Spectroscopic studies on the interaction between carbaryl and calf thymus DNA with the use of ethidium bromide as a fluorescence probe

Journal of Photochemistry and Photobiology B: Biology 108 (2012) 53–61

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Spectroscopic studies on the interaction between carbaryl and calf thymus DNA with the use of ethidium bromide as a fluorescence probe Guowen Zhang ⇑, Xing Hu, Peng Fu State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China

a r t i c l e

i n f o

Article history: Received 18 September 2011 Received in revised form 27 December 2011 Accepted 28 December 2011 Available online 4 January 2012 Keywords: Carbaryl Calf thymus DNA Spectroscopy Parallel factor analysis Ethidium bromide

a b s t r a c t The interaction between carbaryl and calf thymus DNA (ctDNA) was investigated under simulated physiological conditions (Tris–HCl buffer of pH 7.4) using ethidium bromide (EB) dye as a probe by UV–vis absorption, fluorescence and circular dichroism (CD) spectroscopy, as well as DNA melting studies and viscosity measurements. It can be concluded that carbaryl molecules could intercalate into the base pairs of DNA as evidenced by hyperchromic effect of absorption spectra, decreases in iodide fluorescence quenching effect, induced CD spectral changes, and significant increases in melting temperature and relative viscosity of DNA. The binding constants and thermodynamic parameters of carbaryl with DNA were obtained by the fluorescence quenching method. Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way synchronous fluorescence spectral data matrix of the competitive interaction between carbaryl and EB with DNA, and the results provided simultaneously the concentration profiles and corresponding pure spectra for three reaction components (carbaryl, EB and DNA–EB complex) of the kinetic system at equilibrium. This PARAFAC analysis demonstrated the intercalation of carbaryl to the DNA helix by substituting for EB in the DNA–EB complex. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction In recent years, the study on the interaction of pesticides with DNA has been attracting increasingly interest of many research groups in the field of biochemistry and analytical chemistry, because certain pesticides have been categorized as potent genotoxicants and alkylating agents for DNA [1–3]. Several reports have revealed that the pesticides such as heptachlor, endosulfan and cypermethrin interact with DNA and form promutagenic DNA adducts [4–6]. The insecticides, diazinon and aminocarb, have been reported to intercalate between the base pairs of ctDNA to form DNA–Clodinafop-Propargyl and DNA–aminocarb adducts [7,8]. All of these pesticides, which either covalently bind or intercalate in DNA molecule and form DNA adducts may cause gene mutations and initiate carcinogenesis, if the adducts are not repaired or misrepaired before DNA replication occurs [9]. Eisenbrand et al. demonstrated that an increased DNA damage indeed increases the probability of mutations occurring in critical target genes and cells, which may enhance the process of carcinogenesis [10]. Carbaryl (1-naphthylmethylcarbamate) (Fig. 1) is a carbamate pesticide largely used for pest control in agriculture. However, carbaryl is toxic to humans and animals, its mechanism of toxicity is related to inhibition of acetylcholinesterase activity in peripheral ⇑ Corresponding author. Tel.: +86 79188305234; fax: +86 79188304347. E-mail address: [email protected] (G. Zhang). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2011.12.011

and central nervous systems [11]. In addition, some researchers also reported that carbaryl has significant inhibitory effect on the controlling mechanism of reproduction of fishes [12], and can cause heart malformation and irregular heartbeat in medaka [13]. Recently, a risk assessment made by US Environmental Protection Agency (EPA) showed that carbaryl has potential carcinogenicity [14]. Therefore, studies of the binding interaction between carbaryl and DNA are beneficial not only for understanding the mechanism of DNA damage by carbaryl in body, but also for planning the design of new pesticides with low toxicity. It is well known that the interaction of ethidium bromide (EB) with DNA follows the intercalation model and then EB is considered as an intercalator [15,16]. There are competitive or cooperative interactions between drugs and fluorescence probe, and it is important to address this issue in order to obtain a fuller description of the interaction [17]. Many methods are often used to study the DNA binding properties: viscosity measurement; melting measurement; nuclear magnetic resonance (NMR) measurement; X-ray diffraction; agarose gel electrophoresis; spectrometric measurements, including UV– vis spectra, fluorescence spectra, resonance light scattering (RLS) spectra, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectra; electrochemical measurements, including cyclic and differential pulse voltammetry, etc. [18–21]. Among these methods, spectrophotometric ones are common and convincing [22]. In recent years, the application of analytical techniques such

