Radiation-induced micronucleus formation and DNA damage in human lymphocytes and their prevention by antioxidant thiols

Mutation Research 676 (2009) 62–68

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Radiation-induced micronucleus formation and DNA damage in human lymphocytes and their prevention by antioxidant thiols Prabha Tiwari a , Amit Kumar b , S. Balakrishnan a , H.S. Kushwaha a , K.P. Mishra c,∗ a b c

Radiological Physics and Advisory Division, Bhabha Atomic Research Centre, Mumbai 400 085, India Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085, India United Research Center, United Institute of Technology, Allahabad 211 010, India

a r t i c l e

i n f o

Article history: Received 3 November 2008 Received in revised form 6 March 2009 Accepted 29 March 2009 Available online 5 April 2009 Keywords: Antioxidant thiols Ionizing radiation Comet assay DNA damage Oxidative stress Micronucleus

a b s t r a c t Thiol family of antioxidants has been considered to be the most effective class of radio protective agents. Present study reports a comparative evaluation of antioxidant thiols, namely N-acetyl cysteine (NAC), glutathione (GSH) and thioproline (TP), on gamma radiation-induced damage to human lymphocytes DNA as assessed by micronucleus (MN) formation and comet assay parameters. Pretreatment of cells with NAC, GSH and TP showed significant protection against DNA damage and MN frequency in irradiated lymphocytes (2–4 Gy). The magnitude of DNA damage protection was found to be concentration dependent (100–300 ␮M) which followed the order GSH > NAC > TP. Further, antioxidant thiols mediated protection against DNA damage in irradiated lymphocyte showed significant correlation with their ability to decrease intracellular ROS but not to the increase in intracellular GSH. Experiments on the effect of antioxidant thiols on plasmid DNA irradiated under cell free aqueous conditions showed that NAC exerts greater protection than GSH against radiation damage. TP showed similar responses in cellular and plasmid DNA. Greater protection of plasmid DNA by NAC is ascribable to its more potential hydrogen donor ability as revealed by radical chromogen 2,2-diphenyl-1-picrylhydrazyl (DPPH) photometric assay. Thus, present study indicated that radioprotection of lymphocytes DNA by antioxidant thiols are closely correlated to the reduction of cellular oxidative stress, which seems to involve multiple mechanisms. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Protection against ionizing radiation has practical applications in cancer radiotherapy and in the reduction of risk to exposed individuals. Many natural and synthetic/semisynthetic chemicals have been investigated in the recent past for their efficacy to reduce adverse effects of ionizing radiation [1,2]. However, the inherent toxicity for some of the synthetic agents at radioprotective concentration warranted further search of safer and effective compounds [3–5]. Therefore, within these constraints, the strategy to evaluate the radiation protection ability of non-toxic and physiologically acceptable compounds seems promising and warrants investigation. It is widely accepted that most of the radiation induced biological damage arises from the interaction of free radicals with vital cellular biomolecules such as DNA, proteins and lipids [6,7]. Neutralization of ROS is the one mechanism by which antioxidants influence the indirect action of radiation [8]. Hence, modulat-

∗ Corresponding author at: Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India. Tel.: +91 22 25100093, 09320466999 (mobile); fax: +91 22 2550 5151. E-mail addresses: mishra [email protected], [email protected] (K.P. Mishra). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.03.007

ing extracellular and/or intracellular antioxidants may provide a convenient strategy to protect against ionizing radiation-induced damage. Cellular and blood levels of antioxidant thiols were found to be decreased during exposure to ionizing radiation [9–11]. Therefore, additional antioxidant thiol supplements may prove useful in reducing radiooxidation-mediated cellular injury. Antioxidant thiols such as N-acetyl-l-cysteine (NAC) and reduced glutathione (GSH) are considered to be the most “natural” of the thiol protectors and are approved for human use for various purposes [1]. One such thiol antioxidant, thioproline (TP; thiazolidine-4-carboxylic acid) was found to improve mouse life span and stimulates immune system [12]. Earlier studies have demonstrated that NAC and GSH have an ability to inhibit radiation-induced damage in mammalian cells [13–15]. Recently, radioprotective effects of NAC have been demonstrated on radiation toxicity in intestine [16] and in hepatic tissue [17]. NAC acts as a precursor of GSH and, increase in intracellular GSH, a cysteine containing tripeptide (␥-glutamylcysteinylglycine) has been shown to be useful in oxidative stress associated conditions [18]. However, GSH itself does not enter inside the cells efficiently [19]. Therefore, cell permeable esters of GSH have been designed to increase the efficacy of intracellular protection from oxidative stress [20]. Nevertheless, extracellular GSH has been shown to play specific biological

