Radiation Physics and Chemistry 89 (2013) 38–42

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Radiation Physics and Chemistry

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journal homepage: www.elsevier.com/locate/radphyschem

Aspects of γ-radiation induced modification of calf thymus DNA in thepresence of sodium 1,4-dihydroxy-9,10-anthraquinone-2-sulfonateand its transition metal complexes with Cu2+ and Ni2+

Partha Sarathi Guin a, Parikshit Chandra Mandal b,1, Saurabh Das b,n

a Department of Chemistry, Shibpur Dinobundhoo Institution (College), 412/1 G.T. Road (South), Howrah 711 102, Indiab Department of Chemistry, Jadavpur University, Raja S.C. Mullick Road, Kolkata 700032, India

H I G H L I G H T S

� γ-radiation induced DNA modification was studied by ethidium bromide fluorescence.

� In de-aerated and N2O saturated medium NaQSH2 and Ni(II)–NaQSH2 were radioprotectors.� Cu(II)–NaQSH2 showed radiosensitization that was attributed to Fenton reactions.� In aerated medium, NaQSH2 and complexes showed radiosensitization through participation of molecular oxygen.� dOH had the major role in double strand modification of DNA.

a r t i c l e i n f o

Article history:Received 6 July 2012Accepted 2 April 2013Available online 10 April 2013

Keywords:NaQSH2

Cu(II)Ni(II)DNADouble-strand modificationD37

6X/$ - see front matter & 2013 Elsevier Ltd. Ax.doi.org/10.1016/j.radphyschem.2013.04.002

viations: Sodium 1,4-dihydroxy-9,10-anthraqu; Cu(II) complex of NaQSH2, Cu(II)–NaQSH2; NaQSH2; Calf thymus DNA, CT DNA.esponding author. Tel.: +91 33 2457 2148; faxail address: dasrsv@yahoo.in (S. Das).rmerly, Chemical Sciences Division, Saha Insthannagar, Kolkata 700064, India

a b s t r a c t

Radiation-induced double-strand modification of DNA was studied in the absence and presence ofsodium 1,4-dihydroxy-9,10-anthraquinone-2-sulfonate (NaQSH2) and its metal (Cu2+ and Ni2+) com-plexes in aerated, de-aerated (Argon saturated) and N2O saturated aqueous media at pH 7.4. Ethidiumbromide, an established DNA intercalator was used to estimate DNA remaining after interaction withγ-radiation, by measuring loss of fluorescence of the ethidium bromide–DNA adduct. In de-aerated(Argon saturated) and N2O saturated aqueous media radiation-induced double-strand modification ofcalf thymus DNA was comparatively less in presence of NaQSH2 and its Ni(II) complex than standardcontrol indicating the compounds behaved as radio-protectors. However, in presence of the Cu(II)complex radiation-induced double-strand modification increased significantly. In N2O saturated med-ium, double-strand modification of DNA was almost double in all cases than that observed in de-aerated(Argon saturated) medium indicating dOH radicals played a major role in modifying DNA. That dOHradicals were important was verified by repeating experiments using tertiary-butanol that showedsignificant decrease in DNA modification. Another important observation was in aerated mediumNaQSH2, Ni(II)–NaQSH2 did not show radioprotection while Cu(II)–NaQSH2 was an almost equallyeffective radiosensitizer as that observed in N2O saturated medium. Role of molecular oxygen as radio-sensitizer was thus realized.

& 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Ionizing radiation causes several types of DNA damage in livingcells and the extent of damage was seen to vary with oxic andanoxic conditions prevalent in the immediate vicinity of the cells.

ll rights reserved.

inone-2-sulfonate,i(II) complex of NaQSH2,

: +91 33 24146223.

itute of Nuclear Physics,

Radiolysis of water generates radical and molecular species (Hartand Anber, 1970; Draganic, I.G. and Draganic, Z.D., 1971) that reactwith nucleic acid bases in DNA leading to their modification. Suchmodifications initiate changes in the three dimensional structureand orientation of nucleic acids that eventually modify DNAthrough the consequent rupture of hydrogen bonds that areresponsible for holding the strands together. Several reportsindicate during radiotherapy tumor cells are insensitive to ionizingradiation that is seen as a major hurdle in this form of treatment.Chemical compounds that help in getting over the problem andbring about DNA modification or damage have therefore beenimportant. Such compounds are known as radiosensitizers and arevery important in radiotherapy.

