Molecular Mechanism of Lead-Induced Superoxide Dismutase

Dec 12, 2014 - ... both sexes were obtained from the Boning Aquarium market (Jinan, China) ... using Graphpad Prism software (Version 5.01, San Diego,...
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Molecular Mechanism of Lead-Induced Superoxide Dismutase Inactivation in Zebrafish Livers Hao Zhang,† Yang Liu,† Rutao Liu,*,† Chunguang Liu,† and Yadong Chen‡ †

School of Environmental Science and Engineering, China−America CRC for Environment & Health, Shandong University, 27# Shanda South Road, Jinan, Shandong Province 250100, P. R. China ‡ Laboratory of Molecular Design and Drug Discovery, School of Basic Science, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China S Supporting Information *

ABSTRACT: Lead toxicity has been proved to be related with inducing oxidative stress of organisms and causing inactivation of antioxidant enzymes, the mechanism of which remains unknown. This study investigated and compared superoxide dismutase (Cu/Zn SOD) activity inhibited in lead-treated zebrafish livers and explored the mechanism of SOD inactivation by lead at the molecular level using multiple spectroscopic techniques, isothermal titration calorimetric (ITC) measurement, molecular docking study and ICP−AES detection. Results showed lead exposure decreased SOD activities in zebrafish livers due to direct interactions between lead and SOD, resulting in conformational and functional changes of the enzyme. To be specific, Studies at the molecular level indicated that lead bound into the active site channel of SOD, hindered the path of the catalytic substrate (O2−•), damaged its skeleton conformation and secondary structure, and interacted with the enzymatically related residue (Arg 141) through electrostatic forces (ΔH < 0, ΔS > 0), and caused the release of Cu2+ and Zn2+ from the catalytic pocket of SOD. This work shows a correlation between results on organismal and molecular levels, and obtains a possible model hypothesizing mechanisms of lead toxicity using in vitro experiments instead of in vivo ones.

1. INTRODUCTION Lead is a widespread environmental pollutant existing in water, soil, dust and lead-containing products, including leaded gasoline,1 lead paints,2 leaded plumbing components,3 and lead-glazed ceramics.4 Workers in certain occupations are also exposed to high levels of lead.5 Lead can enter the human body mainly through the respiratory and gastrointestinal tracts, causing cognitive dysfunction, hematological disorders, neurological damage, renal impairment, and immunological pathologies, which are known to be linked with oxidative stress,6 an imbalance of oxidant and antioxidant status in target cells and tissues altered by oxidants or chemicals.7 Multiple studies have shown that lead toxicity is related to oxidative stress due to over production of reactive oxygen species (ROS),8,9 depletion of reduced glutathione (GSH)10 and interference of antioxidant enzyme activities, such as superoxide dismutase.11 While the structure of enzyme specifies its function, lead exposure is likely to affect the structure of SOD, resulting in the changes of its activity. Recently numerous studies have focused on changes of SOD signals in lead exposed organisms.12−14 However, the mechanism of lead actions on alterations of SOD structure and function remains unknown. It is worth mentioning that direct interactions between toxic compounds and macromolecules (protein, DNA) at the molecular level can outline the mechanism of conformational changes of macromolecules, which have been systematically investigated by our group and © 2014 American Chemical Society

are helpful to evaluate potential toxicities of xenobiotics in vivo.15−17 Superoxide dismutases (SODs, EC 1.15.1.1), including several distinct isoforms in organisms, such as Cu/Zn SOD, Fe SOD, Mn SOD, and Ni SOD,18,19 are mainly found in the extracellular matrix of tissues,20 and play vital roles in maintaining cellular redox environment and protecting cells and tissues against oxidative damages caused by toxic products through biocatalysis of O2−• into O2 and H2O2.21 Cu/Zn SOD (hereinafter referred to as SOD) is most commonly existed in eukaryotes and worthy to study as a model molecule in our study.22 SOD is a 32 kDa homodimeric metalloenzyme with one copper ion and one zinc ion in the active site and one tyrosine residue (Tyr 108, act as the fluorophore) of each subunit.23 In this work, zebrafish is selected as the toxicological model to study effects of lead exposure on changes of SOD activity and contents in fish livers. Furthermore, the direct interaction between lead and SOD is presented to explore mechanisms on the alteration of SOD activity induced by lead at the molecular level using fluorescence quenching method, UV−vis absorption spectroscopy, circular dichroism spectrosReceived: November 4, 2014 Revised: December 2, 2014 Published: December 12, 2014 14820

