- Email: [email protected]
The effect of Se salts on DNA structure
Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
The effect of Se salts on DNA structure Shohreh Nafisi a,b,⇑, Maryam Montazeri a, Firouzeh Manouchehri a a b
Department of Chemistry, Islamic Azad University, Central Tehran Branch (IAUCTB), Tehran 14676-86831, Iran Research Institute for Islamic and Complementary Medicine, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 13 March 2012 Received in revised form 13 April 2012 Accepted 18 April 2012 Available online 8 May 2012 Keywords: DNA Sodium selenate Sodium selenite FTIR spectroscopy UV–Visible
a b s t r a c t There is considerable interest in the role of selenium in cancer prevention. Various organic and inorganic Se compounds are considered to be antioxidants. In the present study, the binding modes, the binding constants and the stability of Se–DNA complexes have been determined by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed that Na2SeO4 and Na2SeO3 bind to the minor and major grooves of DNA and the backbone phosphate (PO2) with overall binding constants of K(Na2SeO4–DNA) = 5.20 104 M1 and K(Na2SeO3–DNA) = 1.87 103 M1. DNA aggregations occurred at high selenium concentrations. No biopolymer conformational changes were observed upon Na2SeO3 and Na2SeO4 interactions, while DNA remained in the B-family structure. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Selenium is an important micronutrient and an essential trace element for both humans and animals metabolism. Diet containing selenium includes cereals, grains, nuts, vegetables, meat, and seafood [1–3]. Selenium has been extensively studied for its antioxidant activity and cancer prevention [4–8]. It is found in many dietary supplements and multivitamins in forms of sodium selenite (Na2SeO3), sodium selenate (Na2SeO4) or selenomethionine (SeMet) [5,9]. The main inorganic selenium compound used in most cancer treatment studies is sodium selenite (Scheme 1). However, a few studies use other forms, such as sodium selenate (Scheme 1) and selenium oxide [1,6,10–14]. Selenium is incorporated as selenocysteine (SeCys) in selenoproteins P, W, and R, as well as in the active sites of the enzymes such as glutathione peroxidases (GPx) and thioredoxin reductases [1,6,15–17]. In cells, these selenoproteins have important antioxidant activities and protect the mitochondria, plasma membrane, and DNA from oxidative damage by reactive oxygen species (ROS) [18–20]. Sodium selenite inhibits the growth of cells and induces apoptosis in human colonic carcinoma [21], brain tumor [22] and breast cancer cells [23]. It inhibits 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary tumors [24], human fibrosarcoma tumor cell invasiveness [25] and pulmonary metastasis of melanoma cells in mice [26,27]. The majority of clinical trials provide ⇑ Corresponding author at: Department of Chemistry, Islamic Azad University, Central Tehran Branch (IAUCTB), Tehran 14676-86831, Iran. Tel.: +98 21 22426554; fax: +98 21 22432706. E-mail address: drsnafi[email protected] (S. Nafisi). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.04.012
evidence for selenium as a chemopreventive agent for specific cancers: prostate [28,29], lung [30–32], colorectal [33,34], stomach [19] and multiple cancers [35–38]. However, the results of a recent clinical trial showed that selenium and vitamin E taken alone or together did not prevent prostate, lung or colorectal cancers [39]. Even though much is reported about antitumor activities and protective role of Se compounds in DNA damage [40], little is known about the interaction of sodium selenite and selenate with individual DNA. The interaction of some oxoselenium compounds with DNA have been studied before [41]. Their results showed B-DNA stabilization till 1/1 ratio and a partial B to A-DNA transition at higher ratio of 2/1. The aim of this study was to characterize the DNA structural changes in the presence of Na2SeO3 and Na2SeO4. Here, we compared the interactions of Na2SeO3 and Na2SeO4 with DNA in aqueous solution at pH 7 with Se/DNA (P) molar ratios of 1/120–1/1 by FTIR and UV measurements. It is worth mentioning that the concentration of Se used here is much higher than the physiological concentration of Se found in living cell. Since the FTIR and UV spectroscopic methods are not sensitive to very low Se contents, higher concentrations were used in order to determine the effect of Se on DNA structure. Structural analyses regarding the Se binding site, binding constant, and DNA secondary structures are provided here.
2. Materials and methods 2.1. Materials Highly polymerized type I calf-thymus DNA sodium salt (7% Na content) was purchased form Sigma Chemical Co. (St. Louis, MO),
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41
Scheme 1. Chemical structure of Na2SeO4 and Na2SeO3.
37
1662 (thymine), 1610 (adenine), 1492 (cytosine), and 1226 cm1 (PO2 groups) versus Se concentrations were obtained after peak normalization using Ri = Ii/I968, where Ii is the intensity of the absorption peak for DNA in the complex with i as the ligand concentration, and I968 is the intensity of the 968 cm1 peak (DNA internal reference). The plot of intensity was drawn for r = 1/ 120–1/1. 2.4. Absorption spectroscopy
and deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. Na2SeO3 and Na2SeO4 were also purchased from Sigma Chemical (St. Louis, MO) and used without further purification. To check the protein content of DNA solutions, the absorbance bands at 260 and 280 nm were used. The A260/A280 ratio was 2.10 for DNA, showing that DNA samples were sufficiently free from protein [42]. Other chemicals were of reagent grade and used without further purification. 2.2. Preparation of stock solutions DNA was dissolved to 0.5% w/v (0.0125 M) polynucleotide (phosphate) (pH 7) in Tris–HCl at 5 °C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the DNA solution was determined spectrophotometrically at 260 nm using molar extinction coefficient e260 = 66,000 cm1 M1 (DNA) (expressed as molarity of phosphate groups) [43]. The appropriate amounts of sodium selenite and sodium selenate (0.1–12.5 mM) were dissolved in water and added dropwise to DNA solution (12.5 mM) to attain the desired Na2SeO3 and Na2SeO4/DNA(P) molar ratios (r) of 1/120–1/1 with a final DNA(P) concentration of 6.25 mM. The pH values of solutions were adjusted at 7.0 ± 0.2 using NaCl solution. The infrared spectra were recorded 2 h after mixing of the Na2SeO3 and Na2SeO4 with DNA solution. 2.3. FTIR spectroscopy measurements Infrared spectra were recorded on a Bruker ATR spectrometer (Ettlingen, Germany) equipped with an external water cooled high power Hg-arc source and was accessible with a room temperature operated deuterated triglycine sulfate (DTGS) detector and a ZnSe beam splitter. The spectra of Na2SeO3 and Na2SeO4/DNA solutions were recorded using a cell assembled with ZnSe windows. Spectra were recorded using the Opus software supplied by the manufacturer of the spectrophotometer. The spectra of the solutions were recorded after 2 h incubation of the Na2SeO3 and Na2SeO4 with DNA solution, using ZnSe windows. The ratios used for infrared were Se/DNA(P) molar ratios (r) of 1/120, 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1 with a final DNA(P) concentration of 6.25 mM. For each spectrum, 100 scans were recorded with resolution of 4 cm1. The difference spectra [(polynucleotide solution + Na2SeO3 and Na2SeO4 solution) (polynucleotide solution)] were obtained using a sharp DNA band at 968 cm1 as internal Refs. [44,45]. This band, which is due to sugar C–C and C–O stretching vibrations, exhibits no spectral change (shifting or intensity variation) upon Na2SeO3 and Na2SeO4–DNA complexation, and cancelled out upon spectral subtraction. The intensity ratios of the bands due to several DNA in plane vibrations related to A–T, G–C base pairs and the PO2 stretching vibrations were measured with respect to the reference bands at 968 cm1 (DNA) as a function of Se concentrations with an error of ±3%. Similar intensity variations have been used to determine the ligand binding to DNA bases and backbone phosphate groups [46]. The plots of the relative intensity (R) of several peaks of DNA in-plane vibrations related to A–T, G–C base pairs and the PO2 stretching vibrations such as 1710 (guanine),