P.S. Guin et al. / Radiation Physics and Chemistry 89 (2013) 38–42 39

Anthracycline drugs have a place of their own in the clinicalmap for treatment of cancer and are known for their chemother-apeutic action (Gianni et al., 1983; Myers et al., 1988; Hardmanet al., 1996; Umov, 2003). There are only few reports that indicatethem to be radiosensitizers on tumor cells (Jagetia and Nayak,2000; Candelaria et al., 2006; Alvarado-Miranda et al., 2009).Metal complexes of anthracyclines were prepared primarily totake care of aspects of cardiotoxicity that was common to this classof compounds (Beraldo et al., 1985; Fiallo and Garnier-Suillerot,1985, 1986). However, further work on the complexes showed thatthey too had siginificant clinical utility with proven anticanceractivity (Beraldo et al., 1985; Fiallo and Garnier-Suillerot, 1985,1986; Garnier-Suillerot, 1988; Moustatih et al., 1989). Beingsimilar to the anthracycline antibiotics in structure hydroxy-9,10-anthraquinones show close functional similarity with regard tophysicochemical behavior, enzyme assay studies, electrochemicalproperties and biophysical interactions (Das et al., 1996a; Guinet al., 2010; Das et al., 2011; Mukherjee, et al., 2012) that qualifythem to be effective substitutes. Studies on metal complexes ofdihydroxy-9,10-anthraquinones were carried out that made a fewinteresting observations (Das et al., 1996a; Guin et al., 2009, 2012a,2012b).

Still earlier we made some effort to see if metal complexes of1,2-dihydroxy-9,10-anthraquinone with Cu(II), Ni(II) and Fe(III) actas radiosensitizers (Das et al., 1995, 1996b, 1997, 2009). Thesefindings encouraged us to look for the radiosensitizing ability ofsodium 1,4-dihydroxy-9,10-anthraquinone-2-sulfonate (NaQSH2)and its metal complexes [Cu(II) and Ni(II)] on double-strandedDNA. Ethidium bromide (EB) that forms a highly fluorescentcomplex with native DNA through intercalation into base pairswas used as probe to monitor radiation-induced modification ofdouble-stranded calf thymus DNA (Morgan and Pulleyblank, 1974;Waring, 1981). This study describes the interaction of low doses ofγ-radiation with calf thymus DNA in the presence and absence ofNaQSH2 and its transition metal complexes with Cu(II) and Ni(II) inaerated, de-aerated (Argon saturated) and N2O saturated aqueoussolution at pH∼7.4.

1086420Dose (Gray)

% D

oubl

e St

rand

ed D

NA

100

90

Fig. 1. Dose effect curves for radiation induced modification of calf thymus DNA inde-aerated (Argon saturated) medium in absence (●) and presence of NaQSH2 (Δ),Ni(II)–NaQSH2 (▲) and Cu(II)–NaQSH2 (○). [DNA]¼20�10−6 mol dm−3, [NaQSH2]¼[Ni(II)–NaQSH2]¼[Cu(II)–NaQSH2]¼2�10−6 mol dm−3. (Maximum error: 10%).

2. Experimental

Sodium 1,4-dihydroxy-9,10-anthraquinone-2-sulfonate (NaQSH2)and the complexes with Cu(II) and Ni(II) were prepared andcharacterized by methods reported earlier (Guin et al., 2008,2009). Stock solutions of the compounds were prepared in tripledistilled water by weighing a definite amount of each. Uncertaintyin weighing the solid compound was less than 0.3% indicating thaterror in weighing was either negligible or within acceptable limits.Strength of the prepared stock solutions was 2�10−4 mol dm−3.Experimental solutions were prepared by exact dilution of thestock (2�10−4 mol dm−3) using a calibrated micropipette (Pipet-man-M23866E, Gilson, France). In order to verify the concentra-tions of the compounds in experimental solutions a calibrationexperiment (Lambert Beer's Law) was done by measuring theabsorbance of different solutions of the compounds (knownstrength) at 465 nm. The quinone moiety being sensitive to lightsolutions were prepared just before an experiment and kept inthe dark. Concentration of the compounds in the e xperimentalsolutions were 2�10−6 mol dm−3.

