Naringin, a grapefruit flavanone, protects V79 cells against the bleomycin-induced genotoxicity and decline in survival

122 A. JAGETIA ET AL. TOXICOLOGY JOURNAL OF APPLIED J. Appl. Toxicol. 2007; 27: 122–132 Published online 19 December 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jat.1175 Naringin, a grapefruit flavanone, protects V79 cells against the bleomycin-induced genotoxicity and decline in survival Abhinav Jagetia,1,* Ganesh Chandra Jagetia2 and Shalini Jha2 1 2 Department of Metallurgy, Malviya National Institute of Technology, Jaipur-302 017, India Department of Radiobiology, Kasturba Medical College, Manipal-576 104, India Received 28 April 2006; Revised 2 August 2006; Accepted 10 August 2006 ABSTRACT: The effect of naringin, a grapefruit flavonone was studied on bleomycin-induced genomic damage and alteration in the survival of cultured V79 cells. Exposure of V79 cells to bleomycin induced a concentration dependent elevation in the frequency of binucleate cells bearing micronuclei (MNBNC) and a maximum number of MNBNCs were observed in the cells treated with 50 µg ml−1 bleomycin, the highest concentration evaluated. This genotoxic effect of bleomycin was reflected in the cell survival, where a concentration dependent decline was observed in the cells treated with different concentrations of bleomycin. Treatment of cells with 1 mM naringin before exposure to different concentrations of bleomycin arrested the bleomycin-induced decline in the cell survival accompanied by a significant reduction in the frequency of micronuclei when compared with bleomycin treatment alone. The cell survival and micronuclei induction were found to be inversely correlated. The repair kinetics of DNA damage induced by bleomycin was evaluated by exposing the cells to 10 µg ml−1 bleomycin using single cell gel electrophoresis. Treatment of V79 cells with bleomycin resulted in a continuous increase in DNA damage up to 6 h post-bleomycin treatment as evident by migration of more DNA into the tails (% tail DNA) of the comets and a subsequent increase in olive tail moment (OTM), an index of DNA damage. Treatment of V79 cells with 1 mM naringin reduced bleomycin-induced DNA damage and accelerated DNA repair as indicated by a reduction in % tail DNA and OTM with increasing assessment time. A maximum reduction in the DNA damage was observed at 6 h post-bleomycin treatment, where it was 5 times lower than bleomycin alone. Our study, which was conducted on the basis of antioxidant, free radical scavenging and metal chelating properties of naringin demonstrates that naringin reduced the genotoxic effects of bleomycin and consequently increased the cell survival and therefore may act as a chemoprotective agent in clinical situations. Copyright © 2006 John Wiley & Sons, Ltd. KEY WORDS: bleomycin; naringin; micronuclei; cell survival; DNA repair; comet; V79 cells Introduction Bleomycin is a glycopeptide antibiotic that has been isolated from Streptomyces verticillus (Umezawa, 1965; Umezawa et al., 1966). Bleomycin is an S-phaseindependent radiomimetic agent that has been used in the treatment of cancers of the head and neck, squamous cell carcinomas, testicular cancer and some lymphomas (Hay et al., 1991; Stubbe et al., 1996). Bleomycin exerts its action via interaction with DNA in a sequence-specific manner resulting in single- and double-strand breaks through formation of kinetically competent activated bleomycin in the presence of Fe2+ and O2 (Sam and Peisach, 1993; Absalon et al., 1995; Harsch et al., 2000). Bleomycin has been reported to induce DNA damage, * Correspondence to: Ganesh Chandra Jagetia, Department of Radiobiology, Kasturba Medical College, Manipal-576 104, Karnataka, India. E-mail: