Abstract

INTRODUCTION

An antioxidant compound (AO) is any substance present in low concentrations when compared to an oxidizable substrate, which effectively delays or inhibits the oxidation of this substrate (Sies, Stahl, 1995Sies H, Stahl W. Vitamins E and C, β-carotene, and other carotenoids as antioxidants. Am J Clin Nutr. 1995;2(6):315-21.). AOs have been the subject of numerous studies because it is believed that they positively contribute in reducing the risk of many diseases and in delaying cellular aging (Rohenkohl, Carniel, Colpo, 2011Rohenkohl C, Carniel A, Colpo E. Consumo de antioxidantes durante tratamento quimioterápico. Arquivos Brasileiros de Cirurgia Digestiva. 2011;24(1):107-12.; Costa, Monteiro, 2009Costa P, Monteiro A. Benefícios dos antioxidantes na alimentação. Revista Saúde e Pesquisa. 2009;2(1):87-90.). Most AOs can be obtained through diet, by regular consumption of fruits and vegetables. Moreover, AOs can act synergistically at different levels (Hajhashemi et al., 2010Hajhashemi V, Vaseghi G, Pourfarzam M, Abdollahi A. Are antioxidants helpful for disease prevention? Res Pharm Sci. 2010;5(1):1-8.).

On the other hand, studies have shown that in solutions that contain transition metal ions, AO compounds can act as pro-oxidants and thus contribute to the formation of free radicals, which under certain conditions can damage DNA (Win et al., 2002Win W, Cao Z, Peng X, Trush MA, Li Y. Different effects of genistein and resveratrol on oxidative DNA damage in vitro. Mutat Res. 2002;513(1-2):113-20.; Yoshino et al., 1999Yoshino M, Haneda M, Naruse M, Murakami K. Prooxidant activity of flavonoids: copper-dependent strand breaks and the formation of 8-hydroxy-2’-deoxyguanosine in DNA. Mol Genet Metab. 1999;68(4):468-72.). In fact, it was found that melatonin (Sakano et al., 2004Sakano K, Oikawa S, Hiraku Y, Kawanishi S. Oxidative DNA damage induced by a melatonin metabolite, 6-hydroxymelatonin, via a unique non-o-quinone type of redox cycle. Biochem Pharmacol. 2004;68(9):1869-78.), N-acetylcysteine (Oikawa et al., 1999Oikawa S, Yamada K, Yamashita N, Tada-Oikawa S, Kawanishi S. N-acetylcysteine, a cancer chemopreventive agent, causes oxidative damage to cellular and isolated DNA. Carcinogenesis. 1999;20(8):1485-90.), α-tocopherol (Yamashita et al., 1998Yamashita N, Murata M, Inoue S, Burkitt MJ, Milne L, Kawanishi S. Alpha-tocopherol induces oxidative damage to DNA in the presence of copper(II) ions. Chem Res Toxicol. 1998;11(8):855-62.), resveratrol, and ascorbic acid (Sugiyama, Tsuzuki, Ogura, 1991Sugiyama M, Tsuzuki K, Ogura R. Effect of ascorbic acid on DNA damage, cytotoxicity, glutathione reductase, and formation of paramagnetic chromium in Chinese hamster V-79 cells treated with sodium chromate(VI). J Biol Chem. 1991;266(6):3383-6.) can act as pro-oxidants in the presence of Cu(II) and Cr(VI), thereby contributing to the formation of free radicals.

Ascorbic acid (AA) is an AO compound found in fruits and vegetables, which is also used as a food preservative (INS 300). When present in the organism, it takes part in several biochemical processes in the cell, mainly in the hydroxylation of collagen (Cerqueira, Medeiros, Augusto, 2007Cerqueira FM, Medeiros MHG, Augusto O. Antioxidantes dietéticos: controvérsias e perspectivas. Quim Nova. 2007;30(2):441-9.; Manela-Azulay et al., 2003Manela-Azulay M, Mandarim-de-Lacerda CA, Perez MA, Filgueira AL, Cuzzi T. Vitamin C. An Bras Dermatol. 2003;78:265-74.). However, it is known that in a saturated air solution, AA reduces Cu(II) to Cu(I), which can lead to the formation of hydrogen peroxide (H₂O₂) and hydroxyl radicals (HO^•), which can then damage the DNA (Aruoma et al., 1991Aruoma OI, Halliwell B, Gajewski E, Dizdaroglu M. Copper ion-dependent damage to the bases in DNA in the presence of hydrogen peroxide. Biochem J. 1991;273(Pt 3):601-4.).