The absorption spectra were recorded on a LKB model 4054 UV–Visible spectrometer, the quartz cuvettes of 1 cm were used. The absorption spectra recorded with Na2SeO3 and Na2SeO4 concentrations of 0.005–0.5 mM and constant polynucleotide concentration of 0.5 mM. The binding constants of the Na2SeO3 and Na2SeO4–DNA complexes were calculated as reported [47]. It is assumed that the interaction between the ligand [L] and the substrate [S] is 1:1; for this reason a single complex SL (1:1) is formed. The relationship between the observed absorbance change per centimeter and the system variables and parameters is as follow;
DA St K 11 De11 ½L ¼ b 1 þ K 11 ½L
ð1Þ
where DA = A A0 from the mass balance expression St = (S) + (SL), we get (S) = St/(1 + K11(L)). Eq. (1) is the binding isotherm, which shows the hyperbolic dependence on free ligand concentration. The double-reciprocal form of plotting the rectangular hyperbola 1y ¼ df 1x þ de, is based on the linearization of Eq. (1) according to the following equation,
b 1 1 ¼ þ DA St K 11 De11 ½L St De11
ð2Þ
Thus the double reciprocal plot of 1/DA versus 1/(L) is linear and the binding constant can be estimated from the following equation
K 11 ¼
intercept slope
ð3Þ
3. Results and discussion 3.1. FTIR spectra of Na2SeO4–DNA adducts Evidence related to Na2SeO4–DNA complexation comes from the infrared spectroscopic results shown in Figs. 1A and 2A. The spectral changes (intensity and shifting) of several prominent DNA in-plane vibrations at 1710 (G and T, mainly G), 1662 (T, G, A, and C, mainly T), 1610 (A and C, mainly A), 1492 (C and G, mainly C), 1226 (PO2 asymmetric stretch), and 1088 cm1 (PO2 symmetric stretch) [48–52] were monitored at different Na2SeO4–DNA molar ratios, and the results are shown in Figs. 1A and 2A. At r = 1/120, 1/80, major reduction in intensity was observed for the bases and phosphate PO2 vibrations (Figs. 1A and 2A). The intensity of the guanine band at 1710 cm1 decreased by 18%, the thymine band at 1662 cm1 decreased by 15%, the adenine band at 1610 cm1 decreased by 22% and the asymmetric phosphate vibration decreased by 6% (Figs. 1A and 2A). The observed intensity decreases can be related to DNA helix stabilization upon Na2SeO4 complexation. At higher concentrations (r = 1/40, 1/20), intensity of the G, T, A and asymmetric PO2 bands increased. The observed increase in intensity can be related to DNA helix destabilization as a result of extended binding of Na2SeO4 to bases and phosphate group (Fig. 2A). At r = 1/10, 1/5, reduction in the intensity of the bases and phosphate bands can be attributed to DNA aggregation upon Na2SeO4 interaction (Fig. 2A). At r = 1/2, increase
38
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41
Fig. 1. FTIR spectra and difference spectra [(DNA solution + Se salt solution) (DNA solution)] in the region of 1800–600 cm1 for calf thymus DNA (6.25 mM), and Na2SeO4– DNA (A) and Na2SeO3–DNA (B) (0.1–12.5 mM) adducts in aqueous solution at pH = 7. DNA and three complexes spectra obtained at various Na2SeO4, Na2SeO3–DNA (phosphate) molar ratios (top three spectra), three difference spectra (bottom three spectra).
in intensity of the bases and phosphate can be assigned to interaction of Na2SeO4 with bases and backbone phosphate group. Similar intensity changes were observed in the flavonoids–DNA complexes in which increase in intensity of the bands were assigned to interaction of the flavonoids with bases and backbone phosphate group [44]. At r = 1/1, DNA aggregation occurred upon Na2SeO4 interaction. Evidence for this comes from decrease in intensity of the bases and phosphate bands (Figs. 1A and 2A). It should be noted that the intensity changes of the phosphate band were accompanied by shifting of the band at 1226 cm1 to 1231–1233 cm1 (r = 1/120 to r = 1/1). Further evidence regarding Na2SeO4–PO2 interaction comes from intensity ratio variations of the symmetric and asymmetric PO2 bands at 1088/1226 [48]. The ratio of ms/mas was changed from 2.30 (free DNA) to 2.51 (Na2SeO4–DNA complexes) upon Na2SeO4 complexation.
In the difference spectra of Na2SeO4–DNA complexes (r = 1/ 120–1/1), the positive features at 1100–1099 cm1 are coming from increase in intensity of the phosphate vibrational frequency upon Na2SeO4 interaction. It indicates major interaction of Na2SeO4 with DNA backbone phosphate (Fig. 1A). No major spectral changes were observed for the cytosine band at 1492 cm1 upon Na2SeO4 interaction that can be related to minor participation of cytosine bases in Na2SeO4–DNA complexation (Fig. 1A). Similar spectral changes were observed in the biogenic polyamine– and taxol–DNA complexes [51,53,54]. It is worth mentioning that the absorption band with medium intensity at 1640 cm1 in the IR spectrum of the free DNA and at 1653–1651 cm1 for the complexes are due to water deformation mode and they are not coming from DNA vibrations (Fig. 1A) [54].
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41
Fig. 2. Intensity ratio variations for several DNA in-plane vibrations as a function of Na2SeO4 (A) and Na2SeO3 (B) concentrations (0.1–12.5 mM). Intensity ratios for the DNA bands at 1710 (G, T), 1662 (T, G, A, C), 1610 (A), 1492 (C, G) and 1226 (PO2 asymmetric) referenced to the DNA band at 968 cm1.
Additional support for Na2SeO4–DNA interaction comes from decrease in intensity of the UV–Visible band of DNA at 260 nm upon Na2SeO4–DNA complexation (Fig. 3A). 3.2. FTIR spectra of Na2SeO3–DNA adducts Evidence related to Na2SeO3–DNA complexation comes from the infrared spectroscopic results shown in Figs. 1B and 2B. In the Na2SeO3–DNA complex (r = 1/120), major reduction in intensity was observed for the bases and phosphate (PO2) vibrations (Fig. 2B). The intensity of guanine band at 1710 cm1 decreased by 39%, the thymine band at 1662 cm1 decreased by 39%, the adenine band at 1610 cm1 decreased by 58% and the asymmetric phosphate vibration decreased by 22%. The observed reduction in intensity is related to DNA helix stabilization upon Na2SeO3–DNA complexation (Fig. 2B). At r = 1/80, the increase in intensity of the bands related to G, T, A and asymmetric PO2 is due to the helix destabilization as a result of extended binding of Na2SeO3 to bases and phosphate group (Figs. 1B and 2B). At r = 1/40, decrease in intensity of the bases and phosphate bands can be related to DNA aggregation upon Na2SeO3 interaction. At higher concentrations (r = 1/20, 1/10, 1/5), the bases and phosphate vibrations intensified as a result of DNA helix destabilization and external binding of Na2SeO3–DNA. At r = 1/2, DNA aggregation occurred upon Na2SeO3 interaction. Evidence for this comes from reduction in intensity of the bases and phosphate bands (Figs. 1B and 2B). No major interaction was observed at higher concentration (r = 1/1). In the difference spectra of Na2SeO3–DNA complexes (r = 1/ 120–1/1), the positive features at 1087–1082 cm1 are coming
39
Fig. 3. UV–Visible results of free DNA (0.5 mM); free Na2SeO4, Na2SeO4–DNA complexes (0.08 mM) (A) and free DNA, free Na2SeO3, Na2SeO3–DNA complexes (0.08 mM) (B). Plot of 1/(A0 A) versus 1/(ligand concentration) for Na2SeO4 and Na2SeO3 (0.005–0.5 mM) and calf-thymus DNA complexes, where A0 is the initial absorbance of DNA (260 nm) and A is the recorded absorbance at different Na2SeO4, Na2SeO3 concentrations at pH 7.