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functions such as protection of critical thiol groups of membrane transporters and receptor, maintenance of extracellular redox state and acting as a substrate for extracellular GSH-dependent enzymes [21,22]. More recently, oral administration of GSH has been shown to protect radiation induced declining of lymphocytes count in rat [23]. However, studies are rare on comparative evaluation of antioxidant thiols such as NAC, GSH and TP on radiation induced oxidative injury to lymphocytes isolated from whole blood. Therefore, protective efficacy of NAC, GSH and TP against radiation toxicity measured in terms of DNA damage (micronucleus formation and comet assay parameters) and cellular oxidative stress may further advance our knowledge in understanding the mechanism of their protective action. Results of present study indicated that pretreatment of NAC, GSH and TP at equimolar concentration to human peripheral blood lymphocytes exerted different level of protection against gamma radiation induced intracellular oxidative stress and DNA damage. Further, obtained results on activity of antioxidant thiols to DPPH free radical and super coiled plasmid DNA irradiated in hydrated condition have been discussed in view of mechanism of their differential radioprotective response. Our work points to the fact that an approach for the development of antioxidant thiols as an adjuvant for radioprotection may prove useful in practical situations. 2. Materials and methods 2.1. Chemicals NAC, GSH, TP, high melting agarose, low melting point agarose, FicollHistopaque, Na2 -EDTA, Triton X-100, dimethyl sulfoxide (DMSO), Tris-base, ethidium bromide dichlorodihydrofluorescein diacetate (H2 DCFDA), 2,2-diphenyl1-picrylhydrazyl (DPPH) and propidium iodide were obtained from Sigma Chemical Co. Inc. (St. Louis, MO, USA). pBR322 plasmid was obtained from Banglore Genei (India). Glutathione detection kit was purchased from Chemicon (CA, USA). 2.2. Lymphocyte isolation and gamma irradiation Experiments were performed with blood samples from three healthy nonsmoking male volunteers aged between 25 and 35 years. Blood samples were collected in heparinized tubes. Lymphocytes were isolated from whole blood using Ficoll-Histopaque. Blood was diluted 1:1 with phosphate buffered saline (PBS) and layered onto Histopaque at a ratio of 4:3 (blood + PBS:Histopaque). Blood was then centrifuged at 400g for 30 min at room temperature. The lymphocyte layer was removed carefully and washed twice in PBS at 250g for 10 min each, and then used for further experiments [24]. For irradiation Junior Theratron Teletherapy unit (AECL, Otawa, Canada) with a dose rate 0.38–0.5 Gy/min at 38 cm was used.

