomarker changes. A number of recent studies used the concentration of 10% LC50 as the exposure concentration since zebrafish was not visibly affected by these concentrations. Cd concentration ranges from 1 to 50 M when exposing to short-term Cd exposure [14,24,26], in accordance with the naturally occurring level in the aquatic organism [2]. Although the environmental exposure of Cd is much less than the utilized concentration in the literature, Cd can accumulate in organisms through the bioconcentration of food chain after longterm exposure. In consideration of the above reason, we choose 5% of LC50 (10 M) and 20% of LC50 (40 M) [27] as the Cd exposure concentration in the current study to provide representative results. Each treatment was tested in triplicate. 2.2.2. Determination of SOD activity, GSH, GSSG and MDA content The assays of SOD activity, GSH, GSSG and MDA content in the liver of zebrafish were performed according to the protocol provided by the kits (Nanjing Jiancheng Bioengineering Institute, China). Liver tissues (2% w/v) of zebrafish were homogenized in PBS at 4 ◦ C and centrifuged at 13,000 × g for 30 min. The content of GSH and GSSG were measured by DTNB-GR recycling reaction [28]. Absorbance at 405 nm was measured on a Microplate Reader (Thermo Scientific). SOD activity assays were performed at 550 nm using modified xanthine–xanthine oxidase method [29]. MDA assay was performed using the thiobarbituric acid (TBA) colorimetric method (532 nm) [30]. Protein content was determined at 595 nm using bovine serum albumin as a standard [31]. Absorbance at 532 nm, 550 nm and 595 nm were measured on a UV-2450 spectrophotometer (Shimadzu, Japan) equipped with a 10-mm quartz cell. Results are expressed as the percentage of control group with the control group set as 100%. 2.2.3. Determination of CAT Activity CAT activity was measured in 3 mL reaction mixtures containing 150 L of liver homogenates, 0.01 M H2 O2 and 0.01 M PBS. The reaction was initiated by the addition of liver homogenates. The activity of CAT was evaluated by measuring the rate of decreased absorbance at 240 nm [32]. 2.3. In vitro experiments 2.3.1. Determinations of CAT and SOD activity The CAT/SOD–Cd solution was incubated for 30 min before the experiment. Both CAT and SOD activities were measured using the same methods as described for the in vivo experiment, with the exception that liver homogenates were replaced by the CAT/SOD–Cd solution and 0.01 M PBS was replaced by 0.02 M Tris–HCl buffer. 2.3.2. Isothermal titration calorimetry (ITC) The ITC experiment was carried out using a Microcal ITC200 microcalorimeter at 298 K. Typically, buffered enzyme solution was loaded in the calorimeter cell. The injection syringe was loaded
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with Cd(NO3 )2 solution. Following thermal equilibration at 298 K and an initial 1 min delay, the first drop (0.4 L) followed by 26 serial injections of 1.5 L every 2 min were made into the cell from the syringe. The stirring speed was 1000 rpm. Data analysis were performed using Origin 7.0 software (Microcal LLC).
2.3.3. UV–visible absorption measurement UV–visible absorption spectra were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan) equipped with a 10-mm quartz cell. The wavelength was recorded from 190 to 450 nm using a buffer solution containing the equivalent concentration of Cd as the reference.
2.3.4. Circular dichroism (CD) measurement CD spectra were recorded by a J-810 CD spectrometer (Jasco, Japan) using a quartz cell with a path length of 10 mm in a nitrogen atmosphere. The scanning speed was set at 200 nm min−1 . Each spectrum was the average of three successive scans. The secondary structure of proteins was calculated through CDPro software (available at http://lamar.colostate.edu/∼sreeram/CDPro/) [33].
2.3.5. Fluorescence measurements Fluorescence spectra were recorded on an F-4600 fluorophotometer (Hitachi, Japan) equipped with a 10-mm quartz cell and a 150 W xenon lamp. The voltage of photo multiplier tube was 600 V and the excitation and emission slit widths were set at 5.0 nm. The excitation wavelength was set at 280 nm. The scan speed was 1200 nm min−1 . The emission wavelength was recorded from 290 to 450 nm. Synchronous fluorescence spectra were measured at = 15 nm and = 60 nm. The wavelength was recorded from 240 to 320 nm. Time-resolved fluorescence measurements of the enzyme in the absence and the presence of Cd were performed (ex = 280 nm, em = 330 nm) on a FLS920 Combined Fluorescence Lifetime and Steady State spectrometer (Edinburgh, UK).
2.3.6. Molecular docking study The interaction of Cd with CAT and SOD was further confirmed by a molecular docking study using the Molecular Operating Environment (MOE) (Version 2009, Chemical Computing Group Inc., Canada) to determine the possible conformation and preferred binding sites of the enzyme by interacting with Cd. The crystallized structure of CAT (from bovine liver) (PDB code: 1TGU) and SOD (PDB code 2SOD) were downloaded from RSCB Protein Data Bank (http://www.pdb.org/). The 3D structure of the ligand was built using MOE-builder and then the ligand was energy minimized. Essential hydrogen atoms were added to the enzyme model. Gastiger charges were computed and applied to the enzyme model. Docking calculation was carried out using the MOE-Dock with settings: Placement: Triangle Matcher; Rescoring 1: London dG, Refinement: Forcefield. The pose with the best scoring conformer model was selected for further analysis. Interaction within these complexes was visualized using the MOE ligand electrostatic maps simulation.
2.3.7. Data analysis Results were presented as the mean of 3 replicates and reported as means ± SD (standard deviations). Data were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. p < 0.05 was considered as statistically significant.
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3.1. Determination of GSH/GSSG and MDA content
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Antioxidants and antioxidant enzymes are the main components of the antioxidant systems which can protect organisms against xenobiotics-induced oxidative stress [5]. It has been proposed that disturb of the antioxidant defense system will lead to a disturbed oxidant status and the increase of lipid peroxidation [5]. To assess the involvement of Cd-induced oxidative stress, we monitored the modulation of Cd on the level of the antioxidant GSH and its oxidized form GSSG (Fig. S1, Supplementary data). After 24 h exposure, no morbidity or mortality of the zebrafish was observed in any of the control and treatment groups. A decrease in GSH content (82.3% and 38.9% of the initial level) and an increase of GSSG content (257.1% and 380.2% of the initial level) was observed with the increasing exposure concentration, resulting in a significant decrease of GSH/GSSG ratio. Similar results was also reported in the study of Qu et al., in which C. auratus was exposed to 0.1 mg/L Cd for 12 days [34]. In addition to the oxidation of GSH to GSSG through direct neutralization with ROS [35], they suggested this compensatory response might result from the down-regulation of the transcription of the genes involved in GSH biosynthesis. These results suggested that an induction of a temporary shift toward a strongly oxidizing environment occurred within the liver of zebrafish, reflecting an insufficient resistant capacity of the liver to Cd exposure. Lipid peroxidation was assessed by the measurement of MDA concentration, which is linked to ROS production [5,36,37]. As shown in Fig. S1 (Supplementary data), in the liver tissues, exposure to Cd for 24 h generally induced an increase in MDA content (158.2% and 294.8% of the initial level) compared with the control group. Similar results were also obtained in other study of aquatic organisms [15,34]. This result indicated that excessive ROS attacked the membranes, resulting in lipid peroxidation. Altogether these results provided sufficient evidence that Cd altered the redox state and induced oxidative stress in the liver of zebrafish. 3.2. Determination of CAT and SOD activity Lipid peroxidation has been considered as the primary mechanism of Cd toxicity [5,36–38]. The activity change of CAT and SOD generally indicates that excessive ROS needs to be decomposed to maintain the balance of the cell’s intrinsic antioxidant defenses in organisms. Once the balance is disrupted, excessive ROS would attack lipids as manifested by the increased MDA level in our study. As illustrated in Fig. 1, after exposure to 10 M and 40 M Cd(NO3 )2 , the activity of CAT decreased to 77.5% and 40.3% of the initial level; while the activity of SOD increased to 106.3% and 162.0% of the initial level. Similar results were obtained in the study of Shi et al., in which inhibited CAT activity and increased SOD activity was observed in the liver of C. auratus after 24 h Cd (5 mg/L) exposure [11]. Conflicting results have been reported on the activity changes of CAT and SOD involved in Cd-induced oxidative stress in vivo and in vitro. Banni et al. found that CAT and SOD activity were both suppressed in the liver and ovary of zebrafish after 0.4 mg/L Cd exposure [15]. Zeng et al. found that CAT activity exhibited a bell-shaped response and SOD activity increased in a well linear relationship with MDA levels (r2 = 0.964) after Cd exposure in Phanerochaete chrysosporium [39]. These results suggested that the production of CAT and SOD were stimulated to eliminate the excessive ROS. And when the antioxidant system was unable to cope with the excessive ROS, CAT activity decreased and the linear relationship decreased (r2 = 0.873). It has been proposed that the changes of CAT and SOD activity are due to direct interaction
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Fig. 1. Activity changes of CAT and SOD in the liver of zebrafish and in vitro induced by exposure to Cd. Values are means ± SD (n = 3). Differences are considered statistically significant at *p < 0.05, **p < 0.01 and ***p < 0.001, respectively.
