International Journal of

Molecular Sciences Article

Molecular Insights into the Potential Toxicological Interaction of 2-Mercaptothiazoline with the Antioxidant Enzyme—Catalase Zhenxing Huang 1,2,3 , Ming Huang 1 , Chenyu Mi 1 , Tao Wang 1 , Dong Chen 1 and Yue Teng 1,2, * 1

2 3

*

School of Environment and Civil Engineering, Jiangnan University, Wuxi 214122, China; [email protected] (Z.H.); [email protected] (M.H.); [email protected] (C.M.); [email protected] (T.W.); [email protected] (D.C.) Jiangsu Key Laboratory of Anaerobic Biotechnology, Jiangnan University, Wuxi 214122, China Jiangsu Collaborative Innovation Center of Technology and Material of Water Treatment, Suzhou 215009, China Correspondence: [email protected]; Tel./Fax: +86-510-8519-7091

Academic Editor: Stephen C. Bondy Received: 6 July 2016; Accepted: 5 August 2016; Published: 16 August 2016

Abstract: 2-mercaptothiazoline (2-MT) is widely used in many industrial fields, but its residue is potentially harmful to the environment. In this study, to evaluate the biological toxicity of 2-MT at protein level, the interaction between 2-MT and the pivotal antioxidant enzyme—catalase (CAT) was investigated using multiple spectroscopic techniques and molecular modeling. The results indicated that the CAT fluorescence quenching caused by 2-MT should be dominated by a static quenching mechanism through formation of a 2-MT/CAT complex. Furthermore, the identifications of the binding constant, binding forces, and the number of binding sites demonstrated that 2-MT could spontaneously interact with CAT at one binding site mainly via Van der Waals’ forces and hydrogen bonding. Based on the molecular docking simulation and conformation dynamic characterization, it was found that 2-MT could bind into the junctional region of CAT subdomains and that the binding site was close to enzyme active sites, which induced secondary structural and micro-environmental changes in CAT. The experiments on 2-MT toxicity verified that 2-MT significantly inhibited CAT activity via its molecular interaction, where 2-MT concentration and exposure time both affected the inhibitory action. Therefore, the present investigation provides useful information for understanding the toxicological mechanism of 2-MT at the molecular level. Keywords: 2-mercaptothiazoline; catalase; spectroscopic technique; molecular docking; toxicity; protein conformation

1. Introduction Catalase (CAT, hydrogen peroxide oxidoreductase; EC.1.11.1.6) is a common enzyme found in nearly all living organisms exposed to oxygen, which plays a pivotal role in protecting cells from oxidative injuries by catalyzing the detoxification of hydrogen peroxide to oxygen and water (2H2 O2 → O2 ↑ + 2H2 O) [1,2]. Recently, there has been growing evidence that CAT is a major factor against various pathological conditions such as diabetes, inflammation, sickle cell disease, and cancer [3–6]. However, the intake of exogenous environmental chemicals (e.g., organic pollutants, heavy metals, nanomaterials) is likely to trigger the destruction of protein conformation and to further suppress the catalytic activity of the enzyme [7–9]. Hence, as an important antioxidant enzyme, the toxic effects of contaminants on CAT as well as their action mechanisms should be intensively investigated in vivo and in vitro.

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2-Mercaptothiazoline forms Int. J. Mol. Sci. 2016, 17, 1330 (2-MT) is a heterocyclic organic compound with two tautomeric 2 of 12 including a thione form (1,3-thiazolidine-2-thione) and a thiol form (2-thiazoline-2-thiol) [10] 2-Mercaptothiazoline (2-MT) is a heterocyclic organic compound two solution tautomeric[11]. forms (Scheme 1), and 1,3-thiazolidine-2-thione is the predominant form in with aqueous Due to including a thione form (1,3-thiazolidine-2-thione) and a thiol form (2-thiazoline-2-thiol) [10] (Scheme its electron donor properties derived from –(NH)–(C=S)– group, 2-MT has been widely used in 1), and 1,3-thiazolidine-2-thione is the predominant form in aqueous solution [11]. Due to its electron many fields [12]. For instance, 2-MT has been applied to synthesize brightening and stabilization donor properties derived from –(NH)–(C=S)– group, 2-MT has been widely used in many fields [12]. agents the printed wiring as well as the photography industry [13]. In addition, Forfor instance, 2-MT has been board appliedindustry to synthesize brightening and stabilization agents for the printed basedwiring on its adsorption/complexation behaviors on the surface of metal products, 2-MT is commonly board industry as well as the photography industry [13]. In addition, based on its used adsorption/complexation as an organic inhibitorbehaviors against corrosion andofdiffusion by forming on the surface metal products, 2-MT ismetal–chelate commonly usedSchiff as an base complexes In the environmental chemically modified 2-MT can be[14]. used to organic[14]. inhibitor against corrosion and protection diffusion by field, forming metal–chelate Schiff base complexes In the environmental protection field, chemically 2-MTsolutions can be used to remove metal are remove heavy metal ions, such as Hg(II) ions, modified from water [15]. 2-MT heavy derivatives ions, such as Hg(II) ions, from water solutions [15]. 2-MT derivatives areHowever, also used in the medical also used in the medical industry as potent anti-thyroid drugs [16]. 2-MT is resistant industry as potent anti-thyroid drugs [16]. However, 2-MT is resistant to natural and biological to natural and biological degradation, and thus its extensive use can result in significant residue degradation, and thus its extensive use can result in significant residue accumulation in the accumulation in the environment [17]. Many studies have reported that 2-MT residue can be detected environment [17]. Many studies have reported that 2-MT residue can be detected in wastewater in wastewater treatment plants, surface water, as well as soils, and it is considered to be a typical treatment plants, surface water, as well as soils, and it is considered to be a typical organic organic contaminant [18,19]. contaminant [18,19].

Scheme 1. 2-Mercaptothiazoline (2-MT) tautomers: thione form (left) and thiol form (right). The balls

Scheme 1. 2-Mercaptothiazoline (2-MT) tautomers: thione form (left) and thiol form (right). The balls of white, gray, yellow and blue respectively represent the atoms of hydrogen (H), carbon (C), sulfur of white, gray, yellow and blue respectively represent the atoms of hydrogen (H), carbon (C), sulfur (S) (S) and nitrogen (N), respectively. and nitrogen (N), respectively.