Calf thymus DNA was purchased from SRL, India and dissolvedin 0.05 M phosphate buffer containing 120 mM NaCl, 3.5 mM KCl,1 mM CaCl2 and 0.5 mM MgCl2. Purity was checked from theabsorbance ratio A260/A280 and the value was in the range1.8oA260/A280o1.9. This ratio of absorbance (A260/A280) providesan estimate of the purity of DNA with respect to contaminant(mainly protein content) that absorb in the UV (Warburg and

Christian, 1942; Prutz, 1984). DNA sample purified from biologicalsources such as tissue or cells has proteins as contaminant thatabsorb more heavily around 280 nm, primarily due to absorbanceof tryptophan with a lesser contribution from tyrosine residuesthat tend to decrease the ratio (A260/A280) to lower values ifthey be present. An A260/A280 ratio of 1.8–1.9 was considered“clean” in situations concerning DNA purity implying the proteinconcentration was negligible (Warburg and Christian, 1942)and no further protein removal was required. Concentrationof DNA in the prepared stock solution was determined takingε260¼6600 M−1 cm−1 per base.

Strength of the working solutions of calf thymus DNA was20�10−6 mol dm−3. They either contained or did not containNaQSH2 or its Cu(II) and Ni(II) complexes having strengths of2�10−6 mol dm−3. Such solutions in aerated, de-aerated (Argonsaturated) and N2O saturated aqueous media (pH∼7.4) weresubjected to γ-radiation using a 60Co source. In the reactionmixtures the concentration of phosphate buffer was 0.05 M andionic strength was maintained with 120 mM NaCl, 3.5 mM KCl,1 mM CaCl2 and 0.5 mM MgCl2. Maintaining a high ionic strengthis essential to prevent DNA unwinding (Rafi and Wheeler, 1973;Uyesugi and Trumbore, 1983, Grygoryev et al., 2011). The dose rate(0.84 Gy/min) was determined by Fricke dosimeter. Experimentalsolutions were saturated with high purity of either Argon (99%) orN2O (99%) by purging the solutions for 30 min prior to irradiation.The effect of irradiation on the double-strand modification in CTDNA was studied using ethidium bromide as a fluorescent probe(Warburg and Christian, 1942; Prutz, 1984). Ethidium bromide (EB)(AR Grade) was purchased from SRL, India and prepared in tripledistilled water. The investigation uses the aspect of decreasingfluorescence intensity of the ethidium bromide–DNA complex tostudy the radiation-induced double-strand modification in DNA.Fluorescence spectroscopic measurements were carried out onHITACHI S-7000 spectrofluorimeter using a 10�10 mm2

fluores-cence cuvette. As suggested in earlier work that used ethidiumbromide as a probe, concentration was kept around 20 timesthe DNA concentration so that saturation for binding could beachieved (Morgan and Pulleyblank, 1974; von Sonntag et al., 1981).All experiments were repeated four times.

3. Results and discussion

A neutral solution of Ethidium bromide (EB) shows a strongabsorption band at 470 nm. Upon interaction with DNA, this shiftsto 510 nm (Morgan and Pulleyblank, 1974; Prutz, 1984). When asolution of DNA treated with ethidium bromide was excitedat 510 nm fluorescence emission was observed at 590 nm.

