Journal of Photochemistry and Photobiology B: Biology 149 (2015) 9–20

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Exploration of binding of bisphenol A and its analogues with calf thymus DNA by optical spectroscopic and molecular docking methods Yan-Qing Wang ⇑, Hong-Mei Zhang Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng City, Jiangsu Province 224002, People’s Republic of China Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 4 December 2014 Accepted 20 April 2015 Available online 19 May 2015 Keywords: Bisphenols DNA Spectrum Molecular modeling Binding mode

a b s t r a c t Bisphenol A and its analogues have carcinogenic potentials and toxicities. However, there are lacks of studies elucidating gene toxic interactions of bisphenols with DNA. In this work, the binding modes of five bisphenol compounds with calf thymus DNA were characterized. The multi-spectroscopic experimental results indicated that the fluorescence quenching of bisphenols by calf thymus DNA point to groove binding. The ultraviolet visible and circular dichroism spectral data displayed that bisphenols partly induced conformational changes of calf thymus DNA. In addition, the binding constants of bisphenol A, diphenolic acid, bisphenol AF, bisphenol AP, bisphenol fluorine with calf thymus DNA obtained from fluorescence emission spectra were 1.09  104, 3.65  104, 4.46  104, 1.69  104, 4.49  104 L mol1 at 298.15 K, which indicated that the multi-noncovalent binding forces were involved in the binding processes. In silico investigations indicated that DNA has the preferable binding sites binding with bisphenols by minor groove binding and electrons transfer from DNA bases to bisphenols occurred. In addition, the structural differences of these five bisphenols partly affected the binding ability of them with DNA. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Bisphenol A (BPA) and its analogues are chemical compounds used in the production of synthetic polymers and thermal paper, which after degradation may be important sources of these compounds in the environment, food, and water and thus contaminate the food chain [1–8]. Exposures to BPA and its analogues have been associated with various adverse health effects such as endocrine disruption [9–11], hepatotoxicity [12,13] and immunotoxicity [14,15]. Due to increased concerns over the safety of these compounds, many government agencies have banned BPA use in plastic bottles for infants [16]. Such restrictions, criticisms, and public concerns of BPA have led to increased use of alternative bisphenols and ‘‘BPA-free’’ products. However, in some cases, ‘‘BPA-free’’ products also contained estrogen hormone disruptors [7,17]. For example, bisphenol AF (BPAF), an alternative of BPA, is also used as a monomer in the production of many polymer including polyamides, polycarbonates, and food-contact polymers [18]. Analysis from the molecular structure of BPAF, the substitution of the propane bridge of BPA might ⇑ Corresponding author at: Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s Republic of China. Tel./fax: +86 515 88233188. E-mail address: [email protected] (Y.-Q. Wang). http://dx.doi.org/10.1016/j.jphotobiol.2015.04.029 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

result in becoming the more dangerous synthetic estrogenic chemical [19]. Bisphenol AP (BPAP), another analogue of BPA, is also commonly used in polymer materials, the fine chemical industry, and the medicine industry [20]. The United States Environmental Protection Agency (U.S. EPA) has confirmed BPAP to be one of the endocrine disrupting compounds [20]. In addition, fluorene-9-bisphenol (BHPF) has fluorenyl group, which is a rigid skeleton with good thermal stability, so that BPA seem to be substituted with BHPF to improve thermal stability and other chemical properties [21]. Hence, it is necessary to investigate the structure-effect of the toxicity mechanism of BPA and its analogues. Elucidation of the intermolecular interactions between BPA’s analogues with biomacromolecules including proteins, enzymes, and DNA is crucial to understand their toxicity mechanisms [22–30]. Among these biomacromolecules, DNA is a genetic material indicating important biological roles in gene expression, transcription, mutagenesis, and carcinogenesis [31]. Many drugs and toxics compounds reflect their biological and toxically activities through their binding interactions with DNA [32–37]. Above these interactions are likely to interfere with enzymes (e.g. polymerases, topoisomerases) and essential processes of transcription, replication and repair, thus leading to cell death. Therefore, the investigation of the binding interactions of bisphenols with DNA is required for a better understanding of the toxicity mechanism of these compounds.

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Based on the above-mentioned purpose, herein, the interactions of BPA and its four analogues including BPAF, BPAP, diphenolic acid (DPA) and BHPF (Table 1) [38] with DNA were investigated by using absorption, fluorescence, CD spectroscopic methods, and molecular modeling.

Methanol was used to solve bisphenol compounds. All other chemicals were of analytical reagent grade. 2.2. Methods 2.2.1. UV–vis absorption measurements A SPECORD S600 spectrophotometer equipped with 1 cm quartz cells was used to measure the UV–vis absorption spectra of each bisphenol in the absence and presence of ctDNA.

2. Materials and methods 2.1. Materials Calf thymus DNA (ctDNA) was purchased from Sigma–Aldrich. BPA (P99%), DPA P 95%), BPAF P 97%), BPAP P 97%), and BHPF P 97%) were all obtained from Aladdin Industrial Corporation. The stock solution of ctDNA was prepared by dissolving appropriate amount of ctDNA in a 0.05 mol L1 potassium phosphate buffer with pH 7.40 by gentle stirring at room temperature and stored at 4 °C. Its concentration was determined by UV absorption at 260 nm using e260 = 6600 L mol1 cm1 [39].