2-Mercaptothiazoline is classified as a harmful organic compound, according to its Material Safety Data Sheet. Hence,isthe potentialas risk of 2-MT exposure organisms should be investigated. 2-Mercaptothiazoline classified a harmful organic to compound, according to its Material Teng et al. demonstrated that 2-MT could bind into site II (subdomain IIIA) of bovine serum Safety Data Sheet. Hence, the potential risk of 2-MT exposure to organisms should be albumin, investigated. and provided valuable information for understanding the effects of 2-MT on the transportation of Teng et al. demonstrated that 2-MT could bind into site II (subdomain IIIA) of bovine serum albumin, substances in the blood [20]. In addition, Thomes et al. reported that 2-MT could inhibit the activities and provided valuable information for understanding the effects of 2-MT on the transportation of of horseradish peroxidase, lactoperoxidase, and thyroid peroxidase [21]. Nevertheless, the current substances in the blood In addition, Thomesof et2-MT al. reported that 2-MT could thetoactivities knowledge about the[20]. toxicological mechanisms is still severely lacking. Ininhibit addition, the of horseradish peroxidase,there lactoperoxidase, thyroid peroxidasethe [21]. Nevertheless, current best of our knowledge, are no studies and that focus on elucidating biological toxicity ofthe 2-MT knowledge about the toxicological mechanisms on antioxidant enzymes at the molecular level. of 2-MT is still severely lacking. In addition, to the the considerations in thethat present study, the potential toxicological 2- on best of ourGiven knowledge, there are above, no studies focus on elucidating the biological interaction toxicity ofof 2-MT MT with the pivotal antioxidant enzyme—catalase (CAT) was investigated by multiple spectroscopic antioxidant enzymes at the molecular level. techniques and molecular modeling. mechanism as well toxicological as the relevantinteraction binding of Given the considerations above, inThe the quenching present study, the potential parameters, including the number of binding sites, binding constant, and binding forces were 2-MT with the pivotal antioxidant enzyme—catalase (CAT) was investigated by multiple spectroscopic identified. Moreover, the interaction behaviors were further elucidated by molecular docking techniques and molecular modeling. The quenching mechanism as well as the relevant binding simulation and conformation dynamic characterization. Finally, the potential inhibitory effects of parameters, including the number of binding sites, binding constant, and binding forces were identified. 2-MT on CAT activity caused by their interaction were verified by confirmatory experiments.

Moreover, the interaction behaviors were further elucidated by molecular docking simulation and conformation dynamic characterization. Finally, the potential inhibitory effects of 2-MT on CAT activity caused by their interaction were verified by confirmatory experiments.

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2. Results and Discussion 2. Results and Discussion

2.1. Characterization of the Binding Interaction between 2-MT and CAT by Fluorescence Analysis 2.1. Characterization of the Binding Interaction between 2-MT and CAT by Fluorescence Analysis

2.1.1. Fluorescence Quenching Mechanism

2.1.1. Fluorescence QuenchingisMechanism Fluorescence spectroscopy a favorable technique for exploring molecular interactions since it’s has high sensitivity [22]. Fluorescence methodstechnique have beenforwidely used to investigate the interaction Fluorescence spectroscopy is a favorable exploring molecular interactions since it’s has highand sensitivity [22]. haveabout been the widely used tomechanism, investigate binding the between ligands proteins, andFluorescence can providemethods information quenching interaction between ligands and proteins, and can provide information about the quenching constants and binding sites [23,24]. The fluorescence emission spectra of CAT with various doses of binding constants and 1. binding sites [23,24]. The fluorescence emission spectra of CAT 2-MTmechanism, additions are shown in Figure The fluorescence intensity of CAT was remarkably reduced with various doses of 2-MT additions are shown in Figure 1. The fluorescence intensity of CAT was as the 2-MT concentration increased. It is known that tryptophan and tyrosine residues are mainly remarkably reduced as the 2-MT concentration increased. It is known that tryptophan and tyrosine responsible for protein fluorescence, and the fluorescence intensity is associated with the protein residues are mainly responsible for protein fluorescence, and the fluorescence intensity is associated structure. Thus, the CAT fluorescence quenching suggests that 2-MT could bind into CAT and with the protein structure. Thus, the CAT fluorescence quenching suggests that 2-MT could bind into potentially alter its molecular CAT and potentially alter itsstructure. molecular structure.

Figure 1. Fluorescence emission spectra of catalase (CAT) with different doses of 2-MT. Conditions:

Figure 1. Fluorescence emission spectra−1of catalase−5(CAT)−1with different doses of 2-MT. Conditions: CAT: 1.0 × − 107−7 mol·L−1 0 mol·L (a);−11 × 10 mol·L −(b); 2 × 10−5 mol·L−1 (c); 3 × 10−5 mol·L−1 −,12-MT: 5 mol·L−1 (b); 2 × 10−5 mol·L−1 (c); CAT: 1.0 × 10 mol · L , 2-MT: 0 mol · L (a); 1 × 10 (d); 4 × 10−5 mol·L−1 (e); 5 × 10−5 mol·L−1 (f); 6 × 10−5 mol·L−1 (g); pH 7.4; T = 291 K. 3 × 10−5 mol·L−1 (d); 4 × 10−5 mol·L−1 (e); 5 × 10−5 mol·L−1 (f); 6 × 10−5 mol·L−1 (g); pH 7.4; T = 291 K. Quenching mechanisms are usually classified into either dynamic or static quenching. Since a

higher temperature can result in a larger diffusion coefficient, the dynamic quenching constant should increase with increasing temperature. On the other hand, raising or thestatic temperature tends Since to Quenching mechanisms are usually classified into either dynamic quenching. a weaken the complex stability, which can reduce the static quenching constant [25]. Hence, to better higher temperature can result in a larger diffusion coefficient, the dynamic quenching constant should understand the mechanism of CAT fluorescence quenching caused by 2-MT, the fluorescence spectra increase with increasing temperature. On the other hand, raising the temperature tends to weaken the at different temperatures were analyzed according to the Stern-Volmer equation [26].

