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Article Cite This: ACS Omega 2019, 4, 563−572
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Comprehensive Insights into the Interactions of Two Emerging Bromophenolic DBPs with Human Serum Albumin by Multispectroscopy and Molecular Docking Zhenxuan Zhang,† Mengting Yang,*,† Jiaying Yi,† Qingyao Zhu,† Cui Huang,† Yantao Chen,† Juying Li,† Bo Yang,† and Xu Zhao‡ †
ACS Omega 2019.4:563-572. Downloaded from pubs.acs.org by 193.93.194.168 on 01/10/19. For personal use only.
Shenzhen Key Laboratory of Environmental Chemistry and Ecological Remediation, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, China ‡ State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China S Supporting Information *
ABSTRACT: Disinfection byproducts (DBPs) are of high concern due to their ubiquitous existence in disinfected drinking water and their potential adverse effects on human health. In this study, two bromophenolic DBPs 4-bromophenol (4-BrPh) and 2,4-dibromophenol (2,4-DiBrPh) were selected to investigate their binding interactions with human serum albumin (HSA) using spectroscopic techniques and a molecular docking method. The experimental results demonstrated that both of the DBPs could bind with HSA to form bromophenol−HSA complexes with the HSA secondary structure being changed, primarily relying on hydrogen bonding and van der Waals forces, but 2,4DiBrPh showed a higher binding affinity. The binding constants of 4-BrPh−HSA and 2,4-DiBrPh−HSA were 2.66 × 103 and 1.83 × 104 M−1 at 310 K, respectively. Molecular docking results revealed the locations of the binding sites for bromophenols on HSA (locating in subdomain IB). In addition, the comparative toxicity of the two bromophenolic DBPs was evaluated with the mammalian cytotoxicity bioassay and the results showed that the LC50 values of 4-BrPh and 2,4-DiBrPh were 3.08 × 10−5 and 1.09 × 10−5 M, respectively. Notably, 2,4-DiBrPh with a higher binding affinity toward HSA also showed a significantly higher toxic potency than 4-BrPh.
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INTRODUCTION
DBPs in disinfected potable water, there is an increasing concern on the adverse effects of DBPs on human health. Proteins are one group of the most important biological macromolecules participating in a vast array of physiological activities within organisms, such as catalyzing metabolic reactions, transmitting cell signals, binding and targeting antigens for destruction, transporting molecules, and conferring stiffness and resilience to maintain cell shape.14 Investigation of the interactions of xenobiotics/emerging environmental contaminants with proteins could promote better understanding of related toxic mechanisms; such information is important as abnormal proteins in terms of structure and function may induce physiological dysfunctions and diseases in organisms. Among proteins, human serum albumin (HSA) is the most abundant protein in the human blood circulatory system with concentrations of 35−55 mg mL−1 in the bloodstream.15
Disinfection byproducts (DBPs) are a group of chemical substances unintentionally generated from the reactions of disinfectants with organic matter (mainly including natural organic matter, effluent organic matter, and emerging environmental contaminants) during the water disinfection process.1−4 They have generally been considered as one important series of food contaminants owing to their diversity and daily long-term exposure to human beings through ingestion of disinfected drinking water, beverages prepared with tap water (e.g., coffee, tea, and juices) or foods treated using disinfected water (e.g., washed vegetables and fruits).5,6 Previous studies with different types of toxicity bioassays reported that many DBPs were potential cytotoxicants, genotoxicants, mutagens, neurotoxicants, carcinogens, and/or teratogens,7−10 despite the fact that their toxic mechanisms are not fully understood currently. Moreover, human epidemiological studies provided evidence of an association between DBP exposure and adverse birth outcomes/raised health risks of cancers (colorectal and bladder).11−13 Hence, due to the ubiquitous existence of © 2019 American Chemical Society
Received: November 8, 2018 Accepted: December 25, 2018 Published: January 8, 2019 563
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Figure 1. (a) Fluorescence emission spectra of HSA (5 μM) in the absence and presence of increasing concentrations of 4-BrPh (0−7: 0−49 μM with 7 μM intervals) at 300 K. (b) Fluorescence spectra of HSA (5 μM) in the absence and presence of increasing concentrations of 2,4-DiBrPh (0−7: 0−8.4 μM with 1.2 μM intervals) at 300 K. The insets show the Stern−Volmer plots for bromophenol−HSA interactions at three different temperatures.
expected to be beneficial for the understanding of the toxicity, bioavailability, and metabolism of bromophenols inside human body.