gc.jagetia@gmail.com /gc.jagetia@rediffmail.com Copyright © 2006 John Wiley & Sons, Ltd. point mutations, recombination, chromosome aberrations and micronuclei in diverse organisms, including bacteria, bacteriophage, fungi, drosophila and mammals (Vig and Lewis, 1978; Povirk and Austin, 1991; Hoffmann et al., 1993). The metal-binding portion of bleomycin, which determines the sequence selectivity of strand scission, seems to be oriented in the minor groove of DNA (Kane and Hecht, 1994), where activated bleomycin specifically abstracts hydrogen from the 49 position of deoxyribose, forming a free radical (Stubbe et al., 1996; Burger, 1998). The addition of oxygen to the free radical at 49 gives rise to a peroxyl radical whose decomposition causes the release of a base propenal and a strand break with 59-phosphate and 39-phosphoglycolate ends (Povirk and Houlgrave, 1988; Stubbe et al., 1996; Burger, 1998; Charles and Povirk, 1998). About 10% of the strand breaks induced by bleomycin are double-strand breaks, and they are induced with single-hit kinetics (Charles and Povirk, 1998; Povirk and Houlgrave, 1988). After a single-strand break occurs at a primary cleavage site, the J. Appl. Toxicol. 2007; 27: 122–132 DOI: 10.1002/jat NARINGIN, A GRAPEFRUIT FLAVANONE, PROTECTS V79 CELLS 123 same molecule of bleomycin is apparently reactivated in situ and cleaves the complementary strand, resulting in a blunt-ended double-strand break (Steighner and Povirk, 1990; Povirk and Houlgrave, 1988; Stubbe et al., 1996; Charles and Povirk, 1998). Bleomycin reacts with cytochrome P450 reductase in the presence of reduced NADPH to form semiquinone radical intermediates, which in turn react with oxygen to produce superoxide and hydroxyl radicals, which attack DNA and oxidize DNA bases (Hecht, 1986). Bleomycin toxicity is believed to be mediated by redox cycling of the iron–bleomycin complex. Iron has been reported to play an important role in bleomycin-induced free radical generation and oxidative damage (Caspary et al., 1979). Use of bleomycin in the treatment of cancer is associated with significant morbidity and mortality. It induces pulmonary toxicity and fibrosis of the lungs (Kawai and Akaza, 2003). Therefore, it is imperative to screen agents that can reduce the toxicity of bleomycin in cancer patients. Iron chelators may protect normal cells from bleomycin-induced toxic effects and the chemicals that inhibit the cytotoxicity of anticancer drugs in various ways can act as good ‘chemoprotective’ agents. Flavonoids comprise one of the largest and most widely distributed groups of secondary plant metabolites (Kuhnau, 1976) and they possess promising biological activities. Flavonoids are found in practically all photosynthesizing plants, and therefore, all humans consuming foods of plant origin are exposed to them. Naringin (naringenin 7-rhamnoglucoside), a grapefruit flavanone is present in most of the citrus species such as Citrus paradisi, C. sinensis, C. unshiu, C. nobilis, C. tachibana and C. junos, it is also found in Artemisia selengensis, A. stolonifera (Swiader and Zarawska, 1996), roots of Cudrania cochinchinensis var. geronatogea (Li et al., 1956), areal parts of Thymus herba barona (Corticchiato et al., 1995), fruits of Poncirus trifoliata (Kim et al., 1999), Mabea fistulifera (Graciz, 1997) and Swartzia polyphylla (DuBois and Sneden, 1995). Antioxidant, free radical scavenging and metal chelating properties of naringin have been well documented (Chen et al., 1990; Kuo et al., 1998; Russo et al., 2000; Mira et al., 2002; Jagetia et al., 2003, 2004; Jagetia and Reddy, 2005). Naringin showed an appreciable antioxidant activity by inhibiting malonaldehyde formation from lipids (Singh et al., 