Study on the Mechanism of Interaction between Phthalate Acid Esters

Article pubs.acs.org/JAFC

Study on the Mechanism of Interaction between Phthalate Acid Esters and Bovine Hemoglobin Zhenxing Chi,*,†,‡,§ Jing Zhao,‡ Hong You,†,‡ and Mingjing Wang¶ †

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, 73 Huanghe Road, Harbin 150090, P.R. China ‡ School of Marine Science and Technology, Harbin Institute of Technology, Weihai, 2 Wenhua West Road, Weihai 264209, P.R. China § Guangzhou Key Laboratory of Environmental Exposure and Health, School of Environment, Jinan University, Guangzhou 510632, P.R. China ¶ Weihai Blood Center, 28 Qingdao North Road, Weihai 264200, P.R. China ABSTRACT: Phthalate acid esters (PAEs) are widely used in plastic products as a series of chemical softeners. However, PAEs, which now exist in many environmental media such as the atmosphere, water, and soil, have been shown to be environmental endocrine disruptors. Hemoglobin is a functional protein that carries oxygen in the red blood cells of animals. This study aims at revealing the interactions between bovine hemoglobin (BHb) and PAEs using spectroscopic and molecular modeling methods. The results indicate that the selected representative PAEsdimethyl phthalate (DMP), diethyl phthalate (DEP), and dibutyl phthalate (DBP)can interact with BHb to form BHb-PAE complexes with one binding site, mainly relying on hydrophobic forces, with the affinity order DMP > DEP > DBP, opposite to the order of side-chain length. The binding of PAEs can cause conformational and micro-environmental changes in BHb, which may affect the physiological functions of Hb. Furthermore, molecular docking was applied to define the specific binding sites, the results of which show that all the three PAEs can bind into the central cavity of BHb. The study contributes to expound the toxic mechanism of PAEs in vivo from the point of hematological toxicology. KEYWORDS: hemoglobin, phthalate acid esters, multi-spectroscopic techniques, molecular modeling, toxicity evaluation

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INTRODUCTION Phthalate acid esters (PAEs) are a class of chemical softeners that are widely used in plastic products to increase flexibility and toughness. They are also used in paints, adhesives, cosmetics, personal care products, and so on.1,2 However, it is easy for PAEs to be released into the environment due to the weak binding forces between PAEs and plastic products.2 They have been widely detected in environmental media such as the atmosphere,3 water,4 and soil.5 Because of the relatively high log values of the octanol−water partition coefficient (Kow), PAEs have more opportunities to accumulate in the environment and in organisms.6 The toxicological effects and mechanisms of PAEs are long-term concerns in environmental science. PAEs can enter the human body mainly through respiration, skin contact, and ingestion (including diet and dust),7 which is also the essential route of PAEs getting into the blood.8 The largest number of human blood cells are red blood cells (RBCs), which have important physiological functions9 and are important targets for toxic pollutants. PAEs have good lipid solubility, so it is easy for PAEs to enter the biofilm lipids of RBCs and penetrate the cell membrane into the cell interior during the transport and diffusion process in vivo.10,11 Hemoglobin (Hb) is the main component inside RBCs and the only nonmembrane protein. So, the PAEs that get into the RBCs can combine with Hb easily, affecting its structure and function. There have been some studies on the effect of PAEs on Hb. Kwack et al. investigated the influence of PAEs on the RBCs of Sprague−Dawley male rats and found that the Hb level was reduced in the dimethyl phthalate (DMP)-treated group.12 © XXXX American Chemical Society

However, significant increases in mean corpuscular Hb and mean corpuscular Hb concentration levels were observed in the diethylhexyl phthalate (DEHP)-treated group.12 By in vitro and in vivo tests, Fukuoka et al. demonstrated that dibutyl phthalate (DBP) can induce iron release from Hb inside RBCs in the blood of rats.13,14 The research of Watanabe et al. showed that the alkyl length of mono-n-alkyl phthalates (MAP) can determine whether it can cause iron release from Hb in the rat RBCs.15 Iron release was observed with C3−C6 MAP incubation at lower doses and with C1 and C2 MAP incubation at higher doses, but not with C7, C8, and C4(6) MAP incubation. The above research results help us to understand the toxicity of PAEs in RBCs. However, the interaction mechanism of PAEs with Hb is still unknown. The amino acid sequences of bovine and human hemoglobins have up to 85% similarity. In this study, we used bovine hemoglobin (BHb; structure shown in Figure 1) instead of human hemoglobin and investigated the in vitro interaction of PAEs with BHb under simulated physiological conditions. We selected three representative PAEsin order of increasing side-chain length: DMP, diethyl phthalate (DEP), and DBP (molecular structures shown in Figure 2)for investigation. We estimated the binding constants and the numbers of binding sites for the binding of three PAEs with BHb, and also discussed the effect of PAEs on BHb conformation using fluorescence Received: May 15, 2016 Revised: June 30, 2016 Accepted: July 5, 2016