Besides maintaining pH and osmotic blood pressure, it also plays an essential role in the transportation, distribution, and deposition of a broad range of endogenous and exogenous compounds (as ligands) in vivo.16 When toxicants enter into the human blood system, it is generally believed that the binding of toxicants with HSA results in decreased concentrations of free ligands in plasma; thus, the bioavailability of the toxicants can be reduced;17,18 meanwhile, conformational changes of HSA may occur via binding and they could induce dysfunction of HSA. Therefore, it has been extensively reported that binding interactions between serum albumin and ligands can be used to effectively evaluate the bioavailability, toxicity, and health risk of pharmacological drugs and environmental contaminants.19−22 However, although the occurrence of DBPs in blood fluid has been confirmed in several studies,23,24 to the best of our knowledge, interactions of DBPs with serum albumin have not been reported yet. Recently, bromophenols were identified as new aromatic DBPs and drew great attention, as they presented significantly higher toxicity than that of regulated aliphatic DBPs. For example, 4-bromophenol was 181 and 39 times more toxic than bromoacetic acid in terms of developmental toxicity and growth inhibition, respectively.25,26 Their occurrence can cause off-flavor problems from the perspective of high-quality water supply, and the odor threshold concentrations of some bromophenols are in the ng/L level. 27 In addition, bromophenols can be extensively found in various seafoods (such as prawns, fish, and crustaceans) and are considered to be ubiquitous in the lower levels of marine food webs.28,29 Also, they are widely used in various industrial products (e.g., flame retardant intermediates and polymer intermediates) and thus are known as environmental micropollutants in freshwater environments.30−32 Accordingly, two bromophenolic DBPs 4bromophenol (4-BrPh) and 2,4-dibromophenol (2,4-DiBrPh) were chosen as representatives of emerging aromatic DBPs and target compounds in this study. The primary objective of this work was to investigate the binding interactions of the two bromophenolic DBPs with HSA under simulated physiological conditions of human blood, by using multiple spectroscopic techniques (i.e., fluorescence, absorption, and circular dichroism (CD) spectroscopy) and a molecular docking method. Besides, to investigate whether an aromatic DBP showing a higher HSA binding affinity also possesses a higher toxicity, a chronic mammalian cell cytotoxicity assay was conducted to evaluate the comparative toxicity of the two bromophenols. The results gained in the present study were
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RESULTS AND DISCUSSION Fluorescence Quenching. Fluorescence spectroscopy is a useful and convenient method to study the interactions of proteins and ligands as well as related protein conformation information. Generally, HSA possesses three intrinsic fluorophores, i.e., tryptophan (Trp), tyrosine (Tyr), and phenylalanine, but its intrinsic fluorescence is almost contributed by Trp. This is mainly because the quantum yield of phenylalanine is extremely low, whereas the fluorescence of Tyr is almost completely quenched if Tyr is ionized or is near a carboxyl group, an amino group, or a Trp.33 Due to its high sensitivity to the microenvironment of HSA, the intrinsic fluorescence of the protein can be easily quenched through various molecular interactions with quencher molecules, leading to the decrease of fluorescence quantum yields of fluorophores. Figure 1a,b, respectively, shows the effects of 4BrPh and 2,4-DiBrPh addition on the fluorescence intensity of HSA at 300 K. For each of the two bromophenolic DBPs, as DBP concentration increased, the HSA fluorescence intensity decreased regularly, indicating that the intrinsic fluorescence of HSA was quenched and 4-BrPh/2,4-DiBrPh was able to bind with HSA. Quenching Mechanism and Binding Parameters. The fluorescence quenching mechanisms are commonly classified into two types, static quenching (shaping ground-state complexes between fluorophores and quenchers) and dynamic quenching (generated from collisional encounters between fluorophores and quenchers).34 They can be distinguished according to fluorescence lifetime measurements or their dependence on viscosity and temperature.35 When the system temperature rises, the fluorescence quenching constant will increase for dynamic quenching, whereas it generally shows a decreasing trend for static quenching. To characterize the fluorescence quenching mechanism of the 4-BrPh/2,4DiBrPh−HSA system, the quenching experiments were conducted at three different temperatures (i.e., 290, 300, and 310 K). All fluorescence intensity was corrected for the inner filter effect by using the following equation36 Fcor = Fobs 10(A 280 + Aem)/2
(1)
where Fcor and Fobs are the corrected and measured fluorescence data and A280 and Aem are the absorbance of 564
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Table 1. Binding and Thermodynamic Parameters for the Interactions of HSA with the Two Bromophenolic DBPs at Different Temperatures ligand 4-BrPh
2,4-DiBrPh
T (K)
Ksv (M−1)
Kq (M−1 s−1)
K (M−1)
290 300 310 290 300 310
× × × × × ×
× × × × × ×
× × × × × ×
4.81 3.85 3.23 3.54 2.52 1.94
3
10 103 103 104 104 104
4.81 3.85 3.23 3.54 2.52 1.94
11
10 1011 1011 1012 1012 1012
4.72 3.30 2.66 3.72 2.57 1.83
n 3
10 103 103 104 104 104
ΔS0 (J mol−1 K−1)
−24.51
−4.02
−26.58
−4.21
1.00 0.99 0.98 1.00 1.00 0.99
ΔG0 (kJ mol−1) −20.34 −20.30 −20.26 −25.36 −25.32 −25.27
of 4-BrPh−HSA at different temperatures were lower than 1 × 104 M−1, whereas for 2,4-DiBrPh−HSA, those were higher than 1 × 104 M−1, suggesting the existence of moderately strong interaction for 2,4-DiBrPh−HSA and relatively weak interaction for 4-BrPh−HSA. The values of n for both 4BrPh−HSA and 2,4-DiBrPh−HSA were calculated equal to 1 approximately, implying that there was one independent binding site on HSA for either 4-BrPh or 2,4-DiBrPh during interaction. Thermodynamic Analysis. The interaction forces between biomacromolecules and small molecules include hydrophobic interactions, electrostatic forces, hydrogen bonding, and van der Waals forces.41 The thermodynamic parameters, i.e., enthalpy change (ΔH0), entropy change (ΔS0), and change in Gibbs free energy (ΔG0) can be used to characterize the forces involved in the ligand binding to HSA. The values of ΔH0, ΔS0, and ΔG0 for the binding reactions were calculated using the van’t Hoff equation (eq 4) and eq 5 and are listed in Table 1.
