Journal of Photochemistry and Photobiology B: Biology 138 (2014) 109–117

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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

Binding properties of herbicide chlorpropham to DNA: Spectroscopic, chemometrics and modeling investigations Yu Li, Guowen Zhang ⇑, Mo Tao State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, Jiangxi, China

a r t i c l e

i n f o

Article history: Received 1 April 2014 Received in revised form 13 May 2014 Accepted 15 May 2014 Available online 27 May 2014 Keywords: Chlorpropham Calf thymus DNA Multivariate curve resolution-alternating leas squares Binding properties Intercalation

a b s t r a c t The binding properties of chlorpropham (CIPC) to calf thymus DNA (ctDNA) were investigated in vitro by UV–vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy coupled with molecular modeling method. The results obtained from UV–vis absorption, fluorescence and CD spectroscopic methods as well as DNA viscosity and melting measurements indicated that the binding of CIPC to ctDNA was an intercalative mode. The FTIR analysis and molecular modeling showed that CIPC mainly bound to guanine base of ctDNA. The association constant of the ctDNA–CIPC complex was determined to be in the order of 104 L mol1 by fluorescence titration. The calculated enthalpy change and entropy change suggested that hydrophobic forces and hydrogen bonds played prominent roles in the binding process. Furthermore, multivariate curve resolution-alternating least squares (MCR–ALS) approach was used to analyze the combined UV–vis absorption data matrix from the CIPCctDNA reaction system. The concentration profiles of the reaction components (CIPC, ctDNA and CIPC–ctDNA complex) and their pure spectra were successfully obtained to monitor the process of CIPC interaction with ctDNA. This study may contribute to the understanding of the CIPC–ctDNA interaction mechanism and toxicological effect of CIPC at the molecular level. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Chlorpropham (isopropyl N(3chlorophenyl) carbamate (CIPC, structure shown in Fig. 1) is a herbicide and plant growth regulator used for pre-emergence and early post-emergence control of weeds. In the post-harvest treatment of potatoes during storage and transport, it is also used as a sprout suppressant [1]. Residues of CIPC in raw potatoes and the processed products of potatoes were detected. There is a high possibility for human to intake this chemical through diet [2]. Long-term intake of the foods with residue pesticides will seriously affect human health. Previous studies have shown that CIPC is a mitotic disruptor in human and plant cells. Moreover, it displays teratogenic/developmental and neurobehavioral toxicity in mice [3]. The liver, spleen, kidney and erythrocytes were identified as targets of CIPC-induced toxicity in rats and mice [1]. Nakagawa et al. have reported that the cytoxicity of CIPC is caused by the rapid depletion of adenosine triphosphate (ATP) via impairment of mitochondrial function [1]. The toxicity evaluation of CIPC has been well-studied in vivo and

⇑ Corresponding author. Tel.: +86 79188305234; fax: +86 79188304347. E-mail address: [email protected] (G. Zhang). http://dx.doi.org/10.1016/j.jphotobiol.2014.05.011 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

in vitro, whereas there has little information on the DNACIPC interaction. Deoxyribonucleic acid (DNA) is the main components of chromosome in the cell, and serves as the carrier of the genetic information to guide the development and other vital functions of the organism. The double stranded, right-handed helical structure of DNA plays an essential role in DNA’s function, because the message is encoded in the sequence of their monomeric units [4]. Therefore, it is of utmost importance to maintain the integrity of DNA structure. Damage or change in the structure induced by environmental or other external factors may lead to gene mutations which have severe consequences on the organism [5]. The use of pesticides caused some food to contain residues of these compounds, and such contaminated food can readily reach the population and growing concerns have been expressed as to the potential hazards to human health [6]. Epidemiological studies showed that there was a close relationship between chemical pesticides and cancers [7]. In the past few decades, pesticide-DNA binding studies have been of great importance due to their feasible applications for the pesticides’ risk assessment. The formation of DNA adducts may cause gene mutations in the living organisms, which is regarded as the initial step in tumour development. This study may throw light on the mechanism of interaction between CIPC and DNA and be helpful for evaluating the hazard of pesticides.

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Fig. 1. The molecular structure of chlorpropham (CIPC).