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of Cd with the enzyme molecule [12,13,40]. Zhu et al. observed down-regulation of the genes of CAT and SOD but inhibited SOD activity and increased CAT activity in the zebrafish liver cell line after Cd exposure [14]. Cd might directly interact with the antioxidant enzyme resulting in the alterations of the enzyme activity in spite of the down-regulation of the related genes. As Cd might change the activity of enzyme through directly interacting with the enzyme molecule, we investigated the activity change of CAT and SOD interacting with Cd in vitro. As illustrated in Fig. 1, CAT activity decreased to 71.6% and 57.7% of the initial level; while the activity of SOD increased to 184.5% and 215.7% of the initial level. The decline trend of CAT and the upward trend of SOD when interacting Cd in vitro was consistent with that in vivo, indicting that the activity change of CAT and SOD in Cd-induced oxidative stress in vivo was correlated with their direct interaction with Cd. To explore the molecular mechanism of the interaction of CAT and SOD with Cd, further experiments were performed utilizing multiple spectroscopic methods, isothermal titration calorimetry and a molecular docking study.
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3.3.1. Binding affinity The isothermal titration calorimetry (ITC) experiment was utilized to complete the thermodynamic characterization and quantify the stoichiometry and binding affinity associated with the binding process. The results of a typical titration of Cd into CAT and SOD at 298 K after correcting the heats of dilution are shown in Fig. 2. The number of binding sites (n), enthalpy change (H) and binding constant (K) were determined directly from the curvefitting. The experimental titration curve was best fitted with a single
set of binding sites for binding to CAT and two sets of binding sites for binding to SOD, indicating that there existed one type of binding site (nCd-CAT = 8.34 ± 0.434) and two types of binding sites (nCd-SOD,1 = 2.71 ± 0.171, nCd-SOD,2 = 10.7 ± 0.33), respectively. The changes in Gibbs free energy (G) and entropy change (S) were calculated through the equation: G = H − TS = −RTln K, where R is the gas constant and T is the thermodynamic temperature. The correlated parameters are listed in Table 1. The binding processes of CAT and SOD with Cd are both observed to be enthalpically favored and entropically opposed, indicating that the two interactions are mainly driven by electrostatic forces [41]. Negative values of G suggest that the two interaction processes are spontaneous. An order of KCd-CAT (103 ) suggested a weak binding affinity of CAT when interacting with Cd. Although this non-covalent binding process is weak, amounts and varieties of non-covalent bonds may jointly alter the conformation and function of proteins [42]. It is clearly shown that KCd-SOD of both sites were larger compared with KCd-CAT , indicating that the binding between Cd and SOD is comparably stronger than the binding between Cd and CAT. 3.3.2. UV–vis absorption spectroscopy UV–visible absorption spectroscopy was performed to explore the structural change of the enzymes and enzyme–ligand complex formation. The UV–vis absorption spectra of CAT and SOD in the presence and absence of Cd are presented in Fig. 3. Generally, proteins have two major absorption bands. The strong one from 200 to 230 nm reflecting the framework conformation of the protein is attributed to the electronic transitions of the polypeptide bonds →* of the peptide backbone C O [37]. The weaker one from 260 to 290 nm concerning aromatic amino acids
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Fig. 2. ITC profiles of the interaction between Cd with CAT and SOD. Top panels present raw data for the sequential titration of Cd solution into enzymes in Tris–HCl buffer (0.02 M, pH = 7.4) at 298 K. The bottom panels show the integrated heat results of the calorimetric titration after correction of heat of dilution against the molar ratio of Cd/enzyme; the solid line represents the best fit data (A) c(CAT) = 75 M, c(Cd) = 12 mM. (B) c(SOD) = 100 M, c(Cd) = 15 mM. Table 1 Binding parameters of the interaction between Cd and CAT, SOD. Enzyme CAT SOD
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H (cal mol−1 )
Binding site
N
K
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8.34 ± 0.434 2.71 ± 0.171 10.7 ± 0.330
2.69 ± 0.243 × 10 6.07 ± 1.47 × 105 1.02 ± 0.132 × 104 3
(Trp, Tyr, Phe) indicates the transformation of the chromophore microenvironment [43]. With gradual addition of Cd into CAT and SOD solution, the intensity of the peak at 215 nm decreased and was red shifted, indicating that the interaction between the enzyme and Cd led to a loosening and unfolding of the protein skeleton. The intensity of the peak at 280 nm increased in Fig. 3A, indicating that Cd caused reduced hydrophobicity of the microenvironment around the aromatic amino acids in CAT. SOD (Fig. 3B) has no significant absorbance peak at 280 nm because of the absence of Trp residues [44]. Additionally, CAT has another absorption band around 405 nm which is caused by the characteristic absorption of the porphyrinSoret band [45]. Heme groups are located at the active center and participate in the catalytic process. The intensity of the peak at 405 nm increased slightly with no shift, suggesting that Cd affected the activity of CAT but had no profound conformational change of the polypeptides around the heme groups. Since heme groups are deeply embedded 20 A˚ below the molecular surface and 23 A˚ from
−4.16 ± 0.273 × 10 −4.21 ± 0.467 × 102 −1.31 ± 0.0392 × 103 3
S (cal mol−1 K−1 )
G (kcal mol−1 )
1.75 25.0 14.0
−4.68 ± 0.273 −7.87 ± 0.0467 −5.48 ± 0.0392
the molecular center of the tetramer [46], we speculated that there was no direct interaction between Cd and the amino acid residues around the heme group due to steric hindrance [47]. 3.3.3. Circular dichroism spectroscopy CD spectroscopy is widely applied in many fields. Most notably, UV CD is used to investigate the secondary structure of proteins. CD spectroscopy measurements were performed to gain a better understanding of the possible influence on the conformational changes of CAT and SOD after interacting with Cd. Prominent negative bands at 208 nm (Fig. S2, Supplementary data), which are characteristic of the ␣-helix, increased in CAT and decreased in SOD with the addition of Cd. The contents of four secondary structures analyzed by CDPro and listed in Table 2 show that the secondary structures of CAT and SOD were changed. These results indicated that Cd led the proteins to misfold and partially denature. Any structural changes of enzymes may lead to the loss of their normal physiological function since there is a significant
Table 2 Effects of Cd on the percentage of secondary structural content of CAT and SOD. Enzyme
Molar ratio of enzyme to Cd
Secondary structural content in enzyme ␣-helix
-sheet
Turn
Unordered
CAT
1:0 1:10 1:20
27.4% 27.0% 22.0%
20.5% 23.5% 29.8%
21.1% 21.2% 22.4%
30.6% 26.8% 25.1%
SOD
1:0 1:10 1:20
10.9% 11.9% 13.8%
39.5% 38.0% 36.2%
21.9% 20.4% 18.1%
26.5% 30.2% 28.0%
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Wavelength(nm) Fig. 3. UV absorption spectra of CAT and SOD in the presence of different concentration of Cd. Conditions: T = 298 K, pH = 7.4, (A) c(CAT) = 1 × 10−6 M; c(Cd) (×10−5 M) a–e: 0, 1, 2, 3, 4. (B) c(SOD) = 1 × 10−6 M, c(Cd) (×10−5 M) a–e: 0, 1, 2, 3, 4.