HSA in the presence of 4-BrPh/2,4-DiBrPh at the excitation (280 nm) and emission wavelengths, respectively. The corrected fluorescence data were plotted according to the classical Stern−Volmer equation37 F0 = 1 + Kqτ0[Q] = 1 + K sv[Q] F
ΔH0 (kJ mol−1)
(2)
where F and F0 are the fluorescence intensities of HSA with and without DBP, respectively. Kq, is the quenching rate constant of the biomacromolecule; Ksv is the Stern−Volmer quenching constant. τ0 is the average fluorescence lifetime of the biomolecule in the absence of quencher, and the value of the biopolymer equals 10−8 s. [Q] is the concentration of the quencher. On the basis of the inset graphs of Figure 1a,b, the Stern−Volmer plots of HSA fluorescence quenched by 4BrPh/2,4-DiBrPh at different temperatures demonstrated a linear trend and the slopes decreased obviously with the increase in temperature. Accordingly, the calculated Ksv and Kq values at different temperatures are shown in Table 1; the Ksv and Kq values decreased with rising temperature, indicating that the static quenching mechanism dominated in bromophenol−HSA interactions. Moreover, the quenching constant Kq of 4-BrPh and 2,4-DiBrPh was in the range of (3.23−4.81) × 1011 and (1.94−3.54) × 1012 M−1 s−1, respectively; those values are significantly higher than 2.0 × 1010 M−1 s−1, which is the maximum scatter collision quenching constant reported for dynamic quenching.38 Therefore, the relatively high Kq values also support static quenching mechanism responsible for the interactions between bromophenolic DBPs and HSA. The fluorescence data were further analyzed to determine the binding constant and the number of binding sites of the HSA−ligand complexes using eq 339 ÅÄÅ F − F ÑÉÑ ÑÑ = log K + n log[Q] logÅÅÅÅ 0 Ñ ÅÅÇ F ÑÑÑÖ (3)
ln K = −
ΔH 0 ΔS 0 + RT R
ΔG 0 = ΔH 0 − T ΔS 0
(4) (5)
where K is the binding constant at temperature T and R is the universal gas constant (8.3145 J mol−1 K−1). In the present study, the negative ΔG0 values suggest that the bromophenol− HSA complexes formed spontaneously. At the same temperature, compared with the 4-BrPh−HSA complex, a more negative ΔG0 value can be found for the 2,4-DiBrPh−HSA complex, indicating a more stable complex formed during the interaction between 2,4-DiBrPh and HSA. In addition, previous studies35,42 reported that interaction processes mainly involving hydrophobic forces generally show positive values of ΔH0 and ΔS0, whereas van der Waals forces and hydrogen bonding are more important in interactions presenting negative values of ΔH0 and ΔS0; besides, if ΔH0 < 0 and ΔS0 > 0, electrostatic forces play significant roles in the protein association process. Therefore, the negative values of ΔH0 and ΔS0 observed in this study (Table 1) indicated that hydrogen bonding and van der Waals forces are the predominant forces that contributed to the interactions of bromophenols and HSA. Fluorescence Resonance Energy Transfer (FRET) and Binding Distance between Bromophenols and HSA. FRET phenomenon occurs through a nonradiative dipole− dipole coupling due to the transformation of excitation energy from an excited donor molecule (protein) to an acceptor (ligand), being widely used to determine the spatial distance between donor and acceptor. According to Förster’s theory,43 the FRET phenomenon is possible if (1) the UV absorption spectrum of the acceptor suitably overlaps the fluorescence emission spectrum of the donor, (2) the distance between the
where F, F0, and [Q] are the same variables as those in eq 2, K is the binding constant, and n is the number of binding sites. As shown in Figure S1a,c in the Supporting Information (SI), ÄÅ F − F ÉÑ the plot of logÅÅÅÅ 0 F ÑÑÑÑ versus log[Q] gave a straight line for 4Ç Ö BrPh or 2,4-DiBrPh; thus, the K and n values can be obtained from the intercept and slope data. The calculated binding constant and the number of binding site data are presented in Table 1. It was noticed that the determined K values decreased with rising temperature due to destabilization of the bromophenol−HSA complexes at relatively high temperatures, which further confirmed the static quenching mechanism for the interaction between the selected DBPs and HSA. Strong binding between HSA and a small molecule generally shows a K value in the range of 105−107 M−1, and a low-to-moderate binding force between the HSA and ligand has a characteristic K value in the range of 102−104 M−1.40 The binding constants 565
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Figure 2. Overlaps between the fluorescence emission spectra of HSA (5 μM) and the absorption spectra of (a) 4-BrPh (5 μM) and (b) 2,4DiBrPh (5 μM) at 300 K.