Sulfur dioxide (SO₂), another known food preservative (INS 220), is widely used in fruit juices and wines and can also be found as an atmospheric pollutant produced by industrial chemical reactions (Dong-Sheng, Xiao-Ying, Jie- Qing, 2006Dong-Sheng G, Xiao-Ying Y, Jie-Qing L. Influence of Cu(II) on the interaction of sulfite with DNA. Spectrochim. Acta, Part A. 2006;65(2):459-462.). It is well established that free radicals of sulfur oxide (SO₃^•-, SO₄^•- e SO₅^•-) are easily generated when S(IV), which may be present in an aqueous solution as SO₂, HSO₃^- and SO₃^2-, undergoes autoxidation catalyzed by transition metal ions such as Cu(II), Co(II), Mn(II), and Ni(II) (Alipázaga et al., 2009; Aguiar et al., 2007Aguiar A, Ferraz A, Contreras D, Rodríguez J. Mecanismo e aplicações da reação de Fenton assistida por compostos fenólicos. Quim Nova. 2007;30(3):623-8.). Therefore, humans can be exposed to these sulfur derivatives through inhalation, due to the presence of sulfur oxides in the atmosphere, as well as through the consumption of processed foods (Alipázaga et al., 2009Alipázaga VM, Cerchiaro G, Moya HD, Coichev N. Oxidative DNA damage mediated by Cu2+ and Cu+ complexes in the presence of S(IV). J Braz Chem Soc. 2009;20(7):1302-12.; Aguiar et al., 2007Aguiar A, Ferraz A, Contreras D, Rodríguez J. Mecanismo e aplicações da reação de Fenton assistida por compostos fenólicos. Quim Nova. 2007;30(3):623-8.).

Copper ions have an essential role in animal metabolism, as copper-dependent enzymes require traces of this metal to mediate chemical reactions, such as collagen and elastin biosynthesis and blood coagulation (Pedrosa, Cozzolino, 1999Pedrosa LFC, Cozzolino SMF. Alterações metabólicas e funcionais do cobre em diabetes mellitus. Rev Nutr. 1999;12(3):213-224.). A previous study has suggested that the DNA-copper association makes it possible to maintain the chromosome structure and regulate gene expression (Li, Trush, 1994Li Y, Trush MA. Reactive oxygen-dependent DNA damage resulting from the oxidation of phenolic compounds by a copper-redox cycle mechanism. Cancer Res. 1994;54(7 Suppl):1895s-8s.). In a living system, copper ions can also be found in the cell nucleus where they interact with chromosomes, RNA, and DNA, and especially with guanine (Li, Trush, 1994Li Y, Trush MA. Reactive oxygen-dependent DNA damage resulting from the oxidation of phenolic compounds by a copper-redox cycle mechanism. Cancer Res. 1994;54(7 Suppl):1895s-8s.). On the other hand, it was suggested that Cu(II) acts as a catalyst in redox reactions, which gives rise to free radicals that damage biomolecules and cause predisposition towards the development of various diseases (Cerqueira, Medeiros, Augusto, 2007Cerqueira FM, Medeiros MHG, Augusto O. Antioxidantes dietéticos: controvérsias e perspectivas. Quim Nova. 2007;30(2):441-9.).

In the present study, pUC-19 DNA damage was evaluated by electrophoresis, in a saturated air solution ([O₂] ~ 250 μM) containing Cu(II) and the two AOs compounds mentioned above, ascorbic acid and S(IV). DNA damage occurs in the presence of both AA and S(IV), which reduce Cu(II) to Cu(I). This confirms the importance of the Cu(II)/Cu(I) redox cycle. In fact, the presence of Cu(II) and Cu(I) complexing agents, such as EDTA and neocuproine, respectively, inhibits or decreases DNA damage caused by a reducing agent (Roriz, Moya, 2017Roriz BC, Moya HD. Study of DNA damage caused by dipyrone in presence of some transition metal ions. Saudi Pharm J. 2017;25(7):961-6.).

However, when these two AO compounds (AA and S(IV)) are added simultaneously, the damage caused by AA is suppressed. Although from a biological point of view, this inhibition of DNA damage can be a positive event, this may indicate that the antioxidant capacity of a sample (e.g., processed foods) attributed to the sum of these compounds (AA and S(IV)) decreases if they are added simultaneously (mixture at pH 5.5).