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correlation between certain folds and specific functions [48]. Therefore, the activity of CAT and SOD changed due to Cd exposure as manifested in our study. 3.3.4. Fluorescence quenching We utilized fluorescence methods to expound the interaction of the enzyme with Cd, including accessibility and quenching mechanism. Preliminary experiments were performed considering the possible existence of an inner filter effect (IFE) which is caused by the absorption of ligand during the excitation and emission radiation processes. The sum of the ligand absorption at 280 nm (excitation wavelength) and 334 nm (emission wavelength) could cause less than 5% error, so IFE was negligible here [49,50]. Upon addition of Cd to CAT, there was a decrease in intensity and a red shift from 333.8 to 335.8 nm in Fig. 4A, suggesting that Cd interacted with the fluorophores (Trp, Tyr, Phe) of CAT and quenched their intrinsic fluorescence [49]. The red shift indicated that CAT unfolded and the fluorescence chromophores had greater exposure to the aqueous phase [51]. The fluorescence intensity of SOD did not change when Cd concentration was lower than 10−3 M. When the concentration increased to 10−3 M, the fluorescence of SOD was quenched as illustrated in Fig. 4B; that is, the fluorophore of SOD in the native state must be located far away from the binding position. The Cd binding process caused conformational changes in SOD, as UV–vis absorption and CD spectroscopy suggested, and resulted in accessibility of previously shielded fluorescent residues to Cd. Changes in the accessibility due to conformational changes have been reported for other proteins [52,53].
Fig. 4. Emission spectra of CAT and SOD in the presence of different concentrations of Cd. Conditions: T = 298 K, ex = 280 nm, Tris–HCl buffer: pH = 7.4, (A) c(CAT) = 1 × 10−6 M, c(Cd) (×10−4 M), a–g: 0, 0.5, 1, 2, 3, 4, 5. (B) c(SOD) = 5 × 10−6 M, c(Cd) (×10−3 M), a–g: 0, 1, 2, 3, 4, 5, 6.
Fluorescence quenching can be classified into two types: static and dynamic quenching [54]. For the static quenching, 0 / = 1, where 0 and are the fluorescence lifetimes of fluorophore in the absence and presence of quencher. In contrast, for dynamic quenching, F0 /F = 0 /, where F0 and F are the fluorescence intensities in the absence and presence of quencher [49]. Measurement of fluorescence lifetimes was carried out to determine the quenching mechanism of the interaction of CAT and SOD with Cd. The data in Fig. S3 (Supplementary data) were fitted well by a single variable monoexponential decay with 2 values close to 1.00. The interaction of CAT and SOD with Cd should be attributed to static quenching because of 0 / ≈ 1 at different concentrations of Cd (Table 3). These results suggest that complexes are formed between Cd and the enzymes, since Cd quenched the intrinsic fluorescence of both CAT and SOD via static quenching [49].
Table 3 Fluorescence lifetimes of CAT and SOD in Different Concentrations of Cd. Enzyme
Molar ratio of enzyme to Cd
(ns)
2
CAT
1:0 1:50 1:100
3.95 4.05 4.09
1.051 1.089 0.989
SOD
1:0 1:200 1:400
3.34 3.26 3.30
1.001 0.955 0.951
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Fig. 5. (A) Binding interaction between Cd(NO3 )2 and CAT. CAT is shown in cartoon
Q4 mode. Heme groups are shown in black. Cd(NO3 )2 is represented as spheres and labeled in the figure. (B) Electrostatic maps superimposed with the ligand Cd(NO3 )2 and the receptor CAT, positive (charge region) preference is indicated in blue, negative in red and neutral in white. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.3.5. Synchronous fluorescence Synchronous fluorescence experiments were performed to evaluate changes in the microenvironment of Tyr and Trp residues [55]. When is fixed at 15 or 60 nm, synchronous fluorescence spectra supply characteristic information about Tyr residues or Trp residues, respectively [55]. Synchronous fluorescence spectra of CAT and SOD in the presence of different Cd concentration are shown in Fig. S4 (Supplementary data). In Fig. S4A2 (Supplementary data), a slight red shift (from 278.2 to 279.8 nm) was observed in the emission spectra, indicating that Trp residues in CAT resulted in greater exposure to the aqueous phase [56]. In Fig. S4A1 and S4B (Supplementary data), the emission peak did not shift, suggesting that Cd had little effect on the microenvironment of Tyr residues in CAT and SOD [49]. Results from synchronous fluorescence experiments are consistent with the conclusions from UV–vis absorption spectroscopy.
Fig. 6. (A) Binding interaction between Cd(NO3 )2 and SOD. SOD is shown in cartoon mode. Cd(NO3 )2 is represented as spheres and labeled in the figure. The green and yellow spheres represent Cu2+ and Zn2+ , respectively. (B) Electrostatic maps superimposed with the ligand of Cd(NO3 )2 and the receptor SOD, positive (charge region) preference is indicated in blue, negative in red and neutral in white. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3.6. Molecular docking study CAT is composed of four identical subunits which cooperate to perform the function of CAT in organisms. Considering the
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structure of CAT, we focused on one subunit of CAT. The best energy ranked conformer is shown in Fig. 5A. Cd binds to CAT at a cavity surrounded by the wrapping domain, helical domain and barrel, within 13–23 A˚ distance from the active center, which confirmed the results of UV–vis absorption spectroscopy measurement. Electrostatic maps superimposed with the ligand of Cd and the receptor CAT are presented in Fig. 5B. Residues Arg126, Gln 167, Lys 166, Asp 177, Val 181, Trp 185, Leu 198, Phe 199 and His 465 line the binding site. The substrate (H2 O2 ) diffuses to the active center of enzyme mainly through a narrow channel extending from the enzyme surface with Asp127 and Gln167 at the entrance, comprising 14 amino acid residues: Val 73, His 74, Val 115, Asp 127, Pro 128, Asn 147, Phe 152, Phe 153, Phe 160, Phe 163, Phe 164, Gln 167, Trp 185 and Leu 198. Cd interacting with Gln167 and Trp 185 within this major channel [46] hinders the formation of hydrogen bonds between a water molecule and Gln 167—impeding the approach of H2 O2 to the active center and decreasing the CAT activity due to steric hindrance within the channels [57,58]. Additionally, Cd interacting with Trp 185 quenches the fluorescence of CAT and induces a microenvironmental change around Trp residues, in agreement with the conclusion of fluorescence quenching and synchronous fluorescence measurements. As shown in Fig. 6A, the two subunits of SOD are tightly joined by hydrophobic and electrostatic forces. The active site of SOD is located between a barrel and two surface loops [59]. The ITC experiment showed that there are two types of binding sites for Cd binding in SOD via electrostatic forces, and the two best energy ranked conformers were calculated based on this data. As illustrated in Fig. 6A, the two binding sites are both located at the interface of two subunits. Electrostatic maps superimposed with the ligand of Cd and the receptor SOD are presented in Fig. 6B. Isaac Klapper et al. suggested that there exists a strong positive potential at the bottom of the channel around the active site and a weak negative potential surrounding the remainder of the enzyme [60]. In consequence, it is difficult for the positively charged Cd to diffuse to the active center through the negatively charged surface. Cd collides and binds around the surface rather than at the active site of SOD, as the docking study proposed. A possible explanation of the observed increasing enzyme activity is that Cd loosens the protein skeleton and induces conformational changes, making the electrostatically driven superoxide anion easier to diffuse to the active site. Additionally, residues lining the binding position do not contain Trp, Tyr or Phe residues, which confirmed the results obtained from fluorescence quenching and synchronous fluorescence measurement.