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frosted microscope slides (Gold Coin, Mumbai, India) were covered with 200 ␮l of 1% normal melting agarose (NMA) in PBS at 45 ◦ C and immediately coverslipped, and were kept at 4 ◦ C for 10 min to allow the agarose to solidify. Removal of the coverslip from the agar layer was followed by addition of a second layer of 200 ␮l of 0.5% low melting agarose (LMA) containing approximately 105 cells at 37 ◦ C. Cover slips were immediately placed and the slides were put to 4 ◦ C. After solidification of the LMA, the cover slips were removed and slides were placed in a chilled lysing solution containing 2.5 M NaCl, 100 mM Na2 -EDTA, 10 mM Tris–HCl, pH 10 and 1% DMSO, 1% Triton X-100 and 1% sodium sarcosinate for 1 h at 4 ◦ C. The slides were then removed from the lysing solution and placed on a horizontal electrophoresis tank filled with freshly prepared alkaline buffer (300 mM NaOH, 1 mM Na2 -EDTA and 0.2% DMSO, pH ≥13.0). The slides were equilibrated in the same buffer for 20 min and electrophoresis was carried out at 25 V, 180 mA for 20 min. After electrophoresis, the slides were washed gently with 0.4 M Tris–HCl buffer, pH 7.4, to remove the alkali. The slides were stained with 50 ␮l of propidium iodide (PI, 20 ␮g/ml) and visualized using a Carl Ziess fluorescent microscope (Axiosokop) with bright field phase-contrast at an epi-fluorescence facility. The images (100 cells/slide) were captured with a highperformance JVG TK 1208E color video camera. The integral frame grabber used in this system (Cvfb1p) is a PC-based card and it accepts colour composite video output of the camera. The quantification of the DNA strand breaks of the stored images was performed using CASP software by which the percentage of DNA in the tail, tail length, tail moment and Olive tail moment could be obtained directly [28]. 2.5. Intracellular ROS estimation The generation of ROS was detected by dichlorofluorescein diaacetate assay. Separated human lymphocytes (2 × 105 cells/ml) were incubated for 30 min at 37 ◦ C with 100 and 300 ␮M of antioxidants. Cells were then incubated with DCHFDA (10 ␮M) at 37 ◦ C for 15 min and irradiated at 2 and 4 Gy. The change in fluorescence intensity of the resultant 2 ,7 -dichlorofluorescin was measured in a fluorimeter (LS50B, Perkin Elmer, USA) with ex /em = 490/520 nm. The results were compared with the untreated control. The result was expressed as the ratio of the percentage relative increase in the intensity of treated cells to that of untreated controls. Mathematically, the percentage relative increase equaled (Ft /F0 ) × 100, where Ft is the intensity of the treated cells and F0 is the intensity of the untreated cells [29]. 2.6. Intracellular GSH estimation GSH estimation was done using Glutathione Detection Kit (Chemicon, USA). As described previously [30]. Antioxidant thiols were added to isolated lymphocytes (5 × 106 cells) in 1 ml of PBS maintaining final effective concentration of 100 and 300 ␮M. Cells were incubated in CO2 incubator for 30 min at 37 ◦ C and then were exposed to 4 Gy of radiation. After irradiation, Cells were centrifuged and washed with fresh buffer. Cell pellet was lysed in 200 ␮l of 1× lysis buffer on ice for 10 min. After centrifugation cell lysate was transferred into a new microcentrifuge tube. After this 90 ␮l of lysate and 10 ␮l of monochlorobimane (MCB) was added to a 96-well plate. Plate was incubated for 1 h at room temperature in dark. Fluorescence intensity was measured in fluorimeter (Perkin Elmer model LS50B) at ex /em = 380/460 nm. Results were expressed as relative fluorescence unit (RFU) of triplicate samples of two independent experiments [30]. 2.7. Supercoiled plasmid DNA damage assay

2.3. Micronuclei assay Peripheral blood was collected in sterile vacutainers containing heparin as an anti-coagulant. The stock solutions of antioxidants were prepared in medium and filter sterilized using a Millipore syringe filter (0.22 ␮m). Antioxidants were added to the lymphocyte suspensions to achieve a final concentration (100–300 ␮M). Samples were incubated for 30 min at 37 ◦ C in a CO2 incubator and subsequently exposed to 2 and 4 Gy of ␥-radiation. After irradiation, the lymphocytes were washed with culture medium and the cells were cultured with mitogen stimulation to determine radiation-induced genetic damage. Cultures were set up with 5 ml of RPMI 1640 medium supplemented with 15% fetal calf serum, 1% reconstituted phytohaemagglutinin (PHA), and 125 mM L-glutamine for 72 h. Cytochalasin B (6 ␮g/ml) was added to the cultures at 44 h after the initiation of the cultures, and cultures were then terminated at 72 h with mild hypotonic treatment (chilled KCl 75 mM) and fixed in 4:1 methanol:acetic acid. After three washes, cells were gently dropped on a wet slide and stained with 2% Geimsa in Sorenson’s buffer pH 6.8 for 10–12 min. All slides were coded and evaluated for the frequency of micronuclei in cytokinesis blocked binucleated (BN) cells with well-preserved cytoplasms under a light microscope. A total of 1000 BN cells were analysed from each experimental sample [25,26]. 2.4. Single cell gel electrophoresis (SCGE: comet assay) Antioxidants were added to isolated lymphocytes in 1 ml of PBS maintaining the final concentration (100–300 ␮M). Cells were incubated for 25–30 min in a CO2 incubator and were then exposed to 4 Gy of ␥-radiation. Radiation-induced DNA damage in the lymphocytes was measured as strand breaks using single cell gel electrophoresis (comet assay) as described with some modifications [27,28]. In brief,