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Co. Ltd., Hangzhou, China). An electronic thermostat water-bath (Shanghai Yuejin Medical Instrument Company, Shanghai, China) was used for controlling the temperature. All the measurements, unless specified otherwise, were performed at room temperature. 2.2. Chemicals

Fig. 1. Molecular structure of carbaryl.

as fluorescence and UV–vis absorption spectroscopy in conjunction with chemometrics methods in the life science has drawn much attention, because they can provide information well beyond that obtained by conventional methods. Parallel factor analysis (PARAFAC) and multiple curve resolution-alternating least squares (MCR–ALS) are known soft modeling algorithm, which have been applied to analyses of spectroscopic data from biomolecular equilibria in solution, and the equilibrium concentration of each component in the reaction as well as the corresponding pure spectra can be obtained simultaneously. Ni et al. [23] have applied the PARAFAC to study the interaction of the antibiotic, tetracycline (TC), with DNA in the presence of aluminium ions (Al3+) using methylene blue (MB) dye as probe. The three-way synchronous fluorescence data from the interaction system was processed by the PARAFAC, and the concentration information for the three components (TC–Al3+, MB and TC–Al3+–DNA) of the system at equilibrium was obtained simultaneously. Vives et al. [24] investigated the interaction of EB dye and poly(inosinic)–poly(cytidylic) acids with the use of UV–vis absorption, fluorescence and CD spectroscopy. The spectra of all tested samples by these techniques were recorded. The dye concentration ratios and the whole set of spectroscopic data matrices were simultaneously analyzed by the MCR–ALS. In this work, the in vitro interaction between carbaryl and calf thymus DNA was investigated under simulated physiological conditions (Tris–HCl buffer of pH 7.4) using EB dye as a probe with the use of UV–vis absorption, fluorescence and CD spectroscopy, as well as DNA melting temperature and viscosity measurements. The pesticide binding mode and thermodynamic characteristic were explored. Furthermore, the chemometrics method, PARAFAC modeling, was applied to resolve the three-way synchronous fluorescence spectral data of the competitive interaction between carbaryl and EB probe with DNA so as to improve the understanding of complex kinetic processes and extract the equilibrium profiles of the reacting species.

2. Experimental 2.1. Apparatus The fluorescence and synchronous fluorescence spectra were measured on a Hitachi Model F-4500 spectrofluorimeter equipped with a thermostatic bath, and a 1.0 cm quartz cuvette. The excitation and emission slits were set at 5 nm. The UV–vis absorption spectra were recorded on a Shimadzu UV-2450 spectrophotometer using a 1.0 cm cell. The CD spectra were measured on a Bio-Logic MOS 450 CD spectrometer (France) using a 1.0 mm path length quartz cuvette, measurements were taken at wavelengths between 220 and 320 nm with 0.1 nm step resolution and averaged over three scans recorded as a speed of 120 nm min1 purged with nitrogen gas. A pHS-3C digital pH meter (Shanghai Exact Sciences Instrument Co. Ltd., Shanghai, China) was utilized to detect the pH values of the aqueous solutions. The viscosity measurements were carried out using an NDJ-79 viscometer (Yinhua Flowmeter