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Antioxidant and antioxidant enzyme changes are usually the earliest responses used by organisms to protect against oxidative damage in oxidative stress. The results from this study demonstrate that Cd-induced oxidative stress results in lipid peroxidation in the liver of zebrafish through changing the activity of the antioxidant enzymes CAT and SOD and depleting GSH. The direct interactions between the enzyme molecules and Cd contributes to their activity changes in the liver of zebrafish. Furthermore, our study gives insight into the molecular mechanism of the change of CAT and SOD enzyme activity caused by interaction with Cd. The ITC results demonstrated that Cd binds to the two enzymes predominantly via electrostatic forces with one binding site and binding constant of K298K = 2.69 ± 0.243 × 103 M−1 for CAT, with two binding sites and binding constants of K1298K = 6.07 ± 1.47 × 105 M−1 and K2298K = 1.02 ± 0.132 × 104 M−1 for SOD. The UV–vis absorption and CD measurements showed that the secondary structure of CAT and SOD are altered, leading to
enzyme misfolding. Fluorescence spectroscopy results suggested that Cd quenches the intrinsic fluorescence of CAT through static quenching, indicating Cd interacts with fluorophore residues; while Cd does not interact with the fluorophore residues of SOD until the conformational changes of SOD increase the exposure of these residues. Also, synchronous fluorescence showed that Cd changes the microenvironment of Trp residues in CAT; but no change is observed in SOD. A molecular docking study suggested that Cd impedes the substrate diffusing to the active center of CAT through interacting with Gln167 and Trp 185 within the major channel. For SOD, Cd binds to the interface of the two subunits. Together with these observations, our findings suggest that the enzyme misfolding induced by Cd results in changes of CAT and SOD activity. Together with these observations, our findings suggest that the enzyme misfolding induced by Cd results in changes of CAT and SOD activity. This study provides evidence for the mechanism of heavy metalinduced oxidative stress in vivo and introduces a combined in vitro and in vivo method to evaluate the biomacromolecule response involved in biological effects. However, further research is required to interpret the mechanism of the current system more precisely. For example, antioxidant enzymes used in the in vitro experiments should be extracted from the same species as that used as the model organism for the in vivo experiments. Acknowledgments This work is supported by NSFC (20875055, 21277081, Q3 201477067), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education, Ministry of Education of China (708058, 20130131110016), Independent innovation program of Jinan (201202083) and Independent innovation foundation of Shandong University natural science projects (2012DX002) are also acknowledged. All authors thank Dr. Pamela Holt for editing this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijbiomac.2015.02.037. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Molecular mechanism on cadmium-induced activity changes of catalase and superoxide dismutase
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Jing Wang a , Hao Zhang a , Tong Zhang a , Rui Zhang a , Rutao Liu a,∗ , Yadong Chen b a Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment & Health, 27# Shanda South Road, Jinan 250100, Shandong Province, PR China b Laboratory of Molecular Design and Drug Discovery, School of Basic Science, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
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Article history: Received 1 June 2014 Received in revised form 23 August 2014 Accepted 23 February 2015 Available online xxx
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Keywords: Cadmium Oxidative stress Catalase and superoxide dismutase
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1. Introduction
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Cadmium contributes to adverse effects of organisms probably because of its ability to induce oxidative stress via alterations in activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), but their molecular mechanisms remain unclear. We investigated the molecular mechanism of CAT and SOD response under Cd-induced oxidative stress in the liver of zebrafish. The enzyme activity changes observed in vitro were consistent with those seen in vivo, indicating the direct interaction of CAT and SOD with Cd contributes to their activity change in vivo. Further experiments utilizing multiple spectroscopic methods, isothermal titration calorimetry and a molecular docking study were performed to explore the mechanism of molecular interaction of CAT and SOD with Cd. Different interaction patterns were found that resulted in misfolding and changed the enzyme activities. Taken together, we suggest the misfolding of CAT and SOD contributes to their activity change under Cd-induced oxidative stress in vivo. © 2015 Elsevier B.V. All rights reserved.
Cadmium (Cd) is a known environmental contaminant [1]. Prolonged and various application of Cd has resulted in large releases into the environment. Natural levels of Cd found in water bodies range from 0.1 to 10 nM; while Cd level is 0.2–15 g/g in the larvae of Chaoborus punctipennis [2]. Food and tobacco smoking are the main sources of Cd uptake for the non-occupationally exposed population. For the age of 40–50 group, it is estimated that Cd concentration is 1.4–2 g/g and 26–70 g/g in the liver and kidney cortex of human subjects [3]. And human exposure to Cd is predicted to continue to increase in the next decades. Significant attention has been paid to the multiple toxic effects of Cd on human health due to its common presence in the environment. Cd has been officially listed as a potential carcinogenic agent by the International Agency for Research and banned by the European Union’s Restriction on Hazardous Substances directive [4]. Growing evidence indicates that the accumulation of Cd has a significant negative impact on organisms via the induction of oxidative stress mediated by the excessive generation of reactive oxygen species (ROS). Previous investigations have suggested multifactorial mechanisms responsible for Cd-induced oxidative stress.
∗ Corresponding author. Tel.: +86 531 88364868; fax: +86 531 88364868. E-mail address: [email protected] (R. Liu).