A wide variety of techniques have been developed to investigate the interaction of chemical molecules with DNA. A powerful chemometrics approach, multivariate curve resolution-alternating least squares (MCR–ALS) was used to analyze spectroscopic data from the reaction system at equilibrium in solution [8]. As a softmodeling method, MCR–ALS can resolve multiple analyte responses in unknown mixtures [9,10]. With the use of MCR–ALS, the pure spectra and the corresponding concentration profiles of all the chemically analyzed components were extracted from the original measured data. This method offered the valuable information on the changes of the reagents and process of the biomacromolecules–chemical molecules interaction, which cannot be obtained by traditional means. This work was aimed to investigate the binding properties of CIPC to calf thymus DNA (ctDNA) in vitro under simulated physiological conditions (Tris–HCl buffer, pH 7.4) by UV–vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, coupled with DNA melting studies and viscosity measurements as well as molecular docking technique. It was worth noting that the chemometric approach, MCR–ALS was applied to resolve the combined original UV–vis absorption data matrix. The concentration profiles of three species (CIPC, ctDNA and ctDNACIPC complex) in the reaction and their pure spectra were extracted simultaneously, which exhibited remarkable advantages of MCR–ALS algorithm in discussing the interaction of CIPC with ctDNA. The combination of various techniques used in this work was beneficial in the depiction of the mechanism of ctDNACIPC interaction. This study is expected to provide guidance and reference to investigate the binding properties of other small molecule compounds with DNA. 2. Experimental 2.1. Reagents

solution was increased stepwise from 0 to 5.48  10-5 mol L1 at intervals of 2.19  106 mol L1, 26 solutions totally. For Experiment 2: the concentration of CIPC was kept at 2.14  105 mol L1, and ctDNA added in the concentration range of 0–10.5  105 mol L1 at intervals of 4.20  106 mol L1, total of 26 profiles were collected. After each addition, all the solutions were allowed to stand for 4 min to equilibrate, and then the UV–vis absorption spectra (210–300 nm) were collected every 1 nm. Accordingly, two data matrixes DctDNA (26  91) and DCIPC UV UV (26  91) were obtained from the titrations. The original spectroscopic data were then treated by a chemometrics approach, MCR–ALS. Besides that, the absorption spectra titrations were scanned between 200 and 260 nm to provide important information for the ctDNACIPC binding mode. Absorbance was measured by keeping the concentration of CIPC constant, while varying the concentration of ctDNA. ctDNA solutions with the same concentrations as that in mixture solutions were used as blank solutions to eliminate the absorbance of ctDNA itself. The influence of the addition of ctDNA to CIPC solution was obtained by comparing the variations of UV–vis spectra. 2.2.2. Fluorescence quenching measurements The fluorescence was taken with a Hitachi spectrofluorimeter Model F-7000 (Hitachi, Japan) equipped with a 150 W Xenon lamp and a thermostat bath. The CIPC solution (1.02  105 mol L1) was titrated by successive addition of ctDNA to give a final concentration of 1.35  104 mol L1. These solutions were allowed to stand for 4 min to equilibrate, and then the fluorescence spectra were measured at four different temperatures (292, 298, 304 and 310 K) at wavelengths from 270 to 390 nm under the excitation at wavelength of 230 nm. The widths of both the excitation and emission slits were set at 5.0 nm. The fluorescence intensity was corrected to eliminate the impact of re-absorption and inner filter effect. The measured fluorescence and the corrected one met the following relationship [12]:

F c ¼ F m eðA1 þA2 Þ=2

ð1Þ

where Fc and Fm represent the corrected and measured fluorescence, respectively. A1 and A2 are the absorbance of ctDNA at the excitation and emission wavelengths, respectively.

The fibrillar ctDNA and methylene blue (MB) were purchased from Sigma–Aldrich Corporation. CIPC was obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The stock solution of ctDNA was prepared by dissolving an appropriate amount of ctDNA in 0.1 mol L1 NaCl solution, and its concentration was determined at 260 nm by spectrophotometry using a known molar absorption coefficient 6600 L mol1 cm1. The purity was checked by the absorbance ratio at 260 and 280 nm. The ratio of A260/A280 was more than 1.80, indicating that the DNA sample was sufficiently free from protein [11]. CIPC was dissolved in absolute ethanol. MB stock solution was prepared by dissolving its crystals in ultrapure water. Other reagents used in the study were of analytical grade.

2.2.3. Competitive binding between MB and CIPC for ctDNA The mixture of ctDNA and MB in the quartz cuvette was titrated by the increasing concentrations of CIPC, and the fluorescence spectra of the ctDNAMB mixture were recorded in Tris–HCl buffer at 298 K with an excitation wavelength at 630 nm.