The calf thymus DNA (ctDNA) was obtained from Sigma Co., and was dissolved in doubly distilled deionized water containing 0.1 mol L1 NaCl. The purity of the DNA was checked by monitoring the ratio of the absorbance at 260/280 nm. The solution gave a ratio of >1.8 at A260/A280, which indicates that DNA was sufficiently free from protein [25]. The concentration of DNA in stock solution was determined to be 3.30  103 mol L1 by UV absorption at 260 nm using a molar absorption coefficient e260 = 6600 L mol1 cm1 (expressed as molarity of phosphate groups) [26]. The prepared DNA solution was stored at 4 °C. Carbaryl (Chemservice. Inc., USA) stock solution (1.0  103 mol L1) was prepared by dissolving its crystals in 95% (v/v) ethanol solution. The stock solution (1.0  103 mol L1) of ethidium bromide (EB, Sigma) was made up with doubly distilled water. All the solutions used in the experiments were adjusted with the Tris–HCl buffer solution (0.05 mol L1, pH 7.4), which was prepared by mixing and diluting 25 mL of Tris solution (0.2 mol L1) with 45 mL of HCl (0.1 mol L1) and then diluted to 100 mL with water. All chemicals used in this work were of analytical grade, and doubly distilled water was used throughout. 2.3. Procedures 2.3.1. UV–vis measurements The UV–vis absorption spectra of DNA and the mixture of DNA and carbaryl were measured in the wavelength range of 200– 300 nm. All UV–vis measurements were carried out in 0.05 mol L1 Tris–HCl buffer (pH 7.4) at room temperature. 2.3.2. Fluorescence measurements A quantitative analysis of the potential interaction between carbaryl and DNA was carried out by fluorimetric titration. A 3.0 mL solution, containing 1.0  105 mol L1 carbaryl, was titrated by successive additions of a stock solution of DNA (to give a final concentration of 4.71  104 mol L1) with micro-injector. These solutions were allowed to stand for 8 min to equilibrate. The fluorescence emission spectra were measured at 298, 304 and 310 K in the wavelength range of 300–450 nm with an excitation wavelength at 291 nm. Appropriate blanks corresponding to the buffer solution were subtracted to correct background fluorescence. Increasing amounts of carbaryl were micropipetted directly into a 1.0 cm cell containing 2.0  105 mol L1 EB and 8.0  105 mol L1 DNA (total volume 3 mL), and the reaction was performed at room temperature. The synchronous fluorescence spectra was recorded by scanning both excitation and emission wavelengths simultaneously. The spectrofluorometric measurements of nine samples were carried out over the excitation wavelength range of 200–700 nm at 1 nm intervals (total of 501 wavelengths), and Dk (a constant wavelength interval between emission and excitation wavelength) was changed in the range of 10–200 nm at 10 nm intervals (total of 20 Dk increments). The data were arranged in an I  J  K three-way array where the first index (I) refers to the Dk wavelengths, the second (J) to the excitation wavelengths, and the third (K) to the samples. Thus, the size of the experimental threeway array was 20  501  9. 2.3.3. Circular dichroism (CD) studies The CD spectra of DNA incubated with carbaryl at molar ratios ([carbaryl]/[DNA]) value of 0, 1 and 2 were measured at wave-

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lengths between 220 and 320 nm. CD measurements were carried out in a pH 7.4 Tris–HCl buffer at room temperature. The changes in CD spectra were monitored against a blank. The results were taken as ellipticity in mdeg. The optical chamber of the CD spectrometer was deoxygenated with dry nitrogen before use and kept in a nitrogen atmosphere during experiments. Scans were accumulated and automatically averaged. 2.3.4. DNA melting studies DNA melting experiments were carried out by monitoring the absorption of DNA at 260 nm in the absence and presence of carbaryl at different temperatures. The absorbance intensities were then plotted as a function of temperature ranging from 20 to 100 °C. The melting temperature (Tm) of DNA was determined as the transition midpoint. 2.3.5. Viscosity measurements Viscometric titrations were preformed using a viscometer which was immersed in a thermostatic water-bath at 25.0 ± 0.1 °C. The experiments were conducted by adding appropriate amounts of carbaryl into the viscometer to give a certain r (r = [carbaryl]/[DNA]) value while keeping the DNA concentration constant. The flow time of the solution through the capillary was measured with an accuracy of ±0.20 s by using a digital stopwatch. The mean values of three replicated measurements were used to evaluate the average relative viscosity of the samples. The data were presented as (g/g0)1/3 versus the ratios of the concentration of carbaryl to that of DNA [18], where g and g0 are the viscosity of DNA in the presence and absence of carbaryl, respectively. Viscosity values were calculated from the observed flow time of DNA containing solutions (t) and corrected for buffer solution (t0), g = (t  t0)/t0. 2.4. Chemometrics: parallel factor analysis algorithm (PARAFAC) PARAFAC is a chemometrics method for multi-way data decomposition based on the so-called trilinear tensor theory [27]. It was used to resolve the three-way array of synchronous fluorescence spectra data obtained from Section 2.3.2. For a trilinear spectral data array with an appropriate number of selected model factors, PARAFAC will produce unique solutions, corresponding to the true underlying spectra. In addition, the concentrations of analytes can also be extracted [28]. The decomposition of a three-way array of synchronous fluorescence data, X, with dimensions I  J  K is performed. Each PARAFAC component gives three types of loading values: one related to the excitation data (ah), one related to the Dk (bh), and one related to the content of the samples (ch). Thus, three loadings matrices, A, B, and C with elements, aih, bjh, and ckh, can be given for the three dimensions in the data. The aim is to resolve the three-way array so as to obtain the three loadings matrices. The matrix form of the trilinear model can be expressed by the following equation:

xijk ¼

H X

aih bjh ckh þ eijk

55

Therefore, if estimates of b and c are given, it is possible to determine a by the least-squares solution to the model, X = a (b  c). If the vector (b  c) is defined as Z in the case of more than one component, the model defining A is