Generally, as a redox-inactive metal, Cd depletes the cell’s major antioxidants and causes damage to antioxidant enzymes, resulting in ROS accumulation [5]. Furthermore, excessive ROS can overwhelm cell’s intrinsic antioxidant defenses and attack biomolecules including lipids, proteins and DNA, causing lipid peroxidation, protein oxidation and DNA damage [5,6]. The role of antioxidant enzymes has been recognized as an important factor in Cd-induced oxidative stress. Catalase (CAT) and superoxide dismutase (SOD) are essential for maintaining the level of ROS in organisms and are used as biomarkers to indicate ROS production [7,8]. Inactivation of CAT and SOD will allow the generation of excess ROS and then induce oxidative stress in organisms [9]. Conflicting results have been reported on the activity changes of CAT and SOD involved in Cd-induced oxidative stress in vivo and in vitro. López et al. found that in in vitro studies on rat cortical neuron cells SOD activity displayed a dose-dependent increase but CAT activity was not affected by Cd exposure [10]. Shi et al. examined CAT and SOD activity in the liver of Carassius auratus, and their results indicated that Cd inhibited CAT activity and increased SOD activity [11]. Studies by Hussain et al. and Casalino et al. showed that the activity of SOD was inhibited by Cd in the liver of rats both in vivo and in vitro due to direct interaction of Cd with the enzyme molecule [12,13]. Previous research has demonstrated that Cd induce oxidative stress by altering the activity of CAT and SOD both in vitro and in vivo. The modulation of the related genes and the direct
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interaction of Cd with the enzyme might account for the activity change of the enzyme [14,15]. However, the underlying molecular mechanisms have not been fully elucidated [16]. A number of in vitro studies have utilized multiple spectroscopic methods to study the molecular mechanism of the interaction between the enzyme and xenobiotics. Yet, the molecular mechanism of the interaction of Cd with CAT and SOD has not been reported. Zebrafish (Danio rerio) is a popular model in many areas of vertebrate research instead of mammalian model because of its low cost and ease of manipulation. A number of studies have utilized zebrafish as a model system for studying Cd-induced oxidative stress both in vitro and in vivo [14,15,17] due to its capacity to sustain soft water [18], which is a basic requirement for testing biological response of Cd to avoid the interference with other cation [19]. The liver is the primary target organ for Cd accumulation; and it can be critically damaged by chronic Cd exposure from food [20]. The hepatotoxicity of Cd has been extensively studied and widely reported in occupationally and environmentally exposed populations, as well as in various experimental models. Therefore, the liver of zebrafish was selected as a model in this study. This study introduced a combined in vitro and in vivo method to clarify the molecular mechanisms for the alterations of CAT and SOD activity under Cd-induced oxidative stress in the liver of zebrafish. To assess the physiological responses under oxidative stress, we monitored the content of reduced glutathione (GSH), oxidized glutathione (GSSG) and malondiadehyde (MDA), and the activity of CAT and SOD in the liver of zebrafish after exposure to Cd. To evaluate whether the direct interactions of CAT and SOD molecules with Cd resulted in their activity change in vivo, we performed enzyme activity assays on CAT and SOD in vitro. The molecular mechanism of the interaction between Cd and the two antioxidant enzymes was further explored by multiple spectroscopic methods, isothermal titration calorimetry and a molecular docking study. Our study demonstrated that the direct interaction of Cd with CAT and SOD in vitro contributed to the enzyme activity change under Cd-induced oxidative stress in vivo. Different interaction patterns were found that resulted in misfolding and the different activity change of CAT and SOD. Taken together, the activity changes of CAT and SOD in vivo are attributed to the misfolding of the enzymes upon Cd binding. This study can help to clarify the mechanism of oxidative stress and biomacromolecule response induced by heavy metal exposure.
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Cd(NO3 )2 ·4H2 O was purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). CAT from beef liver was obtained from Sigma (USA). Cu/Zn SOD from pig blood was purchased from Biodee Biotechnology Co. (Beijing, China). Tris–HCl (NaCl) buffer (0.02 M, pH = 7.4) was used to control pH in the in vitro experiments. For the in vivo experiment, 0.01 M phosphate buffer saline (PBS, pH = 7.4) was used as the homogenization medium. All reagents were of analytical grade. Ultrapure water (18.25 M cm) was used throughout the experiments. 2.2. In vivo experiment 2.2.1. Model organism and Cd treatment The present study was conducted in accordance with the Guiding Principles outlined in the Use of Animals in Toxicology adopted by the Society of Toxicology in 1989. Adult zebrafish were purchased from the Boning Aquarium market (Jinan, China) and acclimatized in dechlorinated tap water with a natural light/dark
cycle of 12 h:12 h at 26 ± 1 ◦ C [21] in the environmental chamber before experimentation. Zebrafish were fed twice daily with brine shrimp (Artemia) and starved 24 h before exposure to avoid fecal interference in the assay. The water samples used in the current study were filtered prior to use. The pH of the water was 7.4 ± 0.2, and its electrical conductivity was 140 ± 10 S/cm. The dissolved oxygen in the water was 8.9 ± 0.6 mg/L. After adaptive feeding for 15 days, three experimental groups of adult zebrafish were exposed to Cd by adding a solution of Cd(NO3 )2 to each beaker (1 L) for 24 h. The water sample without Cd was set as the control group. The in vitro and in vivo exposure time were selected as 30 min [22,23] and 24 h [24,25] after consulting the literature, which is sufficient to induce oxidative stress and the biomarker changes. A number of recent studies used the concentration of 10% LC50 as the exposure concentration since zebrafish was not visibly affected by these concentrations. Cd concentration ranges from 1 to 50 M when exposing to short-term Cd exposure [14,24,26], in accordance with the naturally occurring level in the aquatic organism [2]. Although the environmental exposure of Cd is much less than the utilized concentration in the literature, Cd can accumulate in organisms through the bioconcentration of food chain after longterm exposure. In consideration of the above reason, we choose 5% of LC50 (10 M) and 20% of LC50 (40 M) [27] as the Cd exposure concentration in the current study to provide representative results. Each treatment was tested in triplicate. 2.2.2. Determination of SOD activity, GSH, GSSG and MDA content The assays of SOD activity, GSH, GSSG and MDA content in the liver of zebrafish were performed according to the protocol provided by the kits (Nanjing Jiancheng Bioengineering Institute, China). Liver tissues (2% w/v) of zebrafish were homogenized in PBS at 4 ◦ C and centrifuged at 13,000 × g for 30 min. The content of GSH and GSSG were measured by DTNB-GR recycling reaction [28]. Absorbance at 405 nm was measured on a Microplate Reader (Thermo Scientific). SOD activity assays were performed at 550 nm using modified xanthine–xanthine oxidase method [29]. MDA assay was performed using the thiobarbituric acid (TBA) colorimetric method (532 nm) [30]. Protein content was determined at 595 nm using bovine serum albumin as a standard [31]. Absorbance at 532 nm, 550 nm and 595 nm were measured on a UV-2450 spectrophotometer (Shimadzu, Japan) equipped with a 10-mm quartz cell. Results are expressed as the percentage of control group with the control group set as 100%. 2.2.3. Determination of CAT Activity CAT activity was measured in 3 mL reaction mixtures containing 150 L of liver homogenates, 0.01 M H2 O2 and 0.01 M PBS. The reaction was initiated by the addition of liver homogenates. The activity of CAT was evaluated by measuring the rate of decreased absorbance at 240 nm [32]. 2.3. In vitro experiments 2.3.1. Determinations of CAT and SOD activity The CAT/SOD–Cd solution was incubated for 30 min before the experiment. Both CAT and SOD activities were measured using the same methods as described for the in vivo experiment, with the exception that liver homogenates were replaced by the CAT/SOD–Cd solution and 0.01 M PBS was replaced by 0.02 M Tris–HCl buffer. 2.3.2. Isothermal titration calorimetry (ITC) The ITC experiment was carried out using a Microcal ITC200 microcalorimeter at 298 K. Typically, buffered enzyme solution was loaded in the calorimeter cell. The injection syringe was loaded
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with Cd(NO3 )2 solution. Following thermal equilibration at 298 K and an initial 1 min delay, the first drop (0.4 L) followed by 26 serial injections of 1.5 L every 2 min were made into the cell from the syringe. The stirring speed was 1000 rpm. Data analysis were performed using Origin 7.0 software (Microcal LLC).
2.3.3. UV–visible absorption measurement UV–visible absorption spectra were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan) equipped with a 10-mm quartz cell. The wavelength was recorded from 190 to 450 nm using a buffer solution containing the equivalent concentration of Cd as the reference.
2.3.4. Circular dichroism (CD) measurement CD spectra were recorded by a J-810 CD spectrometer (Jasco, Japan) using a quartz cell with a path length of 10 mm in a nitrogen atmosphere. The scanning speed was set at 200 nm min−1 . Each spectrum was the average of three successive scans. The secondary structure of proteins was calculated through CDPro software (available at http://lamar.colostate.edu/∼sreeram/CDPro/) [33].