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(ANOVA), followed by Newman-Keuls multiple comparison test, considering p < 0.05 as significant. 2.4. Fluorescence Measurements. (1). Synchronous Fluorescence Measurements. Synchronous fluorescence spectra (Δλ = 15 nm, λex = 275−315 nm) were recorded on an F4600 fluorescence spectrophotometer (Hitachi, Japan). The excitation and emission slit widths were set at 5 nm. Scan speed was 1200 nm/min. PMT (photo multiplier tube) voltage was set at 750 V. (2). Fluorescence Lifetime Measurements. Time-resolved fluorescence measurements were carried out using an FLS920 combined fluorescence lifetime and steady state spectrometer (Edinburgh, U.K.) with λex = 278 nm and λem = 325 nm. 2.5. Isothermal Titration Calorimetry Analysis. Isothermal titration calorimetry (ITC) experiments were carried out with a Microcal ITC200 microcalorimeter (Microcal Inc., Northampton, MA) at 298 K by titrating 200 μL of SOD (0.05 mM) with approximately 40 μL of lead acetate (10 mM) using stirring speed at 1000 rpm. Both SOD and lead acetate were dissolved in HAc−NaAc buffer (0.02 M, pH 5.5) and filtered with 0.22 μm syringe filters, and the spacing time was 120 s between each injection to achieve thermodynamic equilibration. 2.6. Molecular Docking Studies. Direct interactions between lead acetate and SOD were further confirmed by molecular docking studies using the Molecular Operating Environment (MOE) (Version 2009, Chemical Computing Group Inc., Canada) to determine the most dominant binding sites of enzymes. The protein model of SOD (PDB code: 2SOD) was downloaded from the Protein Data Bank (http:// www.pdb.org/). The 3D structure of lead acetate was generated and energy minimized using the MOE Builder module and the Energy Minimize module provided by MOE, respectively. 2.7. UV−Visible Absorption Spectroscopic Studies. UV−vis absorption spectra of SOD were measured on a UV2450 spectrophotometer (Shimadzu, Japan) in the range of 200−300 nm. Slit width was set at 2.0 nm. 2.8. Circular Dichroism (CD) Spectra Measurement. CD spectra of SOD were carried out using a J-810 circular dichroism spectrometer (Jasco, Japan) collected from 190 to 260 nm with the scan speed of 200 nm/min, and two scans were implemented and averaged for each CD spectrum. The secondary structure contents of SOD obtained from CD spectra were analyzed by CDpro software (available at http:// lamar.colostate.edu/~sreeram/CDPro/). 2.9. ICP−AES Measurements. Experiments were utilized to measure Cu2+ and Zn2+ contents substituted by Pb2+ from the active site of SOD by inductively coupled plasma-atomic emission spectrometry (ICP−AES) method (IRIS INTREPID II XSP, Thermo Electron, USA). All instrument operating parameters are summarized in Table 1. Solutions (10 mL) that contained SOD with different concentrations of lead acetate in acetate buffer (pH 5.5) were dialyzed by 14 kDa dialysis bags, and the bags were immersed in 250 mL of the same buffer solution for 24 h.

copy, isothermal titration calorimetric (ITC) measurement, molecular docking study and ICP−AES detection.

2. MATERIALS AND METHODS 2.1. Chemicals. Lead(II) acetate trihydrate (Pb(Ac)2· 3H2O) was purchased from Tianjin Chemical Reagent (Tianjin, China). Superoxide dismutase (Cu/Zn SOD, from pig blood) was bought from Biodee (Beijing, China). The experimental kit for Cu/Zn SOD activities, GSH/GSSG status, and MDA content were all purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China), and the kit for total protein abundance was bought from Biocolor bioscience and biotechnology (Shanghai, China). All the chemicals were analytical-reagent grade. Ultrapure water (18.25 MΩ·cm) was used throughout. 2.2. Animals. Adult and healthy zebrafish of both sexes were obtained from the Boning Aquarium market (Jinan, China), and maintained in a 50-L aquarium filled with continuously aerated, filtered water (pH 7.2−7.6; electrical conductivity, 126−150 μS/cm; dissolved oxygen, 8.3−9.6 mg/ L) for 2 weeks before the experiments. The temperature was kept at 28 ± 2 °C under a 14/10-h light−dark photoperiod. Fishes were fed twice a day with brine shrimp (Huayang aquatic products, Shandong, China) and were free from artificial disturbances. According to the research of Chen et al.,24 zebrafish exposed to a solution of lead(II) acetate had an LC50 of 171 mg/L. After adaptive feeding for 15 days, 30 zebrafish were divided into three groups, and each group contains 10 fishes. One group was set as control, while the other two were incubated at lead acetate concentrations of 10 mg/L (5% of LC50) and 50 mg/L (20% of LC50) for 24 h. Then zebrafish were killed by freezing water and dissected to get livers. Liver homogenates (2% w/v) were prepared in 0.1 M phosphate buffer (pH = 7.4) using a tissue homogenizer at 0−4 °C and centrifuged at 3500 g for 20 min at 4 °C to remove the nuclear and cell debris, and supernatants were collected and kept in a liquid nitrogen tank for assays. 2.3. Assays to Determine SOD Activities. The activity of SOD was evaluated using a modified xanthine-xanthine oxidase method.25 Superoxide radical ions (O2−•) created by the xanthine−xanthine oxidase system can react with hydroxylamine to form nitrite, which can generate a fuchsia product with the absorbance at 550 nm after react with a chromogenic agent. SOD can accelerate the disproportionation of O2−•and reduce the absorption value, which is proportional to the activity of SOD. The SOD activity is calculated as the following equation: USOD =