To examine the effects of irradiation under cell free conditions, the effect of antioxidants on DNA damage was estimated in irradiated plasmid pBR322 DNA using a plasmid relaxation assay. The induction of strand breaks in plasmid pBR322 DNA in vitro by ␥-radiation was studied using the agarose gel electrophoresis method. Plasmid pBR322 DNA (250–300 ng) with or without antioxidants, in 10 ␮l of 100 mM PBS (pH 7.4), were exposed to ␥-radiation (50 Gy). The supercoiled (sc) and open circular (oc) forms of DNA were separated by agarose gel electrophoresis using a 1% agarose gel in 100 mM Tris acetate, 2 mM EDTA buffer of pH 8.3. DNA bands after staining with ethidium bromide were photographed using a SynGene Genius gel documentation system (Biodigital, Switzerland). The images of DNA bands were analyzed using software provided along with the system. Radiation-induced DNA damage was estimated as an increase in the open circular (oc) form of DNA. The percentage of the oc and sc form of each DNA sample was calculated from the total intensities of the oc and sc bands {(oc) OR (sc)/[(sc) + (oc)] × 100}. Furthermore, all values were compared as a percentage of sc in the unirradiated control, [(% sc of test)/(sc of control) × 100] [31]. 2.8. DPPH free radical assay Free radical scavenging capacity was measured using the radical chromogen 2,2-diphenyl-1-picrylhydrazyl (DPPH) photometric assay. Different concentrations of antioxidant thiols (10–1600 ␮M) were maintained in 1 ml of 0.1 mM DPPH solution and were allowed to react at room temperature. After 30 min, the absorbance (Ab) values were spectrophotometrically measured at 517 nm (Jasco V-550, Japan) to monitor the disappearance of the radical chromogen, and were converted into the percentage antioxidant activity by using the following equation: % antioxidant activity = 1 − (Ab of sample/Ab of blank) × 100 [32].

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efficiency of GSH was found to be significantly (p < 0.05) higher than NAC (Fig. 1A) except at 4 Gy of irradiation, NAC (300 ␮M) mediated protection was fairly comparable to GSH (Fig. 1B). Individual treatment of only NAC, GSH and TP to lymphocytes did not result in any significant modification of MN frequency compared to unirradiated control (Fig. 1A). Nuclear division index, NDI = (M1 + 2(M2) + 3(M3) + 4(M4))/N, where M1–M4 represent the number of cells 1–4 nuclei and N represents the total number of viable cells scored was found similar in untreated control (NDI = 2) and antioxidant-treated lymphocytes (NDI = 2). 3.2. Effect of antioxidant thiols on comet parameters of irradiated lymphocyte DNA To investigate the effect of pretreatment of antioxidant thiols on magnitude of DNA damage present in irradiated lymphocytes, single cell gel electrophoresis was performed. Results (Fig. 2) showed an increase in comet parameters, such as tail length (A), % DNA in the tail (B), tail moment (C) and olive tail moment (D) of lymphocytes, which were exposed to 4 Gy of ␥ radiation. These results further revealed that the presence of antioxidant thiols (100–300 ␮M) progressively decreased the comet assay parameters in irradiated lymphocytes. Similar to micronucleus frequency reduction, the pattern of comet assay parameter modification observed for antioxidant thiols was GSH > NAC > TP. Incubation of unirradiated cells with antioxidants at concentrations up to 300 ␮M did not result in any significant alteration of the comet parameters as compared with that from unirradiated control cells. 3.3. Effect of pretreatment of antioxidant thiols on radiation-induced intracellular ROS