2.2.2. Fluorescence spectra measurements A LS-50B Spectrofluorimeter equipped with 1.0 cm quartz cells and a thermostat bath was used for fluorescence spectra measurements of bisphenol compound-ctDNA systems. In the quenching experiments, 20 lL of bisphenol compound (0.0125 mol L1) solutions were transferred into 5 mL brown glass volumetric flasks and solutions of ctDNA with appropriate concentrations were added to the flasks, respectively. The pH 7.40 potassium phosphate buffer was used to fix volume of solution. The fluorescence spectra of

Table 1 The molecular structures of bisphenol compounds. Compounds

Structure and IUPAC name

Bisphenol A (BPA)

CAS

Log Pa

80-05-7

3.4237

126-00-1

3.2686

1478-61-1

4.5085

1571-75-1

5.1788

3236-71-3

5.4609

4-[2-(4-hydroxyphenyl)propan-2-yl]phenol Diphenolic acid (DPA)

4,4-bis(4-hydroxyphenyl)pentanoic acid Bisphenol AF (BPAF)

4-[1,1,1,3,3,3-hexafluoro-2-(4-hydroxyphenyl)propan-2-yl]phenol Bisphenol AP (BPAP)

4-[1-(4-hydroxyphenyl)-1-phenylethyl]phenol Bisphenol fluorine (BHPF)

4-[9-(4-hydroxyphenyl)-9H-fluoren-9-yl]phenol a

Log P: partition coefficient.

Y.-Q. Wang, H.-M. Zhang / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 9–20

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Fig. 1. The absorption spectra (A) and fluorescence spectra (B) of bisphenol compounds. c (BPA) = c (DPA) = c (BPAF) = c (BPAP) = 5.0  105 mol L1, c (BHPF) = 2.5  105 mol L1.

Fig. 2. Absorption spectra of BPA’s analogues, ctDNA, and BPA’s analogues–DNA systems. c (ctDNA) = c (BPA) = c (DPA) = c (BPAF) = c (BPAP) = 5.0  105 mol L1, c (BHPF) = 2.5  105 mol L1.

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Fig. 3. CD spectra of ctDNA (5.0  105 mol L1) in the absence and presence of each bisphenol compounds (50.0  105 mol L1).

above reaction solutions were recorded in the range of 280– 400 nm with the excitation wavelength 270 nm after 1 h equilibration. Three-dimensional fluorescence spectra were recorded under the following conditions. The emission wavelengths range was selected from 250 to 400 nm, the initial excitation wavelength was set to 200 nm with increment of 10 nm, and the scanning number was 15. 2.2.3. Circular dichroism spectra measurements The CD spectra of ctDNA (5.0  105 mol L1) in the absence and presence of bisphenol compounds were acquired on an Applied Photophyysics Ltd. Chirascan spectrometer at 298.15 K. 2.2.4. Molecular modeling The crystal structure of B-DNA (PDB ID 1BNA) was obtained from the Protein Data Bank for molecular modeling [40]. The geometries of bisphenol compound s and DNA bases were optimized at DFT/B3LYP/6-31G++ (d, p) by Gaussian 09 [41]. Autodock 4.2.3 Program was used to perform blind docking calculations of DNA with bisphenol compounds, respectively. In the blind docking calculations, a grid box of 126  126  126 Å with spacing of 0.357 Å was used to enclose DNA and bisphenol compounds. The Lamarckian Genetic Algorithm method was used as the searching algorithm. Then the GA population size, the maximum number of energy evaluation, and the number of GA runs were set at 150, 2,500,000, and 100, respectively. The other AutoDock parameters were set to default. In addition, Pymol software was used to analyze the predicated binding mode [42].

BPAF, the presence of six fluorine atoms results in an obvious blue shift from 277 to 272 nm in the near UV spectra. This is attributed to the decreased electron drift from electron-donating group to the electron-withdrawing group through p-bond of aromatic ring [43]. As for BPAP, the presence of benzene ring results in an obvious red shift from 203 nm to 208 nm in the far UV spectra and a shoulder absorption peak at 228 nm. In addition, compared with the other four compounds, BHPF has the biggest electronic conjugated system, which results in the presence of absorption peak at 309 nm known as R absorption band of aromatic compounds. During the absorption peaks, the peak at about 277 nm coming from p–p⁄ electronic transition of aromatic rings is the most typical absorption band. Just like aromatic amino acids, this p–p⁄ electronic transition is the main contribution of intrinsic fluorescence of these aromatic compounds. Fig. 1(B) shows the fluorescence emission spectra of these five compounds. The maximum fluorescence emission peak of BPA, DPA, BPAF, BPAP, and BHPF located at 307, 306, 300, 309, and 329 nm, respectively. In addition, BHPF also has a shoulder peak at about 320 nm. With the increase of electronic conjugated system, the position of fluorescence emission peak had a red shift. The fluorenyl group in BHPF is a rigid skeleton, which results in the red shift of fluorescence emission peak and the strongest fluorescence emission intensity. The properties of the UV–vis and fluorescence spectra of these five compounds will be used to analyze the binding interactions of them with ctDNA. 3.2. Changes in the UV–vis spectra of bisphenol compounds in the presence of ctDNA The absorption spectra of bisphenol compounds in the presence of ctDNA are shown in Fig. 2. It is apparent that upon addition of ctDNA to bisphenol compound solution, ctDNA induce the absorption spectral changes of them. Seen from ctDNA–BPA’s analogues system, the peak at about 277 nm all increases and have a blue shift. In addition, ctDNA also has absorption peak at about 260 nm coming from the p–p⁄ electronic transition of aromatic rings. Therefore, there is an obvious overlap between ctDNA and BPA’s analogues. The different absorption spectra of BPA and its analogues in the absence and presence of ctDNA are obtained by using the ctDNA solution as a reference. Fig. 2 shows that the spectra of BPA and its analogues, BPA’s analogues–ctDNA systems are remarkably different. The intensity of the absorption peaks of bisphenol compounds all decreases, implying that BPA and its analogues can bind with ctDNA [44]. 3.3. Changes in the CD spectra of ctDNA in the presence of bisphenol compounds