from increase in intensity of the phosphate vibrational frequency upon Na2SeO3 interaction and indicates interaction of Na2SeO3 with DNA phosphate backbone (Figs. 1B and 2B). In addition to major intensity changes of the PO2 stretching at 1226 cm1, the relative intensities of the asymmetric (mas) and symmetric (ms) vibrations were altered upon Na2SeO3 interaction. The ms PO2 (1088 cm1) and mas PO2 (1226 cm1) ratios changed with ms/mas increasing from 2.3 (uncomplexed DNA) to 2.54 (Na2SeO3–DNA complexes) which indicates Na2SeO3–PO2 complexation. Further evidence regarding Na2SeO3–DNA interaction comes from reduction in intensity of the characteristic UV–Visible band of DNA at 260 nm due to Na2SeO3–DNA complexation (Fig. 3B) [55–58]. 3.3. DNA conformation DNA remains in the B-family structure upon Se complexation. This is consistent with the infrared results on the Na2SeO4 and Na2SeO3–DNA complexes that showed no conformational changes for B-DNA with marker B-DNA bands at 1710 (G), 1226 (PO2), and 836 cm1 (phosphodiester modes) (Fig. 1A and B). When a complete B to A transition occurs, the B-DNA marker bands such as 836 cm1 appear at about 820–810 cm1, while the phosphate stretching vibration at 1226 cm1 shifts toward a
40
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41
higher frequency at 1240–1230 cm1 and the guanine band at 1710 appears at 1700 cm1 [50,59]. No major shifting was observed in the B-DNA conformation marker bands upon Na2SeO3 and Na2SeO4 interactions. In Na2SeO4–DNA complexes, the observed shifting of the PO2 asymmetric band at 1226 to 1231–1233 cm1 can be related to PO2 coordination to Na2SeO4 and is not coming from DNA conformational change (Fig. 1A and B). 3.4. Stability of Na2SeO4 and Na2SeO3–DNA complexes The selenium salts–DNA binding constants were determined as described in Section 2 (UV–Visible spectroscopy). The calculations of the overall binding constants were carried out using UV spectroscopy as reported [47]. Concentrations of the complexed ligand were determined by subtracting absorbance of the free DNA at 260 nm from those of the complexed. Concentration of the free ligand was determined by subtraction of the complex ligand from total ligand used in the experiment. Our data of 1/[complexed ligand] almost proportionally increased as a function of 1/[free ligand] (Fig. 3). The double reciprocal plot of 1/(A0 A) versus 1/(selenium salts concentration) is linear, and the binding constant (K) can be estimated from the ratio of the intercept to the slope (Fig. 3), where A0 is the initial absorbance of the free DNA at 260 nm, and A is the recorded absorbance of DNA in the presence of different selenium salts concentrations. The overall binding constant of K for Na2SeO4–DNA = 5.20 104 M1 and Na2SeO3–DNA = 1.87 103 M1 show greater stability for Na2SeO4–DNA complexes. The small K value obtained for Na2SeO3–DNA complex shows weak interaction between Se anions and DNA. However the binding constant for Na2SeO4–DNA showed a strong interaction. Similar binding constants were observed in lipid–DNA complexes in which strong interaction occurred via major and minor grooves and the backbone phosphate group [60]. Weak affinity to DNA was observed for retinol–DNA with overall binding constants of Kretinol–DNA = 3 103 M1 [61]. 4. Summary and conclusions On the basis of our spectroscopic results, the interaction of Na2SeO4 and Na2SeO3 to DNA bases and phosphate backbone group have been concluded. The stabilities of Se salts–DNA adducts are in the order K(Na2SeO4–DNA) = 5.20 104 M1 > K(Na2SeO3– DNA) = 1.87 103 M1. No DNA conformational changes were observed upon Na2SeO4 and Na2SeO3 complexation, while biopolymer aggregation occurred at high selenium contents.
5. Abbreviations
A G C T FTIR UV
Adenine Guanine Cytosine Thymine Fourier transform infrared Ultraviolet
Disclosure statement No competing financial interests exist.
Acknowledgment This work is supported by Grants from Islamic Azad University, Central Tehran Branch (IAUCTB). References [1] H. Tapiero, D.M. Townsend, K.D. Tew, The antioxidant role of selenium and seleno-compounds, Biomed. Pharmacother. 57 (2003) 134.–144. [2] J.A. Pennington, S.A. Schoen, Total diet study: estimated dietary intakes of nutritional elements, Int. J. Vitamin Nutr. Res. 66 (1996) 350–362. [3] J.A. Pennington, S.A. Schoen, Contributions of food groups to estimated intakes of nutritional elements: results from the FDA total diet studies, Int. J. Vitamin Nutr. Res. 66 (1996) 342–349. [4] A. Holmgren, Selenite in cancer therapy: a commentary on ‘‘Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress’’, Free Radical Biol. Med. 41 (2006) 862–865. [5] G.N. Schrauzer, Nutritional selenium supplements: product types, quality, and safety, J. Am. Coll. Nutr. 20 (2001) 1–4. [6] M. Takahashi, T. Sato, F. Shinohara, S. Echigo, H. Rikiishi, Possible role of glutathione in mitochondrial apoptosis of human oral squamous cell carcinoma caused by inorganic selenium compounds, Int. J. Oncol. 27 (2005) 489–495. [7] W. Zhong, T.D. Oberley, Redox-mediated effects of selenium on apoptosis and cell cycle in the LNCaP human prostate cancer cell line, Cancer Res. 61 (2001) 7071–7078. [8] R.R. Ramoutar, J.L. Brumaghim, Antioxidant and anticancer properties and mechanisms of niorganic selenium, oxo-sulfur, and oxo-selenium compounds, Cell Biochem. Biophys. 58 (2010) 1–23. [9] J.W. Finley, C. Ip, D.J. Lisk, C.D. Davis, K.J. Hintze, P.D. Whanger, Cancerprotective properties of high selenium broccoli, J. Agric. Food Chem. 49 (2001) 2679–2683. [10] G. Mantovani, A. Maccio, C. Madeddu, R. Serpe, E. Massa, G. Gramignano, et al., Selenium is effective in inducing lymphocyte progression through cell cycle in cancer patients: potential mechanisms for its activity, J. Exp. Ther. Oncol. 4 (2004) 69–78. [11] G. Spyrou, M. Bjornstedt, S. Skog, A. Holmgren, Selenite and selenate inhibit human lymphocyte growth via different mechanisms, Cancer Res. 56 (1996) 4407–4412. [12] S. Vadhanavikit, C. Ip, H.E. Ganther, Metabolites of sodium selenite and methylated selenium compounds administered at cancer chemoprevention levels in the rat, Xenobiotica 23 (1993) 731–745. [13] C. Ip, The chemopreventive role of selenium in carcinogenesis, J. Am. Coll. Toxicol. 5 (1986) 7–20. [14] J.E. Spallholz, On the nature of selenium toxicity and carcinostatic activity, Free Radical Biol. Med. 17 (1994) 45–64. [15] R.R. Ramoutar, J.L. Brumaghim, Effects of inorganic selenium compounds on oxidative DNA damage, J. Inorg. Biochem. 101 (2007) 101028–101035. [16] K.M. Brown, J.R. Arthur, Selenium, selenoproteins and human health: a review, Public Health Nutr. 4 (2001) 593–599. [17] V.N. Gladyshev, G.V. Kryukov, Evolution of selenocysteine-containing proteins: significance of identification and functional characterization of selenoproteins, Biofactors 14 (2001) 87–92. [18] J.E. Spallholz, L. Mallory Boylan, M.M. Rhaman, Environmental hypothesis: Is poor dietary selenium intake an underlying factor for arsenicosis and cancer in Bangladesh and West Bengal, India?, Sci Total Environ. 323 (2004) 21–32. [19] T.C. Stadtman, Discoveries of vitamin B12 and selenium enzymes, Annu. Rev. Biochem. 71 (2002) 1–16. [20] R.L. Nelson, F.G. Davis, E. Sutter, J.W. Kikendall, L.H. Sobin, J.A. Milner, P.E. Bowen, Serum selenium and colonic neoplastic risk, Dis. Colon Rectum 38 (1995) 1306–1310. [21] M.S. Stewart, R.L. Davis, L.P. Walsh, B.C. Pence, Induction of differentiation and apoptosis by sodium selenite in human colonic carcinoma cells (HT29), Cancer Lett. 117 (1997) 35–40. [22] N. Sundaram, A.K. Pahwa, M.D. Ard, N. Lin, E. Perkins, A.P. Jr, Bowles, selenium causes growth inhibition and apoptosis in human brain tumor cell line, J. Neurooncel. 46 (2000) 125–133. [23] A.M. Watrach, J.A. Milner, M.A. Watrach, K.A. Poirier, Inhibition of human breast cancer cells by selenium, Cancer Lett. 25 (1984) 41–47. [24] H.J. Thompson, L.D. Meeker, P.J. Becci, S. Kokoska, Effect of short-term feeding of sodium selenite on 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in the rat, Cancer Res. 42 (1982) 4954–4958. [25] S.O. Yoon, M.W. Kim, A.S. Chung, Inhibitory effect of selenite on invasion of HT1080 tumor cells, J. Biol. Chem. 94 (2001) 12904–12907. [26] L. Yan, J.A. Yee, M.H. McGuire, G.L. Graef, Effect of dietary supplementation of selenite on pulmonary metastasis of melanoma cells in mice, Nutr. Cancer 28 (1997) 165–169. [27] J.L. Larabee, J.R. Hocker, R.J. Hanas, F.M. Kahn, J.S. Hanas, Inhibition of zinc finger protein–DNA interactions by sodium selenite, Biochem. Pharmacol. 64 (2002) 1757–1765. [28] G.F. Combs, Status of selenium in prostate cancer prevention, Br. J. Cancer 91 (2004) 195–199.