2.3.5. Fluorescence measurements Fluorescence spectra were recorded on an F-4600 fluorophotometer (Hitachi, Japan) equipped with a 10-mm quartz cell and a 150 W xenon lamp. The voltage of photo multiplier tube was 600 V and the excitation and emission slit widths were set at 5.0 nm. The excitation wavelength was set at 280 nm. The scan speed was 1200 nm min−1 . The emission wavelength was recorded from 290 to 450 nm. Synchronous fluorescence spectra were measured at = 15 nm and = 60 nm. The wavelength was recorded from 240 to 320 nm. Time-resolved fluorescence measurements of the enzyme in the absence and the presence of Cd were performed (ex = 280 nm, em = 330 nm) on a FLS920 Combined Fluorescence Lifetime and Steady State spectrometer (Edinburgh, UK).
2.3.6. Molecular docking study The interaction of Cd with CAT and SOD was further confirmed by a molecular docking study using the Molecular Operating Environment (MOE) (Version 2009, Chemical Computing Group Inc., Canada) to determine the possible conformation and preferred binding sites of the enzyme by interacting with Cd. The crystallized structure of CAT (from bovine liver) (PDB code: 1TGU) and SOD (PDB code 2SOD) were downloaded from RSCB Protein Data Bank (http://www.pdb.org/). The 3D structure of the ligand was built using MOE-builder and then the ligand was energy minimized. Essential hydrogen atoms were added to the enzyme model. Gastiger charges were computed and applied to the enzyme model. Docking calculation was carried out using the MOE-Dock with settings: Placement: Triangle Matcher; Rescoring 1: London dG, Refinement: Forcefield. The pose with the best scoring conformer model was selected for further analysis. Interaction within these complexes was visualized using the MOE ligand electrostatic maps simulation.
2.3.7. Data analysis Results were presented as the mean of 3 replicates and reported as means ± SD (standard deviations). Data were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. p < 0.05 was considered as statistically significant.
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Antioxidants and antioxidant enzymes are the main components of the antioxidant systems which can protect organisms against xenobiotics-induced oxidative stress [5]. It has been proposed that disturb of the antioxidant defense system will lead to a disturbed oxidant status and the increase of lipid peroxidation [5]. To assess the involvement of Cd-induced oxidative stress, we monitored the modulation of Cd on the level of the antioxidant GSH and its oxidized form GSSG (Fig. S1, Supplementary data). After 24 h exposure, no morbidity or mortality of the zebrafish was observed in any of the control and treatment groups. A decrease in GSH content (82.3% and 38.9% of the initial level) and an increase of GSSG content (257.1% and 380.2% of the initial level) was observed with the increasing exposure concentration, resulting in a significant decrease of GSH/GSSG ratio. Similar results was also reported in the study of Qu et al., in which C. auratus was exposed to 0.1 mg/L Cd for 12 days [34]. In addition to the oxidation of GSH to GSSG through direct neutralization with ROS [35], they suggested this compensatory response might result from the down-regulation of the transcription of the genes involved in GSH biosynthesis. These results suggested that an induction of a temporary shift toward a strongly oxidizing environment occurred within the liver of zebrafish, reflecting an insufficient resistant capacity of the liver to Cd exposure. Lipid peroxidation was assessed by the measurement of MDA concentration, which is linked to ROS production [5,36,37]. As shown in Fig. S1 (Supplementary data), in the liver tissues, exposure to Cd for 24 h generally induced an increase in MDA content (158.2% and 294.8% of the initial level) compared with the control group. Similar results were also obtained in other study of aquatic organisms [15,34]. This result indicated that excessive ROS attacked the membranes, resulting in lipid peroxidation. Altogether these results provided sufficient evidence that Cd altered the redox state and induced oxidative stress in the liver of zebrafish. 3.2. Determination of CAT and SOD activity Lipid peroxidation has been considered as the primary mechanism of Cd toxicity [5,36–38]. The activity change of CAT and SOD generally indicates that excessive ROS needs to be decomposed to maintain the balance of the cell’s intrinsic antioxidant defenses in organisms. Once the balance is disrupted, excessive ROS would attack lipids as manifested by the increased MDA level in our study. As illustrated in Fig. 1, after exposure to 10 M and 40 M Cd(NO3 )2 , the activity of CAT decreased to 77.5% and 40.3% of the initial level; while the activity of SOD increased to 106.3% and 162.0% of the initial level. Similar results were obtained in the study of Shi et al., in which inhibited CAT activity and increased SOD activity was observed in the liver of C. auratus after 24 h Cd (5 mg/L) exposure [11]. Conflicting results have been reported on the activity changes of CAT and SOD involved in Cd-induced oxidative stress in vivo and in vitro. Banni et al. found that CAT and SOD activity were both suppressed in the liver and ovary of zebrafish after 0.4 mg/L Cd exposure [15]. Zeng et al. found that CAT activity exhibited a bell-shaped response and SOD activity increased in a well linear relationship with MDA levels (r2 = 0.964) after Cd exposure in Phanerochaete chrysosporium [39]. These results suggested that the production of CAT and SOD were stimulated to eliminate the excessive ROS. And when the antioxidant system was unable to cope with the excessive ROS, CAT activity decreased and the linear relationship decreased (r2 = 0.873). It has been proposed that the changes of CAT and SOD activity are due to direct interaction
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Fig. 1. Activity changes of CAT and SOD in the liver of zebrafish and in vitro induced by exposure to Cd. Values are means ± SD (n = 3). Differences are considered statistically significant at *p < 0.05, **p < 0.01 and ***p < 0.001, respectively.
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of Cd with the enzyme molecule [12,13,40]. Zhu et al. observed down-regulation of the genes of CAT and SOD but inhibited SOD activity and increased CAT activity in the zebrafish liver cell line after Cd exposure [14]. Cd might directly interact with the antioxidant enzyme resulting in the alterations of the enzyme activity in spite of the down-regulation of the related genes. As Cd might change the activity of enzyme through directly interacting with the enzyme molecule, we investigated the activity change of CAT and SOD interacting with Cd in vitro. As illustrated in Fig. 1, CAT activity decreased to 71.6% and 57.7% of the initial level; while the activity of SOD increased to 184.5% and 215.7% of the initial level. The decline trend of CAT and the upward trend of SOD when interacting Cd in vitro was consistent with that in vivo, indicting that the activity change of CAT and SOD in Cd-induced oxidative stress in vivo was correlated with their direct interaction with Cd. To explore the molecular mechanism of the interaction of CAT and SOD with Cd, further experiments were performed utilizing multiple spectroscopic methods, isothermal titration calorimetry and a molecular docking study.