V A1 − A 2 ÷ 0.5 × 0 ÷ C A1 V

where A1 and A2 are the absorbance values of the standard tubes and the sample tubes (SOD samples in the absence and presence of lead), respectively. V0 is total volume of reacted solutions and V stands for the volume of added SOD solutions (50 μL). C is the concentration of total protein of 2% liver homogenate in each group. Total protein abundance was determined using bicinchoninic acid (BCA) reaction.26 All statistical analyses and data presentations were accomplished using Graphpad Prism software (Version 5.01, San Diego, CA). Data were expressed as mean ± SEM (standard error of the mean) and analyzed by one-way analysis of variance

Table 1. Operating Conditions for the Determination of Cu2+ and Zn2+ Release Using ICP−AES

14821

nebulizer flow (PSI)

plasma gas (L min−1)

auxiliary gas (L min−1)

sample flow rate (mL min−1)

RF power (W)

pump (RPM)

28.0

13

1.0

1.85

1150

100

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Figure 1. Inhibitions of SOD activity induced by lead at the zebrafish (A) and purified SOD (B) level: (∗) P < 0.05; (∗∗) P < 0.01. The absolute activity of SOD (U/mgprot) was precisely measured in zebrafish livers owning to the detectable values of total protein abundance, and relative activity of SOD in direct Pb−SOD interactions was calculated compared to values from the standard group.

3. RESULTS AND DISCUSSION 3.1. SOD Activity Investigations at the Zebrafish and Molecular (in Vitro) Level. Zebrafish is regarded as a broadly used model organism for toxicological research due to its close homology in human genomes.27,28 Effects of lead exposure on SOD activity in zebrafish liver shown in Figure 1A indicate that there is a significant inhibition of SOD activity with higher incubated concentrations of lead. Results from biomarkers of oxidative stress, including GSH/GSSG status and MDA contents, provide sufficient evidence for oxidative stress of zebrafish under lead treatment (shown in Figure S1, Supporting Information). SOD activity is also regarded as biomarkers of oxidative stress, though there are different views on how to evaluate oxidative stress according to enzyme activities, mainly due to varying exposure times, doses and experimental conditions. Farmand et al. reported increased activities of SOD in the kidney cortex, medulla, and thoracic aorta homogenates of lead-treated Dawley rats.29 Gupta et al. found elevated activities of SOD in the root and shoots of Zea mays seedlings after lead exposure.30 They considered the up-regulation of SOD activities as a compensatory response to oxidative stress from lead exposure. Contrary to their findings, Patra and Swaru demonstrated that SOD in the erythrocytes of lead-treated calves showed decreased activities compared to the control group,31 and Sivaprasad et al. reported lower activities of SOD in the erythrocytes of lead-treated rats.32 In our opinion, methods of measuring SOD activities are mainly by means of changes in the substrate concentrations within a certain period of time, and the decomposition rates of the substrate can be closely related to the alteration of SOD frame structure in zebrafish livers exposed to lead. Results from direct interactions between lead acetate and purified SOD (Figure 1B) show that the activity of SOD decreases significantly with the addition of lead acetate, corresponding with SOD inhibition assay in vivo. So it can be inferred that direct binding interactions between lead acetate and SOD resulted in inhibitions of enzyme activity, the molecular mechanism of which was discussed further below. 3.2. Quenching Mechanism of SOD Fluorescence. Since SOD has one Tyr residue (Tyr 108) and no Trp residues, synchronous fluorescence spectra of SOD at Δλ = 15 nm were applied to probe changes of SOD fluorescence in the presence of lead solutions at 298 K instead of fluorescence emission spectra in order to eliminate Raman signals of solvents (Figure 2). Fluorescence intensities of SOD decreased with no peak shift at the emission wavelength by the addition of lead acetate which indicated lead influenced the structure of SOD (intensity