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41 [29] L. Hardell, A. Degerman, R. Tomic, S.L. Marklund, M. Bergfors, Levels of selenium in plasma and glutathione peroxidase in erythrocytes in patients with prostate cancer or benign hyperplasia, Eur. J. Cancer Prev. 4 (1995) 91– 95. [30] L. Piccinini, P. Borella, A. Bargellini, C.I. Medici, A. Zoboli, A case-control study on selenium, zinc, and copper in plasma and hair of subjects affected by breast and lung cancer, Biol. Trace Elem. Res. 51 (1996) 23–30. [31] B.A. Zachara, E. Marchaluk-Wisniewska, A. Maciag, J. Peplin´ski, J. Skokowski, W. Lambrecht, Decreased selenium concentration and glutathione peroxidase activity in blood and increase of these parameters in malignant tissue of lung cancer patients, Lung 175 (1997) 321–332. [32] P. Knekt, J. Marniemi, L. Teppo, M. Heliövaara, A. Aroma, Is low selenium status a risk factor for lung cancer?, Am J. Epidemiol. 148 (1998) 975–982. [33] P. Ghadirian, P. Maisonneuve, C. Perret, G. Kennedy, D. Krewski, A. Lacroix, A case-control study of toenail selenium and cancer of the breast, colon, and prostate, Cancer Det. Prev. 24 (2000) 305–313. [34] J.T. Salonen, R. Salonen, R. Lappetelainen, P.H. Mäenpää, G. Alfthan, P. Puska, Risk of cancer in relation to serum concentrations of selenium and vitamins A and E: matched case-control analysis of prospective data, Br. Med. J. (Clin. Res. Ed.) 290 (1985) 417–420. [35] W.C. Willett, B.F. Polk, J.S. Morris, M.J. Stampfer, S. Pressel, B. Rosner, J.O. Taylor, K. Schneider, C.G. Hames, Prediagnostic serum selenium and the risk of cancer, Lancet 2 (1983) 130–134. [36] G. Fex, B. Pettersson, B. Akesson, Low plasma selenium as a risk factor for cancer death in middle-aged men, Nutr. Cancer 10 (1987) 221–229. [37] G.W. Comstock, T.L. Bush, K. Helzlsouer, Serum retinol, beta-carotene, vitamin E, and selenium as related to subsequent cancer of specific sites, Am. J. Epidemiol. 135 (1992) 115–121. [38] N.D. Lyn Patrick, Selenium biochemistry and cancer: a review of the literature, Altern. Med. Rev. 9 (2004) 239–258. [39] S.M. Lippman, E.A. Klein, P.J. Goodman, et al., Effect of selenium and vitamin E on risk of prostate cancer and other cancers, JAMA 301 (2009) 39–51. [40] C.L. Shen, W. Song, B.C. Pence, Interactions of selenium compounds with other antioxidants in DNA damage and apoptosis in human normal keratinocytes, Cancer Epidemiol. Biomar. Prev. 10 (2001) 385–390. [41] H.A. Tajmir-Riahi, M. Naoui, S. Diamantoglou, L. Elmahmoudaoui, The Effects 3 3 2 2 2 2 2 of Oxoanions SeO2 4 ; SeO3 ; AsO4 ; AsO3 ; CrO4 ; CO3 ; HCO3 ; SO4 ; SO3 ; 3 PO3 on the Solution Structure of Calf 4 PO3 ; NO3 ; NO2 ; ClO4 , and CH3COO Thymus DNA, Metal Ions in Biology and Medicine, John Liberty Eurotext, Paris, 1994. pp. 53–57. [42] J. Marmur, Thermal renaturation of deoxyribonucleic acids, J. Mol. Biol. 3 (1961) 585–594. [43] R. Vijalakshmi, M. Kanthimathi, V. Subramania, DNA cleavage by a chromium(III) complex, Biochem. Biophys. Res. Commun. 271 (2000) 731– 734. [44] C.D. Kanakis, P.A. Tarantilis, M.G. Polissiou, S. Diamantoglou, H.A. Tajmir-Riahi, DNA interaction with naturally occurring antioxidant flavonoids quercetin, kaempferol, and delphinidin, J. Biomol. Struct. Dyn. 22 (2005) 719–724.
41
[45] C.D. Kanakis, P.A. Tarantilis, M.G. Polissiou, S. Diamantoglou, H.A. Tajmir-Riahi, An overview of DNA and RNA bindings to antioxidant flavonoids, Cell Biochem. Biophys. 49 (2007) 29–36. [46] H. Arakawa, R. Ahmad, M. Naoui, H.A. Tajmir-Riahi, A comparative study of calf thymus DNA binding to Cr(III) and Cr(VI) ions. Evidence for the guanine N-7chromium-phosphate chelate formation, J. Biol. Chem. 275 (2000) 10150– 10153. [47] K. Connors, Binding Constants: The Measurement of Molecular Complex Stability, John Wiley & Sons, NY, 1987. pp. 46–86. [48] S. Alex, P. Dupuis, FT-IR and Raman investigation of cadmium binding by DNA, Inorg. Chim. Acta 157 (1989) 271–281. [49] P.B. Keller, K.A. Hartman, The effect of ionic environment and mercury(II) binding on the alternative structures of DNA; an infrared spectroscopic study, Spectrochim. Acta Part A 42 (1986) 299–306. [50] D.M. Loprete, K.A. Hartman, Conditions for the stability of the B, C and Z, structural forms of poly(dG-dC) in the presence of lithium, potassium, magnesium, calcium and zinc cations, Biochemistry 32 (1993) 4077–4082. [51] A. Ahmed Ouameur, H.A. Tajmir-Riahi, Structural analysis of DNA interactions with biogenic polyamines and cobalt(III)hexamine studied by Fourier transform infrared and capillary electrophoresis, J. Biol. Chem. 279 (2004) 42041–42054. [52] Sh. Nafisi, M. Adelzadeh, Z. Norouzi, M.N. Sarbolouki, Curcumin binding to DNA and RNA, DNA Cell Biol. 28 (2009) 201–208. [53] G.I. Dovbeshko, V.I. Chegel, N.Y. Gridina, O.P. Repnytska, Y.M. Shirshov, V.P. Tryndiak, I.M. Todor, C.I. Solyanik, Surface enhanced IR absorption of nucleic acids from tumor cells: FTIR reflectance study, Biopolymers 67 (2002) 470– 486. [54] A. Ahmed Ouameur, H. Malonga, J.F. Neault, S. Diamantoglou, H.A. TajmirRiahi, Taxol interaction with DNA and RNA-stability and structural features, Can. J. Chem. 82 (2004) 1112–1118. [55] J. Dean, Flavone: The Molecular and Mechanistic Study of How a Simple Flavonoid Protects DNA from Oxidative Damage, PhD Thesis, East Tennessee State University, Jonson City, TN, 2003. [56] D. Freifelder, Physical Biochemistry, W.H. Freeman and Company, NY, 1976 (Chapter 15). [57] J.B. Lambert, H.F. Shurvell, D.A. Lightner, R.G. Cooks, Organic Structural Spectroscopy, Prentice-Hall, Inc., Hillsdale, NJ, 1998 (Chapters 10–11). [58] Sh. Nafisi, F. Manouchehri, H.A. Tajmir-Riahi, M. Varavipour, Structural features of DNA interaction with caffeine and theophylline, J. Mol. Struct. 875 (2008) 392–399. [59] E. Taillandier, J. Liquier, Infrared spectroscopy of DNA, Methods Enzymol. 211 (1992) 307–335. [60] R. Marty, C.N. N’soukpoe´-Kossi, D. Charbonneau, C.M. Weinert, L. Laurent Kreplak, H.A. Tajmir-Riahi, Structural analysis of DNA complexation with cationic lipids, Nucl. Acids Res. 37 (2009) 849–857. [61] J.S. Mandeville, C.N. N’soukpoe´-Kossi, J.F. Neault, H.A. Tajmir-Riahi, Structural analysis of DNA interaction with retinol and retinoic acid, Biochem. Cell Biol. 88 (2010) 469–477.