X ¼ AZ

ð2Þ

The least squares estimate of A is

A ¼ XZ T ðZZ T Þ1

ð3Þ

When the model converges, the results have the same number of triads as the number of factors assumed. Each triad is composed of three orthogonal vectors (column loading vectors a, b, and c) that have the profile of the pure species present in the system. A typical iterative procedure follows the following scheme: (i) estimate the number of chemical components, H; (ii) initialize matrices B and C and estimate A from X, B, and C by least squares regression; (iii) estimate matrices B and C in the sameway as matrix A in step (ii); and (iv) return to step (ii) until convergence. 3. Results and discussion 3.1. Absorption spectra studies Generally, the non-covalent interactions of small molecules with DNA involve three binding modes: intercalation, groove binding and long-range assembly on the molecular surfaces of nucleic acids [30]. Among the three modes, the most effective mode of the drugs targeted to DNA is intercalative binding, which is related to the anti-tumor activity or DNA damage of the compound. The UV–vis absorption spectroscopy is widely used as an effective method in DNA-binding studies [31]. The spectral change process reflects the corresponding changes in the helix structure and the steric configuration of DNA after a compound binding to DNA. Hypochromism results from the contraction of DNA in the helix axis, as well as from the conformational change of DNA, whereas hyperchromism is mainly attributed to damage of the double-helical structure of DNA [32]. As seen in Fig. 2, the absorption peak of DNA at about 260 nm showed gradual increase after addition of different amounts of carbaryl (solid line). These spectra were compared with those calculated from the sum of absorbance of free carbaryl and the free DNA at their different concentrations (dashed lines). If Beer’s law was strictly followed, this calculated set of spectra and the measured spectra should coincide. However, the results displayed that the measured spectra (solid line) were more intense than the calculated spectra (dashed line). The percentage of absorbance increase at 260 nm reached 21% (curves 1–7, Fig. 2). This hyperchromic effect suggested that a DNA–carbaryl binary

ð1Þ

h¼1

where xijk is the original element (i ? I, j ? J, and k ? K) of the trilinear data set X; H is the number of fluorescing species, I indicates the number of Dk, J is the number of excitation wavelength points, K is the number of samples, and eijk represents a residual error term of the three-way array; E and its sum of squares is minimized. Alternating least squares (ALS) can be used to find the solution to the PARAFAC model [29]. The method assumes the loadings in two modes and then estimates the unknown set of parameters of the third mode until the residuals of the model are optimized.

Fig. 2. UV absorption spectra of DNA varying with concentrations of carbaryl at pH = 7.4 and room temperature. c(DNA) = 6.0  105 mol L1 and c(carbaryl) = 0, 0.66, 1.32, 1.96, 2.60, 3.23, and 3.85  105 mol L1 corresponding to the curves from 1 to 7, respectively. Solid line: measured spectra; dashed line: theoretically summed spectra.

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complex had formed and the DNA double-helix structure was damaged after carbaryl bound to DNA through intercalation mode [33,34]. Based on the changes in the absorption spectra of DNA upon binding to carbaryl, Eq. (4) [35] can be utilized to calculate the binding constant, K.

A0 eG eG 1 ¼ þ  A  A0 eHG  eG eHG  eG K½carbaryl

ð4Þ

where A0 and A are the absorbance at 260 nm for the DNA in the absence and presence of carbaryl, eG and eH–G are the absorption coefficients of drug and its complex with DNA, respectively. The plot of A0/(A  A0) versus 1/[carbaryl] was constructed by using the absorption titration data and linear fitting, yielding the binding constant, K = (8.39 ± 0.38)  103 L mol1. 3.2. Fluorescence quenching studies Fluorescence spectroscopy was used to investigated the binding characteristics between chromophore and other molecules [36]. The fluorescence emission spectra of carbaryl in the absence and presence of DNA are shown in Fig. 3. Carbaryl has a maximum emission at 333 nm when excitated at 291 nm. As shown in Fig. 3, a regular decrease in the fluorescence intensity of carbaryl but no shift of fluorescence maximum emission took place with the increase of DNA concentration, which indicated that the binding of carbaryl to DNA indeed exists. The fluorescence quenching data at three different temperatures (298, 304 and 310 K) were analyzed by using Stern–Volmer equation:

F0 ¼ 1 þ K SV ½Q  F

ð5Þ

where F0 and F are the fluorescence intensities of carbaryl in the absence and presence of the quencher, respectively. KSV is the Stern– Volmer dynamic quenching constant, [Q] is the concentration of quencher. Sterm–Volmer equation was applied to determine KSV by linear regression of a plot of F0/F against [Q]. The linear Stern– Volmer quenching plots obtained from the fluorescence titrations of carbaryl by DNA indicated merely one type of quenching process, namely static or dynamic quenching, under the experimental condition [37] (Fig. 4A). The slopes of Stern–Volmer quenching plot, namely KSV, were found to be (2.25 ± 0.02)  103, (1.96 ± 0.03)  103 and (1.73 ± 0.02)  103 L mol1 at 298, 304, 310 K, respectively at pH 7.4. The values of KSV decreased with the increasing temperature, which indicated that the fluorescence quenching of carbaryl by DNA was static [37]. If the static binding reaction between DNA and small molecule happens, and there are the similar and independent binding sites in the DNA, namely, the binding capability of DNA at each binding site is equal. The apparent binding constant (Ka) and the binding

Fig. 4. (A) The Stern–Volmer plots for the quenching of carbaryl by DNA at different temperatures; (B) Plot of log(F0  F)/F versus log[DNA] at different temperatures (pH = 7.4, kex = 291 nm, kem = 333 nm). c(carbaryl) = 1.0  105 mol L1.

stoichiometry (n) of DNA–carbaryl system can be estimated by the following equation using the data from fluorescence titration [38]:

log

F0  F ¼ log K a þ n log½Q  F

The values of Ka were obtained from the intercept of the plot of log[(F0  F)/F] versus log[Q] (Fig. 4B). The corresponding results at different temperatures are summarized in Table 1. The decreasing trend of Ka with increasing temperature is in accordance with KSV’s dependence on temperature as discussed above, a characteristic that accords with the type of static quenching. The value of Ka at 298 K was (8.68 ± 0.06)  103 L mol1, which agrees well with the K value obtained earlier by UV spectroscopy (Section 3.1) and supports the effective role of static quenching [39]. 3.3. Main binding force between carbaryl and DNA The acting forces between small molecule and biomacromolecule include hydrogen bond, hydrophobic interaction, van der Waals force and electrostatic force. If DHh  0, DSh > 0, the main force is hydrophobic interaction; if DHh < 0, DSh > 0, the major force is electrostatic effect; if DHh < 0, DSh < 0, hydrogen bond and Van der Waals force play major role in the reaction [40]. The enthalpy change DHh, entropy change DSh and free energy change DGh for a binding reaction were calculated from the vant’t Hoff equation and Gibbs–Helmholtz equation:

log K a ¼ 

DH h DSh þ 2:303RT 2:303R

DGh ¼ DHh  T DSh Fig. 3. Fluorescence spectra of carbaryl in the presence of DNA (pH = 7.4, T = 298 K, kex = 291 nm, kem = 333 nm). c(carbaryl) = 1.0  105 mol L1 and c(DNA) = 0, 0.54, 1.06, 1.57, 2.06, 2.54, 3.00, 3.45, 3.88, 4.30, and 4.71  104 mol L1 corresponding to the curves from 1 to 11, respectively.

ð6Þ

ð7Þ ð8Þ

In Eq. (7), R is gas constant. The temperatures used are 298, 304 and 310 K. The values of DHh and DSh were obtained from the slope and intercept of the linear van’t Hoff plot based on log K versus 1/T.

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G. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 108 (2012) 53–61 Table 1 The apparent binding constants, binding stoichiometry and thermodynamic parameters of DNA–carbaryl system at different temperatures.

a

T (K)

103Ka (L mol1)

n

Ra

DHh (kJ mol1)

DGh (kJ mol1)

DSh (J k1 mol1)

298 304 310

8.68 ± 0.06 6.53 ± 0.19 5.24 ± 0.21

1.18 ± 0.02 1.17 ± 0.05 1.15 ± 0.06

0.9991 0.9919 0.9902

32.40 ± 0.25

22.45 ± 0.005 22.25 ± 0.001 22.05 ± 0.005

33.40 ± 0.81

R is the correlation coefficient for the Ka values.