complex stability, which can reduce the static quenching constant [25]. Hence, to better understand F0 the mechanism of CAT fluorescence quenching the fluorescence spectra at different (1) Q = 1 +by kq τ2-MT, = 1 + Ksvcaused 0 Q F temperatures were analyzed according to the Stern-Volmer equation [26].

where F and F0 respectively represent the fluorescence intensities with and without the quencher, [Q] is the quencher concentration, τ0 F is0 the average fluorescence lifetime in the absence of quencher (τ0 = = 1 + Ksv [ Q] = 1 + kq τ0 [ Q] (1) 10−8 s [27]), kq is the quenching rate F constant of the biological macromolecule, and KSV is the SternVolmer quenching constant. where F and F0 2respectively represent the fluorescence with andon without the [Q] quencher, Figure showed the fluorescence intensity data that intensities were analyzed based F0/F versus at [Q] isdifferent the quencher concentration, τ0 K, is the fluorescence lifetime thedetermined absence ofusing quencher temperatures (291 K, 300 309 average K, and 318 K). Subsequently, KSVinwas (τ0 = Equation 10−8 s [27]), the quenching of the biological macromolecule, and KSV is the (1) bykqa is linear regression ofrate F0/Fconstant against [Q]. As shown in Table 1, the KSV values reduced 4 4 −1 −1 from 1.59 quenching × 10 to 1.23 constant. × 10 L·mol ·s with the increasing temperature. For dynamic quenching, the Stern-Volmer maximum quenching constant (kintensity q) is 2.0 × data 1010 L·mol ·s−1 [28]. However, the on kq values under [Q] Figure 2 showed therate fluorescence that −1were analyzed based F0 /F versus 10 −1·s−1 in this study (Table 1). Therefore, varioustemperatures temperatures were 2.0 ×318 10 K). L·mol at different (291 much K, 300greater K, 309than K, and Subsequently, KSV was determined using according results above, itofcould fluorescence causedreduced by Equation (1) bytoa the linear regression F /Findicate against that [Q]. the As CAT shown in Table 1,quenching the K values 0

SV

from 1.59 × 104 to 1.23 × 104 L·mol−1 ·s−1 with the increasing temperature. For dynamic quenching, the maximum quenching rate constant (kq ) is 2.0 × 1010 L·mol−1 ·s−1 [28]. However, the kq values under various temperatures were much greater than 2.0 × 1010 L·mol−1 ·s−1 in this study (Table 1).

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Int. J. Mol. Sci. 2016, 17, 1330 4 of 12 Therefore, according to the results above, it could indicate that the CAT fluorescence quenching caused by 2-MT is dominated by the static quenching mechanism, through formation of the 2-MT/CAT 2-MT is dominated by the static quenching mechanism, through formation of the 2-MT/CAT complex, rather thanthan dynamic quenching. complex, rather dynamic quenching.

Figure 2. Stern–Volmer linear regression analysis for CAT fluorescence quenching caused by 2-MT

Figure 2. Stern–Volmer linear regression analysis for CAT fluorescence quenching caused by 2-MT under different temperatures (291 K, 300 K, 309 K, and 318 K). under different temperatures (291 K, 300 K, 309 K, and 318 K). Table 1. Stern–Volmer quenching constants for the interaction of 2-MT with CAT under different

for the interaction of 2-MT with CAT under different Tabletemperatures 1. Stern–Volmer (291 K,quenching 300 K, 309 K,constants and 318 K). temperatures (291 K, 300 K, 309 K, and 318 K). pH T (K) KSV (×104 L·mol−1·s−1) kq (×1012 L·mol−1·s−1) R1 S.D. 2 7.4 291 1.59 1.59 0.9935 0.0429 pH T (K) KSV (×104 L·mol−1 ·s−1 ) kq (×1012 L·mol−1 ·s−1 ) R1 S.D. 2 7.4 300 1.46 1.46 0.9939 0.0384 7.4 291 1.59 1.59 0.9935 0.0429 7.4 309 1.35 1.35 0.9922 0.0398 7.4 300 1.46 1.46 0.9939 0.0384 1.23 1.231.35 0.99100.9922 0.03910.0398 7.4 7.4 309318 1.35 7.4

1

2 S.D. is the standard 1.23 1.23 R is318 the correlation coefficient; deviation for the K0.9910 SV values.0.0391

1

R is the correlation coefficient; 2 S.D. is the standard deviation for the KSV values.

2.1.2. Binding Parameters Determination

2.1.2. Binding Determination WhenParameters small molecules bind independently to equivalent sites on a macromolecule via a static fluorescence quenching mechanism, the relevant parameters including the number of binding sites

When small molecules bind independently to equivalent sites on a macromolecule via a static (n) and the binding constant can be determined based on the following equation [29,30]. fluorescence quenching mechanism, the relevant parameters including the number of binding sites (n) F0 -based F and the binding constant can be determined ona the following equation [29,30]. (2) lg = lgK + n lg Q F

where F0, F, and [Q] are the same as in( FEquation 0 − F ) (1), Ka is the binding constant and n is the number of lg = lgKa + nlg [ Q] (2) binding sites. Using a linear fit of lg[(F0 –FF)/F] versus lg[2-MT] under different temperatures (291 K, 300 K, 309 K and 318 K), the corresponding values of Ka and n can be determined from the slope and whereintercept. F0 , F, and [Q] are the same as in Equation (1), Ka is the binding constant and n is the number of As shown in Table 2, the values of n were approximately 1, which implied that there should binding sites.one Using a linear fitbind of lg[(F versus different temperatures (291 K, 0 – F)/F] be only site for 2-MT to to CAT. Moreover, thelg[2-MT] values ofunder Ka reached an order of magnitude 3 4 300 K,of309 K and 318 K), the corresponding values of K and n can be determined from the slope a 10 to 10 , thereby indicating a strong interaction between 2-MT and CAT. Namely, even at a low and intercept. As shown in Table 2, can the still values of bind n were approximately 1, which implied that there should concentration in cells, 2-MT easily with CAT.