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
The effect of Se salts on DNA structure Shohreh Nafisi a,b,⇑, Maryam Montazeri a, Firouzeh Manouchehri a a b
Department of Chemistry, Islamic Azad University, Central Tehran Branch (IAUCTB), Tehran 14676-86831, Iran Research Institute for Islamic and Complementary Medicine, Tehran University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 13 March 2012 Received in revised form 13 April 2012 Accepted 18 April 2012 Available online 8 May 2012 Keywords: DNA Sodium selenate Sodium selenite FTIR spectroscopy UV–Visible
a b s t r a c t There is considerable interest in the role of selenium in cancer prevention. Various organic and inorganic Se compounds are considered to be antioxidants. In the present study, the binding modes, the binding constants and the stability of Se–DNA complexes have been determined by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed that Na2SeO4 and Na2SeO3 bind to the minor and major grooves of DNA and the backbone phosphate (PO2) with overall binding constants of K(Na2SeO4–DNA) = 5.20 104 M1 and K(Na2SeO3–DNA) = 1.87 103 M1. DNA aggregations occurred at high selenium concentrations. No biopolymer conformational changes were observed upon Na2SeO3 and Na2SeO4 interactions, while DNA remained in the B-family structure. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Selenium is an important micronutrient and an essential trace element for both humans and animals metabolism. Diet containing selenium includes cereals, grains, nuts, vegetables, meat, and seafood [1–3]. Selenium has been extensively studied for its antioxidant activity and cancer prevention [4–8]. It is found in many dietary supplements and multivitamins in forms of sodium selenite (Na2SeO3), sodium selenate (Na2SeO4) or selenomethionine (SeMet) [5,9]. The main inorganic selenium compound used in most cancer treatment studies is sodium selenite (Scheme 1). However, a few studies use other forms, such as sodium selenate (Scheme 1) and selenium oxide [1,6,10–14]. Selenium is incorporated as selenocysteine (SeCys) in selenoproteins P, W, and R, as well as in the active sites of the enzymes such as glutathione peroxidases (GPx) and thioredoxin reductases [1,6,15–17]. In cells, these selenoproteins have important antioxidant activities and protect the mitochondria, plasma membrane, and DNA from oxidative damage by reactive oxygen species (ROS) [18–20]. Sodium selenite inhibits the growth of cells and induces apoptosis in human colonic carcinoma [21], brain tumor [22] and breast cancer cells [23]. It inhibits 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary tumors [24], human fibrosarcoma tumor cell invasiveness [25] and pulmonary metastasis of melanoma cells in mice [26,27]. The majority of clinical trials provide ⇑ Corresponding author at: Department of Chemistry, Islamic Azad University, Central Tehran Branch (IAUCTB), Tehran 14676-86831, Iran. Tel.: +98 21 22426554; fax: +98 21 22432706. E-mail address: drsnafi[email protected] (S. Nafisi). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.04.012
evidence for selenium as a chemopreventive agent for specific cancers: prostate [28,29], lung [30–32], colorectal [33,34], stomach [19] and multiple cancers [35–38]. However, the results of a recent clinical trial showed that selenium and vitamin E taken alone or together did not prevent prostate, lung or colorectal cancers [39]. Even though much is reported about antitumor activities and protective role of Se compounds in DNA damage [40], little is known about the interaction of sodium selenite and selenate with individual DNA. The interaction of some oxoselenium compounds with DNA have been studied before [41]. Their results showed B-DNA stabilization till 1/1 ratio and a partial B to A-DNA transition at higher ratio of 2/1. The aim of this study was to characterize the DNA structural changes in the presence of Na2SeO3 and Na2SeO4. Here, we compared the interactions of Na2SeO3 and Na2SeO4 with DNA in aqueous solution at pH 7 with Se/DNA (P) molar ratios of 1/120–1/1 by FTIR and UV measurements. It is worth mentioning that the concentration of Se used here is much higher than the physiological concentration of Se found in living cell. Since the FTIR and UV spectroscopic methods are not sensitive to very low Se contents, higher concentrations were used in order to determine the effect of Se on DNA structure. Structural analyses regarding the Se binding site, binding constant, and DNA secondary structures are provided here.
2. Materials and methods 2.1. Materials Highly polymerized type I calf-thymus DNA sodium salt (7% Na content) was purchased form Sigma Chemical Co. (St. Louis, MO),
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41
Scheme 1. Chemical structure of Na2SeO4 and Na2SeO3.
37
1662 (thymine), 1610 (adenine), 1492 (cytosine), and 1226 cm1 (PO2 groups) versus Se concentrations were obtained after peak normalization using Ri = Ii/I968, where Ii is the intensity of the absorption peak for DNA in the complex with i as the ligand concentration, and I968 is the intensity of the 968 cm1 peak (DNA internal reference). The plot of intensity was drawn for r = 1/ 120–1/1. 2.4. Absorption spectroscopy
and deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. Na2SeO3 and Na2SeO4 were also purchased from Sigma Chemical (St. Louis, MO) and used without further purification. To check the protein content of DNA solutions, the absorbance bands at 260 and 280 nm were used. The A260/A280 ratio was 2.10 for DNA, showing that DNA samples were sufficiently free from protein [42]. Other chemicals were of reagent grade and used without further purification. 2.2. Preparation of stock solutions DNA was dissolved to 0.5% w/v (0.0125 M) polynucleotide (phosphate) (pH 7) in Tris–HCl at 5 °C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the DNA solution was determined spectrophotometrically at 260 nm using molar extinction coefficient e260 = 66,000 cm1 M1 (DNA) (expressed as molarity of phosphate groups) [43]. The appropriate amounts of sodium selenite and sodium selenate (0.1–12.5 mM) were dissolved in water and added dropwise to DNA solution (12.5 mM) to attain the desired Na2SeO3 and Na2SeO4/DNA(P) molar ratios (r) of 1/120–1/1 with a final DNA(P) concentration of 6.25 mM. The pH values of solutions were adjusted at 7.0 ± 0.2 using NaCl solution. The infrared spectra were recorded 2 h after mixing of the Na2SeO3 and Na2SeO4 with DNA solution. 2.3. FTIR spectroscopy measurements Infrared spectra were recorded on a Bruker ATR spectrometer (Ettlingen, Germany) equipped with an external water cooled high power Hg-arc source and was accessible with a room temperature operated deuterated triglycine sulfate (DTGS) detector and a ZnSe beam splitter. The spectra of Na2SeO3 and Na2SeO4/DNA solutions were recorded using a cell assembled with ZnSe windows. Spectra were recorded using the Opus software supplied by the manufacturer of the spectrophotometer. The spectra of the solutions were recorded after 2 h incubation of the Na2SeO3 and Na2SeO4 with DNA solution, using ZnSe windows. The ratios used for infrared were Se/DNA(P) molar ratios (r) of 1/120, 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1 with a final DNA(P) concentration of 6.25 mM. For each spectrum, 100 scans were recorded with resolution of 4 cm1. The difference spectra [(polynucleotide solution + Na2SeO3 and Na2SeO4 solution) (polynucleotide solution)] were obtained using a sharp DNA band at 968 cm1 as internal Refs. [44,45]. This band, which is due to sugar C–C and C–O stretching vibrations, exhibits no spectral change (shifting or intensity variation) upon Na2SeO3 and Na2SeO4–DNA complexation, and cancelled out upon spectral subtraction. The intensity ratios of the bands due to several DNA in plane vibrations related to A–T, G–C base pairs and the PO2 stretching vibrations were measured with respect to the reference bands at 968 cm1 (DNA) as a function of Se concentrations with an error of ±3%. Similar intensity variations have been used to determine the ligand binding to DNA bases and backbone phosphate groups [46]. The plots of the relative intensity (R) of several peaks of DNA in-plane vibrations related to A–T, G–C base pairs and the PO2 stretching vibrations such as 1710 (guanine),