phate backbone. Owing to a competition for phosphate anion, the addition of the cation will weaken the surface-binding interactions which include electrostatic interaction and hydrogen binding between molecules and DNA [34]. The effect of NaCl on the fluorescence intensity of free carbaryl and DNA–carbaryl system was investigated in a pH 7.4 Tris–HCl buffer at room temperature. The result showed that the fluorescence intensity of both free carbaryl and DNA–carbaryl system did not obvious change with increasing concentration of NaCl (from 0 to 6  102 mol L1). It was deduced that NaCl has no effect on DNA binding with carbaryl. This result indicated that the electrostatic interaction between carbaryl and DNA could be excluded. Fig. 5. Stern–Volmer plots for the quenching of carbaryl by KI in the absence and presence of DNA. c(carbaryl) = 1.0  105 mol L1, cDNA = 3.3  104 mol L1, pH = 7.4. h

h

h

The DG was evaluated from Eq. (8). The values of DH , DS and DGh for carbaryl binding to DNA are given in Table 1. As seen in Table 1, the negative values of DGh revealed that the binding process is spontaneous. The negative DHh and DSh values suggested that the binding of carbaryl to DNA is driven mainly by hydrogen bond and van der Waals forces. Recently, Kashanian et al. studied the in vitro interaction of Clodinafop-Propargyl (CP) herbicide with calf thymus DNA by spectroscopic methods. Their results revealed that the formation of CP–DNA complex causes the negative enthalpy changes and results in a more ordered state of both CP and DNA molecules. The negative entropy is the indication of intercalative binding of CP to DNA [7]. The values of DHh and DSh obtained from the present work are similar to that of Kashanian’s research, thus the binding mode of carbaryl to DNA is intercalation. 3.4. Iodide quenching studies A highly negatively charged quencher is expected to be repelled by the negatively charged phosphate backbone of DNA. An intercalative bound small molecules should be protected from being quenched by anionic quencher, while the free aqueous complex and groove binding molecule should be quenched readily by anionic quencher [41]. Thus, the value of quenching constants (KSV) of the intercalative bound molecule should be lower than that of the molecule bound to DNA by groove binding. The negatively charged I ion was selected to determine the binding mode of carbaryl to DNA in this work. The values of KSV of carbaryl by I ion in the absence and presence of DNA were obtained to be 354.91 and 321.37 L mol1, respectively, using the Stern–Volmer equation (shown in Fig. 5). The results showed that iodide quenching effect was decreased when carbaryl was bound to DNA, suggesting that an intercalation binding should be the interaction mode of carbaryl with DNA.

3.6. Circular dichroism (CD) studies Circular dichroism is a powerful technique for distinguishing the three main DNA-binding modes. Groove binding and electrostatic interaction of small molecules show less or no perturbation on the base stacking and helicity bands, whereas intercalation changes the intensities of both bands, thus stabilizing the righthanded B conformation of DNA [42]. The CD spectrum of DNA exhibits a positive band at 279 nm due to base stacking and a negative band at 246 nm caused by helicity, which are characteristic of DNA in right-handed B form [43]. The interaction of carbaryl with DNA induces a change in the CD spectrum of B DNA (Fig. 6). The intensities of both the positive and negative bands decreased significantly (shifting to zero levels) with increasing [carbaryl]/ [DNA] ratio. Thus the result of the CD studies revealed the effect of intercalation of carbaryl in base stacking and decreased righthandedness of the DNA [31,44]. 3.7. Melting studies Monitoring the change of melting temperature (Tm) of DNA is an efficient method to recognise the binding mode between small molecule and DNA. The previous studies indicated that intercalation binding can increase the stability of helix of DNA, and cause the Tm of DNA increase of about 5–8 °C [45], but the non-intercalation binding causes no obvious increase in Tm. The change in absorbance at 260 nm for the DNA in the absence and presence of

3.5. Effect of the ionic strength on the fluorescence properties DNA double helixes consist of anionic polyelectrolyte and phosphate groups. Increasing the concentration of cation will increase the complexation probability between the cation and DNA phos-

Fig. 6. Circular dichroism spectra of DNA in the presence of increasing amounts of carbaryl at pH 7.4. c(DNA) = 1.0  104 mol L1, c(carbaryl) = 0, 1.0, and 2.0  104 mol L1 corresponding to the curves from 1 to 3, respectively.