1086420Dose (Gray)

% D

oubl

e St

rand

ed D

NA

100

90

Fig. 2. Dose effect curves for radiation induced modification of calf thymus DNA inN2O saturated medium in absence (●) and presence of NaQSH2 (Δ), Ni(II)–NaQSH2

(▲) and Cu(II)–NaQSH2 (○). [DNA]¼20�10−6 mol dm−3, [NaQSH2]¼[Ni(II)–NaQSH2]¼[Cu(II)–NaQSH2]¼2�10−6 mol dm−3. (Maximum error: 10%).

121086420Dose (Gray)

Dou

ble

Stra

nded

DN

A %

100

90

Fig. 3. Dose effect curves for radiation induced modification of calf thymus DNA inaerated medium in absence (●) and presence of NaQSH2 (Δ), Ni(II)–NaQSH2 (▲) andCu(II)–NaQSH2 (○). [DNA]¼20�10−6 mol dm−3, [NaQSH2]¼[Ni(II)–NaQSH2]¼[Cu(II)–NaQSH2]¼2�10−6 mol dm−3. (Maximum error: 10%).

Table 1Radiation induced double-strand damage in calf thymus DNA in the absence andpresence of NaQSH2 and its metal complexes. [DNA]¼20�10−6 mol dm−3, [NaQSH2]¼[Ni(II)–NaQSH2]¼[Cu(II)–NaQSH2]¼2�10−6 mol dm−3. (Maximum error: 10%).

Composition ofExperimentalSolutions

Deaerated media N2O saturatedmedia

Aerated media

% loss ofDNA,(Gray−1)

M.R.

% loss ofDNA,(Gray−1)

M.R.

% loss ofDNA,(Gray−1)

M.R.

DNA 0.0081 – 0.0159 1.96 0.0112 1.38DNA+NaQSH2 0.0037 0.46 0.0073 0.90 0.0119 1.47DNA+(Ni(II)–NaQSH2)

0.0060 0.74 0.0121 1.49 0.0123 1.52

DNA+(Cu(II)–NaQSH2)

0.0114 1.41 0.0210 2.59 0.0168 2.07

P.S. Guin et al. / Radiation Physics and Chemistry 89 (2013) 38–4240

γ-irradiated DNA solution that was made to bind to EB showedlower fluorescence at 590 nm in comparison to that of the un-irradiated control. Morgan and Pulleyblank (1974) showed at pH12.0, hydrogen bonding in single stranded DNA destabilizes anddouble-stranded DNA in the presence of single stranded DNAcould be estimated by measuring the fluorescence of the ethidiumbromide-DNA complex. During irradiation dOH produced destabi-lizes intramolecular hydrogen bonding in single stranded DNA bymodifying or releasing nucleic acid bases. As a result, under theexperimental conditions fluorescence observed with EB for theirradiated DNA might be due to binding of EB with double-stranded DNA at the base-pair region. For the purpose of verifica-tion, fluorescence of the EB complex with irradiated DNA at pH12.0 was measured and it was found that the fluorescenceintensity of the complex formed between EB and irradiated DNAwas identical with that observed in our method. Therefore, it couldbe concluded that fluorescence observed with irradiated DNA wasa measure of double-stranded DNA that was not modified. Resultswere expressed in terms of the percentage of double-strandedDNA remaining intact with absorbed dose.

Dose effect curves for the modification of double-stranded DNAwere exponential with dose (Gray). D37 was calculated as theinverse of the initial slope of the curve. D37 was the dose reducingthe DNA survival (double-stranded DNA remaining intact) to 37%.Figs. 1–3 show dose effect curves for the amount of double-stranded DNA remaining after a certain dose of γ-radiation wasprovided in de-aerated (Argon saturated), N2O saturated andaerated media respectively. D37 values obtained from Figs. 1–3are summarized in Table 1. In de-aerated (Argon saturated) media

D37 values for DNA in the absence and presence of NaQSH2, Ni(II)–NaQSH2 and Cu(II)–NaQSH2 were 123.46, 270.27, 166.67 and87.72 Gy respectively indicating that Cu(II)–NaQSH2 was the mosteffective in modifying DNA.