The absorption spectra were recorded on a LKB model 4054 UV–Visible spectrometer, the quartz cuvettes of 1 cm were used. The absorption spectra recorded with Na2SeO3 and Na2SeO4 concentrations of 0.005–0.5 mM and constant polynucleotide concentration of 0.5 mM. The binding constants of the Na2SeO3 and Na2SeO4–DNA complexes were calculated as reported [47]. It is assumed that the interaction between the ligand [L] and the substrate [S] is 1:1; for this reason a single complex SL (1:1) is formed. The relationship between the observed absorbance change per centimeter and the system variables and parameters is as follow;
DA St K 11 De11 ½L ¼ b 1 þ K 11 ½L
ð1Þ
where DA = A A0 from the mass balance expression St = (S) + (SL), we get (S) = St/(1 + K11(L)). Eq. (1) is the binding isotherm, which shows the hyperbolic dependence on free ligand concentration. The double-reciprocal form of plotting the rectangular hyperbola 1y ¼ df 1x þ de, is based on the linearization of Eq. (1) according to the following equation,
b 1 1 ¼ þ DA St K 11 De11 ½L St De11
ð2Þ
Thus the double reciprocal plot of 1/DA versus 1/(L) is linear and the binding constant can be estimated from the following equation
K 11 ¼
intercept slope
ð3Þ
3. Results and discussion 3.1. FTIR spectra of Na2SeO4–DNA adducts Evidence related to Na2SeO4–DNA complexation comes from the infrared spectroscopic results shown in Figs. 1A and 2A. The spectral changes (intensity and shifting) of several prominent DNA in-plane vibrations at 1710 (G and T, mainly G), 1662 (T, G, A, and C, mainly T), 1610 (A and C, mainly A), 1492 (C and G, mainly C), 1226 (PO2 asymmetric stretch), and 1088 cm1 (PO2 symmetric stretch) [48–52] were monitored at different Na2SeO4–DNA molar ratios, and the results are shown in Figs. 1A and 2A. At r = 1/120, 1/80, major reduction in intensity was observed for the bases and phosphate PO2 vibrations (Figs. 1A and 2A). The intensity of the guanine band at 1710 cm1 decreased by 18%, the thymine band at 1662 cm1 decreased by 15%, the adenine band at 1610 cm1 decreased by 22% and the asymmetric phosphate vibration decreased by 6% (Figs. 1A and 2A). The observed intensity decreases can be related to DNA helix stabilization upon Na2SeO4 complexation. At higher concentrations (r = 1/40, 1/20), intensity of the G, T, A and asymmetric PO2 bands increased. The observed increase in intensity can be related to DNA helix destabilization as a result of extended binding of Na2SeO4 to bases and phosphate group (Fig. 2A). At r = 1/10, 1/5, reduction in the intensity of the bases and phosphate bands can be attributed to DNA aggregation upon Na2SeO4 interaction (Fig. 2A). At r = 1/2, increase
38
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41
Fig. 1. FTIR spectra and difference spectra [(DNA solution + Se salt solution) (DNA solution)] in the region of 1800–600 cm1 for calf thymus DNA (6.25 mM), and Na2SeO4– DNA (A) and Na2SeO3–DNA (B) (0.1–12.5 mM) adducts in aqueous solution at pH = 7. DNA and three complexes spectra obtained at various Na2SeO4, Na2SeO3–DNA (phosphate) molar ratios (top three spectra), three difference spectra (bottom three spectra).
in intensity of the bases and phosphate can be assigned to interaction of Na2SeO4 with bases and backbone phosphate group. Similar intensity changes were observed in the flavonoids–DNA complexes in which increase in intensity of the bands were assigned to interaction of the flavonoids with bases and backbone phosphate group [44]. At r = 1/1, DNA aggregation occurred upon Na2SeO4 interaction. Evidence for this comes from decrease in intensity of the bases and phosphate bands (Figs. 1A and 2A). It should be noted that the intensity changes of the phosphate band were accompanied by shifting of the band at 1226 cm1 to 1231–1233 cm1 (r = 1/120 to r = 1/1). Further evidence regarding Na2SeO4–PO2 interaction comes from intensity ratio variations of the symmetric and asymmetric PO2 bands at 1088/1226 [48]. The ratio of ms/mas was changed from 2.30 (free DNA) to 2.51 (Na2SeO4–DNA complexes) upon Na2SeO4 complexation.
In the difference spectra of Na2SeO4–DNA complexes (r = 1/ 120–1/1), the positive features at 1100–1099 cm1 are coming from increase in intensity of the phosphate vibrational frequency upon Na2SeO4 interaction. It indicates major interaction of Na2SeO4 with DNA backbone phosphate (Fig. 1A). No major spectral changes were observed for the cytosine band at 1492 cm1 upon Na2SeO4 interaction that can be related to minor participation of cytosine bases in Na2SeO4–DNA complexation (Fig. 1A). Similar spectral changes were observed in the biogenic polyamine– and taxol–DNA complexes [51,53,54]. It is worth mentioning that the absorption band with medium intensity at 1640 cm1 in the IR spectrum of the free DNA and at 1653–1651 cm1 for the complexes are due to water deformation mode and they are not coming from DNA vibrations (Fig. 1A) [54].
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41
Fig. 2. Intensity ratio variations for several DNA in-plane vibrations as a function of Na2SeO4 (A) and Na2SeO3 (B) concentrations (0.1–12.5 mM). Intensity ratios for the DNA bands at 1710 (G, T), 1662 (T, G, A, C), 1610 (A), 1492 (C, G) and 1226 (PO2 asymmetric) referenced to the DNA band at 968 cm1.
Additional support for Na2SeO4–DNA interaction comes from decrease in intensity of the UV–Visible band of DNA at 260 nm upon Na2SeO4–DNA complexation (Fig. 3A). 3.2. FTIR spectra of Na2SeO3–DNA adducts Evidence related to Na2SeO3–DNA complexation comes from the infrared spectroscopic results shown in Figs. 1B and 2B. In the Na2SeO3–DNA complex (r = 1/120), major reduction in intensity was observed for the bases and phosphate (PO2) vibrations (Fig. 2B). The intensity of guanine band at 1710 cm1 decreased by 39%, the thymine band at 1662 cm1 decreased by 39%, the adenine band at 1610 cm1 decreased by 58% and the asymmetric phosphate vibration decreased by 22%. The observed reduction in intensity is related to DNA helix stabilization upon Na2SeO3–DNA complexation (Fig. 2B). At r = 1/80, the increase in intensity of the bands related to G, T, A and asymmetric PO2 is due to the helix destabilization as a result of extended binding of Na2SeO3 to bases and phosphate group (Figs. 1B and 2B). At r = 1/40, decrease in intensity of the bases and phosphate bands can be related to DNA aggregation upon Na2SeO3 interaction. At higher concentrations (r = 1/20, 1/10, 1/5), the bases and phosphate vibrations intensified as a result of DNA helix destabilization and external binding of Na2SeO3–DNA. At r = 1/2, DNA aggregation occurred upon Na2SeO3 interaction. Evidence for this comes from reduction in intensity of the bases and phosphate bands (Figs. 1B and 2B). No major interaction was observed at higher concentration (r = 1/1). In the difference spectra of Na2SeO3–DNA complexes (r = 1/ 120–1/1), the positive features at 1087–1082 cm1 are coming
39
Fig. 3. UV–Visible results of free DNA (0.5 mM); free Na2SeO4, Na2SeO4–DNA complexes (0.08 mM) (A) and free DNA, free Na2SeO3, Na2SeO3–DNA complexes (0.08 mM) (B). Plot of 1/(A0 A) versus 1/(ligand concentration) for Na2SeO4 and Na2SeO3 (0.005–0.5 mM) and calf-thymus DNA complexes, where A0 is the initial absorbance of DNA (260 nm) and A is the recorded absorbance at different Na2SeO4, Na2SeO3 concentrations at pH 7.