Fig. 1. Effect of N-acetyl cysteine (NAC), Glutathione (GSH) and Thioproline (TP) on micronucleus frequency in irradiated human lymphocytes. Cells preincubated with or without antioxidants individually (NAC, GSH and TP, 100–300 ␮M) for 30 min at 37 ◦ C were exposed to 2 Gy (A) and 4 Gy (B) of gamma radiation. Cells were processed for the measurement of MN frequency in lymphocytes as described in Section 2. Results are expressed as the mean ± S.D. of duplicate determinations from three independent experiments. NAC 100 (NAC 100 ␮M), NAC 300 (NAC 300 ␮M), GSH 100 (GSH 100 ␮M), GSH 300 (GSH 300 ␮M), TP 100 (TP 100 ␮M) and TP 300 (TP 300 ␮M). *Significantly lower at p < 0.001 compared to respective irradiated control (2 or 4 Gy). # Significantly lower at p < 0.05 compared to respective concentration of NAC (100 or 300 ␮M) and radiation (2 or 4 Gy). 2.9. Statistical analysis Data are presented as the mean ± standard deviation (S.D.). The mean of the treatment group and controls were compared using the Student’s t test. Differences where p < 0.05 were considered to be statistically significant. The significance of correlation between the parameters was determined by Pearson’s correlation coefficient test using Origin 6.0 software.

3. Results 3.1. Effect of antioxidant thiols on  radiation-induced micronuclei formation Results have shown that individual treatment of NAC, GSH and TP significantly (p < 0.001) reduce the frequency of micronuclei in lymphocytes exposed to gamma radiation at 2 and 4 Gy, and such reductions were concentration dependent (Fig. 1A and B) which followed the order GSH > NAC > TP. Among tested antioxidants (at 100 and 300 ␮M), GSH and NAC were found to be significantly (p < 0.001) more effective than TP in lymphocytes exposed to radiation (2 and 4 Gy). When comparing GSH to NAC, the protection

To evaluate the role of oxidative damage in cells and to find possible correlations between oxidative stress modification and the extent of DNA damage reduction after pretreatment with the antioxidant thiols, intracellular ROS in irradiated lymphocytes were measured using a DCH-FDA fluorescence assay. Results (Fig. 3) showed that, at doses of 2–4 Gy of radiation, ROS generation was found to be increase in lymphocytes. Further, pretreatment of lymphocytes with thiols (100–300 ␮M) significantly decreased ROS level in irradiated lymphocytes, which showed concentrationdependent effect. The order of effectiveness of these antioxidant thiols was found to follow GSH > NAC > TP. It was also observed that similar to micronucleus frequency and comet assay parameters, NAC and GSH showed significantly higher protection than TP against radiation-induced ROS generation in lymphocytes. Only antioxidants treatment (300 ␮M) to lymphocytes showed marginal decrease in basal level of ROS compared to unirradiated control. 3.4. Effect of antioxidant thiols on intracellular GSH To evaluate the role of intracellular GSH on ROS reduction in irradiated lymphocytes pretreated with antioxidants thiols, GSH level in lymphocytes was estimated. Results (Fig. 4) showed that GSH level was decreased significantly (p < 0.001) in irradiated lymphocytes (4 Gy) compare to unirradiated control. Only pretreatment of NAC (100–300 ␮M) among studied antioxidant thiols showed protection against radiation induced GSH depletion. NAC, GSH and TP treatment to unirradiated lymphocytes showed marginal increase in GSH level compared to unirradiated control. 3.5. Effect of antioxidant thiols on plasmid DNA against radiation-induced strand breaks To study the protective efficacy of antioxidants thiol on cell free DNA irradiated in aqueous condition, an assay was performed using

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Fig. 2. Comet assay parameters of lymphocytes DNA. Cells were exposed to gamma radiation (4 Gy) preincubated with or without antioxidants individually (NAC, GSH and TP, 100–300 ␮M) for 30 min at 37 ◦ C. After radiation exposure samples were subjected to an alkaline comet assay to monitor DNA damage. Results are expressed as the mean ± S.D. of duplicate determinations from three independent experiments. Comparison was drawn between different antioxidants using the parameters of (A) tail length, (B) DNA (%) in the tail, (C) tail moment and (D) olive tail moment. *Significantly lower at p < 0.001 compared to respective irradiated control (4 Gy). # Significantly lower at p < 0.05 compared to respective concentration of NAC (100 or 300 ␮M) + radiation (4 Gy).