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DOI: 10.1021/acs.jafc.6b02198 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry quenching, fluorescence lifetime spectrometry, UV absorption spectrometry, circular dichroism (CD), and molecular modeling. The results contribute to understanding the mechanism of toxicity of PAEs in vivo from the perspective of hematological toxicology and provide basic data for further study in this field.

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MATERIALS AND METHODS

Reagents. BHb (sigma) was dissolved in ultrapure water to form a 3.0 × 10−5 mol L−1 solution, preserved at 0−4 °C, and diluted as required. Stock solutions of dimethyl phthalate (DMP, 1.0 × 10−3 mol L−1), diethyl phthalate (DEP, 1.0 × 10−3 mol L−1), and dibutyl phthalate (DBP, 1.0 × 10−3 mol L−1) were prepared and diluted as required. Phosphate buffer (0.2 mol L−1, mixture of NaH2PO4·2H2O and Na2HPO4·12H2O, pH 7.4) was used to control the pH. NaH2PO4·2H2O and Na2HPO4· 12H2O were of analytical reagent grade and obtained from Tianjin Damao Chemical Reagent Factory. All other chemicals were of analytical grade. Ultrapure water was used throughout the experiments. Fluorescence Measurements. Fluorescence measurements were performed with a PerkinElmer spectrofluorometer, model LS 45 (PerkinElmer, USA), equipped with a thermostat bath using a 1.0 cm quartz cell. The scan speed was 1200 nm min−1. The excitation and emission slit widths were set at 5 nm. The photomultiplier tube voltage was 700 V. The specific operations for the fluorescence measurements were as follows: Phosphate buffer (1.0 mL, pH 7.4) and 1.0 mL of BHb (3 × 10−5 mol L−1) were added in turn to each of a series of 10 mL colorimetric tubes. Afterward, different amounts of 1.00 × 10−3 mol L−1 stock solutions of the three PAEs were added. The fluorescence spectra were then measured (excitation at 280 nm and emission wavelengths of 280−450 nm). To confirm the quenching mechanism, we analyzed the florescence quenching data with the Stern−Volmer equation,

F0 = 1 + KSV[Q] = 1 + kqτ0[Q] F

(1)

Figure 3. BHb fluorescence under different PAEs concentrations. Conditions: BHb = 3 × 10−6 mol L−1; PAEs (×10−5 mol L−1) = (a) 0, (b) 0.75, (c) 1.5, (d) 2.25, (e) 3, and (f) 4.5; pH 7.4; T = 293 K.

Figure 1. Domain structure of BHb.

Figure 2. Molecular structure of three PAEs (with atom numbering). B


Article

Journal of Agricultural and Food Chemistry

Figure 4. (A) Stern−Volmer plots for the quenching of BHb by three PAEs under different conditions. (B) Plot of log[(F0 − F)/F] vs log[PAE] for the binding of three PAEs to BHb. Conditions: BHb = 3 × 10−6 mol L−1; pH 7.4; T = 293 K.

Table 1. Stern−Volmer Quenching Constants for the Interaction of Three PAEs with BHb KSV (×105 L mol−1) kq (×1012 L mol−1 s−1) DMP DEP DBP

0.15185 0.12894 0.04987

1.5185 1.2894 0.4987

Table 2. Binding Constants and Number of Binding Sites of the BHb-PAE Systems

Ra

SDb

0.9915 0.98563 0.98738

0.0361 0.04003 0.01449

DMP DEP DBP

a R is the correlation coefficient. bSD is the standard deviation for the KSV values.