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carbaryl was measured. The values of Tm of DNA and DNA–carbaryl system were obtained from the transition midpoint of the melting curves based on fss versus temperature (T), where fss = (A  A0)/ (Af  A0), A0 is the initial absorbance intensity, A is the absorbance intensity corresponding to its temperature, Af is the final absorbance intensity [46]. Fig. 7 displays the melting curves of DNA in the absence and presence of carbaryl. As shown in Fig. 7, the Tm of DNA is 66.5 °C, whereas Tm of DNA–carbaryl system is 73.5 °C. These melting experiments strongly supported the intercalation of carbaryl into the DNA helix. 3.8. Viscosity measurements It is known that viscosity measurement is sensitive to the change in the length of the DNA double helix. The viscosity experiment is considered as one of the most unambiguous methods to test the binding mode of small molecules and DNA in solution. Increase in relative viscosity of DNA occurs as a result of a length increase of the duplex following intercalation, but there is little effect on the relative viscosity of DNA if the non-intercalation binding occurs in the binding process [47,48]. Fig. 8 shows the changes of relative viscosity (g/g0)1/3 of DNA at the different molar ratios ([carbaryl]/[DNA]) value. As seen in Fig. 8, the relative viscosity of DNA remarkably increased with the increasing amounts of carbaryl. Therefore, we can conclude that carbaryl molecules have intercalated into the base pairs of DNA. 3.9. Synchronous fluorescence spectra of interaction between carbaryl and DNA–EB EB is a dye and well-known DNA intercalator, so it was employed as molecule probe in this study. Fig. 9A shows the synchronous fluorescence spectra of the EB dye upon addition DNA. EB produced only weak fluorescence in a pH 7.4 Tris–HCl buffer due to quenching by the solvent molecules. However, the fluorescence

intensity of EB at 530 nm was enhanced remarkably with the increasing concentration of DNA because of its intercalation into the DNA, when the D-value (Dk) between excitation wavelength and emission wavelength was set at 80 nm. When the ratio of [DNA]:[EB] reached about 4, the fluorescence intensity stabilized, which suggested that the reaction was complete and a stable DNA–EB complex was formed. With the addition of carbaryl to a DNA–EB solution, some of EB molecules were released into the solution after an exchange with carbaryl, and this resulted in fluorescence quenching (Fig. 9B), indicating that interaction between carbaryl and DNA–EB occurred. In order to evaluate the competitive binding interaction of carbaryl and EB with DNA, the three-way synchronous fluorescence data of the carbaryl– DNA–EB mixture were collected, and the three-way synchronous fluorescence data was then resolved by PARAFAC modeling. In most synchronous fluorescence studies of chemical systems, the selection of Dk is very important with the classical twodimensional synchronous fluorescence. The influence of Dk can be substantial on the shape, location, and signal intensity of a fluorescence peak as well as on interferences attributed to light scattering [49]. In this work, to estimate the optimal value of Dk and to investigate the chemical system thoroughly, three-way synchronous fluorescence data for carbaryl, EB and DNA–EB were sampled. The contour plots produced from such three-way data for carbaryl, EB and DNA–EB showed that the preferred Dk for the three systems were indeed quite different with the value of 50, 120 and 70 nm for carbaryl, EB and DNA–EB complex, respectively (arrows, Fig. 10). It can be clearly seen that the fluorescence intensity varied with the different values of Dk for carbaryl, EB and DNA–EB. Therefore, the advantage of the three-way approach became apparent. With the conventional two-way synchronous

Fig. 7. Melting curves of DNA in the absence and presence of carbaryl at pH 7.4. c(carbaryl) = 6.88  106 mol L1, c(DNA) = 2.27  105 mol L1.

Fig. 8. Effect of increasing amount of carbaryl on the relative viscosity of DNA at pH 7.4. c(DNA) = 3.3  105 mol L1.

Fig. 9. (A) Synchronous fluorescence spectra of ethidium bromide in the presence of DNA at different concentrations. c(EB) = 2.0  105 mol L1 and c(DNA) = 0, 0.66, 1.32, 1.98, 2.63, 3.28, 3.92, 4.56, 5.19, 5.83, 6.45, 7.07, 7.69, 8.31, and 8.92  105 mol L1 corresponding to the curves from 1 to 15, respectively. (B) Synchronous fluorescence spectra of the competition between carbaryl and ethidium bromide with DNA. c(EB) = 2.0  105 mol L1, c(DNA) = 8.0  105 mol L1 and c(carbaryl) = 0, 0.33, 0.66, 0.99, 1.32, 1.64, 1.96, 2.28, 2.60, and 2.91  105 mol L1 corresponding to the curves from 1 to 10, respectively; Dk = 80 nm.