BrPh/2,4-DiBrPh) may occur with high probability. Moreover, the higher r values than the corresponding R0 values can be viewed as another evidence43 to further confirm the static quenching mechanism mentioned above. Interestingly, smaller r value usually indicates that the protein more readily binds and transports the ligand;45 in this study, the distance r in the 2,4-DiBrPh−HSA system (1.62 nm) is less than that in the 4BrPh−HSA system (2.24 nm). Hence, if the two DBPs enter into the human blood system, HSA more readily binds and transports 2,4-DiBrPh than 4-BrPh. Synchronous Fluorescence Analysis. Synchronous fluorescence spectroscopy can be an alternative tool to elucidate characteristic changes of microenvironment around the chromophores in the structure of HSA upon ligand binding.18 When the wavelength intervals Δλ are set at 15 and 60 nm during the synchronous fluorescence analysis, characteristic information of Tyr and Trp residues can be obtained separately, as the shift of the maximum emission wavelength could provide a hint for understanding the polarity and hydrophobicity alteration in the microenvironment surrounding fluorophores.46 Figure 3 shows the effects of the two DBPs on HSA synchronous fluorescence intensity. As the HSA
donor and the acceptor is less than 8 nm, (3) and the transition dipole orientations of the donor and the acceptor are arrayed properly. The energy transfer efficiency (E) from the donor HSA to the acceptor 4-BrPh/2,4-DiBrPh, the distance (r) between the donor HSA and acceptor DBP, and the critical distance (R0) when the energy transfer efficiency equals 50% can be obtained according to the literature44 with details shown in the SI. Figure 2 shows the significant overlap between the fluorescence emission spectrum of HSA and the absorption spectrum of 4-BrPh or 2,4-DiBrPh at a DBP/HSA molar ratio of 1:1 at 300 K. On the basis of the calculation, the parameters R0 and r are listed in Table 2. Since the distance r Table 2. Parameters to Estimate the Spatial Distances between HSA and Bromophenols ligand
J (cm3 M−1)
R0 (nm)
r (nm)
4-BrPh 2,4-DiBrPh
1.13 × 10−15 1.06 × 10−15
1.16 1.15
2.24 1.62
between HSA and each of the two DBPs was less than 8 nm, the energy transfer from the donor (HSA) to the acceptor (4-
Figure 3. Synchronous fluorescence spectra of HSA (5 μM) with the two DBPs at 300 K: (a) 4-BrPh, Δλ = 15 nm; (b) 4-BrPh, Δλ = 60 nm; (c) 2,4-DiBrPh, Δλ = 15 nm; and (d) 2,4-DiBrPh, Δλ = 60 nm. The concentrations of 4-BrPh and 2,4-DiBrPh from 0 to 7 were 0−49 μM with 7 μM intervals and 0−8.4 μM with 1.2 μM intervals, respectively. 566
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Figure 4. (a) UV spectra of HSA (5 μM) in the absence and presence of 4-BrPh (0−7: 0−49 μM with 7 μM intervals) at 290 K. (b) UV spectra of HSA (5 μM) in the absence and presence of 2,4-DiBrPh (0−7: 0−84 μM with 12 μM intervals) at 290 K. (c) CD spectra of HSA (5 μM) in the absence and presence of 4-BrPh (0−3: 0−630 μM with 210 μM intervals) at 290 K. (d) CD spectra of HSA (5 μM) in the absence and presence of 2,4-DiBrPh (0−3: 0−180 μM with 60 μM intervals) at 290 K.