Decrease in DNA damage does not happen due to a previous reaction between sodium sulfite and AA, as it is not thermodynamically favorable at pH 5.5. Moreover, the absorbance measurements using standard solutions of these compounds (sodium sulfite and AA) corroborates this conclusion. In addition, the presence of dissolved O₂, which actively participates in inducing DNA damage (Alipázaga, Moya, Coichev, 2010Alipázaga MV, Moya HD, Coichev N. Effect of some antioxidants on oxidative DNA damage induced by autoxidation of microquantities of sulfite in the presence of Ni(II)/Gly-GlyL-His. J. Coord. Chem. 2010;63(14-16):2450-60.; Alipázaga et al., 2008Alipázaga MV, Moreno RGM, Linares E, Medeiros MHG, Coichev N. DNA damage by sulfite autoxidation catalyzed by cobalt complexes. Dalton Trans. 2008;(41):5636-44.; Dong-Sheng, Xiao-Ying, Jie-Qing, 2006Dong-Sheng G, Xiao-Ying Y, Jie-Qing L. Influence of Cu(II) on the interaction of sulfite with DNA. Spectrochim. Acta, Part A. 2006;65(2):459-462.; Moreno et al., 2005Moreno RGM, Alipázaga MV, Medeiros MHG, Coichev N. DNA damage induced by sulfite autoxidation catalyzed by copper(II)/tetraglycine complexes. Dalton Trans. 2005;(6):1101-7.), might have contributed to the decrease in AA concentration in the previous mixture.

As far as we know, there are currently no studies exploring Cu(II)-mediated DNA damage (present as Cu(II) ion solution) (Dong-Sheng, Xiao-Ying, Jie-Qing, 2006Dong-Sheng G, Xiao-Ying Y, Jie-Qing L. Influence of Cu(II) on the interaction of sulfite with DNA. Spectrochim. Acta, Part A. 2006;65(2):459-462.) in a solution containing the two AO compounds mentioned above (ascorbic acid and sulfur dioxide), which is evaluated in the present study.

MATERIALS AND REAGENTS

The Milli-Q Plus Water System (Millipore®) was used to obtain purified water for use in the preparation of all solutions.

Absorbance measurements were performed with an HP 8453 UV (Agilent) spectrophotometer, using quartz cuvettes (1.0 cm pathlength).

pUC-19 DNA (MM 1.74 × 10⁶ Da, 0.5 μg μL^-1, Fermentas) was used in all experiments. The diluted DNA solution of 18.8 ng μL^-1 was obtained by mixing 6 μL of pUC-19 DNA (0.5 μg μL^-1) with 154 μL of water in an Eppendorf tube.

Cu(NO₃)₂ stock solution (0.20 M, pH 0.6) was prepared by dissolving 1.2719 g of copper wire (Cu, 99.99%, Sigma) in double distilled HNO₃, followed by dilution with water in a 100 mL volumetric flask as previously described (Alipázaga et al., 2009Alipázaga VM, Cerchiaro G, Moya HD, Coichev N. Oxidative DNA damage mediated by Cu2+ and Cu+ complexes in the presence of S(IV). J Braz Chem Soc. 2009;20(7):1302-12.).

Ascorbic acid (AA) stock solution (3.75 mM) was prepared by dissolving 0.0660 g of AA (C₆H₈O₆, 176.13 g mol^-1, Merck) in water in a 100 mL volumetric flask.

S(IV) stock solution (0.15 M, pH 4.2) was prepared by dissolving 0.3565 g of sodium metabisulfite (Na₂S₂O₅, 190.11 g mol^-1, 98.0%, Sigma) in water in a 25 mL volumetric flask.

TBE buffer (pH 8.0) stock solution (Tris 446 mM, boric acid 448 mM, and EDTA 10 mM) was prepared by dissolving 27 g of tris-(hydroxymethyl)-aminomethane (C₄H₁₁NO₃, 121.14 g mol^-1, 99.8%, Synth), 13.75 g of boric acid (H₃BO₃, 61.83 g mol^-1, 99.0%, Sigma Aldrich), and 1.861 g of EDTA (C10H16N2O8, 372.24 g mol-1, 99.0%, Neon) in water in a 500 mL volumetric flask. TBE buffer working solution (Tris 89.5 mM, boric acid 89.6 mM, and EDTA 2.00 mM) was prepared by diluting 100 mL of stock solution in a 500 mL volumetric flask.