2.2. Procedures

2.2.5. Viscosity measurements The viscosity of solution relied on the time flowed through the capillary viscometer. The viscosity measurements were conducted by keeping the viscometer immersed in a water-bath maintained at 25 ± 0.1 °C. Flow times were measured and the relative viscosity (g) was obtained by the equation: g ¼ ðt  t 0 Þ=t0 , where t and t0 represent the observed flow time of ctDNA containing solutions and buffer solution alone, respectively. The data was presented

2.2.1. UV–vis absorption measurements Absorbance spectra were recorded using a Shimadzu UV-2450 spectrophotometer (Shimadzu, Japan). Two different titration measurements were conducted in Tris–HCl buffer, pH 7.4. For Experiment 1: the concentration of ctDNA was fixed at 5.42  10-5 mol L1, and the concentration of CIPC added to the

2.2.4. Melting studies The measurements of melting temperatures (Tm) were performed by continuous heating the solutions from 20 to 100 °C and recorded the absorbance at 260 nm of ctDNA every 5 °C. The Tm value was obtained from the transition midpoint of the curves [13].

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as (g/g0)1/3 versus the ratios of the concentration of CIPC to that of ctDNA ([CIPC]/[ctDNA]) [14]. 2.2.6. Circular dichroism (CD) spectroscopy CD spectra were recorded on a Bio-Logic MOS 450 CD spectrometer (Bio-Logic, Claix, France). The CD measurements of ctDNA incubated with CIPC at molar ratios ([CIPC/[ctDNA] of 0:1, 1:20, 1:10 and 1:5 were performed over the range of 230–310 nm in Tris–HCl buffer at room temperature under nitrogen atmosphere. All the observed CD spectra were corrected for buffer signal. 2.2.7. FT–IR spectroscopic measurements FT–IR spectra were carried out with Thermo Nicolet-5700 spectrometer (Thermo Nicolet Co., USA), equipped with a germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector and a KBr beam splitter. After 2 h incubation of ctDNA with CIPC, the spectra were recorded between 4000 and 800 cm1 with a resolution of 4 cm1 in Tris–HCl buffer at room temperature. Interferograms were accumulated and a good water subtraction was achieved as shown by a flat baseline around 2200 cm1 where the water combination mode is located. This method is a rough estimate but removes the water in a satisfactory way [15]. The difference spectra [(CIPC solution + ctDNA solution) – CIPC solution] were obtained to compare the spectral changes before and after the interaction. The ctDNA peak at 968 cm1 was regarded as internal reference to evaluate the variation of ctDNA characteristic peaks [16]. The plots of the relative intensity (Ri ¼ Ii =I968 ) versus different CIPC concentrations were obtained, where Ii is the intensity of peak at i cm1 for pure ctDNA and ctDNA in the complexes, and I968 is the intensity of the 968 cm1 peak [17]. 2.2.8. Docking study The interaction of CIPC with DNA was simulated to forecast their possible binding conformation with the use of Autodock 4.2. docking software. Structure of B-form DNA dodecamer d(CGCGAATTCGCG)2 (PDB code 1BNA) was downloaded from protein data bank. The water molecules were removed from the 1BNA, and Gasteiger charges were added by Autodock Tools (ADT) before performing docking calculations [18]. The structure of CIPC was generated by Sybyl  1.1 and subsequently optimized to minimal energy. The Lamarckian genetic algorithm was selected to perform docking calculations. A total population of 100 positioned individuals was performed at the end of each autodock execution. 2.3. Theory of MCRALS MCRALS allows the mathematical analysis of more than one data matrix simultaneously [19]. It has been applied to decompose measured response signals such as spectra into the concentration and pure spectra profiles for each species in a mixture and determine the molecular interaction process. The use of this algorithm includes the following four basic steps: (1) data arrangement; (2) determination of number of species; (3) initial estimate of the concentration profiles or pure spectra; (4) alternating least squares constrained optimization. Its theory has been described in detail in previous reports [20–22]. 3. Results and discussion 3.1. Analysis of absorption spectra by MCR–ALS The UV–vis absorption spectra collected from Experiment 1 and Experiment 2 are displayed in Fig. 2. ctDNA exhibited a strong UV