Table 3. Effects of 4-BrPh and 2,4-DiBrPh on the HSA Secondary Structure CHSA (M) 5.0 5.0 5.0 5.0 5.0 5.0 5.0
× × × × × × ×
10−6 10−6 10−6 10−6 10−6 10−6 10−6
Cligand (M) 0 2.1 4.2 6.3 0.6 1.2 1.8
× × × × × ×
10−4 10−4 10−4 10−4 10−4 10−4
ligand
α-helix (%)
β-sheet (%)
β-turn (%)
random coil (%)
4-BrPh 4-BrPh 4-BrPh 2,4-DiBrPh 2,4-DiBrPh 2,4-DiBrPh
62.1 60.2 57.7 54.0 57.2 52.8 50.7
4.9 5.0 6.3 8.6 6.8 7.0 7.6
12.3 13.0 13.9 14.7 14.2 16.6 17.9
21.7 22.7 23.8 24.6 24.2 25.9 27.7
fluorescence intensity is highly related to Trp and Tyr, the trend of synchronous fluorescence intensity generally coincides with the aforementioned fluorescence spectra (Figure 1a,b), illustrating quenching effects on fluorophores upon addition of either 4-BrPh or 2,4-DiBrPh. Noticeably, as 4-BrPh concentration increased, there was no discriminable change in the emission maximum of Trp, whereas the emission peaks corresponding to Tyr showed a slight redshift (1 nm); for 2,4-DiBrPh, both emission maxima of Trp and Tyr were unchanged. This phenomenon implies that binding of 4-BrPh or 2,4-DiBrPh to HSA had little effect on the change of microenvironment around Trp residue (Trp 214), which located in subdomain IIA. Interactions of 4-BrPh−HSA and 2,4-DiBrPh−HSA also cannot quite effectively perturb the environment of Tyr residues since only the addition of 4-BrPh slightly increased the polarity around Tyr residues of HSA. UV Absorption and CD Analysis. UV−vis absorption measurement is a simple and applicable method to explore the structural alterations of biomolecules and to confirm the formation of protein−ligand complexes.47 The UV absorption spectra of HSA with different concentrations of 4-BrPh/2,4DiBrPh were measured to further analyze the conformation changes of HSA by addition of DBP. Figure 4a,b displays the UV absorption spectra for interactions of the two bromophenols with HSA. The absorption peak at around 279 nm reflects
the absorption of the aromatic amino acid residues (Trp, Tyr, and phenylalanine) in the HSA structure.35 As the concentration of 4-BrPh/2,4-DiBrPh increased, the intensity of HSA absorption peak at 277 nm increased, exhibiting an obvious hyperchromic effect, and the absorption peak shifted slightly toward the longer wavelength region (from 277 to 278 nm for 4-BrPh and from 277 to 281 nm for 2,4-DiBrPh). These changes in UV absorption spectra indicated that the bromophenolic DBPs could interact with the protein to form complexes, leading to the decrease in the hydrophobicity of the HSA microenvironment. CD spectroscopy is a well-established technique to determine the secondary structural changes of proteins.48 To detect the effects of the two DBPs on the secondary structure of HSA, CD spectra of HSA with various amounts of the DBPs were measured (Figure 4c,d). In general, two negative bands at around 208 and 222 nm represent the characteristic absorption of the α-helix structure in HSA.49 As can be seen from the graph, the intensities of the two negative bands changed evidently upon the addition of 4-BrPh/2,4-DiBrPh, implying the alterations of the secondary structure of HSA. The CDPro software was adopted to analyze the CD spectra and to calculate the fractions of α-helix, β-sheet, β-turn, and random coil by using SELCON3 method.50 The results indicated that as the DBP concentration increased, the α-helical content in 567
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Figure 5. (a) Lowest docking energy conformation of 4-BrPh−HSA and 2,4-DiBrPh−HSA complexes (dashed circle shows the binding sites). (b, c) Three-dimensional (3D) and two-dimensional (2D) views show the interaction between 4-BrPh and its neighboring residues. (d, e) 3D and 2D views show the interaction between 2,4-DiBrPh and its neighboring residues. The bright yellow dashed lines represent hydrogen bonding.
4), indicating that van der Waals forces and hydrogen bonding play dominant roles for bromophenol−HSA complex formation, which supported the conclusion gained from the thermodynamic analysis. Besides, as can be seen from Figure 5b,d, one hydrogen bond can be found for both 4-BrPh−HSA and 2,4-DiBrPh−HSA interactions (with a length of 2.2 and 2.0 Å, respectively) contributed by the polar interactions between hydrophilic moieties of 4-BrPh/2,4-DiBrPh and residue Leu 135. Third, 2,4-DiBrPh−HSA interaction has a lower value (−0.42 kJ mol−1) of electrostatic binding energy compared with 4-BrPh−HSA interaction (−0.25 kJ mol−1) (Table 4). This could be explained by the difference in orientation of the two bromophenols in subdomain IB. 2,4-DiBrPh is sandwiched between two Tyr residues (Tyr 138 and Tyr 161) by a relatively stronger π−π stacking interaction, whereas 4-BrPh lies closer to only Tyr 161 (Figure 5b,e). This orientation could also explain the observed synchronous fluorescence results. 