Ka (×104 L mol−1)

n

Ra

SDb

0.77 0.59 0.11

0.92675 0.91476 0.84317

0.99606 0.99056 0.99628

0.02838 0.04351 0.02508

a

R is the correlation coefficient for the Ka values. bSD is the standard deviation.

Figure 5. Time-resolved fluorescence decay profile of BHb and BHb-PAE systems. Conditions: BHb = 3 × 10−6 mol L−1; PAE = 1.5 × 10−5 mol L−1; pH 7.4; T = 293 K. C


Article

Journal of Agricultural and Food Chemistry where F0 and F are the fluorescence intensity in the absence and presence of the quencher, respectively, [Q] is the concentration of the quencher, τ0 is the fluorescence lifetime in the absence of the quencher, kq is the quenching rate constant of the biological macromolecule, and KSV is the Stern−Volmer quenching constant. For static quenching, we can also obtain the binding constant (Ka) and number of bonding sites (n) per BHb molecule according to the following equation when small molecules independently bind to a set of equivalent sites on a macromolecule:16

lg

(F0 − F ) = lg K a + n lg [Q] F

where Cp is the molar concentration of the protein, n is the number of amino acid residues, l is the path length of the cell (1 cm), MRE208 is the observed mean residue ellipticity value at 208 nm, 4000 is the MRE of the β-form and random coil conformation cross at 208 nm, and 33 000 is the MRE value of a pure α-helix at 208 nm.17 Molecular Docking. Docking calculations were carried out using Docking Server.18 The MMFF94 force field19 was used for energy minimization of the ligand molecule (DMP) using Docking Server. Gasteiger partial charges were added to the ligand atoms. Nonpolar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out using the BHb protein model. Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of AutoDock tools.20 Affinity (grid) maps of 100 × 100 × 100 Å grid points with 0.375 Å spacing were generated using the Autogrid program.20 AutoDock parameter set- and distance-dependent dielectric functions were used in the calculation of the van der Waals and electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm and the Solis and Wets local search method.21 The initial positions, orientations, and torsions of the ligand molecules were randomly set. Each docking experiment was derived from 10 different runs that were set to terminate after a maximum of

(2)

The fluorescence lifetime spectra were recorded using an FLS920 spectrophotometer (Edinburgh Instruments, UK). While the excitation wavelength was 280 nm, the emission wavelength was 341 nm. UV−Vis Absorption Spectra Measurements. The UV−vis absorption spectra of BHb in the presence and absence of the three PAEs were recorded at room temperature (293 K) using a UV-520 spectrophotometer (Hitachi, Japan) equipped with 10 mm quartz cells in the range of 190−350 nm. Circular Dichroism (CD) Measurements. CD spectra were recorded at room temperature (293 K) and measured from 200 to 260 nm on a Jasco 810 spectropolarimeter with a quartz cell of 1 cm path length. The concentration of BHb was fixed at 3.0 × 10−7 mol L−1, and the scanning speed was set at 200 nm min−1. Each spectrum was the average of two successive scans. Generally speaking, the α-helix of proteins has two characteristic negative peaks at 208 and 222 nm, which reflect the secondary structure of the protein. The α-helical contents of BHb in the absence and presence of the three PAEs were calculated from eqs 3 and 4,

MRE =

obsd CD (mdeg) C pnl × 10

α‐helix (%) =

− MRE 208 − 4000 × 100 33 000 − 4000

Table 3. Atoms Involved in the Hydrophobic Interaction of PAEs with BHb and the Distances between Them, Analyzed from the Molecular Docking Result PAE atom

BHb atom

d (Å)

DMP

C10 C1 C5 C14 C2 C3 C14

β1 TYR 35 (CE1) β1 PRO 36 (CD) β1 TRP 37 (CZ3) α2 PRO 95 (CB, CG) α2 TYR 140 (CB) α2 TYR 140 (CB, CD2) α2 TYR 140 (CE2)

3.65 3.35 3.74 3.56 3.48 3.28 3.65

DEP

C16 C15 C10 C15

β1 PRO 36 (CD) β1 TRP 37 (CZ3) β1 TRP 37 (CH2, CZ3) α2 TYR 140 (CB, CD2)

3.79 3.80 3.24 3.48

DBP

C20 C11 C4 C11 C3 C12 C13

α1 VAL 1 (CG1) β1 PRO 36 (CD) β1 TRP 37 (CH2, CZ3) β1 TRP 37 (CZ3) β1 TRP 37 (CH2) α2 TYR 140 (CB, CD2, CG) α2 TYR 140 (CB)

3.67 3.64 3.58 3.64 3.85 3.14 3.43

(3)

(4)

Figure 6. Binding mode between PAEs and BHb. BHb is displayed in cartoon. The atoms of PAEs are color-coded as follows: O, red; C, green.