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absorption peak at about 258 nm (Fig. 2A) arising from p–p* transitions in the aromatic purine (Ade, Gua) and pyrimidines (Cyt, Thy) rings of the nucleotide basis [23] and a weak peak at about 207 nm due to the deoxyribose and phosphoric acids of keleton structure. Upon successive addition of CIPC, the intensity of ctDNA spectrum centered at 207 nm increased and the peak position shifted to 210 nm accompanied by a new peak at about 238 nm. As shown in Fig. 2B, the original spectra of CIPC also changed with increasing the concentration of ctDNA. The peak of CIPC centered at 238 nm became a broader band (240–280 nm), and the intensity of the band and another peak at 208 nm increased markedly. It clearly showed a high spectral overlap between ctDNA and CIPC. Further information cannot be extracted from the profiles of mixture. Therefore, two individual data matrices from Experiment 1 and 2 were combined into an augmented data matrix, and then the matrix was analyzed by MCR–ALS. Fig. 3 shows the recovered pure absorption spectra and concentration profiles for different species by MCR–ALS. With the application of SVD model to the augmented data matrix, the extracted three pure spectra are assigned to free CIPC, free ctDNA and ctDNA–CIPC complex [24] (Fig. 3A). The resolved spectra (shown in dashed line) of the free ctDNA and CIPC by MCR–ALS agreed well with the measured spectra (solid line). The good matching between the predicted and the measured spectra demonstrated that the results were persuasive [25]. And importantly, the spectrum of the interaction product, ctDNA–CIPC was obtained by MCR–ALS analysis, which was difficult to acquire by conventional methods. The equilibrium concentration profiles of free CIPC, free ctDNA and ctDNA–CIPC complex are displayed in Fig. 3B and C. With the addition of CIPC, the concentration of the complex ctDNA–CIPC increased gradually along with the decrease of the concentration of free ctDNA (Fig. 3B). In contrast, the successive titration of ctDNA solution led to a decrease in the concentration of free CIPC and an increase in the concentration of the ctDNA–CIPC complex (Fig. 3C). The concentration changes of the three reaction components described visually the kinetic interaction process. Thus, the results from MCRALS offered a strong evidence of the interaction of CIPC with ctDNA. 3.2. Changes in UV–vis spectra of CIPC and binding mode The changes in UV–vis spectra of small molecules before and after interaction with DNA can provide important information about small molecules–DNA binding modes and complexes formation [26]. Generally, the binding of an intercalative molecule to DNA is accompanied by hypochromism and/or red shift (bathochromism) in the absorption spectra due to the strong stacking interaction between the aromatic chromophore of the ligand and DNA base pairs, and the extent of hypochromism and red-shift is commonly consistent with the strength of the intercalative interaction [27]. There are two prominent bands in the UV–vis spectrum of CIPC at 207 and 238 nm (Fig. 4). Upon the addition of ctDNA, the absorption intensity of the bands was weakened significantly with a slight red shift at 207 nm. The observed hypochromicity along with minor bathochromic shift for the absorption band of CIPC suggested that the ctDNA–CIPC interaction could be an intercalative mode. The changes in UV–vis spectra of CIPC may be explained as follows: the empty p*–orbital of CIPC coupled with the p*–orbital of ctDNA base pairs, which caused the decrease of p–p* transition energy. That is the reason for the red shift of CIPC absorbance. At the same time, the empty p*–orbital was partially filled with electrons to reduce the transition probability, which led to the hypochromic effect [28].

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Fig. 2. (A) UV–vis spectra obtained from Experiment 1, ctDNA in the presence of CIPC, c(ctDNA) = 5.42  105 mol L1, the concentration of CIPC was 0–5.48  105 mol L1 for curves from a to z. (B) UV–vis spectra for CIPC with the addition of ctDNA in Experiment 2, c(CIPC) = 2.14  105 mol L1, the concentrations of ctDNA was 0– 10.5  105 mol L1 for curves from a to z.

Fig. 4. UV–vis difference spectra [(ctDNA solution + CIPC solution) – ctDNA solution] for CIPC with various concentrations of ctDNA. Inset: Absorption spectra of the system with increasing the concentration of ctDNA in the wavelength range of 230–245 nm. c(CIPC) = 4.39  105 mol L1; c(ctDNA) = 0, 0.84, 1.69, 2.53, 3.37, 4.21, 5.06, 5.90, 6.74, 7.58 and 8.43 mol L1 corresponding to the curves from a to k, respectively.