2,4-DiBrPh is sandwiched between Tyr 138 and Tyr 161, making it difficult to perturb the microenvironment of Tyr; thus, no blue- or redshift can be observed in the spectra of 2,4-DiBrPh (Δλ = 15 nm) (Figure 3), whereas 4-BrPh−HSA has a relatively weaker π−π stacking interaction, making it effectively enough to perturb the microenvironment of Tyr; thus, a slight redshift (1 nm) can be observed in the spectra of 4-BrPh (Δλ = 15 nm) (Figure 3). Fourth, the predicted binding energy of 2,4-DiBrPh−HSA interaction (−23.26 kJ mol−1) is lower than that of 4-BrPh− HSA interaction (−20.13 kJ mol−1) (Table 4), similar with the experimental results, confirming that 2,4-DiBrPh could form a more stable complex with HSA compared with 4-BrPh. In addition, the calculated inhibition constants are 298.6 and 84.0 μM for 4-BrPh−HSA and 2,4-DiBrPh−HSA interactions, respectively (Table 4). As the inhibition constant can indicate the effectiveness of the ligand in inhibiting biological function of the protein and a smaller value means a greater inhibition potency,53 2,4-DiBrPh may have a higher toxicity potential toward human health than 4-BrPh. Mammalian Cytotoxicity of 4-BrPh and 2,4-DiBrPh. The comparative cytotoxicity of the two bromophenolic DBPs 4-BrPh and 2,4-DiBrPh were evaluated with the mammalian Chinese Hamster Ovary (CHO)-K1 cells and the results are shown in Figure 6. It was observed that 2,4-DiBrPh exhibited an apparently higher toxic potency than 4-BrPh and LC50
HSA exhibited a decreasing trend, accompanied with increases of β-sheet, β-turn, and random coil contents in HSA (Table 3). Therefore, the CD results clearly demonstrated that the binding of either 4-BrPh or 2,4-DiBrPh to HSA can cause the alterations in the secondary structure and 2,4-DiBrPh has a greater impact than 4-BrPh. It should be noted that secondary structure is highly associated with the biological activity of biomacromolecules and a decrease in the α-helix percentage may induce a loss of the physiological function of HSA.35 Molecular Docking Analysis. Molecular docking was performed to further investigate the binding mechanism at a molecular level so as to present a reasonable explanation for our experimental results. The docking results of 4-BrPh or 2,4DiBrPh with HSA are shown in Figure 5 and Table 4, and Table 4. Estimated Molecular Binding Energies of Bromophenol−HSA Interactions ligand
binding energy (kJ mol−1)
electrostatic energy (kJ mol−1)
VDW force and H bondinga energy (kJ mol−1)
inhibition constant (μM)
4-BrPh 2,4-DiBrPh
−20.13 −23.26
−0.25 −0.42
−21.13 −24.10
298.6 84.0
a
van der Waals force and hydrogen bonding.
some important insights could be drawn. First, the overall three-dimensional images (Figure 5a) illustrate that either 4BrPh or 2,4-DiBrPh can enter into the central channel of subdomain IB with surrounding residues of Leu 135, Leu 139, Ile 142, Tyr 138, Ala 158, and Tyr 161 for 4-BrPh and residues of Leu 135, Leu 139, Tyr 138, Ala 158, and Tyr 161 for 2,4DiBrPh. A few studies have reported ligands docked into the subdomain IB of HSA.51,52 These results differed from the popular viewpoint that ligands were more favorably located at or near two major binding sites of HSA (Sudlow’s site I in hydrophobic cavities in subdomain IIA and Sudlow’s site II in subdomain IIIA).16 The binding sites for the two bromophenolic DBPs are located in the subdomain IB, which is relatively far from residue Trp 214 in subdomain IIA; that is why no blue- or redshift of the emission peak corresponding to Trp can be observed for both 4-BrPh−HSA and 2,4-DiBrPh−HSA interactions during the synchronous fluorescence analysis. Second, the van der Waals force and hydrogen bonding energy is significantly lower than the electrostatic energy for both 4-BrPh−HSA and 2,4-DiBrPh−HSA interactions (Table 568
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HSA than 4-BrPh. Accordingly, the binding constant and inhibition constant for the binding with HSA could potentially be used to predict toxicity potentials for DBPs. As an important toxicity evaluation method, mammalian cytotoxicity bioassays with CHO cells have successfully evaluated the comparative toxicities of more than 100 DBPs by Plewa’s group.59 The LC50 values of most DBPs reported in the previous studies vary by 6 orders of magnitude, from 8.46 × 10−2 M for N-nitromorpholine (the least toxic) to 6.78 × 10−7 M for diiodoacetamide (the most toxic); compared with their studies, 4-BrPh and 2,4-BrPh in the present study showed moderate toxic potencies. It can be expected that the binding affinity and inhibition potency of those more than 100 DBPs may also show similar rank orders as those of cytotoxicity. As our study confirmed that bromophenolic DBPs can bind with HSA and change the conformation of HSA, the interactions of other DBPs with HSA are worth further investigation, especially for those DBPs with higher cytotoxicity than the two bromophenolic DBPs.
Figure 6. CHO-K1 cell chronic cytotoxicity concentration−response curves of 4-BrPh and 2,4-DiBrPh.