Modification of radiation induced double-strand modificationin DNA was expressed in terms of modification ratio (M.R.), whichwas the ratio of the slope of the dose effect curves obtained indifferent medium in the absence or presence of compounds to thatin the absence of any compound in de-aerated (Argon saturated)medium (Das et al., 1996b, 1997). In de-aerated (Argon saturated)media the ratio for NaQSH2 and Ni(II)–NaQSH2 were 0.46 and 0.74respectively while for Cu(II)–NaQSH2 it was 1.41 indicating mod-ification of calf thymus DNA in the presence of NaQSH2 and Ni(II)–NaQSH2 was significantly less than that in the absence of anycompound under the same experimental conditions. In the pre-sence of Cu(II)–NaQSH2, there was an increase in modification ofDNA compared to the situation where DNA was irradiated alone inde-aerated (Argon saturated) medium.

In N2O saturated medium, D37 values in the absence andpresence of NaQSH2, Ni(II)–NaQSH2 and Cu(II)–NaQSH2 as calcu-lated from the dose effect curves (Fig. 2) were 62.89, 136.99, 82.64and 47.62 Gy respectively. The modification ratio calculated fordouble-strand modification of DNA in the absence and presence ofNaQSH2, Ni(II)–NaQSH2 and Cu(II)–NaQSH2 complexes were 1.96,0.90, 1.49 and 2.59 respectively. Under the experimental condi-tions the value obtained for the modification of double stranded inthe presence of NaQSH2 was slightly lower than that obtained forDNA in the absence of any compound under deaerated (Argonsaturated) medium (Table 1) clearly indicates that NaQSH2 pre-vented a γ-radiation induced modification of DNA. When the DNAwas irradiated in the presence of Ni(II)–NaQSH2, the observedmodification ratio (1.49) was lower than that obtained for DNAirradiated in the absence of any compound under N2O saturatedconditions. This indicated once again that Ni(II)–NaQSH2 preventsa γ-radiation induced modification of DNA although the effect wasnot as pronounced as that of Ni(II)–NaQSH2 that was irradiated indeaerated (Ar saturated medium). When DNAwas irradiated in thepresence of Cu(II)–NaQSH2 there was a significant increase inmodification ratio clearly indicating that the Cu(II) complex wasable to sensitize γ-radiation induced modification of DNA. The D37

values obtained in N2O saturated media were almost half of thatobtained in de-aerated (Argon saturated) media and likewise themodification ratio was almost double to that obtained in de-aerated media clearly indicating the participation of dOH. Cu(II)–NaQSH2 probably participated in Fenton reaction shown earlier(Das et al., 1997) as a consequence of which the modification ratiowas significantly higher.

P.S. Guin et al. / Radiation Physics and Chemistry 89 (2013) 38–42 41

Radiolysis of water produces the following primary species insolution (Hart and Anber, 1970; Draganic, I.G. and Draganic, Z.D.,1971):

H2O-Hd, dOH, eaq−, H2, H2O2, H3O+

Nitrous oxide used during irradiation enabled the conversion ofhydrated electrons (eaq−) to hydroxyl-radicals.

eaq−+N2O—-N2+dOH+OH−

Therefore, reactions with DNA bases following irradiation weredue to hydroxyl-radicals only (Roots et al., 1982). It was estab-lished earlier that among the radical and molecular species dOHplays a vital role in degrading target molecules (Dizdaroglu et al.,1975; Koppenol et al., 1976; Bertinchamps et al., 1978; von Sonntaget al., 1981; Bhattacharya and Mandal, 1983; Bhattacharya et al.,1990) that was also obtained in the present study. From D37 andmodification ratio values (Table 1) it was evident that double-strand modification of calf thymus DNA was greatly enhanced inN2O saturated medium in comparison to de-aerated (Argonsaturated) medium that was attributed to an increased presenceof dOH in the medium which increased the formation ofC-4΄ radical in deoxyribose unit through the release of a proton(Dizdaroglu et al., 1975).