from increase in intensity of the phosphate vibrational frequency upon Na2SeO3 interaction and indicates interaction of Na2SeO3 with DNA phosphate backbone (Figs. 1B and 2B). In addition to major intensity changes of the PO2 stretching at 1226 cm1, the relative intensities of the asymmetric (mas) and symmetric (ms) vibrations were altered upon Na2SeO3 interaction. The ms PO2 (1088 cm1) and mas PO2 (1226 cm1) ratios changed with ms/mas increasing from 2.3 (uncomplexed DNA) to 2.54 (Na2SeO3–DNA complexes) which indicates Na2SeO3–PO2 complexation. Further evidence regarding Na2SeO3–DNA interaction comes from reduction in intensity of the characteristic UV–Visible band of DNA at 260 nm due to Na2SeO3–DNA complexation (Fig. 3B) [55–58]. 3.3. DNA conformation DNA remains in the B-family structure upon Se complexation. This is consistent with the infrared results on the Na2SeO4 and Na2SeO3–DNA complexes that showed no conformational changes for B-DNA with marker B-DNA bands at 1710 (G), 1226 (PO2), and 836 cm1 (phosphodiester modes) (Fig. 1A and B). When a complete B to A transition occurs, the B-DNA marker bands such as 836 cm1 appear at about 820–810 cm1, while the phosphate stretching vibration at 1226 cm1 shifts toward a
40
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41
higher frequency at 1240–1230 cm1 and the guanine band at 1710 appears at 1700 cm1 [50,59]. No major shifting was observed in the B-DNA conformation marker bands upon Na2SeO3 and Na2SeO4 interactions. In Na2SeO4–DNA complexes, the observed shifting of the PO2 asymmetric band at 1226 to 1231–1233 cm1 can be related to PO2 coordination to Na2SeO4 and is not coming from DNA conformational change (Fig. 1A and B). 3.4. Stability of Na2SeO4 and Na2SeO3–DNA complexes The selenium salts–DNA binding constants were determined as described in Section 2 (UV–Visible spectroscopy). The calculations of the overall binding constants were carried out using UV spectroscopy as reported [47]. Concentrations of the complexed ligand were determined by subtracting absorbance of the free DNA at 260 nm from those of the complexed. Concentration of the free ligand was determined by subtraction of the complex ligand from total ligand used in the experiment. Our data of 1/[complexed ligand] almost proportionally increased as a function of 1/[free ligand] (Fig. 3). The double reciprocal plot of 1/(A0 A) versus 1/(selenium salts concentration) is linear, and the binding constant (K) can be estimated from the ratio of the intercept to the slope (Fig. 3), where A0 is the initial absorbance of the free DNA at 260 nm, and A is the recorded absorbance of DNA in the presence of different selenium salts concentrations. The overall binding constant of K for Na2SeO4–DNA = 5.20 104 M1 and Na2SeO3–DNA = 1.87 103 M1 show greater stability for Na2SeO4–DNA complexes. The small K value obtained for Na2SeO3–DNA complex shows weak interaction between Se anions and DNA. However the binding constant for Na2SeO4–DNA showed a strong interaction. Similar binding constants were observed in lipid–DNA complexes in which strong interaction occurred via major and minor grooves and the backbone phosphate group [60]. Weak affinity to DNA was observed for retinol–DNA with overall binding constants of Kretinol–DNA = 3 103 M1 [61]. 4. Summary and conclusions On the basis of our spectroscopic results, the interaction of Na2SeO4 and Na2SeO3 to DNA bases and phosphate backbone group have been concluded. The stabilities of Se salts–DNA adducts are in the order K(Na2SeO4–DNA) = 5.20 104 M1 > K(Na2SeO3– DNA) = 1.87 103 M1. No DNA conformational changes were observed upon Na2SeO4 and Na2SeO3 complexation, while biopolymer aggregation occurred at high selenium contents.
5. Abbreviations
A G C T FTIR UV
Adenine Guanine Cytosine Thymine Fourier transform infrared Ultraviolet
Disclosure statement No competing financial interests exist.
Acknowledgment This work is supported by Grants from Islamic Azad University, Central Tehran Branch (IAUCTB). References [1] H. Tapiero, D.M. Townsend, K.D. Tew, The antioxidant role of selenium and seleno-compounds, Biomed. Pharmacother. 57 (2003) 134.–144. [2] J.A. Pennington, S.A. Schoen, Total diet study: estimated dietary intakes of nutritional elements, Int. J. Vitamin Nutr. Res. 66 (1996) 350–362. [3] J.A. Pennington, S.A. Schoen, Contributions of food groups to estimated intakes of nutritional elements: results from the FDA total diet studies, Int. J. Vitamin Nutr. Res. 66 (1996) 342–349. [4] A. Holmgren, Selenite in cancer therapy: a commentary on ‘‘Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress’’, Free Radical Biol. Med. 41 (2006) 862–865. [5] G.N. Schrauzer, Nutritional selenium supplements: product types, quality, and safety, J. Am. Coll. Nutr. 20 (2001) 1–4. [6] M. Takahashi, T. Sato, F. Shinohara, S. Echigo, H. Rikiishi, Possible role of glutathione in mitochondrial apoptosis of human oral squamous cell carcinoma caused by inorganic selenium compounds, Int. J. Oncol. 27 (2005) 489–495. [7] W. Zhong, T.D. Oberley, Redox-mediated effects of selenium on apoptosis and cell cycle in the LNCaP human prostate cancer cell line, Cancer Res. 61 (2001) 7071–7078. [8] R.R. Ramoutar, J.L. Brumaghim, Antioxidant and anticancer properties and mechanisms of niorganic selenium, oxo-sulfur, and oxo-selenium compounds, Cell Biochem. Biophys. 58 (2010) 1–23. [9] J.W. Finley, C. Ip, D.J. Lisk, C.D. Davis, K.J. Hintze, P.D. Whanger, Cancerprotective properties of high selenium broccoli, J. Agric. Food Chem. 49 (2001) 2679–2683. [10] G. Mantovani, A. Maccio, C. Madeddu, R. Serpe, E. Massa, G. Gramignano, et al., Selenium is effective in inducing lymphocyte progression through cell cycle in cancer patients: potential mechanisms for its activity, J. Exp. Ther. Oncol. 4 (2004) 69–78. [11] G. Spyrou, M. Bjornstedt, S. Skog, A. Holmgren, Selenite and selenate inhibit human lymphocyte growth via different mechanisms, Cancer Res. 56 (1996) 4407–4412. [12] S. Vadhanavikit, C. Ip, H.E. Ganther, Metabolites of sodium selenite and methylated selenium compounds administered at cancer chemoprevention levels in the rat, Xenobiotica 23 (1993) 731–745. [13] C. Ip, The chemopreventive role of selenium in carcinogenesis, J. Am. Coll. Toxicol. 5 (1986) 7–20. [14] J.E. Spallholz, On the nature of selenium toxicity and carcinostatic activity, Free Radical Biol. Med. 17 (1994) 45–64. [15] R.R. Ramoutar, J.L. Brumaghim, Effects of inorganic selenium compounds on oxidative DNA damage, J. Inorg. Biochem. 101 (2007) 101028–101035. [16] K.M. Brown, J.R. Arthur, Selenium, selenoproteins and human health: a review, Public Health Nutr. 4 (2001) 593–599. [17] V.N. Gladyshev, G.V. Kryukov, Evolution of selenocysteine-containing proteins: significance of identification and functional characterization of selenoproteins, Biofactors 14 (2001) 87–92. [18] J.E. Spallholz, L. Mallory Boylan, M.M. Rhaman, Environmental hypothesis: Is poor dietary selenium intake an underlying factor for arsenicosis and cancer in Bangladesh and West Bengal, India?, Sci Total Environ. 323 (2004) 21–32. [19] T.C. Stadtman, Discoveries of vitamin B12 and selenium enzymes, Annu. Rev. Biochem. 71 (2002) 1–16. [20] R.L. Nelson, F.G. Davis, E. Sutter, J.W. Kikendall, L.H. Sobin, J.A. Milner, P.E. Bowen, Serum selenium and colonic neoplastic risk, Dis. Colon Rectum 38 (1995) 1306–1310. [21] M.S. Stewart, R.L. Davis, L.P. Walsh, B.C. Pence, Induction of differentiation and apoptosis by sodium selenite in human colonic carcinoma cells (HT29), Cancer Lett. 117 (1997) 35–40. [22] N. Sundaram, A.K. Pahwa, M.D. Ard, N. Lin, E. Perkins, A.P. Jr, Bowles, selenium causes growth inhibition and apoptosis in human brain tumor cell line, J. Neurooncel. 46 (2000) 125–133. [23] A.M. Watrach, J.A. Milner, M.A. Watrach, K.A. Poirier, Inhibition of human breast cancer cells by selenium, Cancer Lett. 25 (1984) 41–47. [24] H.J. Thompson, L.D. Meeker, P.J. Becci, S. Kokoska, Effect of short-term feeding of sodium selenite on 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in the rat, Cancer Res. 42 (1982) 4954–4958. [25] S.O. Yoon, M.W. Kim, A.S. Chung, Inhibitory effect of selenite on invasion of HT1080 tumor cells, J. Biol. Chem. 94 (2001) 12904–12907. [26] L. Yan, J.A. Yee, M.H. McGuire, G.L. Graef, Effect of dietary supplementation of selenite on pulmonary metastasis of melanoma cells in mice, Nutr. Cancer 28 (1997) 165–169. [27] J.L. Larabee, J.R. Hocker, R.J. Hanas, F.M. Kahn, J.S. Hanas, Inhibition of zinc finger protein–DNA interactions by sodium selenite, Biochem. Pharmacol. 64 (2002) 1757–1765. [28] G.F. Combs, Status of selenium in prostate cancer prevention, Br. J. Cancer 91 (2004) 195–199.