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values of 4-BrPh and 2,4-DiBrPh were calculated to be 3.08 × 10−5 and 1.09 × 10−5 M respectively. Interestingly, the DBP 2,4-DiBrPh with higher cytotoxicity also showed a higher binding affinity to HSA compared with 4-BrPh. Panigrahi et al.54 reported that the toxicity of five anthraquinones (toxic compounds originated from a toxic weed Cassia occidentalis) in rat primary hepatocyte and HepG2 cells is directly proportional to the binding affinity of the toxic compounds toward bovine serum albumin and anthraquinones exhibiting a higher binding affinity to bovine serum albumin showed a higher toxicity. Comparing with binding constants gained from previous studies focused on toxicant−HSA binding would help us understand the toxicity level in terms of binding behaviors. Xie et al.55 investigated the binding of two endocrine disruptors 4-tert-octylphenol and 4-nonylphenol to HSA, and the results indicated a lower binding constant for 4tert-octylphenol (4.37 × 103 M−1) and a higher binding constant for 4-nonylphenol (4.72 × 104 M−1) at 300 K. Lv et al.56 studied binding behaviors of methyl-triclosan to HSA and gained its binding constant value as 6.32 × 103 M−1 at 298 K. Zhuang et al.57 selected six benzotriazole (BZT) UV stabilizers and evaluated the interactions with HSA of the six BZTs; the binding constants were in the range of (0.32−2.96) × 104 M−1 at 298 K. Zhang et al.58 probed the interaction of four UV filters with HSA, and the binding constants were in the range of (1.91−12.96) × 104 M−1 at 296 K. The binding constants of 4-BrPh (3.30 × 103 M−1) and 2,4-DiBrPh (2.57 × 104 M−1) with HSA at 300 K acquired from the present study lied within the level and range of toxicants from the above studies. This indicated that toxicological effects of bromophenols toward humans should not be neglected. The distribution, free concentration, and metabolism of the ligands strongly depend on their binding constants with serum albumin.17,18 The binding of toxicants to serum albumin can impede the transport of endogenous substances and cause conformational changes of protein.55 Thus, binding with HSA could lower down the free concentration of 4-BrPh and 2,4DiBrPh, which as a result lowers down their bioavailability. This effect is more obvious for 2,4-DiBrPh than 4-BrPh as the former exhibited higher binding affinity. Moreover, the DBP 2,4-DiBrPh with a higher toxicity also showed a higher HSA inhibition constant than 4-BrPh; 2,4-DiBrPh was 2.83 times more toxic than 4-BrPh in terms of cytotoxicity, and 2,4DiBrPh exhibited 3.55 times higher inhibition potency toward
CONCLUSIONS In conclusion, interactions of two bromophenolic DBPs with HSA were investigated using multispectroscopic techniques and the molecular docking method. The experimental results obtained under simulative physiological conditions demonstrated that both of the two DBPs could bind with HSA to form a bromophenol−HSA complex with one binding site, whereas 2,4-DiBrPh showed a higher binding affinity toward HSA. The binding interactions occurred spontaneously, and van der Waals interactions and hydrogen bonding played dominant roles in forming the complex. Binding of the two DBPs to HSA resulted in alterations of microenvironment and the conformation of HSA, as analyzed by UV absorption and CD spectroscopy. Results of molecular docking were in accordance with the experimental results, and the modeling study also revealed the locations of the most probable binding sites for bromophenols on HSA (locating in domain IB). Furthermore, the comparative cytotoxicity of the two bromophenolic DBPs was evaluated with the mammalian CHO-K1 cells; notably, 2,4-DiBrPh with a higher binding affinity toward HSA also showed a significantly higher toxic potency than that of 4-BrPh. These results will facilitate a better understanding of toxicity mechanisms of DBPs.