Figure 7. Molecular modeling of the interaction between PAEs and BHb. The atoms of PAEs are color-coded as blue, and the atoms of amino acid residues of BHb are gray. D


Article

Journal of Agricultural and Food Chemistry 250 000 energy evaluations. The population size was 150. During the search, a translational step of 0.2 Å and quaternion and torsion steps of 5 were applied.

The detailed results (Table 3) indicate that the three PAEs can interacts with BHb primarily via hydrophobic forces (DMP with β1 TRP 35, β1 PRO 36, β1 TYR 37, α2 PRO 95, and α2 TYR 140 residues; DEP with β1 PRO 36, β1 TYR 37, and α2 TYR 140 residues; and DBP with α1 VAL 1, β1 PRO 36, β1 TYR 37, and α2 TYR 140 residues). In addition, there are some other interaction forces except for hydrophobic forces. DMP can form hydrogen bonds with α1 ASP 126, and some other forces (DMP with α1 ASP 126, α1 LYS 127, α1 ALA 130, β1 TRP 37, α2 THR 137, α2 TYR 140) are also observed. There are no hydrogen bonds between DEP and BHb, while

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RESULTS AND DISCUSSION Fluorescence Measurements. The intrinsic fluorescence of proteins can provide considerable information about their structure and dynamics.22,23 BHb can emit intrinsic fluorescence, primarily due to tryptophan (Trp) and tyrosine (Tyr) residues. In this study, we measured the fluorescence spectra of BHb under different concentrations of DMP, DEP, and DBP at a constant BHb concentration and temperature of 293 K. The results are shown in Figure 3. The fluorescence emission maximum of BHb was at approximately 341 nm under the experimental conditions. With increasing PAEs (DMP, DEP, or DBP) concentration, the fluorescence intensity of BHb decreased gradually, indicating that the fluorescence of BHb can be quenched by the three PAEs. In addition, a blue shift of the fluorescence peak was also observed, revealing that the three PAEs caused the hydrophobicity of the micro-environment around the amino acid residues (Trp or Tyr) to increase.24 Since PAEs can quench the intrinsic fluorescence of BHb, the fluorescence quenching data were further analyzed according to the Stern−Volmer equation (eq 1) to determine the quenching mechanism and examine whether PAEs interact with BHb to form BHb-PAE complexes. The Stern−Volmer plots for the quenching of BHb by the three PAEs are given in Figure 4A. The values of KSV and kq are listed in Table 1. The KSV values decreased following the order DMP > DEP > DBP, opposite to the order of side-chain length and log(Kow). The kq was greater than 2.0 × 1010 L mol−1 s−1, indicating that the quenching was not initiated from dynamic collision but from the formation of a complex.25,26 We then measured the fluorescence lifetime to directly determine the quenching mechanism of BHb fluorescence by PAEs. Figure 5 shows the time-resolved decay of BHb and BHb-PAE systems. The data fit well to the sum of a singleexponential decay with a χ2 value close to 1.00. There is no obvious difference in the lifetime values of BHb with and without PAEs. Thus, we conclude that the quenching mechanism is static and involves the formation of BHb-PAE complexes.24 Then, the binding constant (Ka) and number of binding sites (n) were calculated according to eq 2. A plot of log[(F0 − F)/F] versus log[Q] gives a straight line (Figure 4B) using least-squares analysis; the slope is equal to n, and the Y-intercept is log Ka. The calculated n and Ka are shown in Table 2. The number of binding sites n is approximately 1, indicating that there is one binding site in BHb for PAEs during their interaction. In summary, all three PAEs can interact with BHb with one binding site to form BHb-PAE complexes. The interaction affinity decreases with increasing side-chain length of PAEs. The hydrophobicity of the micro-environment around the amino acid residues of BHb (Trp or Tyr) was increased. Computational Modeling of the BHb-PAE Complexes. After determining that the three PAEs interacted with BHb at one binding site to form BHb-PAE complexes, we employed the molecular docking method to identify the specific binding sites of the PAEs on BHb. The best energy ranked results are given in Figures 6 and 7. Hb is conmposed of two α subunits and two β subunits, with a central cavity (Figure 1). Figure 6 shows that all the three PAEs bind into the central cavity of Hb. Figure 7 provides the binding modes of the three PAEs with the residues of BHb.