The preparation of a 0.8% (m/v) agarose gel and the Ficoll/Bromophenol blue mixture is described in Supplementary Material 1.

EXPERIMENTAL PROCEDURE

DNA with a final concentration of 5.0 ng mL^-1 was used in all solutions (the general procedure for agarose gel electrophoresis, AGE, is described in Supplementary Material 1).

All AGE experiments (triplicates) were conducted in saturated air solutions ([O₂] ~ 250 μM) under controlled temperature (21 ± 2) ºC in a PowerPac Basic Bio-Rad horizontal mini-tank (BioAgency®), with constant current (30 mA) for 80 minutes.

The gels were placed in a UV Transilluminator ZT-21 (Cristal BioGlow®) and photographed with a PowerShot G10 (Canon®) digital camera after electrophoresis. The photographs were analyzed with AlphaEase® FC software version 6.0. The DNA damage was verified by the conversion of the native form (SC = supercoiled) into an open circular (OC) form, due to single chain breaks, and a linear (L) form, due to double chain breaks.

RESULTS AND DISCUSSION

Influence of Cu(II) concentration

Under the experimental conditions used throughout this work, there was no DNA damage observed in the presence of Cu(II) concentrations ranging from 10 to 120 μM (Figure 1A). Likewise, other studies have shown that there were no lesions to the DNA in an air-saturated solution ([O₂] ~ 250 μM) containing only Cu(II) (Alipázaga et al., 2009; Dong-Sheng, Xiao-Ying, Jie-Qing, 2006Dong-Sheng G, Xiao-Ying Y, Jie-Qing L. Influence of Cu(II) on the interaction of sulfite with DNA. Spectrochim. Acta, Part A. 2006;65(2):459-462.; Kawanishi, Yamamoto, Inoue, 1989Kawanishi S, Yamamoto K, Inoue S. Site-specific DNA damage induced by sulfite in the presence of cobalt(II) ion. Role of sulfate radical. Biochem Pharmacol. 1989;38(20):3491-96.). This confirms that the presence of a reducing agent is required to promote the Cu(II)/Cu(I) redox cycle and the consequent DNA damage. In fact, it is well known that Cu(I) can decompose organic peroxides (RO₂H) into alkoxyl radicals (RO•) and organic peroxyl (RO₂•) (Aguiar et al., 2007Aguiar A, Ferraz A, Contreras D, Rodríguez J. Mecanismo e aplicações da reação de Fenton assistida por compostos fenólicos. Quim Nova. 2007;30(3):623-8.) and subsequently react with H₂O₂ to produce OH^●, thereby regenerating Cu(II) and resuming the Cu(II)/Cu(I) redox cycle.

However, there is no agreement regarding the Cu(II) concentration necessary (5.0, 10, 20, 50, 100, and 150 μM) to induce DNA damage (Moreno, 2005Moreno RGM. Estudo da lesão no DNA promovido pela autoxidação de S(IV) na presença de complexos de Cu(II)/ tetraglicina. [tese]. São Paulo: Instituto de Química, USP; 2005.; Frelon et al., 2003Frelon S, Douki T, Favier A, Cadet J. Hydroxyl radical is not the main reactive species involved in the degradation of DNA bases by copper in the presence of hydrogen peroxide. Chem Res Toxicol. 2003;16(2):191-7.; Lesniak et al., 2003Lesniak W, Harris WR, Kravitz JY, Schacht J, Pecoraro VL. Solution chemistry of copper(II)-gentamicin complexes: relevance to metal-related aminoglycoside toxicity. Inorg Chem. 2003;42(5):1420-9.; Iwamoto et al., 2003Iwamoto T, Hiraku Y, Oikawa S, Mizutanoi H, Kojima M, Kawanishi S. Oxidative DNA damage induced by photodegradation products of 3’-azido-3’-deoxythymidine. Arch Biochem Biophys. 2003;416(2):155-63.; Ogawa et al., 2003Ogawa K, Hiraku Y, Oikawa S, Murata M, Sugimura Y, Kawamura J, et al. Molecular mechanisms of DNA damage induced by procarbazine in the presence of Cu(II). Mutat. Res. 2003;539(1-2):145-155.; Husain, Hadi, 1995Husain S, Hadi SM. Strand scission in DNA induced by L-DOPA in the presence of Cu(II). FEBS Lett. 1995;364(1):75-8.).