be only one site for 2-MT to bind to CAT. Moreover, the values of Ka reached an order of magnitude 2. Binding constantsaand thermodynamic parameters from 2-MT/CAT interaction (pH 7.4). 4 , thereby of 103 to 10Table indicating strong interaction between 2-MT and CAT. Namely, even at a low 4 −1 1 −1 −1 −1 concentration with CAT. ) ΔS° (J·mol ·K ) ΔG° (kJ·mol−1) T (K) inKcells, a (× 102-MT mol·Lcan ) still R bindΔH° (kJ·mol n easily 291 2.97 1.14 0.9987 −24.84 Table constants and thermodynamic parameters from 2-MT/CAT interaction (pH 7.4). 3002. Binding 1.07 1.07 0.9973 −23.11 −80.79 −192.26 309 0.41 0.96 0.9955 −21.38 T (K) 318 Ka (× 104 mol n R1 ∆H ◦ (kJ·mol−1 ) ∆S◦ (J·mol−1 ·K−1 ) ∆G◦ (kJ·mol−1 ) 0.17·L−1 ) 0.83 0.9956 −19.65 291 300 309 318

2.97 1.07 0.41 0.17

1.14 1.07 0.96 0.83

R is the correlation coefficient. 0.9987 0.9973 −80.79 0.9955 0.9956

1

1

R is the correlation coefficient.

−192.26

−24.84 −23.11 −21.38 −19.65

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2.1.3. Identification of Binding Forces Non-covalent intermolecular forces primarily cover Van der Waals’ forces, hydrogen bonding, Int. J. Mol. Sci. 2016, 17, 1330 5 of 12 electrostatic forces, and hydrophobic interactions, which can be identified by the calculation of thermodynamic parameters [31].Forces For moderate temperature change under a constant pressure, the 2.1.3. Identification of Binding enthalpy change (∆H ◦ ) of a specific reaction can be regarded as a constant [32]. Thus, the values of ∆H ◦ Non-covalent intermolecular forces primarily cover Van der Waals’ forces, hydrogen bonding, and ∆S◦ can be approximately calculated by the linear fit of lnK versus 1/T based on the Van’t Hoff electrostatic forces, and hydrophobic interactions, which can be identified by the calculation of ◦ ) under different temperatures equation (Equation (3)) [33]. Meanwhile, the free-energy change (∆G thermodynamic parameters [31]. For moderate temperature change under a constant pressure, the can be acquired according to athe classic thermodynamic equation (Equation (4)). enthalpy change (ΔH°) of specific reaction can be regarded as a constant [32]. Thus, the values of ΔH° and ΔS° can be approximately calculated by the ◦ linear ◦fit of lnK versus 1/T based on the Van't ∆Hthe free-energy ∆S Hoff equation (Equation (3)) [33]. Meanwhile, change (ΔG°) under different lnK = − + RT thermodynamic R temperatures can be acquired according to the classic equation (Equation (4)). ◦

°

◦°

ΔH ΔS ∆G ln K= = -∆H − + T∆S RT

◦

R

(3)

(3) (4)

where K is analogous to the binding constant° (Ka ) °in Equation (2) at a specific temperature(4)(291 K, ∆G = ∆H - T∆S° 300 K, 309 K, and 318 K in this study). R is the universal gas constant (8.314 J·mol−1 ·K−1 ). where K is analogous to the binding constant (Ka) in Equation (2) at a specific temperature (291 K, 300 The linear regression of ln K versus 1/T for the interaction between 2-MT and CAT is shown K, 309 K, and 318 K ◦in this study). R is the universal gas constant (8.314 J·mol−1·K−1). in Figure 3. The ∆H and ∆S◦ were obtained from the slope and intercept, listed in Table 2. The linear regression of ln K versus 1/T for the interaction between 2-MT and CAT is shown in◦ The thermodynamic laws were used to evaluate the types of non-covalent forces: If ∆H < 0, Figure 3. The ΔH° and ΔS° were obtained from the slope and intercept, listed in Table 2. The ∆S◦ 0, ∆S◦ > 0, hydrophobic interactions play a pivotal role in the binding reaction; bonding; If ∆H intermolecular interactions are dominated by Van der Waals’ force and hydrogen bonding; If ΔH° > 0, ◦ < 0, ∆S◦ > 0, electrostatic interaction is the dominant force [33]. In this study, the values of If ∆HΔS° > 0, hydrophobic interactions play a pivotal role in the binding reaction; If ΔH° 0, ◦ −1 ) and −1 ·In 1 ) were −1) interaction is ∆S the◦dominant [33]. study,both the values of ΔH° (−80.79 kJ·mol ∆H electrostatic (−80.79 kJ·mol (−192.26force J·mol K−this negative, implying that Van der −1 −1 and ΔS° (−192.26 J·mol ·K ) were both negative, implying that Van der Waals’ forces and hydrogen Waals’ forces and hydrogen bonding are primarily responsible for the interaction between 2-MT and are primarily responsible for the interaction between 2-MT andthat CAT. addition, the CAT. bonding In addition, the negative ∆G◦ calculated from Equation (4) indicated theIninteraction process negative ΔG° calculated from Equation (4) indicated that the interaction process should be should be spontaneous. Taken together, the results demonstrate that 2-MT could spontaneously bind spontaneous. Taken together, the results demonstrate that 2-MT could spontaneously bind to CAT at to CAT at one binding site to form the 2-MT/CAT complex, mainly through the use of Van der Waals’ one binding site to form the 2-MT/CAT complex, mainly through the use of Van der Waals’ forces forcesand and hydrogen bonding. hydrogen bonding.

Figure 3. Van’t Hoff linear regression analysis for the interaction of 2-MT with CAT.

Figure 3. Van’t Hoff linear regression analysis for the interaction of 2-MT with CAT.