2004; Ali and Kader, 2004). Naringin supplementation has been reported to upregulate mRNA expression for certain antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase (Jeon et al., 2001, 2002). Naringin significantly ameliorates hypoglycemia and antioxidant status in streptozotocin-induced diabetic rats (Ali and Kader, 2004). Naringin has been reported to protect against the oxidative stress in ischemia reperfusion or glycerolinduced renal injury in rats (Singh and Chopra, 2004; Singh et al., 2004). It has also been shown to protect Copyright © 2006 John Wiley & Sons, Ltd. against the iron-induced toxicity in mice (Jagetia et al., 2004). Naringin supplementation lowers plasma lipids and enhances erythrocyte antioxidant enzyme activities in hypercholesterolemic subjects (Kim et al., 2004). It has also been reported to inhibit the activity of cytochrome P450 enzymes like CYP1A1, 1A2, 3A4 and other enzymes (Wilcox et al., 1999; Ueng et al., 1999; Bear and Teel, 2000; Le Marchand et al., 2000; AlvarezGonzalez et al., 2001; Schwarz et al., 2005). Flavonoids are generally considered to have low toxicity and a single 2 g oral dose of naringin to a human volunteer had no deleterious effects (Wilcox et al., 1999). The antioxidant, free radical scavenging and metal chelating properties of naringin stimulated us to evaluate its chemoprotective activity against the bleomycininduced genomic damage in cultured V79 cells. Materials and Methods Chemicals Naringin (Fig. 1) was obtained from Across Organics, Geel, Belgium, whereas bleomycin sulphate was procured from Dabur Pharmaceuticals, Mumbai, India. CytochalasinB, MEM, L-glutamine, gentamicin sulfate, fetal calf serum and DMSO were obtained from Sigma Chemical, St Louis, USA. Naringin and bleomycin were dissolved in MEM as required, whereas cytochalasin-B (Cat. No. C-6762) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mg ml−1, stored at −80 °C and diluted with sterile PBS immediately before use. Cell Line and Culture V79, Chinese hamster lung fibroblast cells, procured from the National Center for Cell Sciences, Pune, India, were Figure 1. Chemical structure of naringin, 7-(2-O-(6deoxy-alpha-L-mannopyranosyl)-β -D-glucopyranosyloxy)2,3-dihydro-4′,5,7-trihydroxyflavone J. Appl. Toxicol. 2007; 27: 122–132 DOI: 10.1002/jat 124 A. JAGETIA ET AL. used throughout the study. The cells were routinely grown in 75 cm2 flasks (Falcon, Becton Dickinson, USA) with loosened caps, containing Eagle’s minimum essential medium (MEM) supplemented with 10% fetal calf serum, 1% L-glutamine and 50 µg ml−1 gentamycin sulfate at 37 °C in a CO2 incubator (NuAire, Plymouth, MN, USA) in a humidified atmosphere of 5% CO2 in 95% air. Experimental A fixed number (3 × 106) of exponentially growing V79 cells were inoculated into several culture dishes and divided into the following groups. Bleomycin group: The cultures of this group were treated with 0, 1, 2.5, 5, 10, 25 or 50 µg ml−1 of bleomycin sulfate. Naringin + bleomycin group: This group of cultures was treated with 1 mM naringin (Jagetia et al., 2004) before exposure to different concentrations of bleomycin and a matching control of naringin was also included. After 1 h of naringin treatment the naringin containing media was replaced with a fresh media containing different concentrations of bleomycin for 1 h. The cells were dislodged using trypsin-EDTA treatment and cytotoxicity and genotoxicity was estimated as follows. Clonogenic Assay The modulation of bleomycin-induced cytotoxicity by naringin was measured using a colony-forming assay (Puck and Marcus, 1955). Usually 200 cells were plated on to several culture dishes (Greiner, Bio-One, Frickenhausen, Germany) in 5 ml medium in quintuplicate for each