Previous studies that evaluated the role of S(IV) showed that DNA damage was observed when Cu(II) concentration was fixed at 100 μM (Moreno, 2005Moreno RGM. Estudo da lesão no DNA promovido pela autoxidação de S(IV) na presença de complexos de Cu(II)/ tetraglicina. [tese]. São Paulo: Instituto de Química, USP; 2005.). Thus, in the present study, we evaluated DNA damage in the presence of Cu(II) and AA (in the absence and presence of S(IV)) at a fixed Cu(II) concentration of 100 μM.

DNA damage in the presence of Cu(II) and AA (0.5 to 100 μM).

Addition of AA to a solution containing 100 μM Cu(II) promoted pUC-19 DNA damage, which can be observed by the appearance of the open circular (OC) configuration at AA concentrations ranging from 1.0 µM (Figure 1 B; Lane 4) to 10 µM (Figure 1 B; Lane 6). At concentrations above 50 μM AA (Figure 1 B, Lane 8), it is no longer possible to observe any of the pUC-19 DNA configurations (OC, L or SC) as shown in Table I. No lesions were observed when Cu(II) was maintained at a concentration of 5.0 μM (Figure 1 C) or 10 μM (data not shown).

Previous studies have shown that the addition of S(IV), as SO₃^2-, can induce DNA damage in the presence of free or complexed transition metal ions (Dong-Sheng, Xiao-Ying, Jie-Qing, 2006Dong-Sheng G, Xiao-Ying Y, Jie-Qing L. Influence of Cu(II) on the interaction of sulfite with DNA. Spectrochim. Acta, Part A. 2006;65(2):459-462.; Jameton, Muller, Burrows, 2002Jameton RA, Muller JG, Burrows CJ. Oxidative DNA damage from sulfite autoxidation catalyzed by manganese(III). C R Chimie. 2002;5(5):461-6.; Kawanishi, Yamamoto, Inoue, 1989Kawanishi S, Yamamoto K, Inoue S. Site-specific DNA damage induced by sulfite in the presence of cobalt(II) ion. Role of sulfate radical. Biochem Pharmacol. 1989;38(20):3491-96.).

In the present study, the presence of S(IV) in a solution containing 100 μM Cu(II) (in the absence of complexing agents) revealed that DNA damage occurs at concentrations of 100 μM S(IV) (Figure 1 D, Lane 4) and remains constant up to concentrations of 500 μM (Figure 1 D, Lane 6).

After reaching concentration of 1000 μM S(IV), an increase in the OC form is noted (Figure 1D, Lane 7), which remains constant until a concentration of 2000 μM S(IV), (Figure 1D, Lane 7 and 8). The % of (OC + L) and SC forms are shown in Figure 2.

The results presented above confirm that under these experimental conditions, S(IV) reduces free Cu(II) to Cu(I), which then effectively contributes to pUC-19 DNA damage.

Indeed, when Cu(II) 100 μM is added as a Cu(II)/ EDTA complex (log β₄ = 18.8) (Smith, Martell, 2004Smith RM, Martell AE. Version 8.0. NIST critically selected stability constants of metal complexes database 46. [Computer program]. Gaithersburg: National Institute of Standards Technology; 2004.) in the solution containing S(IV) (5.0 to 1000 μM), pUC19 DNA damage does not occur (data not shown). This indicates the importance of free Cu(II) in the process that promotes DNA damage. In addition, the decrease in DNA damage caused by drugs such as dipyrone (Roriz, Moya, 2017Roriz BC, Moya HD. Study of DNA damage caused by dipyrone in presence of some transition metal ions. Saudi Pharm J. 2017;25(7):961-6.) and bilirubin/biliverdin (Asad et al., 2002Asad SF, Singh S, Ahmad A, Hadi SM. Bilirubin/biliverdinCu(II) induced DNA breakage; reaction mechanism and biological significance. Toxicol Lett. 2002;131(3):181-9.) also decreases significantly when Cu(II) 100 μM is in a solution containing neocuproine (NC), in a ratio of 1Cu(II):2NC. In this case, the decrease in DNA damage is related to the formation of the Cu(I)/NC complex (log β₂ = 19.1) (Lee et al., 2011Lee G, Rossi MV, Coichev N, Moya HD. The reduction of Cu(II)/neocuproine complexes by some polyphenols. Analytical application in wine samples. Food Chemistry. 2011;126:679-86.), which prevents the return of Cu(I) to Cu(II). As such, this confirms that DNA damage occurs only in the presence of the Cu(II)/Cu(I) redox cycle.