3. Results and discussion 3.1. The UV–vis absorption and fluorescence spectral properties of bisphenols The UV–vis absorption and fluorescence spectra of organic molecules can represent their internal structures and functions. Fig. 1(A and B) shows the UV–vis absorption and fluorescence spectra of bisphenol compounds. Seen from their molecular structures, aromatic rings and –OH are their common feature. The p–p⁄ electronic transition of aromatic ring attributes to spectral absorption of bisphenol compounds. These five compounds all have three common absorption peaks at about 203, 226, and 277 nm. In addition, BHPF not only has these three absorption peaks, but also has other absorption peak at about 309 nm. Among these five compounds, the absorption peaks’ position of BPA and DPA are the same, implying that the carboxyl group of DPA does not remarkably effect the electronic transition of aromatic rings of it. As for

The destruction of ctDNA structure after binding to bisphenol compounds was measured by CD spectroscopy. It can be seen from Fig. 3 that ctDNA has a negative peak of 246 nm and a positive peak of 278 nm, the former is due to the helical structure of DNA and the later is due to the stacking of base pairs of DNA [45]. In addition, the values of the above two bands are both decreased in the presence of BPA or its analogues, which can be contributed to the groove binding of them. The groove binding perhaps induces the partial aggregation of DNA and decreases the loss of intensity of the band at 246 and 278 nm of DNA [46]. Some conformational changes of ctDNA are induced by the BPA or its analogues. 3.4. Changes in the three-dimensional fluorescence spectra of bisphenol compounds in the presence of ctDNA The three-dimensional fluorescence spectrum is a new technique for providing an overall view of all the fluorescence features

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Fig. 4. The three-dimensional fluorescence spectra of bisphenol compounds and bisphenol compounds-DNA (BPAP) = 5.0  105 mol L1, c (BHPF) = 2.5  105 mol L1; the concentration of ctDNA (B, D, F, H, J): 18.0  105 mol L1.

systems.

c

(BPA) = c

(DPA) = c

(BPAF) = c

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Table 2 Three-dimensional fluorescence spectral characteristics of BPA and its analogues in the absence and presence of ctDNA. System

Peak a (kex/kem)

Dk (nm)

Intensity

Intensity ratio Peak a/Peak b

Peak b (kex/kem)

Dk (nm)

Intensity

BPA (A) BPA-ctDNA (B) DPA (C) DPA-ctDNA (D) BPAF (E) BPAF-ctDNA (F) BPAP (G) BPAP-ctDNA (H) BHPF (I) BHPF-ctDNA (J)

230/309 230/309 230/308 230/308 230/300 230/302 230/310 230/312 240/328 230/328

79 79 78 78 70 72 80 82 88 98

510.68 358.24 674.23 438.76 883.28 496.63 693.97 436.98 512.72 214.55

1.54:1 1.93:1 1.60:1 2.00:1 3.07:1 5.45:1 1.97:1 2.46:1 1.05:1 0.87:1

280/309 280/310 280/309 280/310 270/299 270/301 280/310 280/312 280/329 290/329

29 30 29 30 29 31 30 32 49 39

330.76 185.63 422.32 219.38 288.10 91.09 353.15 177.74 487.69 246.88

Fig. 5. The fluorescence emission spectra of bisphenol compounds in the absence and presence of ctDNA, respectively. c (BPA) = c (DPA) = c (BPAF) = c (BPAP) = 5.0  105 mol L1, c (BHPF) = 2.5  105 mol L1. c (ctDNA) (105 mol L1) (from top to bottom) (A–D), 0, 1.5, 3.0, 4.5.0, 6.0, 7.5, 9.0, 10.5, 12.0, and 15.0, respectively. c (ctDNA) (105 mol L1) (from top to bottom) (E), 0, 3.0, 4.5.0, 6.0, 7.5, 9.0, 12.0, 15.0, 18.0, and 24.0, respectively.

of the fluorescence functional groups [47,48]. The three-dimensional fluorescence spectra of bisphenol compounds in the absence and presence of ctDNA are shown in Fig. 4. It could

be seen from Fig. 4 that there are two kinds of fluorescence spectral peaks including the endogenous fluorophores peaks (Peak a and Peak b) and the Raleigh scattering peaks (Peak c). Peak a and peak

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fluorescence. The effects of ctDNA on the intrinsic fluorescence of these five compounds could give some information about their binding interactions. Fig. 5 indicates the changes in the steady state fluorescence spectra of bisphenol compounds in the absence and presence of ctDNA. The intensity of the characteristic broad emission of them all decrease regularly with the increasing concentration of ctDNA, but no wavelength shift is observed. These results implied that BPA and its analogues could bind to ctDNA by intercalating between adjacent base pairs of ctDNA and p–p stacking might be enhanced consequently [22]. The photoelectrons would be transferred from base pairs of ctDNA to the excited state of BPA and its analogous [49]. 3.6. Binding Nature of bisphenol compounds with ctDNA Fig. 6. The log (F0–Fcor)/Fcor vs. log [ctDNA] for the binding of bisphenol compounds with ctDNA, c (BPA) = c (DPA) = c (BPAF) = c (BPAP) = 5.0  105 mol L1, c (BHPF) = 2.5  105 mol L1.