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 113 (2012) 36–41 [29] L. Hardell, A. Degerman, R. Tomic, S.L. Marklund, M. Bergfors, Levels of selenium in plasma and glutathione peroxidase in erythrocytes in patients with prostate cancer or benign hyperplasia, Eur. J. Cancer Prev. 4 (1995) 91– 95. [30] L. Piccinini, P. Borella, A. Bargellini, C.I. Medici, A. Zoboli, A case-control study on selenium, zinc, and copper in plasma and hair of subjects affected by breast and lung cancer, Biol. Trace Elem. Res. 51 (1996) 23–30. [31] B.A. Zachara, E. Marchaluk-Wisniewska, A. Maciag, J. Peplin´ski, J. Skokowski, W. Lambrecht, Decreased selenium concentration and glutathione peroxidase activity in blood and increase of these parameters in malignant tissue of lung cancer patients, Lung 175 (1997) 321–332. [32] P. Knekt, J. Marniemi, L. Teppo, M. Heliövaara, A. Aroma, Is low selenium status a risk factor for lung cancer?, Am J. Epidemiol. 148 (1998) 975–982. [33] P. Ghadirian, P. Maisonneuve, C. Perret, G. Kennedy, D. Krewski, A. Lacroix, A case-control study of toenail selenium and cancer of the breast, colon, and prostate, Cancer Det. Prev. 24 (2000) 305–313. [34] J.T. Salonen, R. Salonen, R. Lappetelainen, P.H. Mäenpää, G. Alfthan, P. Puska, Risk of cancer in relation to serum concentrations of selenium and vitamins A and E: matched case-control analysis of prospective data, Br. Med. J. (Clin. Res. Ed.) 290 (1985) 417–420. [35] W.C. Willett, B.F. Polk, J.S. Morris, M.J. Stampfer, S. Pressel, B. Rosner, J.O. Taylor, K. Schneider, C.G. Hames, Prediagnostic serum selenium and the risk of cancer, Lancet 2 (1983) 130–134. [36] G. Fex, B. Pettersson, B. Akesson, Low plasma selenium as a risk factor for cancer death in middle-aged men, Nutr. Cancer 10 (1987) 221–229. [37] G.W. Comstock, T.L. Bush, K. Helzlsouer, Serum retinol, beta-carotene, vitamin E, and selenium as related to subsequent cancer of specific sites, Am. J. Epidemiol. 135 (1992) 115–121. [38] N.D. Lyn Patrick, Selenium biochemistry and cancer: a review of the literature, Altern. Med. Rev. 9 (2004) 239–258. [39] S.M. Lippman, E.A. Klein, P.J. Goodman, et al., Effect of selenium and vitamin E on risk of prostate cancer and other cancers, JAMA 301 (2009) 39–51. [40] C.L. Shen, W. Song, B.C. Pence, Interactions of selenium compounds with other antioxidants in DNA damage and apoptosis in human normal keratinocytes, Cancer Epidemiol. Biomar. Prev. 10 (2001) 385–390. [41] H.A. Tajmir-Riahi, M. Naoui, S. Diamantoglou, L. Elmahmoudaoui, The Effects 3 3 2 2 2 2 2 of Oxoanions SeO2 4 ; SeO3 ; AsO4 ; AsO3 ; CrO4 ; CO3 ; HCO3 ; SO4 ; SO3 ; 3 PO3 on the Solution Structure of Calf 4 PO3 ; NO3 ; NO2 ; ClO4 , and CH3COO Thymus DNA, Metal Ions in Biology and Medicine, John Liberty Eurotext, Paris, 1994. pp. 53–57. [42] J. Marmur, Thermal renaturation of deoxyribonucleic acids, J. Mol. Biol. 3 (1961) 585–594. [43] R. Vijalakshmi, M. Kanthimathi, V. Subramania, DNA cleavage by a chromium(III) complex, Biochem. Biophys. Res. Commun. 271 (2000) 731– 734. [44] C.D. Kanakis, P.A. Tarantilis, M.G. Polissiou, S. Diamantoglou, H.A. Tajmir-Riahi, DNA interaction with naturally occurring antioxidant flavonoids quercetin, kaempferol, and delphinidin, J. Biomol. Struct. Dyn. 22 (2005) 719–724.
41
[45] C.D. Kanakis, P.A. Tarantilis, M.G. Polissiou, S. Diamantoglou, H.A. Tajmir-Riahi, An overview of DNA and RNA bindings to antioxidant flavonoids, Cell Biochem. Biophys. 49 (2007) 29–36. [46] H. Arakawa, R. Ahmad, M. Naoui, H.A. Tajmir-Riahi, A comparative study of calf thymus DNA binding to Cr(III) and Cr(VI) ions. Evidence for the guanine N-7chromium-phosphate chelate formation, J. Biol. Chem. 275 (2000) 10150– 10153. [47] K. Connors, Binding Constants: The Measurement of Molecular Complex Stability, John Wiley & Sons, NY, 1987. pp. 46–86. [48] S. Alex, P. Dupuis, FT-IR and Raman investigation of cadmium binding by DNA, Inorg. Chim. Acta 157 (1989) 271–281. [49] P.B. Keller, K.A. Hartman, The effect of ionic environment and mercury(II) binding on the alternative structures of DNA; an infrared spectroscopic study, Spectrochim. Acta Part A 42 (1986) 299–306. [50] D.M. Loprete, K.A. Hartman, Conditions for the stability of the B, C and Z, structural forms of poly(dG-dC) in the presence of lithium, potassium, magnesium, calcium and zinc cations, Biochemistry 32 (1993) 4077–4082. [51] A. Ahmed Ouameur, H.A. Tajmir-Riahi, Structural analysis of DNA interactions with biogenic polyamines and cobalt(III)hexamine studied by Fourier transform infrared and capillary electrophoresis, J. Biol. Chem. 279 (2004) 42041–42054. [52] Sh. Nafisi, M. Adelzadeh, Z. Norouzi, M.N. Sarbolouki, Curcumin binding to DNA and RNA, DNA Cell Biol. 28 (2009) 201–208. [53] G.I. Dovbeshko, V.I. Chegel, N.Y. Gridina, O.P. Repnytska, Y.M. Shirshov, V.P. Tryndiak, I.M. Todor, C.I. Solyanik, Surface enhanced IR absorption of nucleic acids from tumor cells: FTIR reflectance study, Biopolymers 67 (2002) 470– 486. [54] A. Ahmed Ouameur, H. Malonga, J.F. Neault, S. Diamantoglou, H.A. TajmirRiahi, Taxol interaction with DNA and RNA-stability and structural features, Can. J. Chem. 82 (2004) 1112–1118. [55] J. Dean, Flavone: The Molecular and Mechanistic Study of How a Simple Flavonoid Protects DNA from Oxidative Damage, PhD Thesis, East Tennessee State University, Jonson City, TN, 2003. [56] D. Freifelder, Physical Biochemistry, W.H. Freeman and Company, NY, 1976 (Chapter 15). [57] J.B. Lambert, H.F. Shurvell, D.A. Lightner, R.G. Cooks, Organic Structural Spectroscopy, Prentice-Hall, Inc., Hillsdale, NJ, 1998 (Chapters 10–11). [58] Sh. Nafisi, F. Manouchehri, H.A. Tajmir-Riahi, M. Varavipour, Structural features of DNA interaction with caffeine and theophylline, J. Mol. Struct. 875 (2008) 392–399. [59] E. Taillandier, J. Liquier, Infrared spectroscopy of DNA, Methods Enzymol. 211 (1992) 307–335. [60] R. Marty, C.N. N’soukpoe´-Kossi, D. Charbonneau, C.M. Weinert, L. Laurent Kreplak, H.A. Tajmir-Riahi, Structural analysis of DNA complexation with cationic lipids, Nucl. Acids Res. 37 (2009) 849–857. [61] J.S. Mandeville, C.N. N’soukpoe´-Kossi, J.F. Neault, H.A. Tajmir-Riahi, Structural analysis of DNA interaction with retinol and retinoic acid, Biochem. Cell Biol. 88 (2010) 469–477.