2.2. Molecular Docking Simulation

2.2. Molecular Docking Simulation

In this study, the molecular docking simulation was performed using AutoDock 4.2 to further

In this study, molecular docking simulation wasCAT. performed using AutoDock 4.2 to further characterize thethe interaction behaviors between 2-MT and The CAT model was obtained from the Protein Bank (PDB Code: 1TGU). As the predominant tautomeric form in aqueous solution,from characterize theData interaction behaviors between 2-MT and CAT. The CAT model was obtained the thione form (1,3-thiazolidine-2-thione) was selected as the ligand for molecular docking, andsolution, its the Protein Data Bank (PDB Code: 1TGU). As the predominant tautomeric form in aqueous 3D structure was downloaded from the ZINC Database (http://zinc.docking.org/). The binding the thione form (1,3-thiazolidine-2-thione) was selected as the ligand for molecular docking, and its stereostructure with the lowest energy state is shown in Figure 4, which represents the optimum 3D structure was downloaded from the ZINC Database (http://zinc.docking.org/). The binding binding mode and the most likely binding sites. It was found that 2-MT could bind to the junctional

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stereostructure with the lowest energy state is shown in Figure 4, which represents the optimum Int. J. Mol. Sci. 2016, 17, 1330 6 of 12 binding mode and the most likely binding sites. It was found that 2-MT could bind to the junctional region of the the CAT CAT subdomains subdomains(Figure (Figure4a), 4a),and andthe thebinding bindingsite site was close CAT active sites region of was close to to thethe CAT active sites on on chain A and chain D. The detailed docking results (Figure 4b) further revealed that the amino chain A and chain D. The detailed docking results (Figure 4b) further revealed that the amino acid acid residues link the binding sitecomposed were composed of (chain ILE 68A), (chain (chain A), residues used used to linktothe binding site were of ILE 68 GLUA), 70 GLU (chain70A), SER 119 SER 119 (chain A),(chain ILE 68D), (chain D), and (chain D). The molecular also indicated (chain A), ILE 68 and GLU 70 GLU (chain70D). The molecular docking docking also indicated that the that the interaction between 2-MT and CAT was dominated by Van der Waals’ forces and hydrogen interaction between 2-MT and CAT was dominated by Van der Waals’ forces and hydrogen bonding, bonding, which was in accordance withfluorescence the fluorescence analysis results above (Section2.1.3). 2.1.3).Figure Figure 4b which was in accordance with the analysis results above (Section 4b illustrates that the hydrogen bond exists between the hydrogen atoms (HN) of 2-MT and the oxygen illustrates that the hydrogen bond exists between the hydrogen atoms (HN) of 2-MT and the oxygen atom atom (OE1) (OE1) of of GLU GLU 70 70 (chain (chain A). A).

Figure 4. Molecular docking for the interaction between CAT and 2-MT. (a) Binding site of 2-MT to Figure 4. Molecular docking for the interaction between CAT and 2-MT. (a) Binding site of 2-MT to CAT; (b) Detailed illustration of the binding between CAT and 2-MT. CAT; (b) Detailed illustration of the binding between CAT and 2-MT.

2.3. CAT Conformation Dynamic Characterization 2.3. CAT Conformation Dynamic Characterization 2.3.1. UV–Vis Absorption Spectroscopy Spectroscopy 2.3.1. UV–Vis Absorption UV–Vis absorption absorption spectroscopy the structural changes of UV–Vis spectroscopy is isaawidely widelyused usedtechnique techniquetotoexplore explore the structural changes proteins. The UV–Vis absorption spectra of CAT in the absence and presence of 2-MT is shown in of proteins. The UV–Vis absorption spectra of CAT in the absence and presence of 2-MT is shown in Figure 5. 5. Depending Figure Depending on on its its backbone backbone conformation, conformation, the the maximum maximum absorption absorption of of CAT CAT approximately approximately appeared at 205 nm. However, with increasing 2-MT addition, the UV–Vis absorbance was gradually gradually appeared at 205 nm. However, with increasing 2-MT addition, the UV–Vis absorbance was reduced, and the position of the absorption peak was red-shifted to roughly 208 nm. The dynamic reduced, and the position of the absorption peak was red-shifted to roughly 208 nm. The dynamic changes implied implied that that the the interaction interaction with with 2-MT 2-MT could could lead lead to to the changes the loosening loosening and and unfolding unfolding of of the the CAT CAT peptide chains [34]. Thus, the hydrophobicity of the CAT skeleton decreased. peptide chains [34]. Thus, the hydrophobicity of the CAT skeleton decreased.

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Figure 5. UV-Vis spectra of CAT with different concentrations of 2-MT (versus the same concentration

Figure 5. UV-Vis spectra of CAT with different concentrations of 2-MT (versus the same concentration −1; 2-MT: 0 mol·L−1 (a); 1 × 10−5 mol·L−1 (b); 2 × 10−5 of 2-MT solution). Conditions: CAT: 1.0 × 10−7−mol·L 7 − 1 of 2-MT solution). Conditions: CAT: 1.0 × 10 mol·L ; 2-MT: 0 mol·L−1 (a); 1 × 10−5 mol·L−1 (b); mol·L−1 (c); 3 × 10−5 mol·L−1 (d); pH 7.4; T = 291 K. − 5 5 mol·L−1 (d); pH 7.4; T = 291 K. 2 × 10 mol L−1 (c);spectra 3 × 10of−CAT Figure 5. ·UV-Vis with different concentrations of 2-MT (versus the same concentration

2.3.2.ofSynchronous Fluorescence Spectroscopy 2-MT solution). Conditions: CAT: 1.0 × 10−7 mol·L−1; 2-MT: 0 mol·L−1 (a); 1 × 10−5 mol·L−1 (b); 2 × 10−5