concentration of bleomycin in each group. The cells were allowed to grow for 8 days. The resultant colonies were stained with 1% crystal violet in methanol and clusters containing 50 or more cells were scored as a colony. The plating efficiency of cells was determined and the surviving fraction was fitted on a linear quadratic equation: 2 SF = expγ -α D+β D Micronucleus Assay The left over cells were used for the micronucleus assay where 1 × 106 cells were inoculated in triplicates for each concentration of bleomycin in each group. The micronuclei were prepared according to the modified method of Fenech and Morley (1985). Six hours after the cell attachment, the cells were treated with 3 µg ml−1 of cytochalasin-B to inhibit cytokinesis, left undisturbed and were allowed to grow for another 16 h (Jagetia and Adiga, 1995). Thereafter, the medium containing Copyright © 2006 John Wiley & Sons, Ltd. cytochalasin-B was removed and the cells were washed once with PBS. Finally, the cells were dislodged by trypsin EDTA treatment, centrifuged, subjected to mild hypotonic treatment (0.7% ammonium oxalate) for 5 min at 37 °C, centrifuged again and the resultant cell pellet was fixed in Carnoy’s fixative (3: 1 methanol, acetic acid). The cells were centrifuged again, resuspended in a small volume of fixative and spread on to precleaned coded slides to avoid observer bias. The protection factor (PF) was calculated for each bleomycin concentration as follows: PF = (Bleomycin alone − MEM) [(Naringin + Bleomycin) − Naringin alone] Single Cell Gel Electrophoresis A separate experiment was carried out to assess the repair kinetics of bleomycin-induced DNA damage and its alteration by NIN at different post-bleomycin treatment times. The grouping and other conditions remained similar to that described for the micronucleus assay, except that the cells were treated with 10 µg ml−1 bleomycin and harvested at 0, 0.5, 1, 2, 4 or 6 h post-bleomycin treatment. The comet assay was performed under alkaline conditions essentially according to the procedure of Singh et al. (1988) with minor modifications (Collins et al., 1993). Before analysis of DNA repair kinetics the cells from both the groups were dislodged by trypsin EDTA treatment, washed and resuspended in fresh, drug-free medium and kept on ice to inhibit repair if any. Briefly, frosted slides were covered with 100 µl of 0.6% low melting agarose (Sigma-Aldrich Co., St Louis, USA; Cat No. A-4718) prepared in Ca- and Mg-free PBS at 37 °C and the agarose was allowed to solidify under a cover slip on ice after which the cover slips were removed. One ml aliquots containing 1 × 105 harvested V79 cells in culture medium were centrifuged at 1500 rpm for 5 min. The pelleted cells were resuspended in 80 µl of 1.2% low melting agarose layered on to the first layer and allowed to solidify under a cover slip on ice. All the steps described above were carried out under diffused light to avoid additional DNA damage. The slides embedded with cells were placed in cold lysis buffer containing 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Trizma base, pH 10 and 1% Triton X-100 (added fresh) to solubilize cellular proteins leaving DNA as nucleoids, at 4 °C for 2 h. After cell lysis, the slides were drained of lysis buffer and placed into a horizontal gel electrophoresis tank filled with fresh electrophoresis buffer containing 300 mM NaOH, 1 mM Na2EDTA, pH 13, to a level of ~0.25 cm above the slides. Slides were kept in buffer for 20 min to allow DNA unwinding. Electrophoresis was then carried out for 20 min at 1.25 V cm−1 and 300 mA under cold conditions. The slides were J. Appl. Toxicol. 