FIGURE 1
Gel electrophoresis of pUC-19 DNA in the presence of Cu(II) and AA. Saturated air solution ([O₂] ~ 250 μM). pH = 5.5; T = (21 ± 2) ºC. All lanes (1) contain only pUC-19 DNA= 5.0 ng μL^-1.

FIGURE 2
% of (OC + L) and SC configurations of pUC-19 DNA according to the experimental conditions described in Figure 1D (Lanes 1 to 8).

DNA damage in the presence of Cu(II), AA, and S(IV).

In solutions containing either 100 μM Cu(II) or AA/S(IV) as AO agents (Figure 3, lanes 2, 3 and 4), no DNA damage was observed, lanes results which were previously described (Zheng et al., 2006Zheng LF, Wei QY, Cai YJ, Fang JG, Zhou B, Yang L, Liu ZL. DNA damage induced by resveratrol and its synthetic analogues in the presence of Cu (II) ions: mechanism and structure-activity relationship. Free Radic Biol Med. 2006;41(12):1807-16.; Dong-Sheng, Xiao-Ying, Jie-Qing, 2006Dong-Sheng G, Xiao-Ying Y, Jie-Qing L. Influence of Cu(II) on the interaction of sulfite with DNA. Spectrochim. Acta, Part A. 2006;65(2):459-462.; Kawanishi, Yamamoto, Inoue, 1989Kawanishi S, Yamamoto K, Inoue S. Site-specific DNA damage induced by sulfite in the presence of cobalt(II) ion. Role of sulfate radical. Biochem Pharmacol. 1989;38(20):3491-96.). Figure 3 shows that DNA damage occurs in the presence of 100 μM Cu(II), in a solution containing either 500 μM AA or 500 μM S(IV) (Figure 3, Lane 5 and 6). Moreover, DNA damage also occurs in the mixture containing the two AO agents (500 μM AA + 500 S(IV)) (Figure 3, Lane 8).

Sodium sulfite, a preservative in the food industry in Brazil (INS 220), is used in quantities ranging from 0.0020 g to 0.30 g SO₂/100 g food (Favero, Ribeiro, Aquino, 2011Favero DM, Ribeiro CSG, Aquino AD. Sulfitos importância na indústria alimentícia e seus possíveis malefícios à população. Segurança Alimentar e Nutricional. 2011;18(1):11-20.), which is equivalent to concentrations ranging from 320 μM to 47 mM, respectively.

As it was previously described, DNA damage in the presence of free or complexed Cu(II) can occur at S(IV) concentrations ranging from 50 μM to 20 mM (Alipázaga et al., 2009; Dong-Sheng, Xiao-Ying, JieQing, 2006Dong-Sheng G, Xiao-Ying Y, Jie-Qing L. Influence of Cu(II) on the interaction of sulfite with DNA. Spectrochim. Acta, Part A. 2006;65(2):459-462.; Kawanishi, Yamamoto, Inoue, 1989Kawanishi S, Yamamoto K, Inoue S. Site-specific DNA damage induced by sulfite in the presence of cobalt(II) ion. Role of sulfate radical. Biochem Pharmacol. 1989;38(20):3491-96.). Therefore, to evaluate the effect of the simultaneous addition of both S(IV) and AA on DNA damage, the S(IV) concentration was fixed at 500 μM. Under these conditions, approximately 20% of the pUC-19 DNA is damaged (Figure 1 D, Lane 6 and Table II). Notably, the S(IV) concentration used corresponds to 0.0032 g SO₂/100 g food, which is close to the minimum concentration of S(IV) (350 μM), used in Brazil (Favero, Ribeiro, Aquino, 2011Favero DM, Ribeiro CSG, Aquino AD. Sulfitos importância na indústria alimentícia e seus possíveis malefícios à população. Segurança Alimentar e Nutricional. 2011;18(1):11-20.).