2.3.2. Synchronous Spectroscopy mol·L−1 (c); 3Fluorescence × 10−5 mol·L−1 (d); pH 7.4; T = 291 K. Synchronous fluorescence spectroscopy can provide important information on the polarity changes of the microenvironments around molecular fluorophores [35]. The technologyon is based on Synchronous fluorescence spectroscopy can provide important information the polarity 2.3.2. Synchronous Fluorescence Spectroscopy simultaneous scanning of the excitation and emission monochromators while keeping a constant changes of the microenvironments around molecular fluorophores [35]. The technology is based Synchronous fluorescence spectroscopyinterval can provide on synchronous the polarity wavelength interval. When the wavelength (Δλ) isimportant fixed at 15information or 60 nm, the on simultaneous scanning of the excitation and emission monochromators while keeping a constant changes of the around molecular fluorophores [35]. The technology is based on fluorescence is microenvironments characterized by tyrosine residues or tryptophan residues, respectively [36]. wavelength interval. When the wavelength interval (∆λ) is fixed at 15while or 60keeping nm, thea synchronous simultaneous scanning of the excitationspectra and emission constant The CAT synchronous fluorescence with andmonochromators without 2-MT addition is shown in Figure 6. fluorescence characterized tyrosine residues or tryptophan residues, respectively [36]. wavelength interval. the wavelength interval (Δλ)ofisthe fixed atconcentration 15 or 60 nm, did the not synchronous When theisΔλ was keptWhen at 15by nm (Figure 6A), the variation 2-MT obviously The CAT synchronous fluorescence spectra with and without 2-MT is shown in Figure 6. fluorescence is characterized by tyrosine residues or suggested tryptophan residues, respectively [36]. on change the position of the emission peaks, which that 2-MTaddition had fewer effects the Whenmicroenvironment theThe ∆λ CAT was kept at 15 nm (Figure the variation of the 2-MT didinnot obviously synchronous fluorescence spectra with and However, without 2-MT addition of is shown Figure 6. around the tyrosine6A), residues of CAT. the concentration position the emission peak When Δλ at 15 nmfrom (Figure 6A), variation 2-MT concentration did not effects obviously change the=the position ofred-shifted the emission peaks, that 2-MT hadincreased fewer on the at Δλ 60 nmwas waskept 338 nm the towhich 345 nmsuggested as of thethe 2-MT concentration from 0 to −1 (Figure change the position of 6B). the emission which suggested that 2-MT had fewer effects on the peak 6 × 10−5 mol·L resultspeaks, indicated the However, CAT interaction with 2-MT could increase microenvironment around theThe tyrosine residues ofthat CAT. the position of the emission microenvironment around the tyrosine residues of CAT. However, the position of the emission peakfrom the exposure of its tryptophan residues to a more polar microenvironment [37], thereby further at ∆λ = 60 nm was red-shifted from 338 nm to 345 nm as the 2-MT concentration increased at Δλ =−60 nm was red-shifted from 338conformation. nm to 345 nm as the 2-MT concentration increased from 0 to confirming the 2-MT impacts on CAT 5 − 1 0 to 6 × 10 mol·L (Figure 6B). The results indicated that the CAT interaction with 2-MT could 6 × 10−5 mol·L−1 (Figure 6B). The results indicated that the CAT interaction with 2-MT could increase increase the exposure of its tryptophan residues to a more polar microenvironment [37], thereby further the exposure of its tryptophan residues to a more polar microenvironment [37], thereby further confirming the 2-MT impacts on CAT conformation. confirming the 2-MT impacts on CAT conformation.

Figure 6. Synchronous fluorescence spectra of CAT with different concentrations of 2-MT. Conditions: (A) Δλ = 15 nm and (B) Δλ = 60 nm; CAT: 1.0 × 10−7 mol·L−1; 2-MT: 0 mol·L−1 (a); 1 × 10−5 mol·L−1; (b) 2 × 10−5 mol·L−1 (c); 3 × 10−5 mol·L−1 (d); 4 × 10−5 mol·L−1 (e); 5 × 10−5 mol·L−1; (f) 6 × 10−5 mol·L−1 (g); pH 7.4;6.TSynchronous = 291 K. Figure fluorescence spectra of CAT with different concentrations of 2-MT. Conditions:

Figure 6. Synchronous fluorescence spectra of CAT with different concentrations of 2-MT. Conditions: −7 mol·L−1; 2-MT: 0 mol·L−1 (a); 1 × 10−5 mol·L−1; (A) Δλ = 15 nm and (B) Δλ = 60 nm; CAT: 1.0 ×−10 7 mol (A) ∆λ = 15 nm and (B) ∆λ = 60 nm; CAT: 1.0 × 10 ·L−1 ; 2-MT: 0 mol·L−1 (a); 1 × 10−5 mol·L−1 (b) 2 × 10−5 mol·L−1 (c); 3 × 10−5 mol·L−1 (d); 4 × 10−5 mol·L−1 (e); 5 × 10−5 mol·L−1; (f) 6 × 10−5 mol·L−1 (g); − 5 − 1 − 5 − 1 (b); 2pH × 7.4; 10 T = mol ·L (c); 3 × 10 mol·L (d); 4 × 10−5 mol·L−1 (e); 5 × 10−5 mol·L−1 (f); 291 K. − 5 − 1 6 × 10 mol·L (g); pH 7.4; T = 291 K.

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The results above preliminarily indicated the CAT conformational changes are induced by 2-MT. 2.3.3. Circular Dichroism In order to further investigate the 2-MT effects on the secondary structure of CAT, circular dichroism The results above preliminarily indicated the CAT conformational changes are induced by 2-MT. (CD) measurements were performed in the absence and presence of 2-MT. Shown in Figure 7, there In order to further investigate the 2-MT effects on the secondary structure of CAT, circular dichroism were two peakswere (at 208 and 221 in the CD (CD)negative measurements performed in nm) the absence and spectra presenceof ofCAT, 2-MT.which Shownisincharacteristic Figure 7, there of the protein α-helix [38]. Subsequently, the CD spectra was analyzed using the CDPro software package, were two negative peaks (at 208 and 221 nm) in the CD spectra of CAT, which is characteristic of the and the results are summarized in Table was shown that CAT contained thesoftware secondary structures protein α-helix [38]. Subsequently, the 3. CDItspectra was analyzed using the CDPro package, and the results are summarized in Table It was shown CAT contained the secondary structures of α-helix (19.7%), β-sheet (31.1%), turns3.(23.3%), andthat unordered (25.8%). However, the addition of α-helix (19.7%), β-sheet (31.1%), (23.3%), unordered (25.8%). However, addition of of 2-MT into the CAT solution (molarturns ratio: 10:1) and reduced α-helix proportion bythe 0.7%. Meanwhile, 2-MT into the CAT solution (molar ratio: 10:1) reduced α-helix proportion by 0.7%. Meanwhile, the the proportions of β-sheet and turns were respectively increased by 0.4% and 0.3%. The comparison proportions of β-sheet and turns were respectively increased by 0.4% and 0.3%. The comparison demonstrated that 2-MT could cause some secondary structural changes in CAT. The decrease of demonstrated that 2-MT could cause some secondary structural changes in CAT. The decrease of αα-helixhelix proportion suggests anddenaturation denaturation of CAT induced by 2-MT [9], which proportion suggestsa acertain certain unfolding unfolding and of CAT induced by 2-MT [9], which is in accordance with the results from the UV–Vis absorption and synchronous fluorescence analysis. is in accordance with the results from the UV–Vis absorption and synchronous fluorescence analysis.