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3.3.1. Binding affinity The isothermal titration calorimetry (ITC) experiment was utilized to complete the thermodynamic characterization and quantify the stoichiometry and binding affinity associated with the binding process. The results of a typical titration of Cd into CAT and SOD at 298 K after correcting the heats of dilution are shown in Fig. 2. The number of binding sites (n), enthalpy change (H) and binding constant (K) were determined directly from the curvefitting. The experimental titration curve was best fitted with a single
set of binding sites for binding to CAT and two sets of binding sites for binding to SOD, indicating that there existed one type of binding site (nCd-CAT = 8.34 ± 0.434) and two types of binding sites (nCd-SOD,1 = 2.71 ± 0.171, nCd-SOD,2 = 10.7 ± 0.33), respectively. The changes in Gibbs free energy (G) and entropy change (S) were calculated through the equation: G = H − TS = −RTln K, where R is the gas constant and T is the thermodynamic temperature. The correlated parameters are listed in Table 1. The binding processes of CAT and SOD with Cd are both observed to be enthalpically favored and entropically opposed, indicating that the two interactions are mainly driven by electrostatic forces [41]. Negative values of G suggest that the two interaction processes are spontaneous. An order of KCd-CAT (103 ) suggested a weak binding affinity of CAT when interacting with Cd. Although this non-covalent binding process is weak, amounts and varieties of non-covalent bonds may jointly alter the conformation and function of proteins [42]. It is clearly shown that KCd-SOD of both sites were larger compared with KCd-CAT , indicating that the binding between Cd and SOD is comparably stronger than the binding between Cd and CAT. 3.3.2. UV–vis absorption spectroscopy UV–visible absorption spectroscopy was performed to explore the structural change of the enzymes and enzyme–ligand complex formation. The UV–vis absorption spectra of CAT and SOD in the presence and absence of Cd are presented in Fig. 3. Generally, proteins have two major absorption bands. The strong one from 200 to 230 nm reflecting the framework conformation of the protein is attributed to the electronic transitions of the polypeptide bonds →* of the peptide backbone C O [37]. The weaker one from 260 to 290 nm concerning aromatic amino acids
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Fig. 2. ITC profiles of the interaction between Cd with CAT and SOD. Top panels present raw data for the sequential titration of Cd solution into enzymes in Tris–HCl buffer (0.02 M, pH = 7.4) at 298 K. The bottom panels show the integrated heat results of the calorimetric titration after correction of heat of dilution against the molar ratio of Cd/enzyme; the solid line represents the best fit data (A) c(CAT) = 75 M, c(Cd) = 12 mM. (B) c(SOD) = 100 M, c(Cd) = 15 mM. Table 1 Binding parameters of the interaction between Cd and CAT, SOD. Enzyme CAT SOD
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H (cal mol−1 )
Binding site
N
K
1 1 2
8.34 ± 0.434 2.71 ± 0.171 10.7 ± 0.330
2.69 ± 0.243 × 10 6.07 ± 1.47 × 105 1.02 ± 0.132 × 104 3
(Trp, Tyr, Phe) indicates the transformation of the chromophore microenvironment [43]. With gradual addition of Cd into CAT and SOD solution, the intensity of the peak at 215 nm decreased and was red shifted, indicating that the interaction between the enzyme and Cd led to a loosening and unfolding of the protein skeleton. The intensity of the peak at 280 nm increased in Fig. 3A, indicating that Cd caused reduced hydrophobicity of the microenvironment around the aromatic amino acids in CAT. SOD (Fig. 3B) has no significant absorbance peak at 280 nm because of the absence of Trp residues [44]. Additionally, CAT has another absorption band around 405 nm which is caused by the characteristic absorption of the porphyrinSoret band [45]. Heme groups are located at the active center and participate in the catalytic process. The intensity of the peak at 405 nm increased slightly with no shift, suggesting that Cd affected the activity of CAT but had no profound conformational change of the polypeptides around the heme groups. Since heme groups are deeply embedded 20 A˚ below the molecular surface and 23 A˚ from
−4.16 ± 0.273 × 10 −4.21 ± 0.467 × 102 −1.31 ± 0.0392 × 103 3
S (cal mol−1 K−1 )
G (kcal mol−1 )
1.75 25.0 14.0
−4.68 ± 0.273 −7.87 ± 0.0467 −5.48 ± 0.0392
the molecular center of the tetramer [46], we speculated that there was no direct interaction between Cd and the amino acid residues around the heme group due to steric hindrance [47]. 3.3.3. Circular dichroism spectroscopy CD spectroscopy is widely applied in many fields. Most notably, UV CD is used to investigate the secondary structure of proteins. CD spectroscopy measurements were performed to gain a better understanding of the possible influence on the conformational changes of CAT and SOD after interacting with Cd. Prominent negative bands at 208 nm (Fig. S2, Supplementary data), which are characteristic of the ␣-helix, increased in CAT and decreased in SOD with the addition of Cd. The contents of four secondary structures analyzed by CDPro and listed in Table 2 show that the secondary structures of CAT and SOD were changed. These results indicated that Cd led the proteins to misfold and partially denature. Any structural changes of enzymes may lead to the loss of their normal physiological function since there is a significant
Table 2 Effects of Cd on the percentage of secondary structural content of CAT and SOD. Enzyme
Molar ratio of enzyme to Cd
Secondary structural content in enzyme ␣-helix
-sheet
Turn
Unordered
CAT
1:0 1:10 1:20
27.4% 27.0% 22.0%
20.5% 23.5% 29.8%
21.1% 21.2% 22.4%
30.6% 26.8% 25.1%
SOD
1:0 1:10 1:20
10.9% 11.9% 13.8%
39.5% 38.0% 36.2%
21.9% 20.4% 18.1%
26.5% 30.2% 28.0%
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Wavelength(nm) Fig. 3. UV absorption spectra of CAT and SOD in the presence of different concentration of Cd. Conditions: T = 298 K, pH = 7.4, (A) c(CAT) = 1 × 10−6 M; c(Cd) (×10−5 M) a–e: 0, 1, 2, 3, 4. (B) c(SOD) = 1 × 10−6 M, c(Cd) (×10−5 M) a–e: 0, 1, 2, 3, 4.
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correlation between certain folds and specific functions [48]. Therefore, the activity of CAT and SOD changed due to Cd exposure as manifested in our study. 3.3.4. Fluorescence quenching We utilized fluorescence methods to expound the interaction of the enzyme with Cd, including accessibility and quenching mechanism. Preliminary experiments were performed considering the possible existence of an inner filter effect (IFE) which is caused by the absorption of ligand during the excitation and emission radiation processes. The sum of the ligand absorption at 280 nm (excitation wavelength) and 334 nm (emission wavelength) could cause less than 5% error, so IFE was negligible here [49,50]. Upon addition of Cd to CAT, there was a decrease in intensity and a red shift from 333.8 to 335.8 nm in Fig. 4A, suggesting that Cd interacted with the fluorophores (Trp, Tyr, Phe) of CAT and quenched their intrinsic fluorescence [49]. The red shift indicated that CAT unfolded and the fluorescence chromophores had greater exposure to the aqueous phase [51]. The fluorescence intensity of SOD did not change when Cd concentration was lower than 10−3 M. When the concentration increased to 10−3 M, the fluorescence of SOD was quenched as illustrated in Fig. 4B; that is, the fluorophore of SOD in the native state must be located far away from the binding position. The Cd binding process caused conformational changes in SOD, as UV–vis absorption and CD spectroscopy suggested, and resulted in accessibility of previously shielded fluorescent residues to Cd. Changes in the accessibility due to conformational changes have been reported for other proteins [52,53].
Fig. 4. Emission spectra of CAT and SOD in the presence of different concentrations of Cd. Conditions: T = 298 K, ex = 280 nm, Tris–HCl buffer: pH = 7.4, (A) c(CAT) = 1 × 10−6 M, c(Cd) (×10−4 M), a–g: 0, 0.5, 1, 2, 3, 4, 5. (B) c(SOD) = 5 × 10−6 M, c(Cd) (×10−3 M), a–g: 0, 1, 2, 3, 4, 5, 6.
Fluorescence quenching can be classified into two types: static and dynamic quenching [54]. For the static quenching, 0 / = 1, where 0 and are the fluorescence lifetimes of fluorophore in the absence and presence of quencher. In contrast, for dynamic quenching, F0 /F = 0 /, where F0 and F are the fluorescence intensities in the absence and presence of quencher [49]. Measurement of fluorescence lifetimes was carried out to determine the quenching mechanism of the interaction of CAT and SOD with Cd. The data in Fig. S3 (Supplementary data) were fitted well by a single variable monoexponential decay with 2 values close to 1.00. The interaction of CAT and SOD with Cd should be attributed to static quenching because of 0 / ≈ 1 at different concentrations of Cd (Table 3). These results suggest that complexes are formed between Cd and the enzymes, since Cd quenched the intrinsic fluorescence of both CAT and SOD via static quenching [49].