Figure 2. Influence of lead on synchronous fluorescence spectra of SOD (Δλ = 15 nm). Conditions: SOD, 5.0 × 10−6 mol L−1; lead acetate (mg L−1) (A−E), 0, 10, 30, 50, 70; pH = 5.5; T = 293 K.

decrease) without interacting near Tyr 108 (no peak shift). Fluorescence lifetime measurement is used to determine quenching mechanisms (dynamic quenching and static quenching), which can precisely distinguished by time-resolved fluorescence spectrometry.33 The time-resolved decays of SOD in different lead doses were measured at λex = 278 nm and λem = 325 nm. The data fit well to the sum of a single exponential decay with a χ2 value close to 1.00. As shown in Figure 3, the fluorescence lifetime of SOD presents no obvious changes under different doses of lead acetate, which confirms that the quenching mode of SOD fluorescence caused by lead could be attributed to the static one, meaning the formation of the Pb− SOD complex.34 3.3. Binding Mode Investigations by ITC and Molecular Docking Studies. Isothermal titration calorimetry (ITC) can be used to investigate the binding affinity constant (k), binding stoichiometry (n), enthalpy (ΔH) and entropy (ΔS) effects, and binding forces between SOD and lead acetate, and free-energy change (ΔG) can be calculated by the thermodynamic equation ΔG = −RT ln K a = ΔH − T ΔS

(R is the universal gas constant and T is the absolute temperature).35 The ITC results shown in Figure 4 were corrected for dilution effects by subtracting the reference data measured in identical series of injections into buffer, and fit the 14822

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Figure 3. Time-resolved fluorescence decay profile of SOD induced by lead. Conditions: SOD, 1.0 × 10−5 mol L−1; lead acetate (mg L−1) (A−C), 0, 10, 50; pH = 5.5; T = 293 K.

Table 2. Thermodynamic Parameters for Interaction of Lead Acetate with SOD by ITC According to Figure 4 thermodynamic parameters

SOD−Pb(Ac)2

Ka (L mol−1) ΔH (kJ mol−1) ΔS (J mol−1 K−1) ΔG (kJ mol−1)

3.87(±0.92) × 104 −23.1(±0.34) 14.96 −27.6 ± 4.8

104 magnitude, which is comparable to other binding interactions between lead and macromolecules, such as PbBSA (Ka = 7.5 × 104 M−1) and Pb-HSA (Ka=8.2 × 104 M−1) bindings.36 Molecular operating environment (MOE) is a powerful molecular modeling tool to perform computational simulations of binding mechanisms between small ligands and macromolecules.37 The molecular modeling of Pb(Ac)2−SOD complex was further investigated through MOE software. The electrostatic loop area that acts as the active site channel of SOD and accelerates the substrate O2−• into the active site,38 was selected as the potential binding pocket and the lowest energy result in the dominant site of SOD are illustrated in Figure 5. Lead acetate entered into the active site channel of SOD, hindered the path of the catalytic substrate (O2−•), and electrostatically bound with an oxygen atom (carboxyl oxygen with negative charge, 2.48 Å) of Arg 141, a residue that plays a key role in properly docking with O2−• near the catalytic metal (Cu2+), resulting the inhibition of SOD activity.39 Moreover, since Tyr residue locates far from the binding site, the microenvironment surrounding Tyr 108 remain constant, which is accordance with results from synchronous fluorescence spectra of SOD (no peak shift). 3.4. Conformational Changes of SOD by UV−Vis Absorption and CD Spectra. UV−vis absorption spectroscopic technique can be employed to explore the structural changes of protein and to investigate formations of macromolecule-ligand complex.40 The absorption spectra of SOD in the absence and presence of lead acetate are shown in Figure 6. The strong absorption peak at 208 nm reflects the framework conformation of SOD, resulting from the π−π* transition of protein’s characteristic polypeptide backbone structure C O.41 With gradual addition of lead acetate to SOD solution, the absorption peak decreased and underwent a red shift, which indicated that the interaction caused the main skeletons of SOD loosening and unfolding.42

Figure 4. ITC studies of titrating Pb(Ac)2 (10 mM) into SOD solutions (0.05 mM) for the binding of Pb(Ac)2 to SOD at 298 K, pH = 5.5. The upper section shows heat flow of each titration (μcal/s) in a period of time (min), and the bottom part illustrates integrated heats in terms of kcal/mol of injectant plotted against molar ratio of Pb(Ac)2/SOD.