Figure 7. CD spectra of CAT in the absence and presence of 2-MT. Conditions: CAT: 1 × 10−7 mol·L−1;

Figure 7. CD spectra of CAT in the absence and presence of 2-MT. Conditions: CAT: 1 × 10−7 mol·L−1 ; −1 (a); 1 × 10−6 mol·L−1 (b); pH 7.4; T = 291 K. 2-MT: 0 mol·L − 1 2-MT: 0 mol·L (a); 1 × 10−6 mol·L−1 (b); pH 7.4; T = 291 K. Table 3. Effects of 2-MT on the proportion of secondary structural elements in CAT at 291 K.

Table 3. Effects of 2-MT on the proportion of secondary structural elements in CAT at 291 K. Secondary Structural Elements in CAT Molar Ratio of CAT to 2-MT α-Helix (%) β-Sheet (%) Turns (%) Unordered (%) Secondary Structural Elements in CAT Molar Ratio of 1:0 19.7 31.1 23.3 25.8 CAT to 2-MT α-Helix19.0 (%) β-Sheet Turns Unordered (%) 1:10 31.5(%) 23.6 (%) 25.9 1:0 19.7 31.1 23.3 25.8 1:10of 2-MT Inhibitory 19.0 31.5 23.6 25.9 2.4. Evaluation Effect on CAT Activity According to the experimental results above, it was found that 2-MT could induce CAT

2.4. Evaluation of 2-MT Inhibitory Effecttoonthe CAT Activity conformational changes by binding junctional region of the CAT subdomains, and the binding site was close to the enzyme active sites on chain A and chain D (Sections 2.2 and 2.3). In order to

According to the experimental results above, it was found that 2-MT could induce CAT verify the potential toxic effects of 2-MT caused by the interaction, the CAT activities under different conformational changes by binding to the junctional region of the CAT subdomains, and the binding 2-MT concentrations and exposure times were determined. As shown in Figure 8, since there was a site was close to the enzyme sitesCAT on chain andbinding chain constant D (Sections 2.2 2.1), and the 2.3).enzyme In order to strong interaction betweenactive 2-MT and with aA high (Section verify activity the potential toxic effects of 2-MT caused bytothe interaction, the CAT activities under different was immediately inhibited when exposed 2-MT for just 10 min. The relative activities were −5 mol·L−1 and 1 × 10−4 decreased 83.3% and 66.7%were whendetermined. 2-MT concentrations were 3in × 10 2-MT significantly concentrations and to exposure times As shown Figure 8, since there was −1, respectively. In addition, the CAT activity gradually reduced as the exposure time increased, mol·L a strong interaction between 2-MT and CAT with a high binding constant (Section 2.1), the enzyme −4 mol·L−1) for 120 min. Therefore, and the activityinhibited was only 56.1% with 2-MT inhibition × 10just activity was residual immediately when exposed to 2-MT(1for 10 min. The relative activities these experiments demonstrated that both 2-MT concentration and exposure time could influence the were significantly decreased to 83.3% and 66.7% when 2-MT concentrations were 3 × 10−5 mol·L−1 inhibition of CAT activity by 2-MT. and 1 × 10−4 mol·L−1 , respectively. In addition, the CAT activity gradually reduced as the exposure time increased, and the residual activity was only 56.1% with 2-MT inhibition (1 × 10−4 mol·L−1 ) for 120 min. Therefore, these experiments demonstrated that both 2-MT concentration and exposure time could influence the inhibition of CAT activity by 2-MT.

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Figure 8. Inhibitory effect of 2-MT on CAT activity. Conditions: CAT: 1.0 × 10−6 mol·L−1; 2-MT: Figure 8. Inhibitory effect of 2-MT on CAT activity. Conditions: CAT: 1.0 × 10−6 mol·L−1 ; 2-MT: (A); 3 × 10−5 mol·L−1 (B); 1 × 10−4 mol·L−1 (C); pH 7.4, T = 291 K. 0 mol·L−1 0 mol·L−1 (A); 3 × 10−5 mol·L−1 (B); 1 × 10−4 mol·L−1 (C); pH 7.4, T = 291 K.

3. Materials and Methods 3. Materials and Methods 3.1. Reagents 3.1. Reagents Catalase (from bovine liver) was purchased from Sigma and was dissolved in ultrapure water Catalase (from bovine liver) was purchased from Sigma and was dissolved in ultrapure water to prepare stock solution (1.0 × 10−−55 mol·L−1−),1 which was then preserved at 0–4 °C. All the other to prepare stock solution (1.0 × 10 mol·L ), which was then preserved at 0–4 ◦ C. All the other chemicals were of analytical grade, and were purchased from Sinopharm Chemical Reagent Co., Ltd. chemicals were of analytical grade, and were purchased from Sinopharm Chemical Reagent Co., Ltd. The stock solution (1.0 × 10−3 mol·L−1) of 2-mercaptothiazoline (2-MT) was prepared. The reaction pH The stock solution (1.0 × 10−3 mol·L−1 ) of 2-mercaptothiazoline (2-MT) was prepared. The reaction pH was controlled using 0.2 mol·L−1 phosphate buffer (mixture of NaH2PO4·2H2O and Na2HPO4·12H2O, was controlled using 0.2 mol·L−1 phosphate buffer (mixture of NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O, pH 7.4). The NaCl stock solution (0.5 mol·L−1) was used to simulate the osmotic pressure in organisms. pH 7.4). The NaCl stock solution (0.5 mol·L−1 ) was used to simulate the osmotic pressure in organisms. All solutions were prepared with ultrapure water (18.25 MΩ). All solutions were prepared with ultrapure water (18.25 MΩ). 3.2. Apparatus 3.2. Apparatus and and Measurements Measurements 3.2.1. Fluorescence Measurements