Table 3 Fluorescence lifetimes of CAT and SOD in Different Concentrations of Cd. Enzyme
Molar ratio of enzyme to Cd
(ns)
2
CAT
1:0 1:50 1:100
3.95 4.05 4.09
1.051 1.089 0.989
SOD
1:0 1:200 1:400
3.34 3.26 3.30
1.001 0.955 0.951
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Fig. 5. (A) Binding interaction between Cd(NO3 )2 and CAT. CAT is shown in cartoon
Q4 mode. Heme groups are shown in black. Cd(NO3 )2 is represented as spheres and labeled in the figure. (B) Electrostatic maps superimposed with the ligand Cd(NO3 )2 and the receptor CAT, positive (charge region) preference is indicated in blue, negative in red and neutral in white. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.3.5. Synchronous fluorescence Synchronous fluorescence experiments were performed to evaluate changes in the microenvironment of Tyr and Trp residues [55]. When is fixed at 15 or 60 nm, synchronous fluorescence spectra supply characteristic information about Tyr residues or Trp residues, respectively [55]. Synchronous fluorescence spectra of CAT and SOD in the presence of different Cd concentration are shown in Fig. S4 (Supplementary data). In Fig. S4A2 (Supplementary data), a slight red shift (from 278.2 to 279.8 nm) was observed in the emission spectra, indicating that Trp residues in CAT resulted in greater exposure to the aqueous phase [56]. In Fig. S4A1 and S4B (Supplementary data), the emission peak did not shift, suggesting that Cd had little effect on the microenvironment of Tyr residues in CAT and SOD [49]. Results from synchronous fluorescence experiments are consistent with the conclusions from UV–vis absorption spectroscopy.
Fig. 6. (A) Binding interaction between Cd(NO3 )2 and SOD. SOD is shown in cartoon mode. Cd(NO3 )2 is represented as spheres and labeled in the figure. The green and yellow spheres represent Cu2+ and Zn2+ , respectively. (B) Electrostatic maps superimposed with the ligand of Cd(NO3 )2 and the receptor SOD, positive (charge region) preference is indicated in blue, negative in red and neutral in white. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3.6. Molecular docking study CAT is composed of four identical subunits which cooperate to perform the function of CAT in organisms. Considering the
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structure of CAT, we focused on one subunit of CAT. The best energy ranked conformer is shown in Fig. 5A. Cd binds to CAT at a cavity surrounded by the wrapping domain, helical domain and barrel, within 13–23 A˚ distance from the active center, which confirmed the results of UV–vis absorption spectroscopy measurement. Electrostatic maps superimposed with the ligand of Cd and the receptor CAT are presented in Fig. 5B. Residues Arg126, Gln 167, Lys 166, Asp 177, Val 181, Trp 185, Leu 198, Phe 199 and His 465 line the binding site. The substrate (H2 O2 ) diffuses to the active center of enzyme mainly through a narrow channel extending from the enzyme surface with Asp127 and Gln167 at the entrance, comprising 14 amino acid residues: Val 73, His 74, Val 115, Asp 127, Pro 128, Asn 147, Phe 152, Phe 153, Phe 160, Phe 163, Phe 164, Gln 167, Trp 185 and Leu 198. Cd interacting with Gln167 and Trp 185 within this major channel [46] hinders the formation of hydrogen bonds between a water molecule and Gln 167—impeding the approach of H2 O2 to the active center and decreasing the CAT activity due to steric hindrance within the channels [57,58]. Additionally, Cd interacting with Trp 185 quenches the fluorescence of CAT and induces a microenvironmental change around Trp residues, in agreement with the conclusion of fluorescence quenching and synchronous fluorescence measurements. As shown in Fig. 6A, the two subunits of SOD are tightly joined by hydrophobic and electrostatic forces. The active site of SOD is located between a barrel and two surface loops [59]. The ITC experiment showed that there are two types of binding sites for Cd binding in SOD via electrostatic forces, and the two best energy ranked conformers were calculated based on this data. As illustrated in Fig. 6A, the two binding sites are both located at the interface of two subunits. Electrostatic maps superimposed with the ligand of Cd and the receptor SOD are presented in Fig. 6B. Isaac Klapper et al. suggested that there exists a strong positive potential at the bottom of the channel around the active site and a weak negative potential surrounding the remainder of the enzyme [60]. In consequence, it is difficult for the positively charged Cd to diffuse to the active center through the negatively charged surface. Cd collides and binds around the surface rather than at the active site of SOD, as the docking study proposed. A possible explanation of the observed increasing enzyme activity is that Cd loosens the protein skeleton and induces conformational changes, making the electrostatically driven superoxide anion easier to diffuse to the active site. Additionally, residues lining the binding position do not contain Trp, Tyr or Phe residues, which confirmed the results obtained from fluorescence quenching and synchronous fluorescence measurement.
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Antioxidant and antioxidant enzyme changes are usually the earliest responses used by organisms to protect against oxidative damage in oxidative stress. The results from this study demonstrate that Cd-induced oxidative stress results in lipid peroxidation in the liver of zebrafish through changing the activity of the antioxidant enzymes CAT and SOD and depleting GSH. The direct interactions between the enzyme molecules and Cd contributes to their activity changes in the liver of zebrafish. Furthermore, our study gives insight into the molecular mechanism of the change of CAT and SOD enzyme activity caused by interaction with Cd. The ITC results demonstrated that Cd binds to the two enzymes predominantly via electrostatic forces with one binding site and binding constant of K298K = 2.69 ± 0.243 × 103 M−1 for CAT, with two binding sites and binding constants of K1298K = 6.07 ± 1.47 × 105 M−1 and K2298K = 1.02 ± 0.132 × 104 M−1 for SOD. The UV–vis absorption and CD measurements showed that the secondary structure of CAT and SOD are altered, leading to
enzyme misfolding. Fluorescence spectroscopy results suggested that Cd quenches the intrinsic fluorescence of CAT through static quenching, indicating Cd interacts with fluorophore residues; while Cd does not interact with the fluorophore residues of SOD until the conformational changes of SOD increase the exposure of these residues. Also, synchronous fluorescence showed that Cd changes the microenvironment of Trp residues in CAT; but no change is observed in SOD. A molecular docking study suggested that Cd impedes the substrate diffusing to the active center of CAT through interacting with Gln167 and Trp 185 within the major channel. For SOD, Cd binds to the interface of the two subunits. Together with these observations, our findings suggest that the enzyme misfolding induced by Cd results in changes of CAT and SOD activity. Together with these observations, our findings suggest that the enzyme misfolding induced by Cd results in changes of CAT and SOD activity. This study provides evidence for the mechanism of heavy metalinduced oxidative stress in vivo and introduces a combined in vitro and in vivo method to evaluate the biomacromolecule response involved in biological effects. However, further research is required to interpret the mechanism of the current system more precisely. For example, antioxidant enzymes used in the in vitro experiments should be extracted from the same species as that used as the model organism for the in vivo experiments. Acknowledgments This work is supported by NSFC (20875055, 21277081, Q3 201477067), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education, Ministry of Education of China (708058, 20130131110016), Independent innovation program of Jinan (201202083) and Independent innovation foundation of Shandong University natural science projects (2012DX002) are also acknowledged. All authors thank Dr. Pamela Holt for editing this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.ijbiomac.2015.02.037. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
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