Figure 3 presents the results obtained from the addition of the mixture containing AA and S(IV) to a solution containing Cu(II) and DNA. The importance of this metallic ion is highlighted by the absence of DNA damage when these two AOs are together in the absence of Cu(II) (Figure 3, lane 7). As expected, DNA damage occurs in presence of Cu(II) at 100 μM and S(IV) at 500 μM, leading to the formation of an OC conformation (Figure 3, lane 5). On the other hand, total degradation of DNA takes place in the presence of Cu(II) at 100 μM and AA at 500 μM (Figure 3, lane 6). When AA and S(IV) (both at 500 μM) are added simultaneously, the DNA damage is also observed through the appearance of the OC and L forms (Figure 3, lane 8 and Table II). This is noteworthy as the two AO compounds do not act synergistically, whereas the native pUC-19 DNA form (SC) was preserved. This shows that the presence of S(IV) in a solution containing Cu(II) inhibited the AAinduced pUC-19 DNA damage.

By keeping Cu(II) constant at 100 μM and S(IV) at 500 μM, DNA damage (% OC) decreases as the concentration of AA decreases from 20 to 5.0 μM (Figure 4, lanes 3 to 8 and Table III). Under these experimental conditions, DNA integrity was 32% preserved, provided that the [S(IV)]:[AA] ratio was 50: 1 (Figure 4, lane 6, Table III). This can be better observed when comparing lane 4, Figure 4 (Cu(II) 100 μM + AA 500 μM) with lane 8, Figure 3 (Cu(II) 100 μM + AA 500 μM + S(IV) 500 μM). The addition of S(IV) maintains the [S(IV)]:[AA] ratio (1:1) and prevents the total degradation of the DNA originating from the OC (90%) and L (10%) forms, as shown in Table III.

By maintaining Cu(II) 100 μM and AA at 50 μM but varying the concentration of S(IV) from 10 to 1000 μM (Figure 5), we observed an inhibition of DNA damage. At an AA concentration of 50 μM, total DNA degradation occurs (Figure 1B, lane 7 and Figure 5, lane 4). However, from the first addition of the mixture containing 10 μM S(IV) (Figure 5, lane 5), inhibition of DNA damage can be observed, giving rise to (54 ± 2) % of L form. When S(IV) is added at a concentration of 50 μM in the mixture (Figure 5, lane 6), the formation of the SC conformation (23 ± 5) % is observed, which reaches a maximum of (75 ± 3) % at a concentration of 500 μM S(IV) (Figure 5, lane 7) and remains constant up to a concentration of 1000 μM S(IV) (Figure 5, lane 8), as shown in Table IV.

Agarose gel electrophoresis does not enable us to elucidate the mechanisms by which the preservation of pUC-19 DNA is achieved. However, the results presented in this study show that the [S(IV)]:[AA] ratio (ranging from (1:1) to (20:1)) is essential for this inhibition of pUC-19 DNA damage to occur.

These in vitro studies cannot be accurately applied to more complex mixtures (e.g. fruit juice, wines or canned food). However, it can be inferred that the presence of S(IV) and AA (in the ratio described above) would allow the association of the antioxidant capacity of these compounds with a low level of DNA damage.

Stability of the mixture containing AA and S(IV)

The inhibition of pUC-19 DNA damage in the mixture containing S(IV), AA and Cu(II) in a saturated air solution ([O₂] ≈ 250 μM) suggested a reaction between these two AO compounds, which would result in a decrease in the concentration of AA.

Figure 6 shows the overlap of the absorption spectra of the AOs mixture (S(IV) 500 μM + AA 50 μM) and that of the standard solutions of 500 μM S(IV) and 50 μM AA. This seems to indicate that there was no reaction between AA and S(IV) in the mixture.

FIGURE 3
Gel electrophoresis of pUC-19 DNA in the presence of Cu(II) and AA. Saturated air solution ([O₂] ~ 250 μM). pH = 5.5; T = (21 ± 2) ºC. All lanes (1) contain only pUC-19 DNA = 5.0 ng μL^-1.

FIGURE 4
Gel electrophoresis of pUC-19 DNA in the presence of Cu(II) and AA. Saturated air solution ([O₂] ~ 250 μM). pH = 5.5; T = (21 ± 2) ºC. All lanes (1) contain only pUC 19 DNA = 5.0 ng μL^-1.

FIGURE 5
Gel electrophoresis of pUC-19 DNA in the presence of Cu(II) and AA. Saturated air solution ([O₂] ~ 250 μM). pH = 5.5; T = (21 ± 2) ºC. All Lane (1) contain only pUC-19 DNA = 5.0 ng μL^-1.