Figure 8. UV−vis spectra of BHb under different PAEs concentrations (vs the same concentration of PAEs solution). Conditions: BHb = 3 × 10−6 mol L−1; PAEs (×10−5 mol L−1) = (a) 0, (b) 0.75, (c) 1.5, (d) 2.25, (e) 3, and (f) 4.5; pH 7.4; T = 293 K. E


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Journal of Agricultural and Food Chemistry some other forces (DEP with α1 ASP 126, α1 LYS 127, β1 TRP 37, α2 PRO 95, α2 THR 137, α2 SER 138, α2 TYR 140) are present. DBP interacts with BHb through hydrogen bonds (DBP with α1 ASP 126) and some other forces (DBP with α1 ASP 126, α1 LYS 127, β1 TRP 37, α2 PRO 95, α2 THR 134, α2 THR 137, α2 SER 138, α2 TYR 140). However, the hydrophobic forces play a major role in the binding of the three PAEs to BHb. To sum up, the molecular docking results revealed that all the three PAEs can bind to the central cavity of BHb mainly through hydrophobic forces. Effect of PAEs on the Conformation of BHb. To study the effect of PAEs on the conformation of BHb, we employed UV−vis absorption and CD spectroscopy. UV−Vis Absorption Spectroscopy. UV−vis absorption spectroscopy can be utilized for exploring protein structural changes and investigating protein−ligand complex formation.27 The UV−vis spectra of BHb under different PAEs concentrations are shown in Figure 8. BHb has a strong absorption peak at approximately 209 nm, which reflects the framework conformation of the protein.28 When the PAEs concentration in BHb solution increased, the intensity of the absorption peak at 209 nm decreased with red-shifted (Figure 8) and the degree of decrease in peak intensity followed the order DMP > DEP > DBP. These results indicate that the interactions between the three PAEs and BHb can lead to the loosening and unfolding of the protein skeleton.29 Circular Dichroism Spectroscopy. CD spectroscopy is a quantitative technique used to investigate the secondary structure changes of proteins.30 The CD spectra of BHb with and without PAEs are shown in Figure 9. The α-helical content

with the order DBP > DEP > DMP, contrary to the effect on the protein skeleton.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: +86 6315687231; fax: +86 6315687231. Funding

This work was supported by the Shandong Provincial Natural Science Foundation, China (ZR2014BQ033), the Guangzhou Key Laboratory of Environmental Exposure and Health (No. GZKLEEH201613), and a China Postdoctoral Science Foundation funded project (2013M540297). The Natural Scientific Research Innovation Foundation in Harbin Institute of Technology (HIT.NSRIF.2014126) is also acknowledged. Notes

The authors declare no competing financial interest.

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REFERENCES

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Figure 9. CD spectra of BHb under different PAEs. Conditions: BHb = 3.0 × 10−7 mol L−1; PAEs = 6 × 10−6 mol L−1; pH 7.4; T = 293 K.

calculated using eqs 3 and 4 successively increases in the order of BHb, BHb-DMP, BHb-DEP, and BHb-DBP, being 31.89%, 33.46%, 34.14%, and 34.94%, respectively. The binding of PAEs to BHb can change the secondary structures of BHb, with the α-helical content of BHb lower than those of BHb-PAE complexes.29 As mentioned above, the conformation of BHb was altered for its interaction with PAEs. The binding of all the three PAEs to BHb can lead to the loosening and unfolding of the protein skeleton with the order DMP > DEP > DBP, opposite to the order of side-chain length. The α-helical content of the secondary structure of BHb increased because of bound PAEs F


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Study on the Mechanism of Interaction between Phthalate Acid Esters

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