2007; 27: 122–132 DOI: 10.1002/jat NARINGIN, A GRAPEFRUIT FLAVANONE, PROTECTS V79 CELLS 125 then drained and flooded slowly with three changes of neutralization buffer (0.4 M Trizma base, pH 7.5) for 5 min each and subsequently the slides were stained with 50 µl of ethidium bromide (2 mg ml−1) and covered with a cover slip for immediate analysis. Ethidium bromide stained DNA on each slide was visualized at 40× magnification using fluorescence microscopy as ‘comets’ with a fluorescent head and a tail. The comet images were captured using an epifluorescence microscope (Olympus BX51, Olympus Microscopes, Tokyo, Japan) equipped with a 515–535 nm excitation filter, a 590 nm barrier filter, and a CCD camera (CoolSNAP-Procf Digital Color Camera Kit Ver 4.1, Media Cybergenetics, Silver Spring, Maryland, USA). A total of 50 cells per slide was analysed to give a representative result for the population of cells (Price et al., 2000). The comets thus captured were analysed using Komet Software (Version 5.5, Kinetic Imaging Ltd, Bromborough, UK). Mean olive tail moment (defined as the distance between the profile centers of gravity for DNA in the head and tail) or OTM and percent tail DNA provide a good correlation of genotoxicity (Kumaravel and Jha, 2006) therefore, data for OTM and percent tail DNA were collected from three independent experiments, each containing quintuplicate measures and presented as mean ± SEM (standard error of mean). Figure 2. Alteration in survival of V79 cells treated with 1 mM naringin before exposure to different concentrations of bleomycin. Upper curve: Naringin + Bleomycin and Lower curve: Bleomycin alone Statistical Analyses The statistical analyses were performed using GraphPad Prism 2.01 statistical software (GraphPad Software, San Diego, CA, USA). The significance among all groups was determined by one-way ANOVA and Bonferroni’s post-hoc test was applied for multiple comparisons. Fisher’s exact test was used for micronucleus assay. Results The results are expressed as surviving fraction (Fig. 2), frequency of micronucleated binucleate cells (Fig. 3) and DNA damage (Table 1). Cell Survival Treatment of V79 cells with different concentrations of bleomycin caused a concentration dependent decline in the cell survival expressed as surviving fraction (Fig. 2). Treatment of V79 cells with 10 µg ml−1 bleomycin resulted in a 50% decline in the cell survival and this was considered as the IC50 concentration. A further increase in bleomycin concentration caused a corresponding decline in the survival and approximately 77% of the cells were killed at a concentration of 50 µg ml−1, the highest Copyright © 2006 John Wiley & Sons, Ltd. Figure 3. Alteration in the micronuclei frequency of V79 cells by 1 mM of naringin before exposure to different doses of bleomycin. Upper curve: Bleomycin alone and Lower curve: Naringin + Bleomycin J. Appl. Toxicol. 2007; 27: 122–132 DOI: 10.1002/jat 85.94 ± 0.27a 8.06 ± 0.27a 8.81 ± 0.20a 89.94 ± 0.27a 10.06 ± 0.27a 7.81 ± 0.21a concentration of bleomycin evaluated. Treatment of V79 cells with 1 mM naringin before bleomycin exposure resulted in a significant elevation in the surviving fraction, where an increase of 27% in surviving fraction was observed for 10 µg ml−1 bleomycin. The greater protection against bleomycin-induced decline by naringin was discernible in cells exposed to the higher concentration of bleomycin (Fig. 2). A 55% elevation in the cell survival was observed for V79 cells treated with 1 mM naringin before exposure to 50 µg ml−1 bleomycin. 