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MATERIALS AND METHODS Chemicals and Reagents. HSA (≥96%) was purchased from J&K Chemical (Beijing, China) and used without further purification. 4-BrPh (99%) and 2,4-DiBrPh (95%) were purchased from Sigma (St Louis). Tris−HCl buffer (pH 7.4) was obtained from Solarbio (Beijing, China). Ham’s F-12K medium and fetal bovine serum were obtained from Hyclone. All other reagents were of analytical grade. Ultrapure water was produced by a Milli-Q water purification system. Sample Preparation. Stock solutions of 4-BrPh (1.0 mM) and 2,4-DiBrPh (1.0 mM) were prepared by dissolving the required amount of standards in ethanol. An HSA stock solution (1.0 mM) was prepared in the Tris−HCl buffer solution (pH 7.4), and a stock solution of NaCl (1.0 M) was prepared by directly dissolving the required amount of NaCl in ultrapure water. All of the above stock solutions were kept in the refrigerator at 4 °C and diluted as required before use. All spectroscopy measurements were performed by keeping the 569
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concentration of HSA at 5 μM in Tris−HCl buffer (pH 7.4) with NaCl concentration of 0.9% (w/v) to maintain the ionic strength of the simulative physiological condition, while varying 4-BrPh/2,4-DiBrPh concentrations with different ranges. Fluorescence Spectroscopy Measurements. All fluorescence spectra were measured on a spectrofluorimeter (Thermo Lumina, Germany) equipped with a 150 W xenon lamp, using a 1 cm path-length quartz cell. The excitation and emission slit widths were both set at 5.0 nm. The scan speed and photomultiplier tube voltage were set at 600 nm min−1 and 700 V, respectively. The fluorescence emission spectra of HSA in the absence and presence of 4-BrPh/2,4-DiBrPh were recorded in the range of 300−500 nm with an excitation wavelength of 280 nm at 290, 300, and 310 K, respectively. The required temperature was controlled with a circulating water bath (Laptop, China). The synchronous fluorescence spectra of HSA in the absence and presence of 4-BrPh/2,4DiBrPh were scanned from 280 and 370 nm with wavelength intervals set at Δλ = 15 and 60 nm at 300 K. UV−Visible Absorption Measurements. The UV− visible absorption spectra of HSA in the absence and presence of 4-BrPh/2,4-DiBrPh were recorded on a Mapada UV-3200 spectrophotometer (Shanghai, China) by using a 1 cm quartz cuvette in the wavelength range of 200−400 nm at 290 K. The slit width was set at 1.0 nm. CD Spectra Measurements. By utilizing a Chirascan spectropolarimeter (Applied Photophysics, U.K.) equipped with a thermostated cell holder, the CD spectra were obtained over a wavelength range of 200−260 nm at 290 K with a 1 mm quartz cuvette. The scan rate speed was set at 100 nm min−1. Each CD spectrum was obtained on the basis of three successive scans. Results were expressed as ellipticity values (mdeg), and the values were acquired in mdeg directly from the instrument. Molecular Docking Study. To explore the interaction mechanism at a molecular level, the binding interactions between the two bromophenolic DBPs and HSA were further simulated by the molecular docking method with the program Autodock 4.2.60 The structures of 4-BrPh and 2,4-DiBrPh were initially constructed with the software ChemOffice (CambridgeSoft). The structures with optimized energy and geometry were obtained on the basis of density functional theory method at the B3LYP/6-311+G(d,p) level, as performed in Gaussian 03.61 The three-dimensional structure of HSA was retrieved from the RCSB Protein Data Bank (PDB code: 1H9Z).62 Polar hydrogens and Gasteiger charges were added to the HSA macromolecule, and water molecules were removed prior to docking. The torsion bonds of the ligands were set in default values. Blind docking was achieved by using a grid box of 100 × 100 × 100 Å3 with 1.000 Å spacing, and grid maps were produced with Autogrid 4.2. The effective and reliable method Lamarckian genetic algorithm was chosen to implement molecular docking and perform ligand conformational searching.63 The docking parameters were set as follows: docking trials, 100, population size, 150; and energy evaluations, 2 500 000 (Table S1). Binding energy could indicate whether the interaction between the protein and ligand molecules is strong or weak.64 Among the 100 positions generated for each bromophenol−HSA complex via molecular docking, the conformation with the lowest energy was further visualized and analyzed by using Pymol 1.765 and LigPlot+ v.1.4.5.66
Mammalian Cell Cytotoxicity Assay. As numerous human epidemiological studies have already demonstrated the correlation between DBP exposure and several human health impacts, including bladder cancer, birth defects, and fetal loss,11−13 CHO cells that have been frequently used in DBP toxicity study67 were selected in the present study to comparatively assess the in vitro toxicity of bromophenols. CHO-K1 cell line68 acquired from the American Type Culture Collection was used in the present study to comparatively evaluate the cytotoxicity of 4-BrPh and 2,4-DiBrPh. This assay measures the reduction in cell density on flat-bottom 96-well microplates as a function of DBP concentration over a period of 72 h (three cell cycles) compared with untreated controls. The experimental procedures were according to a published literature,59 with some modifications, and the details are shown in the SI. Each experiment was repeated 2 to 3 times. On the basis of the data, a concentration−response curve was generated and a nonlinear regression analysis was adopted by using Sigma-Plot 12. The LC50 (% C1/2) values were obtained from the regression analysis and represent the bromophenol concentration that induced a 50% reduction in cell density as compared to the concurrent negative controls.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03116.
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Experimental procedures for the mammalian cell cytotoxicity assay and calculation of parameters characterizing the FRET phenomenon; input parameters and roles of each software for molecular docking (Table S1); double-log plots and van’t Hoff plots for bromophenol−HSA interactions (Figure S1) (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +86 139 2843 2825. ORCID
Mengting Yang: 0000-0003-4352-558X Yantao Chen: 0000-0001-8686-1116 Juying Li: 0000-0003-1294-241X Bo Yang: 0000-0001-6739-4997 Xu Zhao: 0000-0003-1180-8956 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Grant 51508335), Natural Science Foundation of Guangdong Province (Grant 2016A030310061), and Shenzhen Basic Research Project (Grant JCYJ20170818091859147 and Grant JCYJ20150525092940987).
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