supercoiled plasmid circular DNA (ccc) which gets converted into open circular form (oc) due to strand breaks. It can be seen (Fig. 5) that ∼55% of the ccc form of plasmid DNA was converted into the oc form at a dose of 50 Gy. Further, results showed that presence of antioxidant thiols (100–300 ␮M) significantly protected (p < 0.001) radiation-induced relaxation of ccc form of plasmid DNA. These antioxidants at 300 ␮M treatment showed higher protection than 100 ␮M treatment, but the significance level was small. NAC was found to be more effective than GSH and TP in protecting plasmid DNA against radiation damage. Incubation of only NAC, GSH and TP did not affect supercoiled status of plasmid DNA compared to untreated control.

3.6. DPPH radical activity of NAC, GSH and TP In order to estimate antiradical activity of NAC, GSH and TP to organic radical, a standard DPPH assay was performed by incubating DPPH with increasing concentrations of NAC, GSH and TP (0–1800 ␮M). Results showed that NAC was more effective to neutralize DPPH free radicals compared to GSH and TP (Fig. 6). An inhibitory concentration (IC50 : concentration required to neutralize DPPH radicals by 50%) was 25 ␮M for NAC, 200 ␮M for GSH and 1600 ␮M for TP.

4. Discussion Considerable efforts have been devoted to explore the potential of plant products and extracts, as well as synthetic products, to reduce the undesirable adverse effects of ionizing radiation. However, several potential compounds have failed to pass the transition from bench to bedside because of unacceptable behavioral toxicity and side effects [3–5,33]. Therefore, present study evaluated non-toxic and physiologically acceptable compounds [34–36]. Very recently, protective effects of NAC and GSH were found comparable to the clinically used radioprotector, amifostine (WR2721) in rodents [15,37,38]. The micronucleus test and comet assay have been used as established methods and become a most reliable indicator of radiation-induced genetic damage [26,39]. Therefore, radiation damage to DNA was determined by micronucleus frequency and comet assay parameters in human peripheral blood lymphocytes exposed to ␥ radiation and the effect of antioxidant thiols as modulators of the induced damage. Similar nuclear division index (NDI = 2) of untreated control and antioxidanttreated lymphocytes suggests that these antioxidant thiols in the employed concentration range were without significant cytotoxic effects. In present study, antioxidant thiols (NAC, GSH and TP) have shown different level of protection against radiation-induced dam-

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Fig. 5. Protection of plasmid DNA irradiated in presence of antioxidant thiols. Supercoiling of control plasmid was considered as 100% to calculate supercoiling (%) in irradiated sample in presence or in absence of antioxidant thiols. The values are expressed as mean ± S.D. of triplicates from two independent experiments. *Significantly higher at p < 0.001 compare to radiation control (50 Gy). # Significantly higher compared to 50 Gy + GSH 100 or 300 at p < 0.001.

Fig. 3. Effect of pretreatment of NAC, GSH and TP on intracellular ROS in lymphocytes exposed to 2 Gy (A) or 4 Gy (B) of radiation as measured by the DCH-FDA method. The values are expressed as mean ± S.D. of triplicates. Significantly lower compared to respective irradiated control (2 or 4 Gy) at p < 0.001 (*) or at p < 0.05 (#).

Fig. 4. Cellular GSH level in human lymphocytes. Cells were untreated (control), or treated with only antioxidant thiols (NAC 300, GSH 300, TP 300) or pretreated with antioxidant thiols followed by irradiation (4 Gy). The values are expressed as mean ± S.D.*Significantly lower at p < 0.001 compare to unirradiated control.

age to DNA. At 300 ␮M concentration, NAC showed protective effect comparable to GSH against MN frequency induced by 4 Gy dose of radiation (Fig. 1B). Taking together with the protection of comet assay parameters by NAC and GSH (300 ␮M at 4 Gy), which revealed that GSH effectively protected radiation damage to lymphocytes DNA than NAC (Fig. 2). Thus, this study further support that comet assay is capable to detect the DNA damage more sensitively than MN assay [39]. Results (Figs. 1 and 2) showed that GSH was significantly more effective than NAC and TP against radiation induced DNA damage, which could be due to its greater ability to attenuate ROS than NAC and TP as evidenced by the pattern of intracellular ROS reduction in lymphocytes. TP was found to be least effective in MN frequency reduction as consistent with the extent of ROS reduction in TP-treated lymphocytes. Statistical analysis showed a significant correlation (r = 0.97, p < 0.001) between the percentage ROS reduction (Fig. 3) and DNA damage modification measured in terms of MN frequency (Fig. 1) and comet parameters (Fig. 2), suggesting a significant role for antioxidant action of these molecules