3.3. Fluorescence titration The effects of different concentrations of ctDNA on the fluorescence emission spectra of CIPC are shown in Fig. 5A. The fluorescence of CIPC was remarkably quenched by ctDNA. The reduction in fluorescence intensity indicated that the interaction between ctDNA and CIPC occurred. Whereas, there were no changes in both the position and the shape of the fluorescence spectra of CIPC, suggesting that the non-covalent bonds rather than covalent bonds formed between CIPC and ctDNA [29]. The fluorescence quenching can be classified into dynamic quenching and static quenching. To further study their interaction, the influence of temperature on the fluorescence quenching was conducted. The dynamic quenching is caused by the contact between the fluorophore and the quencher, and the static quenching is due to the formation of the ground state complex [30]. The two quenching processes can be distinguished by their dependence on temperatures. Dynamic quenching depends upon diffusion, higher temperature results in larger quenching, and quenching constant is expected to increase with increasing temperature. Reversely, the static quenching constant decreased with the increase in temperature [31]. To reveal the nature of the quenching process, the Stern–Volmer equation was used to analyze the fluorescence quenching data: Fig. 3. Recovered results by MCR–ALS: (A) the recovered UV–vis spectra obtained by MCR–ALS (solid line–measured and dashed line–recovered). (B) the equilibrium concentration profiles for Experiment 1. (C) for Experiment 2.

F0 ¼ 1 þ K SV ½ctDNA F

ð2Þ

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where F0 and F denote the fluorescence intensities in the absence and presence of ctDNA, respectively. KSV is the Stern–Volmer quenching constant, which was obtained by plotting F0/F against [ctDNA]. As shown in the inset in Fig. 5A, the Stern–Volmer plots for the quenching of CIPC by ctDNA at four different temperatures (292, 298, 304 and 310 K) exhibited a good linear relationship, suggesting the predominance of a single quenching mechanism. The KSV values receded with rising temperature (Table 1), indicating that the probable fluorescence quenching mechanism of CIPC by ctDNA was a static quenching procedure. The association constant can be estimated by the modified Stern–Volmer equation [32]:

F0 1 1 1 þ ¼ F 0  F fa K a ½ct DNA fa

ð3Þ

where Ka is the modified Stern–Volmer association constant for the accessible fluorophores and fa denotes the fraction of accessible fluorescence. The dependence of F0/(F0F) on the reciprocal value of the quencher concentration 1/[ctDNA] is linear and Ka is the quotient of an ordinate 1/fa and slope 1/faKa. The Ka values for the ctDNACIPC complex at different temperatures are summarized in Table 1. The Ka values followed a trend of decline with an increasing temperature as KSV, which was the characteristic of static quenching mechanism [33]. The value of Ka was determined to be in the order of 104 L mol1, which corresponded to other intercalative compounds, such as clodinafop-propargyl [34], permethrin [35] and hesperitin [36]. 3.4. Thermodynamic parameters and the nature of the binding forces The interaction forces between small molecules and biomolecules mainly include hydrogen bonds, van der Waals force, hydrophobic forces and electrostatic interactions. The thermodynamic parameters such as enthalpy change (DH), entropy change (DS) and free energy change (DG) can be determined from the van’t Hoff equation:

log K a ¼ 

Fig. 5. Fluorescence spectra of CIPC in the presence of ctDNA. c(CIPC) = 1.02  105 mol L1 and c(ctDNA) = 0, 0.12, 0.24, 0.36, 0.47, 0.59, 0.71, 0.82, 0.94, 1.05, 1.16, 1.27 and 1.44  104 mol L1 corresponding to the curves from a to m, respectively. Inset: Stern–Volmer plots for ctDNA–CIPC at different temperatures. (B) Fluorescence spectra of ctDNA–MB system in the presence of CIPC at different concentrations (kex = 630 nm, kem = 692 nm). c(ctDNA) = 9.57  105 mol L1, c(MB) = 9.66  106 mol L1, and c(CIPC) = 0, 0.80, 1.60, 2.39, 3.19, 3.99, 4.79, 5.59, 6.39, 7.18, 7.98 and 8.78  105 mol L1 corresponding to the curves from a to k, respectively. The dashed line represents the spectrum of free MB. (C) Fluorescence quenching plots of CIPC by ds ctDNA and ss ctDNA. c(CIPC) = 2.14  105 mol L1.