fluorescencemeasurements measurements were conducted a fluorescence spectrophotometer (RFThe fluorescence were conducted on a on fluorescence spectrophotometer (RF-5301PC, 5301PC, Shimadzu, Kyoto, Japan) equipped with 1 cm cell. Thewavelength excitation wavelength was at Shimadzu, Kyoto, Japan) equipped with a 1 cm cell.aThe excitation was set at 280 nm,set and 280 emission nm, and the emission were recorded the range 290–500 nmspeed with of a scan of 1200 the spectra were spectra recorded in the range ofin290–500 nmofwith a scan 1200speed nm/min and and a photomultiplier tube (PMT) of 700 V. and Theemission excitation and emission widths anm/min photomultiplier tube (PMT) voltage of 700 voltage V. The excitation slit widths wereslit both 5 nm. were both 5 nm. 3.2.2. Molecular Docking Investigation 3.3.2.Docking Molecular Docking Investigation calculations were performed on a CAT protein model (PDB code 1TGU) using AutoDock 4.2. As the predominant tautomeronofa2-MT solutionmodel [11], the thione was using used Docking calculations were performed CATinprotein (PDB codeform 1TGU) for the molecular docking. The 3Dtautomer structureofof2-MT 2-MTinwas downloaded ZINC AutoDock 4.2. As the predominant solution [11], the from thionethe form wasDatabase used for (http://zink.docking.org/). aid of AutoDock, the ligand root of 2-MT and the the molecular docking. TheWith 3D the structure of 2-MT was downloaded from was the detected ZINC Database rotatable bonds were defined. In addition, H O molecules were removed from the CAT model, while (http://zink.docking.org/). With the aid of AutoDock, the ligand root of 2-MT was detected and the 2 polar hydrogen and Compute Gasteiger were added. Alongfrom the X-, andmodel, Z-axeswhile with rotatable bonds atoms were defined. In addition, H2Ocharges molecules were removed theY-, CAT 0.375 grid spacing, theand molecular docking was first performed with the grid the size X-, set Y-, to 126, and polar Å hydrogen atoms Compute Gasteiger charges were added. Along and126 Z-axes 126 Å. On this basis, the grid size was subsequently shrunk to 86, 86 and 86 Å in order to improve with 0.375 Å grid spacing, the molecular docking was first performed with the grid size set to 126, the accuracy. were carried out shrunk by the Lamarckian algorithm 126 docking and 126 Å. On thisDocking basis, thesimulations grid size was subsequently to 86, 86 andgenetic 86 Å in order to (LGA) search method. Each run of the docking experiment was set to terminate after a maximum improve the docking accuracy. Docking simulations were carried out by the Lamarckian genetic of 250,000 (LGA) energysearch evaluations, and the run population size was set to 150.was By set the to molecular algorithm method. Each of the docking experiment terminatedocking after a simulation, the conformation with the lowest free energy for150. further analysis. maximum of 250,000 energy evaluations, andbinding the population sizewas wasused set to By the molecular docking simulation, the conformation with the lowest binding free energy was used for further analysis.

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3.2.3. UV–Visible Absorption Measurements The UV–Visible absorption was measured using a double beam spectrophotometer (UV-6100, Mapada, Shanghai, China), with 1 cm quartz cells and a slit width of 2 nm. The wavelength range was set at 200–240 nm. The reference was the corresponding 2-MT solution without CAT addition. 3.2.4. Synchronous Fluorescence Measurements The synchronous fluorescence spectra of CAT with different 2-MT concentrations were measured (∆λ = 15 nm, λex = 260–340 nm and ∆λ = 60 nm, λex = 300–400 nm, respectively) using a fluorescence spectrophotometer (RF-5301PC, Hitachi, Japan). The excitation and emission slit widths were set at 5 nm. The scan speed and PMT voltage were 1200 nm/min and 700 V, respectively. 3.2.5. Circular Dichroism (CD) Measurements The CD measurements were performed on a spectrophotometer (MOS-450/AF-CD, Bio-Logic, Claix, France). The CAT spectra in the absence and presence of 2-MT were recorded in the wavelength range of 200–260 nm, with a bandwidth of 4 nm and a scanning speed of 1 nm/2 s. 3.2.6. CAT Activity Determination The CAT catalyzes the decomposition of hydrogen peroxide into water and oxygen, and its activity can be assayed by monitoring the absorbency decrease of hydrogen peroxide at 240 nm [39]. The determination was carried out in a pH 7.4 phosphate buffer (20 mM) containing 9 mM hydrogen peroxide. The reaction was initiated by adding the CAT that was exposed to various doses of 2-MT for different times. The relative activity of CAT was calculated by the following equation: Relative activity =

∆A1 × 100% ∆A0

(5)

where ∆A0 represents the absorbency reduction (240 nm) in 2 min after adding CAT without any exposure to 2-MT; ∆A1 represents the absorbency reduction in 2 min after adding the CAT that was exposed to various doses of 2-MT for different time. 4. Conclusions In this study, the toxicological interaction of 2-mercaptothiazoline (2-MT) with the pivotal antioxidant enzyme—catalase (CAT) was investigated at molecular level by multiple spectroscopic techniques and molecular modeling. 2-MT could quench the CAT fluorescence via a static quenching mechanism through formation of the 2-MT/CAT complex. Van der Waals’ forces and hydrogen bonding were primarily responsible for the interaction of 2-MT with CAT at one binding site. The molecular docking simulation indicated that 2-MT could bind to the junctional region of the CAT subdomains, and the binding site was close to the CAT active sites on chain A and chain D. The interaction had few effects on the microenvironment around the tyrosine residues of CAT, but increased the exposure of tryptophan residues to a more polar microenvironment. The presence of 2-MT also induced secondary structural changes, and caused a certain unfolding and denaturation in CAT. Due to the molecular binding, the activity of CAT was significantly inhibited by 2-MT, where 2-MT concentration and exposure time both affected the inhibitory action. Taken together, this work provides valuable insight into the interaction between 2-MT and CAT at the molecular level, and helps us to understand the mechanism of 2-MT toxicity on the environment and human health. Acknowledgments: The work was financially supported by the National Nature Science Foundation of China (NSFC 21307043, 21506076, 21276114), the China Postdoctoral Science Foundation (2016M590411), and the Scientific Practice Project for University Graduate of Jiangsu Province (1125210232141500). Author Contributions: Zhenxing Huang and Yue Teng conceived and designed the experiments. Zhenxing Huang and Ming Huang carried out the experiments, analyzed the data, and drafted the paper. Chenyu Mi participated

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in fluorescence measurements and CAT activity determination. Tao Wang and Dong Chen assisted in molecular docking investigation and circular dichroism measurements. Zhenxing Huang and Yue Teng revised the manuscript. All authors have read and approved the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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