“one set of sites” model, which indicated that lead acetate entered into one binding site of SOD. The upper section in the figure shows heat flow of each titration (μcal/s) in a period of time (min), and the bottom part illustrates integrated heats in terms of kcal/mol of the reaction plotted against molar ratio of Pb(Ac)2/SOD. The ITC responses of the SOD−Pb(Ac)2 complex presented negative heat deflections, indicating that the binding interactions belonged to exothermic effects. Thermodynamic parameters calculated from ITC measurement were summarized in Table 2. More specifically, electrostatic binding between SOD and Pb(Ac)2 was confirmed by negative ΔH and positive ΔS values. Negative ΔG value revealed that binding interactions of lead acetate with SOD occurred spontaneously. The binding constants (Ka) is in the range of 14823

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Figure 7. CD spectra of SOD (5.0 × 10−6 mol/L) after lead exposures (A−C: 0, 10, 50 mg/L) at 298 K, pH = 5.5.

addition of Pb(Ac)2 at 10 mg/L and 50 mg/L, the α-helix contents decreased to 9.6% and 8.2%, β-sheet contents increased to 42.7% and 43.3%, β-turn decreased to 20.5% and 19.4%, and unordered coil increased to 27.2% and 29.1%, respectively (shown in Table 3), which indicate that the Table 3. Effects of Lead on the Percentage of Secondary Structural Elements of SOD Calculated by CDpro Software According to Figure 7

Figure 5. Molecular docking results of Pb−SOD complex by MOE show one binding site of SOD for Pb(Ac)2 (A), and the interacted scheme in binding site is presented using “ligand interactions” module of MOE (B), which shows lead acetate electrostatically binds to the oxygen atom of Arg 141 with the distance of 2.48 Å. Tyr 108 and Arg 141 are shown in ball and stick, and lead acetate is illustrated using spheres. Different types of the secondary structure of SOD are colorcoded as follows: α-helix, red; β-pleated sheet, yellow; β-turn and random coil, green.

secondary structure content in SOD (%) lead concentrations (mg/L)

α-helix

β-sheet

β-turn

unordered

0 10 50

0.109 0.096 0.082

0.422 0.427 0.433

0.219 0.205 0.194

0.250 0.272 0.291

binding interaction of lead with SOD causes alterations on the secondary structure of SOD. It has been proved that α-helix locates near the electrostatic loop unit (shown in Figure 5), and closely relates to SOD activity.44 So the decreased proportion of α-helical structure may be another possible reason causing inhibition of SOD activity. 3.5. Determination of Cu2+ and Zn2+ Release from Lead-Treated SOD Using ICP−AES. Cu2+ and Zn2+ located in the active site of SOD play important roles in acting as the catalytic cofactor and maintaining the structure integrity of SOD.45 ICP−AES experiments have been carried out to determine whether Cu2+ and Zn2+ could be released during inactivation of SOD after incubation with toxic compound. Lead acetate decreased SOD activity and entered into the active site according to the enzyme assay and molecular docking study, so we wonder whether Cu2+ and Zn2+, binding with His and Asp residues in the active site of SOD,46 can be released into solution affected by lead. The contents of free Cu2+ and Zn2+ were quantified by ICP−AES, and results from Table 4 confirm the hypothesis that 12.2% and 15.3% of Cu2+ and 21.5% and 25.0% of Zn2+ were released from active site of SOD after incubated in 10 and 50 mg/L of lead, respectively, which probably induced inactivation of SOD. Combined with the MOE docking and CD spectra studies, we can safely draw the conclusion that lead acetate entered into the active site channel of SOD, interacted with Arg 141, hindered the path for O2−•, and competitively substituted Cu2+ and Zn2+, causing decrease of SOD activity.

Figure 6. UV−vis spectra of SOD in different concentrations of lead acetate. Conditions: SOD, 1.0 × 10−6 mol L−1; lead acetate (mg L−1) (a−c), 0, 10, 50; pH = 5.5; T = 293 K.