In aerated media, D37 values were 89.29 Gy (in absence) and84.03, 81.30, 59.52 Gy in the presence of NaQSH2, Ni(II)–NaQSH2

and Cu(II)–NaQSH2 respectively. Modification ratio as calculatedfor γ-radiation induced modification of DNA in the absence andpresence of NaQSH2, Ni(II)–NaQSH2 and Cu(II)–NaQSH2 were 1.38,1.47, 1.52 and 2.07 respectively. Results in aerated medium do notshow any tendency on the part of Ni(II)–NaQSH2 and NaQSH2 thatsuggest they prevent the γ-radiation induced modification of DNAas was observed for these two compounds in de-aerated (Argonsaturated) and N2O saturated medium, although to differentextent. The reason for this was attributed to the presence ofmolecular oxygen in the medium which is a recognized radio-sensitizer itself and whose absence makes tumor cells becomeresistant to radiotherapy (Wouters et al., 2007; Anoopkumar-Dukie et al., 2009). Hence the radiation protection behavior ofNaQSH2 and Ni(II)–NaQSH2 as seen in the two other media wasprobably operative here also but the radiosensitizing ability ofmolecular oxygen masked their radioprotective action as a resultof which both compounds appeared radiosensitizing. The Cu(II)complex in aerated medium was an almost comparable radio-sensitizer to that found in N2O saturated medium.

NaQSH2 and Ni(II)–NaQSH2 showed that they could prevent theγ-radiation induced modification of DNA in dearated (Argonsaturated) as well as N2O saturated medium which was differentfrom the behavior of 1,2-dihydroxy-9,10-anthraquinone and its Ni(II) complex (Das et al., 1995, 1996b, 1997, 2009). The mechanismfor the radioprotection of DNA that was observed could be due totwo reasons. One being kinetic, where the rate of interaction ofNaQSH2 and Ni(II)–NaQSH2 with dOH was faster than with DNAalone thereby resulting in less DNA damage than when DNA wasirradiated alone. The other aspect could be that dOH initiallyinteracts with a nucleic acid base leading to base modificationwhich is reversed by the action of NaQSH2 and its Ni(II) complex.Details of the mechanism involved would be taken up later.

In order to confirm that the radiation induced damage ofdouble-stranded DNA occurred due to dOH produced during theradiolysis of water the radiation chemical studies were repeated inthe absence and presence of NaQSH2 and its metal complexes inde-aerated (Argon saturated) and N2O saturated medium in thepresence of a radical scavengers like tertiary-butanol. It wasobserved that the loss of fluorescence of the irradiated DNA–EB

complex was negligible indicating that dOH was involved as theprincipal damaging agent.

4. Conclusion

It was observed that in deaerated (Argon saturated) and N2Osaturated aqueous media in the presence of NaQSH2 and Ni(II)–NaQSH2 double-strand modification of calf thymus DNA was muchless than when DNAwas irradiated alone indicating that these twocompounds acted as radioprotectors. In the presence of Cu(II)–NaQSH2 double-strand modification of DNA increased significantlyindicating that the Cu(II) complex was an efficient radiosensitizer.In N2O saturated medium, double-strand modification was almostdouble to that observed in de-aerated media indicating dOHproduced during radiolysis of water plays a major role in the basedegradation process. In aerated media, the property of radio-protection due to NaQSH2 diminished to a considerable extent incomparison to that observed in de-aerated (Argon saturated)medium while Ni(II)–NaQSH2 behaved as a radiosensitizer withalmost equal efficiency as that observed in N2O saturated medium.The Cu(II)–NaQSH2 complex was an even better radiosensitizer inaerated medium than in de-aerated (Argon saturated) medium butslightly less effective compared to its action in N2O saturatedmedium. Results of the aerated medium reiterate the role ofmolecular oxygen in the sensitization process.

From this study, it could be concluded that sodium 1,4-dihydroxy-9,10-anthraquinone-2-sulfonate and its Cu(II) complexcould act as radiosensitizers on target tumor cells which isimportant as hydroxy-9,10-anthraquinones act as mimic mole-cules to the anthracyclines. With this advantage, hydroxy-9,10-anthraquinones could be used both in chemotherapy as well asradiotherapy of cancer.

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