DH DS þ 2:303RT 2:303R

ð4Þ

where R is the gas constant, and four different temperatures of 292, 298, 304 and 310 K were used. The values of DH and DS were obtained from the slope and intercept of the equation. The value of free energy change (DG) can be obtained from the Gibbs– Helmholtz equation:

DG ¼ DH  T DS

ð5Þ

The thermodynamic parameters for the interaction of ctDNA with CIPC are listed in Table 1. The negative values of DG revealed that the interaction is spontaneous, and the positive value of DS is frequently considered to be a symbol for hydrophobic forces [32]. The negative DH value showed that the binding process is mainly

Table 1 The Stern–Volmer quenching constants KSV, association constants Ka, and thermodynamic parameters for the interaction of ctDNA–CIPC at different temperatures.

a b

T(K)

Ksv (103 L mol1)

Ra

Ka (104 L mol1)

Rb

DH (kJ mol1)

DS (J mol1K1)

DG (kJ mol1)

292 298 304 310

6.82 5.95 5.55 5.39

0.9983 0.9993 0.9994 0.9978

2.20 2.11 1.86 1.75

0.9903 0.9922 0.9970 0.9911

12.56

40.34

24.34 24.58 24.82 25.07

R is the correlation coefficient for the KSV values. R is the correlation coefficient for the Ka values.

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enthalpy driven and by means of hydrogen binding interactions [37]. Thus, both hydrophobic forces and hydrogen bonds are the main contributors in the CIPCctDNA interaction. 3.5. Competitive interaction of CIPC and MB with ctDNA The competitive interaction was carried out by the addition of CIPC to the ctDNAMB complex. MB is a photosensitizing agent and can bind to duplex DNA due to its planar structure. Thus, MB has been used as a probe in the competitive interaction with fluorophore for DNA binding [38]. Previous studies have shown that at a lower molar ratio of MB to DNA (R < 0.125), the major binding mode was intercalation, while electrostatic interaction dominated at higher R values (R > 0.125) [39,40]. In this study, the ctDNAMB solution was prepared by keeping R value at 0.101(in this case, an intercalative binding between MB and ctDNA occurred). As shown in Fig. 5B, with the addition of CIPC, the fluorescence intensity of the ctDNAMB complex at 692 nm increased gradually and there was no effect of CIPC on MB emission spectrum. The increased fluorescence intensity of the ctDNAMB system may be due to the intercalation of the CIPC molecule into ctDNA by replacing the bound MB to the solution, increasing the concentration of free MB and fluorescence intensity accordingly. 3.6. Effect of native and denatured ctDNA on fluorescence quenching The binding mode of CIPC to ctDNA was further investigated by comparing the fluorescence quenching effect of both singlestranded ctDNA (ss ctDNA) and double-stranded ctDNA (ds ctDNA) on CIPC. When small molecules bind to the phosphate backbone of DNA, the quenching effects of ds DNA and ss DNA on the fluorescence should be uniform [41]. If there was a groove binding, the quenching effect on the compound should be strengthened compared to ds DNA. Intercalative mode should cause a weaker quenching effect of ss DNA than that of ds DNA [32]. Native ds ctDNA was heated in a boiling water-bath and followed by sudden cooling in the ice-bath, then gradually up to 25 °C to separate the strands for the preparation of ss ctDNA [42]. The fluorescence quenching effect of CIPC by ss ctDNA was inferior to that of ds ctDNA (Fig. 5C). This result provided further support for the intercalation of ctDNACIPC interaction. 3.7. DNA melting studies The double helical structure of DNA undergone a transition into a randomly single-stranded form at the condition of heating. The temperature at which half of the double-stranded DNA dissociated is defined as melting temperature (Tm). Tm is strictly related to the stability of the macromolecule, the intercalation of small molecules