CD spectra were utilized to study alterations on secondary structure of SOD after binding with Pb(Ac)2, which are shown in Figure 7 that the negative band in the far-UV region at 208 nm show α-helical properties of SOD secondary structure.43 The calculated secondary structure content of SOD according to SELCON program of CDpro software was 10.9% α-helix, 42.2% β-sheet, 21.9% β-turn, and 25% unordered coil. With the 14824

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Table 4. Contents of Free Cu2+ and Zn2+ Released from SOD after Lead Incubation Detected by ICP−AES

associated to high lead in blood levels in two Mexican rural communities. J. Toxicol. Environ. Health A 1994, 42, 45−52. (5) Levin, S. M.; Goldberg, M. Clinical evaluation and management of lead-exposed construction workers. Am. J. Ind. Med. 2000, 37, 23− 43. (6) Patrick, L. Lead toxicity part II: the role of free radical damage and the use of antioxidants in the pathology and treatment of lead toxicity. Altern. Med. Rev. 2006, 11, 114−127. (7) Hailwell, B. Environmental Stressors in Health and Diseas; CRC Press: New York, 2001. (8) Chen, L.; Yang, X.; Jiao, H.; Zhao, B. Tea catechins protect against lead-induced ROS formation, mitochondrial dysfunction, and calcium dysregulation in PC12 cells. Chem. Res. Toxicol. 2003, 16, 1155−1161. (9) An, H.; Zhai, Z.; Yin, S.; Luo, Y.; Han, B.; Hao, Y. Coexpression of the superoxide dismutase and the catalase provides remarkable oxidative stress resistance in Lactobacillus rhamnosus. J. Agric. Food. Chem. 2011, 59, 3851−3856. (10) Jozefczak, M.; Remans, T.; Vangronsveld, J.; Cuypers, A. Glutathione is a key player in metal-induced oxidative stress defenses. Int. J. Mol. Sci. 2012, 13, 3145−3175. (11) Vaziri, N. D.; Lin, C. Y.; Farmand, F.; Sindhu, R. K. Superoxide dismutase, catalase, glutathione peroxidase and NADPH oxidase in lead-induced hypertension. Kidney Int. 2003, 63, 186−194. (12) Patil, A. J.; Bhagwat, V. R.; Patil, J. A.; Dongre, N. N.; Ambekar, J. G.; Jailkhani, R.; Das, K. K. Effect of lead (Pb) exposure on the activity of superoxide dismutase and catalase in battery manufacturing workers (BMW) of Western Maharashtra (India) with reference to heme biosynthesis. Int. J. Environ. Res. Publ. Health 2006, 3, 329−337. (13) Srivastava, R. K.; Pandey, P.; Rajpoot, R.; Rani, A.; Dubey, R. S. Cadmium and lead interactive effects on oxidative stress and antioxidative responses in rice seedlings. Protoplasma 2014, 251, 1047−1065. (14) Semedo, M.; Reis-Henriques, M. A.; Rey-Salgueiro, L.; Oliveira, M.; Delerue-Matos, C.; Morais, S.; Ferreira, M. Metal accumulation and oxidative stress biomarkers in octopus (Octopus vulgaris) from Northwest Atlantic. Sci. Total Environ. 2012, 433, 230−237. (15) Chi, Z.; Liu, R.; Zhang, H. Noncovalent interaction of oxytetracycline with the enzyme trypsin. Biomacromolecules 2010, 11, 2454−2459. (16) Zhang, H.; Liu, Y.; Zhang, R.; Liu, R.; Chen, Y. Binding Mode Investigations on the Interaction of Lead (II) Acetate with Human Chorionic Gonadotropin. J. Phys. Chem. B 2014, 118, 9644−9650. (17) Zhao, X.; Liu, R. Recent progress and perspectives on the toxicity of carbon nanotubes at organism, organ, cell, and biomacromolecule levels. Environ. Int. 2012, 40, 244−255. (18) Uchiyama, S.; Shimizu, T.; Shirasawa, T. CuZn-SOD deficiency causes ApoB degradation and induces hepatic lipid accumulation by impaired lipoprotein secretion in mice. J. Biol. Chem. 2006, 281, 31713−31719. (19) Perry, J.; Shin, D.; Getzoff, E.; Tainer, J. The structural biochemistry of the superoxide dismutases. BBA-Proteins Proteom. 2010, 1804, 245−262. (20) Petersen, S. V.; Oury, T. D.; Ostergaard, L.; Valnickova, Z.; Wegrzyn, J.; Thøgersen, I. B.; Jacobsen, C.; Bowler, R. P.; Fattman, C. L.; Crapo, J. D. Extracellular superoxide dismutase (EC-SOD) binds to type I collagen and protects against oxidative fragmentation. J. Biol. Chem. 2004, 279, 13705−13710. (21) Karunakaran, C.; Zhang, H.; Joseph, J.; Antholine, W. E.; Kalyanaraman, B. Thiol oxidase activity of copper, zinc superoxide dismutase stimulates bicarbonate-dependent peroxidase activity via formation of a carbonate radical. Chem. Res. Toxicol. 2005, 18, 494− 500. (22) Rosen, D. R.; Siddique, T.; Patterson, D.; Figlewicz, D. A.; Sapp, P.; Hentati, A.; Donaldson, D.; Goto, J.; O’Regan, J. P.; Deng, H. X.; et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993, 362, 59−62. (23) Francis, J. W.; Hosler, B. A.; Brown, R. H., Jr.; Fishman, P. S. CuZn superoxide dismutase (SOD-1): tetanus toxin fragment C