b are caused by the p ? p⁄ transition of aromatic rings in BPA and its analogues. Seen from the shapes of these five compounds, BPA (Fig. 4A and B), DPA (Fig. 4C and D), BPAF (Fig. 4E and F), and BPAP (Fig. 4G and H) have close similarities that there are two circular fingerprints in the three-dimensional fluorescence spectra of them. Yet it is seen from Fig. 4(I and J) that BHPF has slender fingerprints. In addition, the three-dimensional fluorescence spectral characteristics of BPA and its analogues in the absence and presence of ctDNA are also shown in Table 2. As the data shows, the intensities of peak a are stronger than those of peak b for BPA, DPA, BPAF, and BPAP. Upon addition of trace amount of ctDNA to BPA and its analogues solution, the intensities of peak a and peak b for these five compounds all decreases. In addition, some remarkable shape changes of peak a and peak b are observed. The values of the radio peak a/peak b of BPA, DPA, BPAF, BPAP and BHPF change from 1.54, 1.60, 3.07, 1.97 and 1.05 to 1.93, 2.00, 5.45, 2.46, and 0.87, respectively. The results show that the change trend of BHPF is different from those of the other four compounds. This difference implies that the fluorenyl group in BHPF plays an important role in the binding interaction of BHPF with ctDNA. The above result could be also obtained from the changes of Stroke shift (Dk). The Stroke shift of peak a and peak b of BHPF in the absence of ctDNA is 88 and 49 nm, respectively (Table 2). With addition of ctDNA in BHPF, the values of Stroke shift are remarkably different and change to 98 and 39 nm, respectively. 3.5. Changes in the steady state fluorescence spectra of bisphenol compounds in the presence of ctDNA As seen from Fig. 1(B), BPA and its analogues have aromatic benzene rings known as endogenous fluorophores emitting

To determine the binding constant (KA) and binding sites (n) for the complex formation of BPA or its analogues with ctDNA, the fluorescence quenching data from Fig. 4 have been taken. The following Eq. (1) was used to estimate the values of KA and n [50].

log

  F 0  F cor ¼ log K A þ n log½ctDNA F cor

ð1Þ

where F0 and Fcor are the corrected fluorescence intensities of BPA or its analogues in the absence and presence of ctDNA, respectively. KA, n, and [ctDNA] are the binding constant, binding sites, and the concentration of ctDNA, respectively. In addition, fluorescence intensity was corrected using Eq. (2) to decrease the inner filter effect due to ctDNA solution absorbance at excitation wavelength and the maximum emission wavelength [51],

F cor ¼ F obs  eðAex þAem Þ=2

ð2Þ

where Fcor and Fobs are the fluorescence intensity corrected and observed, respectively; Aex and Aem are the absorbance of ctDNA at excitation and emission wavelength, respectively. The plots of log (F0–Fcor)/Fcor vs. log [ctDNA] are shown in Fig. 6. The values of KA and n can be obtained from the intercept and slope of the plots. The results are listed in Table 3. It can be seen from Table 3 that the KA values at 298.15 K decrease in the order, KA (BHPF)  KA(BPAF) > KA (DPA) > KA (BPAP) > KA (BPA), indicating that the replacements of the methyl groups in BPA partly affect the binding ability of BPA’s analogues with ctDNA. Especially BPAF and BHPF, the six fluorine atoms and the fluorenyl group remarkably enhance the binding interactions of these two compounds with ctDNA. In addition, the values of binding sites (n) are about one, indicating the existence of one main binding site in ctDNA for bisphenol compounds. Herein, the binding forces of bisphenol compound with ctDNA, the enthalpy change (DH°), the entropy (DS°) and Gibbs energy change (DG°) are calculated by using Eqs. 3–5.

Table 3 Thermodynamic parameters of the binding interactions of bisphenol compounds with ctDNA at pH 7.40.

a

Compounds

T (K)

KA (L mol1)

n

Ra

DH° (kJ mol1)

DG° (kJ mol1)

BPA

298.15 310.15

1.09  104 0.82  104

1.02 0.94

0.9995 0.9961

18.22

23.03 23.23

16.14

DPA

298.15 310.15

3.65  104 2.57  104

1.15 1.04

0.9996 0.9985

22.52

26.03 25.62

11.78

BPAF

298.15 310.15

4.46  104 3.81  104

1.13 1.23

0.9994 0.9991

10.08

26.52 27.18

55.17

BPAP

298.15 310.15

1.69  104 2.08  104

1.04 1.14

0.9987 0.9985

13.67

24.12 25.63

126.81

BHPF

298.15 310.15

4.49  104 5.83  104

1.25 1.27

0.9896 0.9919

16.71

26.54 27.69

145.13

The correlation coefficient.

DS° (J mol1 K1)

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Fig. 7. The surface of the molecular orbital plots (HOMO and LUMO) of bisphenol compounds and DNA bases.

ln

  ðK A Þ2 DH 1 1 ¼  ðK A Þ1 R T1 T2

ð3Þ

DG ¼ RT ln K A

ð4Þ

DH   DG  T

ð5Þ

DS ¼

The DG°, DH°, and DS° are shown in Table 3. Firstly, the negative values of DG° for the binding interactions of bisphenol compounds with ctDNA reveal that the binding process is spontaneous. In addition, according to the views of Ross and Subramanian [52], more than one interaction forces are involved in the binding processes of bisphenol compound with ctDNA. As for BPA, DPA, and BPAF, the DH° is the large contribution of DG° value, implying that van der Waals and hydrogen binding interactions are the mainly binding forces stabilizing the complex between BPA, DPA, or BPAF and ctDNA, but the hydrophobic interactions should not be excluded. In addition, as for BPAP and BHPF,

Table 4 Calculated conceptual density functional reactivity descriptors in a.u. for the series of bisphenol compounds and DNA bases. Molecule

ELUMO (a.u.)