Fig. 6. Antiradical activity of antioxidant thiols (NAC, GSH and TP) to 2,2-diphenyl1-picrylhydrazyl (DPPH) at different concentrations as described in Section 2. Values represent the mean ± S.D. of triplicates.

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in modifying radiation-induced oxidative damage to DNA. Intracellular level of GSH (Fig. 4) did not show significant correlation with the extent of ROS reduction and DNA damage protection in irradiated lymphocytes preincubated with antioxidant thiols. Taking together, this analysis indicates the potential role of extracellular antioxidant thiols in protection of intracellular oxidative stress and associated DNA damage in lymphocytes. Present observations further get support from previously reported study [40], which revealed that NAC and glutathione ethylester mediated radioprotection showed correlation to the concentration of thiols in medium but not to the intracellular level of GSH in UV irradiated human cultured cells. Further, deeper studies are required to understand the mechanism of intracellular protection due to extracellular presence of thiols and its impact on redox conditions in the local environment. Most of the radiation-induced damage to biomolecules is mediated through ROS derived from radiolysis of water (indirect effect). Thus, irradiation of DNA under dilute aqueous condition, where indirect effect becomes dominant, may provide better understanding of comparative efficacy of antioxidants (e.g. NAC, GSH and TP) to scavenge primary water radicals. Greater radioprotective effect of NAC than GSH and TP on plasmid DNA (Fig. 5) can be explained by in part due to its higher potential of H donation from –SH function to radiation induced *OH radicals and to damaged deoxyriboses of DNA as evidenced by DPPH assay (Fig. 6). DPPH assay is based on the direct transfer of H atom from antioxidants(s) to DPPH radical, which have no other interference such as enzymatic inhibition, or the presence of multiple radicals [41]. In conclusion, the present work has shown that the examined antioxidant thiols (NAC, GSH and TP) have a characteristic level of capability in protecting against radiation-induced damage to cellular DNA in human blood lymphocytes. These observations seem associated with their differential ability to suppress radiationinduced free radicals in lymphocytes. Strategies using compounds or combination of compounds that could up regulate or maintain cellular and extracellular thiol may serve as an effective modulator of radiation damage, which warrants further mechanistic studies. Conflict of interest statement Authors declare that there are no conflicts of interest. Acknowledgements The authors thank Mr. Manjoor Ali for his technical help in irradiating the samples. We further extend our thanks to Dr. Mayya, Head, RP&AD for providing an opportunity to work in RPAD. Ms. Prabha Tiwari would like to acknowledge CSIR-UGC, Government of India for providing her a Fellowship during the course of this work. References [1] J.F. Weiss, M.R. Landauer, Protection against ionizing radiation by antioxidant nutrients and phytochemicals, Toxicology 189 (2003) 1–20. [2] J.M. Gutteridge, B. Halliwell, Free radicals and antioxidants in the year: a historical look to the future, Ann. N. Y. Acad. Sci. 899 (2000) 136–147. [3] M.R. Landauer, H.D. Davis, K.S. Kumar, J.F. Weiss, Behavioral toxicity of selected radioprotectors, Adv. Space Res. 12 (1992) 273–283. [4] H. Monig, O. Messerschmidt, C. Streffer, E. Scherer, C. Streffer, K.R. Trott (Eds.), Chemical Radioprotection in Mammals and in Man. Radiation Exposure and Occupational Risks, Springer-Verlag, Berlin, 1990, pp. 97–143. [5] P.U. Devi, Normal tissue protection in cancer therapy: progress and prospects, Acta Oncol. 37 (1998) 247–252. [6] E.J. Hall, A.J. Giaccia (Eds.), Radiobiology for the Radiologist, Williams & Wilkins, Lippincott, Philadelphia, 2006.

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