content (mg/L)a element

0 mg/L of Pb

10 mg/L of Pb

50 mg/L of Pb

Cu2+ Zn2+

0 0

0.0310 0.0560

0.0392 0.0652

a

Contents of Cu2+ and Zn2+ listed in table are the average value of three parallel tests.

4. CONCLUSIONS The present work explores the mechanisms of SOD inactivity in lead-treated zebrafish livers at the molecular level. SOD activity inhibited by lead showed the same trends both at the zebrafish and purified enzyme levels due to direct interactions between lead and SOD. So mechanisms on SOD inactivity in lead-treated zebrafish livers can be explained through direct interactions between lead and SOD at the molecular level, results of which showed that lead could form complex with SOD through electrostatic effect, enter into the active channel of SOD, hinder the access of the substrate, interact with enzymatically related residue (Arg 141), alter the secondary structure of SOD, and cause the release of Cu2+ and Zn2+ from the active site of SOD, resulting in a decrease of enzyme activity.

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ASSOCIATED CONTENT

S Supporting Information *

Determination of the biomarkers of oxidative stress (GSH/ GSSG status, MDA contents, activities of related enzyme activities) in the liver of zebrafish after lead exposure (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(R.L.) Telephone/Fax: 86-531-88365489. E-mail: rutaoliu@ sdu.edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by NSFC (21277081, 21477067), the Cultivation Fund of the Key Scientific and Technical Innovation Project, the Research Fund for the Doctoral Program of Higher Education, Ministry of Education of China (708058, 20130131110016), the Science and Technology Development Plan of Shandong Province (2014GSF117027), and the Independent Innovation Program of Jinan (201202083) are also acknowledged.

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REFERENCES

(1) Patrick, L. Lead toxicity, a review of the literature. Part 1: Exposure, evaluation, and treatment. Altern. Med. Rev. 2006, 11, 2−22. (2) Jacobs, D. E.; Clickner, R. P.; Zhou, J. Y.; Viet, S. M.; Marker, D. A.; Rogers, J. W.; Zeldin, D. C.; Broene, P.; Friedman, W. The prevalence of lead-based paint hazards in US housing. Environ. Health Perspect. 2002, 110, A599. (3) Russell Jones, R. The continuing hazard of lead in drinking water. Lancet 1989, 2, 669−670. (4) Rojas-López, M.; Santos-Burgoa, C.; Ríos, C.; Hernández-Avila, M.; Romieu, I. Use of lead-glazed ceramics is the main factor 14825