EHOMO (a.u.)

Chemical potential (l)

Chemical hardness (g)

BPA DPA BPAF BPAP BHPF Adenine Guanine Cytosine Thymine

0.19446 0.19486 0.19500 0.20156 0.21722 0.16580 0.15526 0.17436 0.17653

0.33819 0.33825 0.33519 0.33955 0.33974 0.31671 0.30831 0.33518 0.33241

0.26633 0.26656 0.26510 0.27056 0.27848 0.24126 0.23179 0.25477 0.25447

0.071865 0.071695 0.070095 0.068995 0.061260 0.075455 0.076525 0.080410 0.077940

the hydrophobic interactions are the mainly binding force. Based on the hydrophobicity of the groove of ctDNA and the bulk hydrophobic group benzene ring of BPA and its analogues, it is tempting to speculate that bisphenol compound enter into the

Y.-Q. Wang, H.-M. Zhang / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 9–20 Table 5 The calculated charge transfer between bisphenol compounds and DNA bases. Molecule

BPA

DPA

BPAF

BPAP

BHPF

Adenine Guanine Cytosine Thymine

0.085087 0.116383 0.037958 0.039585

0.085967 0.117292 0.038756 0.040398

0.081896 0.113593 0.034318 0.035904

0.101419 0.133212 0.052843 0.054752

0.136123 0.169431 0.083680 0.086243

17

Herein, the quantum chemistry descriptors including lowest unoccupied molecular orbital’s energy (ELUMO) and highest occupied molecular orbital’s energy (EHOMO) of these five compounds and four DNA bases were obtained alongside the optimized calculation. In addition, chemical potential (l), chemical harness (g), fraction number of electrons (DN) from system A (BPA and its analogous) to B (Adenine, Guanine, Cytosine, and Thymine) were obtained by using Eqs. 6–8 according to Ref [53].

grooves of ctDNA and water molecules are released from the shell of grooves of ctDNA [45]. Just like the binding interaction of BPA with human serum albumin, BPA could enter into the hydrophobic cavity of protein by hydrophobic and hydrogen binding interactions [23,24].

l¼

ELUMO þ EHOMO 2

ð6Þ

g¼

ELUMO  EHOMO 2

ð7Þ

3.7. Molecular modeling

DN ¼

lB  lA 2ðgA þ gB Þ

ð8Þ

Molecular modeling studies can gain some important insight into the binding interactions between ligands and DNA [50].

where lA, lB and gA, gB are the chemical potentials and chemical harnesses of system A and B, respectively.

Fig. 8. The overview structure and the best docked results of each bisphenol compounds in the binding site of DNA, respectively.

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Table 6 Docking results of bisphenol compounds with DNA. Compounds

Rank

Run

DG (kcal/mol)

Einter-mol (kcal/mol)

EVHD (kcal/mol)

Eelec (kcal/mol)

Etotal (kcal/mol)

Etorsional (kcal/mol)

BPA DPA BPAF BPAP BHPF

1 1 1 1 1

23 50 62 17 18

7.01 7.10 7.15 7.05 8.12

8.20 9.49 7.94 8.55 9.31

7.85 8.37 7.69 8.20 8.90

0.35 1.12 0.25 0.35 0.41

0.34 0.83 0.31 0.91 1.52

+1.19 +2.39 +1.79 +1.49 +1.19

The molecular orbital plots of bisphenol compounds and DNA bases are shown in Fig. 7. As seen from Fig. 7(A–C), the charge densities in HOMO of BPA, DPA, and BPAF were mainly accumulated on the benzene ring and –OH. However, in case of their LUMO, more charge densities moved from –OH to the benzene rings and the charge densities are accumulated on their two benzene rings. As for BPAP and BHPF (Fig. 7D and E), the replacements of the methyl groups by aromatic rings remarkably affected the charge densities in their HOMO and LUMO orbital. The charge densities in HOMO of BPAP are mainly accumulated on benzene rings with –OH. However, in case of its LUMO, more charge densities moved from –OH to the three benzene rings and delocalized over them. Different form BPA, DPA, BPAF and BPAP, the charge densities in HOMO of BHPF are mainly accumulated on one of benzene rings with –OH and the fluorenyl group replacing the methyl groups of BPA. However, the charge densities are only accumulated on the fluorenyl group of BHPF in LUMO. The distribution of charge densities of BPA and its analogues might affect their binding interactions with ctDNA. For Adenine, Guanine, Cytosine, and Thymine (Fig. 7F–I), the charge densities focused on the aromatic-ring in LUMO and HOMO. In addition, l and g of bisphenol compounds, DNA bases are presented in Table 4. The negative of the chemical potential could be called the absolute electro-negativity indicating a transfer of electrons from less electronegative systems to more electronegative systems [53]. Eq. (8) is used to calculate the charge transfer between bisphenol compounds and DNA bases. The results are shown in Table 5, which displayed that BPA and its analogues acted as electron acceptors and DNA bases acted as electron donors. According to these theory calculations, the electron transfer from DNA bases to BPA and its analogues occurred, which might be one main reason for the fluorescence quenching of BPA and its analogues induced by ctDNA. When BPA and its analogues interact with the bases pairs of ctDNA, electrons would be transferred from the bases to the excited state of them, which could result in quenching of the fluorescence intensity of them [48]. In addition, the positive values of BPAP and BHPF with DNA bases are larger than that of BPA, DPA and BPAP, implying that the electron transport ability enhanced with the increasing number of aromatic rings. Herein, molecular docking studies are also performed to predict the binding sites and binding energy of bisphenol compounds [54]. The predicted binding model with the lowest docking energy analysis was used for binding orientation analysis. The results are shown in Fig. 8, indicating that DNA offered several points of close contact with the surface of BPA or its analogues and these five compounds preferred to intercalate into the double helix of DNA and to sit in the minor groove of DNA [55]. There are three binding modes including electrostatic binding, groove binding, and intercalative binding existing in which molecules can be noncolvalently attached to DNA [54]. Analyzed from the molecular structures of bisphenol compounds, it can be found that they all have aromatic rings and two –OH groups, which could play different roles in their binding with DNA. The aromatic rings system could make them enter into the narrower regions of DNA groove, however, two – OH groups could form van der Waals and/or hydrogen bond