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fluorescence approach, where Dk is fixed at one value, errors will clearly arise in any simultaneous investigation of carbaryl, EB and DNA–EB complex systems. These errors may be avoided with the use of the three-way synchronous fluorescence method for the selection of Dk values. 3.10. Decomposition of the three-way synchronous fluorescence spectra by PARAFAC A three-way data array was obtained (Section 2.3.2). When carbaryl with nine different concentrations was added sequentially to a solution of DNA–EB, a total of nine two-way data matrices were produced. Then these data were arranged into a 3D array stack (Dk [A-way]  excitation wavelength [B-way]  sample [C-way]). This matrix was decomposed into the loading matrices, A, B and C, with the use of the PARAFAC method. The synchronous fluorescence data matrix, X (20  501  9) (obtained from the experiment in Section 2.3.2) was processed with the aid of the SVD model [50], and the extracted seven eigenvalues were 19.7542, 3.5414, 0.2243, 0.0063, 0.0041, 0.0025, and 0.0017, indicating that there are three significant factors for the prediction of the three separate chemical components in the system, i.e., free carbaryl, EB and DNA–EB complex. As previously indicated, the spectra of these three components in mixtures overlapped, especially those of EB and DNA–EB complex, it was difficult to estimate the equilibrium concentrations of each component during the titration process by conventional methods. Consequently, the data matrix of X (20  501  9) was resolved by PARAFAC analysis to extract more information for further investigation. The extracted spectra (solid line, Fig. 11A) of free carbaryl, EB and DNA–EB complex, agreed well with their measured counterparts (dashed line, Fig. 11A). The pure spectra of the DNA–EB complex, which was difficult to extract by conventional methods, was readily obtained by the PARAFAC method. This suggested that the results of the PARAFAC model were unique and reliable and that the correct number of components for the model was chosen. From the resolved concentration profiles (Fig. 11B), it can be seen that with the addition of carbaryl, the concentration of the

Fig. 11. (A) Comparison of the measured excitation synchronous fluorescence spectra for carbaryl, EB and DNA–EB complex with those resolved from the PARAFAC analysis. Solid line: resolved spectra from PARAFAC; dashed line: measured spectra. (B) Equilibrium concentrations of carbaryl, EB and DNA–EB complex resolved by the PARAFAC.

DNA–EB complex decreased gradually accompanied by the increasing of the concentration of EB. The displacement of EB in the DNA–EB complex by carbaryl can be visualized clearly, indicating that carbaryl intercalates into the same base sites of DNA, releasing the bound EB. This PARAFAC analysis further verified the intercalation binding of carbaryl to DNA obtained by above methods.

Fig. 10. Synchronous fluorescent spectra (contour mode) and the selection of the preferred value of Dk for carbaryl (A), EB probe (B) and DNA–EB complex (C).

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4. Conclusion In this paper, the interaction between carbaryl and ctDNA was studied using the EB dye as a probe by UV–vis absorption, fluorescence and CD spectroscopy, as well as DNA melting studies and viscosity measurements.  The intrinsic fluorescence of carbaryl was quenched by DNA through static quenching mechanism. The negative DHh and DSh suggested that hydrogen bond and van der Waals forces play major roles in the binding of carbaryl to DNA and contribute to the stability of the complex.  The intercalation binding of carbaryl to DNA was demonstrated by hyperchromic effect of absorption spectra, decreases in iodide fluorescence quenching effect, induced CD spectral changes, and significant increases in melting temperature and relative viscosity of the DNA.  By PARAFAC method, the concentration information (quantitative information) for the three reaction components, carbaryl, EB and DNA–EB, in the system at equilibrium and the corresponding pure spectra (qualitative information) were obtained simultaneously. This indicated that the intercalation of carbaryl molecule into the double helix of DNA by substituting for EB probe in the DNA–EB complex.  Studies demonstrate that planarity of a molecule is one of the necessary conditions for efficient intercalation into the DNA double helix [51]. The previous studies indicated that some carbamate pesticides including aminocarb, carbofuran, isoprocarb and pirimicarb can bind to DNA by intercalation [2,8,52,53]. Since these pesticides molecules contain a planar aromatic ring, which can stack between DNA bases. The results obtained from this study are similar to those of previous reports, which might be attributed to the similar chemical structures of the carbamate pesticides.

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