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hybrid protein for targeted delivery of SOD-1 to neuronal cells. J. Biol. Chem. 1995, 270, 15434−15442. (24) Chen, J.; Hu, G.; Qu, J. Study on joint toxicity of lead and chromium. J. Zhejiang College Fisheries 1997, 17, 169−173. (25) Nandi, A.; Chatterjee, I. Assay of superoxide dismutase activity in animal tissues. J. Biosciences 1988, 13, 305−315. (26) Smith, P.; Krohn, R. I.; Hermanson, G.; Mallia, A.; Gartner, F.; Provenzano, M.; Fujimoto, E.; Goeke, N.; Olson, B.; Klenk, D. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76−85. (27) Lam, S. H.; Ung, C. Y.; Hlaing, M. M.; Hu, J.; Li, Z.-H.; Mathavan, S.; Gong, Z. Molecular insights into 4-nitrophenol-induced hepatotoxicity in zebrafish: transcriptomic, histological, and targeted gene expression analyses. BBA-Gen. Subjects 2013, 1830, 4778−4789. (28) Barbazuk, W. B.; Korf, I.; Kadavi, C.; Heyen, J.; Tate, S.; Wun, E.; Bedell, J. A.; McPherson, J. D.; Johnson, S. L. The syntenic relationship of the zebrafish and human genomes. Genome Res. 2000, 10, 1351−1358. (29) Farmand, F.; Ehdaie, A.; Roberts, C. K.; Sindhu, R. K. Leadinduced dysregulation of superoxide dismutases, catalase, glutathione peroxidase, and guanylate cyclase. Environ. Res. 2005, 98, 33−39. (30) Gupta, D. K.; Nicoloso, F. T.; Schetinger, M. R.; Rossato, L. V.; Pereira, L. B.; Castro, G. Y.; Srivastava, S.; Tripathi, R. D. Antioxidant defense mechanism in hydroponically grown Zea mays seedlings under moderate lead stress. J. Hazard. Mater. 2009, 172, 479−484. (31) Patra, R.; Swarup, D. Effect of lead on erythrocytic antioxidant defence, lipid peroxide level and thiol groups in calves. Res. Vet. Sci. 2000, 68, 71−74. (32) Sivaprasad, R.; Nagaraj, M.; Varalakshmi, P. Combined efficacies of lipoic acid and meso-2,3-dimercaptosuccinic acid on lead-induced erythrocyte membrane lipid peroxidation and antioxidant status in rats. Hum. Exp. Toxicol. 2003, 22, 183−192. (33) Van de Weert, M.; Stella, L. Fluorescence quenching and ligand binding: a critical discussion of a popular methodology. J. Mol. Struct. 2011, 998, 144−150. (34) Chi, Z.; Liu, R. Phenotypic characterization of the binding of tetracycline to human serum albumin. Biomacromolecules 2010, 12, 203−209. (35) Gruner, S.; Neeb, M.; Barandun, L. J.; Sielaff, F.; Hohn, C.; Kojima, S.; Steinmetzer, T.; Diederich, F.; Klebe, G. Impact of protein and ligand impurities on ITC-derived protein-ligand thermodynamics. BBA-Gen. Subjects 2014, 1840, 2843−2850. (36) Belatik, A.; Hotchandani, S.; Carpentier, R.; Tajmir-Riahi, H. A. Locating the binding sites of Pb(II) ion with human and bovine serum albumins. PloS one 2012, 7, e36723. (37) Vilar, S.; Cozza, G.; Moro, S. Medicinal chemistry and the molecular operating environment (MOE): application of QSAR and molecular docking to drug discovery. Curr. Top. Med. Chem. 2008, 8, 1555−1572. (38) Getzoff, E. D.; Tainer, J. A.; Weiner, P. K.; Kollman, P. A.; Richardson, J. S.; Richardson, D. C. Electrostatic recognition between superoxide and copper, zinc superoxide dismutase. Nature 1983, 306, 287−290. (39) Polticelli, F.; Battistoni, A.; O’Neill, P.; Rotilio, G.; Desideri, A. Role of the electrostatic loop charged residues in Cu,Zn superoxide dismutase. Protein Sci. 1998, 7, 2354−2358. (40) Tong, C.; Xiang, G.; Bai, Y. Interaction of paraquat with calf thymus DNA: a terbium(III) luminescent probe and multispectral study. J. Agric. Food. Chem. 2010, 58, 5257−5262. (41) Li, Z.; Abramavicius, D.; Zhuang, W.; Mukamel, S. Two dimensional electronic correlation spectroscopy of the npi* and pipi* protein backbone transitions: a simulation study. Chem. Phys. 2007, 341, 29−36. (42) Zhao, X.; Liu, R.; Chi, Z.; Teng, Y.; Qin, P. New insights into the behavior of bovine serum albumin adsorbed onto carbon nanotubes: comprehensive spectroscopic studies. J. Phys. Chem. B 2010, 114, 5625−5631. (43) Sreerama, N.; Woody, R. W. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN,

SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 2000, 287, 252−260. (44) Ma, L.; Ze, Y.; Liu, J.; Liu, H.; Liu, C.; Li, Z.; Zhao, J.; Yan, J.; Duan, Y.; Xie, Y. Direct evidence for interaction between nano-anatase and superoxide dismutase from rat erythrocytes. Spectrochim. Acta, Part A 2009, 73, 330−335. (45) Teoh, M. L.; Walasek, P. J.; Evans, D. H. Leporipoxvirus Cu, Zn-superoxide dismutase (SOD) homologs are catalytically inert decoy proteins that bind copper chaperone for SOD. J. Biol. Chem. 2003, 278, 33175−33184. (46) Auchère, F.; Capeillère-Blandin, C. Oxidation of Cu, Znsuperoxide dismutase by the myeloperoxidase/hydrogen peroxide/ chloride system: functional and structural effects. Free Radical Res. 2002, 36, 1185−1198.

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dx.doi.org/10.1021/jp511056t | J. Phys. Chem. B 2014, 118, 14820−14826