interactions of them with the helix of DNA. Just as above analysis, the hydrogen bond interactions exist between –OH groups of BPA or its analogues and DNA. Meanwhile, the number and length of hydrogen bonds are different because of the difference in the molecular structures of BPA and its analogues. For example, hydrogen bonds interactions could play important role in the binding interaction of DPA with DNA due to the substitution of one –CH3 of BPA by –COOH in DPA. In addition, the orientation of BPA and its analogues inside the minor groove is remarkably different. As for BPA and BPAF, their two benzene rings with –OH face toward the interface of DNA. As for DPA, –COOH and one benzene ring with –OH face toward the interface of DNA. The benzene without –OH and one of two benzene rings with –OH in BPAP face toward the interface of DNA. In addition, there is a fluorenyl group in BHPF, which is a rigid skeleton that is important for it to be placed into the minor groove of DNA. Fig. 8(E) shows that the fluorenyl group in BHPF intercalates the minor groove and is arranged in an almost coplanar configuration. The p-stacking interactions between the aromatic rings of BPA or its analogues and the nucleic bases of DNA may be involved in their binding processes [54]. Beside the cluster analyses and the binding sites, the binding energies in rank 1 with the lowest docking energy are also obtained from the Autodock molecular modeling data. The overview structures are shown in Fig. 8. Meanwhile, the values of above different kinds of binding energy are shown in Table 6. It can be seen from Table 6 that EVHD energy is the main part of binding free energies between bisphenol compounds and DNA, implying that vdw, hydrogen bonding and hydrophobic forces are the main forces in their binding processes. In addition, electrostatic forces are also involved in the binding interactions of them, but are not the mainly driving forces. Therefore, a conclusion was obtained that multi-noncovalently binding forces stabilize the complexes of bisphenol compounds with DNA. 4. Conclusion In this work, five bisphenol compounds were selected to study the binding interactions of them with DNA by in vitro and in silico analyses. The spectroscopic and theoretical calculation studies confirmed the binding interactions of them with ctDNA as mainly minor groove binding by multi-noncovalently binding forces. The molecular structure differences of these five compounds partly affected the binding ability of them with ctDNA. The results from this paper clearly displayed the binding modes of BPA and its analogues with DNA. These studies could provide influential insight into the ecotoxicology of BPA and its analogues. Acknowledgments We gratefully acknowledge financial support of the Fund for the National Natural Science Foundation of China (Project No. 21201147), the Natural Science Foundation of Jiangsu Province (Grant No. BK2012671), Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection (JLCBE11008), the Jiangsu Fundament of ‘‘Qilan Project’’ and the

Y.-Q. Wang, H.-M. Zhang / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 9–20

sponsorship of Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents.