A more efficient way to verify whether AA and S(IV) react with each other in the mixture is by performing a multiwavelength linear regression analysis (Blanco et al., 1989Blanco M, Iturriaga H, Maspoch S, Tarin P. A simple method for spectrophotometric determination of two-components with overlapped spectra. J Chem Educ. 1989;66(2):178-80.) (the mathematical deduction is described in Supplementary Material 2). Considering that S(IV)m, S(IV)p, AAm, and AAp represent sodium sulfite and ascorbic acid concentrations in the mixture (m) and in the standard solutions (p), the [S(IV)] and [AA] concentrations in the mixture (pH = 5.5) can be calculated. Using the absorbance values of these solutions (S(IV)m, S(IV)p, AAm and AAp) and the mathematical formulas presented in the Supplementary Material 3, we obtained the graph shown in Figure 6 (inserted).

The a value (0.95) represents the ratio of [S(IV)_m]/ [S(IV)_p], suggesting that sodium sulfite consumption should not occur. The b value (0.73), represents the ratio of [AA_m]/[AAp], which indicates a 27% loss in [AA] content. However, this should not be attributed to the direct reaction with S(IV) in these experimental conditions (pH = 5.5).

According to the standard reduction potential (E⁰) values of dehydroascorbic acid (DAA/AA) (Harris, 2005Harris DC. Aplicações da espectrofotometria. In: Harris DC, editor. Análise Química Quantitativa. 6th ed. Rio de Janeiro: LTC. 2005. p. 424-450.), E⁰ = 0.390 V vs. NHE, and sulfate/sulfite (SO₄^2-/ SO₃^2-) (Lurie, 1978Lurie J. Handbook of Analytical Chemistry. 2nd. ed. Moscow: Mir Publishers; 1978. p. 300-313.), E⁰ =-0.936 V vs. NHE, the reaction between the two AOs should not occur under these experimental conditions (pH 5.5).

As such, the mechanism by which DNA damage decreases in the presence of AA and SO₃^2- remains unclear. The addition of sodium sulfite to a solution of AA increases the pH value. Therefore, at the pH value of the AA + S(IV) mixture used in the present study (5.5), the AA reduction capacity seems to decrease. In addition, autoxidation of AA in a solution containing SO₃^2- due to the presence of dissolved oxygen ([O₂] - 250 μM) may be a possible explanation for the decrease in DNA damage.

Although the gel electrophoresis analysis performed here clearly demonstrates that the presence of S(IV) in the AA solution inhibits DNA damage, studies with other techniques (e.g., HPLC or ion chromatography) should be performed to unequivocally identify the mechanism underlying this process.

CONCLUSIONS

AA (at concentrations above 0.5 μM) and S(IV) (at concentrations above 100 μM) induce damage to pUC-19 DNA in the presence of Cu(II) 100 μM in an air-saturated solution ([O₂] ~ 250 μM). Under the same experimental conditions, AA at 50 μM causes total defragmentation of pUC-19 DNA. However, the simultaneous addition of AA at 50 μM and S(IV) at 500 μM minimizes pUC-19 DNA damage caused by AA. The results showed that inhibition of pUC-19 DNA damage occurs when the ratio of [S(IV)]:[AA] ranges from (1:1) to (20:1).

Absorbance measurements and thermodynamic data show that no reaction occurs between AA and S(IV) when the previously mentioned mixture (pH 5.5) is added to pUC-19 DNA. Moreover, the presence of dissolved oxygen ([O₂] ≈ 250 μM) may be the cause of AA consumption in the mixture of these two AOs, which subsequently decreases DNA damage.

The results obtained by gel electrophoresis allowed us to observe the occurrence of pUC-19 DNA damage in the solution containing Cu(II), AA or S(IV). However, it is not possible to clarify the mechanism by which the reduction in this damage occurs after the simultaneous addition of AA and S(IV). Studies using other techniques, such as HPLC, should be performed to unequivocally identify the mechanism underlying this process.

FIGURE 6
Absorption spectra of solutions containing 50 μM AA, 500 μM S(IV) and mixture (50 μM AA + 500 μM S(IV)). pH = 5.5. Water as reference solution. 1.0 cm pathlength (quartz cuvette). Figure inserted: A_mixture/A_S(IV)p = 1.00 + 0.727×A_AAp/AS(IV)p; r2 = 0.996.

ACKNOWLEDGEMENTS

The authors acknowledge the financial support from Brazilian agencies FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico).

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