can stabilize the double helix structure and increase Tm, but the non-intercalative binding cause no obvious increase in Tm [43]. The value of Tm can be obtained from the midpoint of the melting curves (Fig. 6A), the value of Tm for ctDNA was 82 °C and that of the ctDNACIPC system was 86 °C. The Tm value of ctDNA was found to be increased by 4 °C upon binding to CIPC. The increase in Tm revealed the intercalative binding and more stable structure of ctDNA. 3.8. Viscosity measurements The viscosity measurement can provide crucial proof to clarify the interaction mode of small molecules with DNA because it is sensitive to the changes in the length of DNA molecule [44]. In the case of classic intercalation, the compounds inserted into the adjacent DNA base pairs and needed a large space to be accommodated, which resulted in the increase of the length of DNA helix and the viscosity [45]. In contrast, a partial and non-classical intercalation often cause a slight decrease or little change in the viscosity because of the existence of a bend or kink in DNA helix and reduction of its effective length [46]. As shown in Fig. 6B, it was found that with the addition of CIPC, the relative viscosity of ctDNA increased gradually. This result provided strong evidence for the intercalative binding of CIPC to ctDNA, which conformed well to the one derived from UV–vis spectroscopic and competitive binding studies. 3.9. FTIR analysis FTIR spectroscopy is an effective technique in the study of DNA with small molecules in solution. The technique is fast and nondestructive, and requires only small amounts of samples, and is thus ideal for systematic DNA studies [47]. FTIR spectra of free ctDNA and ctDNA incubated with varying concentrations of CIPC were monitored. The infrared absorption characteristic peaks of free B-form DNA are situated in the region of 1800–700 cm1. The band at 1720 cm1 is attributed mainly to guanine (G) C7@N in plane stretching vibrations. The band observed at position 1658 cm1 is mainly due to vibrations caused by thymine (T) C2@O stretching. The 1610 cm1 band occurs owing to adenine (A) stretching. The band at 1489 cm1 corresponds to vibrations of cytosine (C) bases [48,49]. Bands at 1220 and 1088 cm1 denote phosphate asymmetric and symmetric vibrations, respectively [48]. All of the bands mentioned above are the prominent bands of DNA, which are concerned during the interaction of ctDNA with CIPC in this study. The difference spectra (Fig. 7) provided ctDNACIPC interaction information at different concentrations of CIPC. The peak at position 1720 cm1 shifted to 1714–1717 cm1, and the other bases

Fig. 6. (A) Melting curves of ctDNA in the absence and presence of CIPC. c(CIPC) = 2.49  105 mol L1, c(ctDNA) = 3.61  105 mol L1. (B) The influence of CIPC concentration on the relative viscosity of ctDNA. c(ctDNA) = 2.41  105 mol L1.

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Fig. 8. Circular dichroism spectra of free ctDNA and the ctDNA–CIPC complex. c(CIPC) = 4.33  104 mol L1 (dashed line). c(ctDNA) = 4.33  104 mol L1, the molar ratios ([CIPC]/[ctDNA]) were 0:1(a), 1:20(b), 1:10(c) and 1:5(d), respectively.

the negative band decreased (shifting to zero level). The results suggested that the binding of CIPC to ctDNA reduced the righthanded helicity of ctDNA and increased the base stacking degree of ctDNA [54]. Kashanian et al. [34] believed that the increased CD signal around 275 nm with increasing the concentration of clodinafop-propargyl herbicide is important to state their intercalation binding mode. Based on this, the recorded similar CD changes demonstrated an intercalating interaction mode between CIPC and ctDNA. 3.11. Molecular modeling of CIPC–DNA interaction Fig. 7. (A) FT–IR spectra for free ctDNA and difference spectra [(ctDNA + CIPC) – (CIPC)] for ctDNA–CIPC complexes in the region of 1800–800 cm1. (B) Relative intensity variations for characteristic peaks of ctDNA at different CIPC/ctDNA molar ratios.

To get detailed information for the interaction of CIPC with ctDNA, molecular docking studies were carried out. The information

vibrations showed only 1–2 cm1 shift. The position changes of these bands were also accompanied by the increases in their vibrational intensity. Among the four bases, the band of guanine exhibited the most manifest changes in terms of peak position and intensity variation. Such spectral changes may be related to the fact that the CIPC molecule intercalates into ctDNA by guanine bases [50]. The intensity of the phosphate stretching peaks (both asymmetric and symmetric) showed an increasing trend, and no major shift in wavenumber was observed, which suggested noninteraction of CIPC with phosphate group of ctDNA backbone [47], but the existence of CIPC may arouse some interference. 3.10. CD spectroscopic studies CD measurement is very sensitive to detect minor conformational variations of DNA induced by small molecule binding [51]. The observed CD spectrum of free DNA shows a positive band at 277 nm due to base stacking and a negative band at 245 nm from the right-handed helicity. The two bands are the known features of a right-handed B form DNA [52]. The characteristic bands of DNA are quite sensitive to the mode of DNA interactions with small molecules. Simple groove binding and electrostatic interaction do not cause any appreciable changes in the base-stacking and helicity bands, while intercalation influences the intensities of the two bands [53]. The CD spectra were measured in the presence of CIPC with various ratios of CIPC to ctDNA (Fig. 8). It was found that the CD spectra of ctDNA displayed obvious changes in both positive and negative bands when CIPC was incubated with ctDNA, while the free CIPC showed no CD signal in the measuring wavelength range. The intensity of the positive band increased while the ellipticity of