References [1] T. Vega-Morales, Z. Sosa-Ferrera, J.J. Santana-Rodríguez, Determination of alkylphenol polyethoxylates, bisphenol-A, 17a-ethynylestradiol and 17bestradiol and its metabolites in sewage samples by SPE and LC/MS/MS, J. Hazard. Mater. 183 (2010) 701–711. [2] J. Li, B. Zhou, Y. Liu, Q. Yang, W. Cai, Influence of the coexisting contaminants on bisphenol A sorption and desorption in soil, J. Hazard. Mater. 151 (2008) 389–393. [3] H.T. Wan, P.Y. Leung, Y.G. Zhao, X. Wei, M.H. Wong, Chris K.C. Wong, Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations, J. Hazard. Mater. 261 (2013) 763–769. [4] J. Yonekubo, K. Hayakawa, J. Sajiki, Concentrations of bisphenols A, bisphenol A diglycidyl ether, and their derivatives in canned foods in Japanese markets, J. Agric. Food Chem. 56 (2008) 2041–2047. [5] J. Michalowicz, Bisphenol A-sources, toxicity and biotransformation, Environ. Toxicol. Pharmacol. 37 (2014) 738–758. [6] S. Lee, X. Liu, S. Takeda, K. Choi, Genotoxic potentials and mechanisms of bisphenol A and other bisphenol compounds: a comparison study employing chicken DT40 cells, Chemosphere 93 (2013) 434–440. [7] Y.X. Feng, J. Jin, Z.H. Jiao, J.C. Shi, M. Li, B. Shao, Bisphenol AF may cause testosterone reduction by directly affecting testis function in adult male rats, Toxicol. Let. 211 (2012) 201–209. [8] L. Vandenberg, I. Chowhound, J. Heindel, V. Padmanabhan, F. Paumgartten, G. Schoenfelder, Urinary, circulating, and tissue biomonitoring studies indicate widespread exposure to bisphenol A, Cien. Saude. Colect. 17 (2012) 407–434. [9] S. Flint, T. Markle, S. Thomson, E. Wallace, Bisphenol A exposure, effects, and policy: a wildlife perspective, J. Environ. Manage. 104 (2012) 19–34. [10] M.Y. Chen, M. Ike, M. Fujita, Acute toxicity, mutagenicity, and estrogenicity of bisphenol-A and other bisphenols, Environ. Toxicol. 17 (2002) 80–86. [11] H. Okada, T. Tokunage, X. Liu, S. Takayanagi, A. Matsushima, Y. Shimohigashi, Direct evidence revealing structural elements essential for the high binding ability of bisphenol A to human estrogen-related receptor-gamma, Environ. Health Perspect. 116 (2008) 32–38. [12] L. Huc, A. Lemari, F. Guéraud, C. Héliès-Toussaint, Low concentrations of bisphenol A induce lipid accumulation mediated by the production of reactive oxygen species in the mitochondria of HepG2 cells, Toxicol. In Vitro 26 (2012) 709–717. [13] M.K. Moon, M.J. Kim, I.K. Jung, Y.D. Koo, H.Y. Ann, K.J. Lee, S.H. Kim, Y.C. Yoon, B.J. Cho, K.S. Park, H.C. Jang, Y.J. Park, Bisphenol A impairs mitochondrial function in the liver at doses below the no observed adverse effect level, J. Korean Med. Sci. 27 (2012) 644–652. [14] J. Lee, K. Lim, Plant-originated glycoprotein suppresses interleukin-4 and -10 in bisphenol A-stimulated primary culture mouse lymphocytes, Drug Chem. Toxicol. 33 (2010) 421–429. [15] M. Goto, Y. Takano-Ishikawa, H. Ono, M. Yoshida, K. Hamaki, H. Shinmoto, Orally administered bisphenol A disturbed antigen specific immunoresponses in the naive condition, Biosci. Biotech. Biochem. 71 (2007) 2136–2143. [16] L. Peyre, P. Rouimi, G.D. Sousa, C. Helies-Toussaint, B. Carre, S. Barcellini, M.C. Chagnon, R. Rahmani, Comparative study of bisphenol A and its analogue bisphenol S on human hepatic cells: a focus on their potential involvement in non-alcoholic fatty liver disease, Food Chem. Toxicol. 70 (2014) 9–18. [17] R. Viñas, C.S. Watson, Bisphenol S disrupts estradiol-induced nongenomic signaling in a rat pituitary cell line: effects on cell functions, Environ. Health Perspect. 121 (2013) 352–358. [18] P. Wei, M. Cakmak, Y.W. Chen, X.H. Wang, Y.P. Wang, Y.M. Wang, The influence of bisphenol AF unit on thermal behavior of thermotropic liquid crystal copolyesters, Thermochim. Acta 586 (2014) 45–51. [19] Y. Shimohigashi, Y. Akahori, K. Yamasaki, M. Takatsuki, Relation between the results of in vitro receptor binding assay to human estrogen receptor a and in vivo uterotrophic assay: comparative study with 65 selected chemicals, Toxicol. In. Vitro. 22 (2008) 225–231. [20] L. Zhang, P. Fang, L. Yang, J. Zhang, X. Wang, Rapid method for the separation and recovery of endocrine-disrupting compound bisphenol AP from wastewater, Langmuir 29 (2013) 3968–3975. [21] W. Liu, F. Wang, Q.H. Qiu, L. Fi, C.Y. Wang, M.L. Zhang, Synthesis and characterisation of 9,9-bis(4-hydroxyphenyl)-fluorene catalysed by cation exchanger, Pigm. Resin Technol. 37 (2008) 9–15. [22] Y.L. Zhang, X. Zhang, X.C. Fei, S.L. Wang, Binding of bisphenol A and acrylamide to BSA and DNA: Insights into the comparative interactions of harmful chemicals with functional biomacromolecules, J. Hazard. Mater. 182 (2010) 877–885. [23] X.Y. Xie, X.R. Wang, X.M. Xu, H.J. Sun, X.G. Chen, Investigation of the interaction between endocrine disruptor bisphenol A and human serum albumin, Chemosphere 80 (2010) 1075–1080. [24] Z.H. Zhang, D.P. Xu, M. Tie, F.Y. Li, Z.J. Chen, J. Wang, W. Gao, X.T. Ji, Y. Yao, Spectroscopic study on interaction between bisphenol A or its degraded solution under microwave irradiation in the presence of activated carbon and human serum albumin, J. Lumin. 131 (2011) 1386–1392. [25] X. Wang, J.H. Yang, Y.J. Wang, Y.H. Li, F. Wang, L. Zhang, Studies on electrochemical oxidation of estrogenic disrupting compound bisphenol AF

[26]

[27]

[28] [29]

[30]

[31]

[32] [33]

[34]

[35]

[36]

[37]

[38] [39]

[40] [41]

[42] [43]

[44]

[45]

[46]

[47]

[48]

[49] [50]

[51]

[52]

19

and its interaction with human serum albumin, J. Hazard. Mater. 276 (2014) 105–111. A. Izzotti, S.KanitzF. Agostini, A. Camoirano, S.D. Flors, Formation of adducts by bisphenol A, an endocrine disruptor, in DNA in vitro and in liver and mammary tissue of mice, Mutati. Res-Gen. Tox. En. 679 (2009) 28–32. M. Mathew, S. Sreedhanya, P. Manoj, C.T. Aravindakumar, U.K. Aravind, Exploring the interaction of bisphenol-S with serum albumins: a better or worse alternative for bisphenol A?, J Phys. Chem. B 118 (2014) 3832–3843. K. Kolsek, J. Mavri, M.S. Dolenc, Reactivity of bisphenol A-3,4-quinone with DNA. A quantum chemical study, Toxicol. In Vitro 26 (2012) 102–106. H.M. Zhang, J. Cao, Z.H. Fei, Y.Q. Wang, Investigation on the interaction behaviour between bisphenol A and pepsin by spectral and docking studies, J. Mol. Struct. 1021 (2012) 34–39. Y.Q. Wang, T.T. Chen, H.M. Zhang, Investigation of the interactions of lysozyme and trypsin with biphenol A using spectroscopic methods, Spectrochim. Acta. A 73 (2010) 1130–1137. G.W. Zhang, Y. Ma, Spectroscopic studies on the interaction of sodium benzoate, a food preservative, with calf thymus DNA, Food Chem. 141 (2013) 41–47. M.C. Poirier, S. Gupta-Burt, C.L. Litterst, E. Reed, Detection of cisplatin—DNA adducts in humans, ACS Sym. Ser. 451 (1990) 300–307. S. Agarwal, D.K. Jangir, P. Singh, R. Mehrotra, Spectroscopic analysis of the interaction of lomustine with calf thymus DNA, J. Photochem. Photobiol., B 130 (2014) 281–286. J. Mehta, E. Rouah-Martin, B.V. Dorst, B. Maes, W. Herrebout, M. Scippo, F. Dardenne, R. Blust, J. Robbens, Selection and characterization of PCB-binding DNA aptamers, Anal. Chem. 84 (2012) 1669–1676. P. Basu, D. Bhowmik, G.S. Kumar, The benzophenanthridine alkaloid chelerythrine binds to DNA by intercalation: photophysical aspects and thermodynamic results of iminium versus alkanolamine interaction, J. Photochem. Photobiol., B 129 (2013) 57–68. S. Agarwal, D.K. Jangir, R. Mehrotra, Spectroscopic studies of the effects of anticancer drug mitoxantrone interaction with calf-thymus DNA, J. Photochem. Photobiol., B 120 (2013) 177–182. G.W. Zhang, L.H. Wang, X.Y. Zhou, Y. Li, D.M. Gong, Binding characteristics of sodium saccharin with calf thymus DNA in vitro, J. Agric. Food Chem. 62 (2014) 991–1000. . M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, A further examination of the molecular weight and size of desoxypentose nucleic acid, J. Am. Chem. Soc. 76 (1954) 3047–3053. . M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09 Revision D. W.L. Delano, The PyMol Molecular Graphic System, DeLano Scientific, San Carlos, CA, USA, 2004 . Y.Q. Wang, H.M. Zhang, Comparative studies of the binding of six phthalate plasticizers to pepsin by multispectroscopic approach and molecular modeling, J. Agric. Food Chem. 61 (2013) 11191–11200. Y.Q. Wang, H.M. Zhang, J. Cao, B.P. Tang, Binding of a new bisphenol analogue, bisphenol S to bovine serum albumin and calf thymus DNA, J. Photochem. Photobiol., B 138 (2014) 182–190. F. Ahmadi, K. Ghanbari, Proposed model for binding of permethrin and deltamethrin insecticides with ct-DNA, a structural comparative study, Ecotoxicol. Environ. Saf. 106 (2014) 136–145. C.N. Nsoukpoé-Kossi, A.A. Ouameur, T. Thomas, A. Shirahata, T.J. Thomas, H.A. Tajmir-Riahi, DNA interaction with antitumor polyamine analogues: a comparison with biogenic polyamines, Biomacromolecules 9 (2008) 2712– 2718. Y.Q. Wang, C.Y. Tan, S.L. Zhuang, P.Z. Zhai, Y. Cui, Q.H. Zhou, H.M. Zhang, Z.H. Fei, In vitro and in silico investigations of the binding interactions between chlorophenols and trypsin, J. Hazard. Mater. 278 (2014) 55–65. S. Hazra, M. Hossain, G. Suresh Kumar, Binding of isoquinoline alkaloids berberine, palmatine and coralyne to hemoglobin: structural and thermodynamic characterization studies, Mol. Biosyst. 27 (2013) 143–153. C. Ozluer, H.E. Kara, In vitro DNA binding studies of anticancer drug idarubicin using spectroscopic techniques, J. Photochem. Photobiol., B 138 (2014) 36–42. D. Sahoo, P. Bhattacharya, S. Chakravorti, Quest for mode of binding of 2-(4(dimethylamino)styryl)-1-methylpyridinium iodide with calf thymus DNA, J. Phys. Chem. B 114 (2010) 2044–2050. S. Hazra, G. Suresh Kumar, Structural and thermodynamic studies on the interaction of iminium and alkanolamine forms of sanguinarine with hemoglobin, J. Phys. Chem. B 118 (2014) 3771–3784. P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096–3102.

20

Y.-Q. Wang, H.-M. Zhang / Journal of Photochemistry and Photobiology B: Biology 149 (2015) 9–20

[53] J. Padmanabhan, R. Parthasarathi, V. Subramanian, P.K. Chattaraj, Group philicity and electrophilicity as possible descriptors for modeling ecotoxicity applied to chlorophenols, Chem. Res. Toxicol. 19 (2006) 356–364. [54] J.J. Nogueira, L. González, Molecular dynamics simulations of binding modes between methylene blue and DNA with alternating GC and AT sequences, Biochemistry 53 (2014) 2391–2412.

[55] Y.Q. Wang, H.M. Zhang, J. Cao, Quest for the binding mode of tetrabromobisphenol A with Calf thymus DNA, Spectrochim. Acta A 131 (2014) 109–113.