Fig. 9. (A) Molecular modeling of CIPC interaction with DNA (the formed hydrogen bond and distance have been marked). (B) The sketch map of the specific binding site.

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may get insight into their specific binding and validate above experimental results. Among the 100 distinct individuals, the lowest energy ranked interaction model of CIPC with DNA was elected and displayed in Fig. 9A. The CIPC molecule was selectively bound to the G–C rich region of DNA helix and surrounded by residues DC9A, DG10A, DC15B and DG16B. The simulative conformation revealed that CIPC interacted with ctDNA via guanine base. As shown in Fig. 9B, a hydrogen bond was formed between the oxygen atom O11 of CIPC and the second hydrogen atom H22 associated with N2 of G16 on B-chain, and the length of hydrogen bond was 2.242 Å. The estimated binding energy (3.81 kcal mol1) by Autodock was comparable to the Gibbs free energy (5.87 kcal mol1) in the thermodynamic analysis, suggesting that the results obtained from the simulated analysis were convincing. Considering the fruitful information about the mechanism of interaction and the conformation of adduct from different aspects, the molecular docking can be regarded as a vital complement to the spectroscopic techniques in the study of the interaction between small molecules with biomacromolecules [55]. 4. Conclusions This study for the first time focuses on the binding of the herbicide chlorpropham to ctDNA through spectroscopic, viscometric, molecular modeling and chemometrics techniques. The concentration changes and the spectra for the three reaction components (CIPC, ctDNA and CIPC–ctDNA) were obtained simultaneously to quantitatively analyse the reaction process by resolving the combined UV–vis spectroscopic data matrix with the use of MCR–ALS algorithm. CIPC was found to be an intercalator of ctDNA, which was supported by the emergence of hypochromism for CIPC absorption, the competitive binding between CIPC and MB, less quenching effect by denatured ctDNA, and increases in the relative viscosity and melting temperature of ctDNA. Hydrophobic forces and hydrogen bonds played predominant roles in the binding reaction. Moreover, the FT–IR analysis revealed that the interaction occurred preferably at guanine base, and the result was confirmed by the molecular simulation. The study showed significant potential for understanding the interaction mechanism of target DNA with small compounds at the molecular level. Acknowledgements We are grateful for financial support provided by the National Natural Science Foundation of China (Nos. 21167013 and 31060210), the Program of Jiangxi Provincial Department of Science and Technology (20141BBG70092), and the Research Program of State Key Laboratory of Food Science and Technology of Nanchang University (SKLF–ZZB–201305, SKLF–ZZA–201302 and SKLF–KF–201203). References [1] Y. Nakagawa, K. Nakajima, T. Suzuki, Chlorpropham induces mitochondrial dysfunction in rat hepatocytes, Toxicology 200 (2004) 123–133. [2] T. Fujitani, Y. Tada, M. Yoneyama, Chlorpropham-induced splenotoxicity and its recovery in rats, Food Chem. Toxicol. 42 (2004) 1469–1477. [3] T. Fujitani, Y. Tada, A.T. Noguchi, M. Yoneyama, Effects of chlorpropham (CIPC) on the hemopoietic system of rats, Food Chem. Toxicol. 39 (2001) 253–259. [4] D. Voet, J.G. Voet, Biochemistry, second ed., John Wiley & Sons Inc., New York, 1995. [5] R.R. Sinden, DNA Structure and Function, Gulf Professional Publishing, Woburn, 1994. [6] Y. Picó, G. Font, M.J. Ruiz, M. Fernández, Control of pesticide residues by liquid chromatography-mass spectrometry to ensure food safety, Mass Spectrom. Rev. 25 (2006) 917–960. [7] J. Dich, S.H. Zahm, A. Hanberg, H.O. Adami, Pesticides and cancer, Cancer Causes Control 8 (1997) 420–443.

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