60.16 ± 0.44 39.83 ± 0.44 39.91 ± 0.34 59.36 ± 1.02 40.64 ± 1.03 40.55 ± 0.99 Bleomycin + Naringin Bleomycin + Naringin Bleomycin Bleomycin + Naringin Bleomycin Micronuclei 85.35 ± 0.49a 8.37 ± 1.05a 5.57 ± 0.96a 80.02 ± 0.03a 7.34 ± 0.05a 4.33 ± 0.96a 90.68 ± 1.05a 9.32 ± 1.05a 6.29 ± 0.47a The effect of naringin on bleomycin-induced DNA damage was investigated by evaluating micronuclei frequency in binucleate cells. The frequency of micronucleated binucleate cells (MNBNC) increased in a concentration dependent manner in V79 cells treated with different concentrations of bleomycin (Fig. 3). The frequency of MNBNCs was greater than two fold in the cells receiving 10 µg ml−1 bleomycin than that of spontaneous MNBNC frequency. A maximum number of MNBNCs was observed in the cells treated with 50 µg ml−1 bleomycin, the highest concentration used, where the frequency of MNBNCs was three fold greater than the baseline frequency. Exposure of V79 cells to 1 mM naringin consistently reduced the bleomycin induced MNBNC frequency (Fig. 3). This decline in MNBNC frequency was significantly lower in the cells receiving 1 mM naringin before treatment with 2.5 to 50 µg ml−1 bleomycin. The protection factor was approximately 1.2 for all bleomycin concentrations except 2.5 and 10 µg ml−1, where it was 1.3. Treatment of V79 cells with 1 mM naringin resulted in a significant reduction in MNBNC frequency. Copyright © 2006 John Wiley & Sons, Ltd. Single Cell Gel Electrophoresis a p < 0.0001 (Bleomycin + Naringin group compared to Bleomycin alone). MEM = Minimum Essential Medium. 99.23 ± 0.05 0.77 ± 0.05 0.26 ± 0.01 Head DNA (%) Tail DNA (%) OTM 99.23 ± 0.05 0.77 ± 0.05 0.26 ± 0.01 95.79 ± 0.39 4.21 ± 0.39 1.56 ± 0.08 90.16 ± 0.09a 0.84 ± 0.09a 0.98 ± 0.03a 75.02 ± 0.05 9.34 ± 1.05 5.31 ± 0.96 82.48 ± 0.05 9.72 ± 0.75 6.58 ± 0.43 89.94 ± 0.27 10.05 ± 0.27 7.85 ± 0.21 Bleomycin + Naringin Bleomycin Bleomycin + Naringin Bleomycin Naringin MEM Bleomycin Bleomycin + Naringin Bleomycin 1 0.5 0 Control Assessment time (hours) Table 1. Repair of bleomycin-induced DNA damage in V79 cells by naringin assessed by comet assay 6 4 A. JAGETIA ET AL. 2 126 Evaluation of DNA damage by single cell gel electrophoresis revealed that most of the DNA was in the cell nucleus (head of the comet) in the cells treated or not with naringin alone indicating that naringin treatment did not increase the spontaneous DNA damage (Table 1). Exposure of V79 cells to 10 µg ml−1 bleomycin resulted in a time dependent elevation in the DNA damage as evidenced by a greater migration of DNA from the head of the comets to the tail. The greatest DNA damage was observed at 6 h post-bleomycin treatment, however, the difference between 4 and 6 h was statistically nonsignificant (Table 1). Approximately 40% of the DNA migrated into the comet tails at 4 h post bleomycin treatment. This was reflected in the OTM, which also increased with assay time, and a maximum OTM was reported at 6 h post-bleomycin treatment. Treatment of V79 cells with 1 mM naringin before bleomycin J. Appl. Toxicol. 2007; 27: 122–132 DOI: 10.1002/jat NARINGIN, A GRAPEFRUIT FLAVANONE, PROTECTS V79 CELLS 127 exposure showed a pattern similar to that of bleomycin alone except that the naringin pretreatment inhibited the bleomycin-induced DNA damage and only 10% DNA was found in the comet tails as against 40% in bleomycin treatment alone after 4 h of bleomycin treatment. This was also indicated by a reduction in OTM, which was approximately five times lesser at 4 and 6 h in the naringin pretreated group (Table 1). The steady reduction in bleomycin-induced DNA damage by naringin indicated an efficient repair of the bleomycin-induced DNA strand breaks and alkali labile sites. This reduction in DNA damage is translated into the increased survival of cells, where a 38% increase in the cell survival was observed in the naringin + bleomycin group compared with the bleomycin treatment alone (Fig. 2). Biological Response The biological response of bleomycin or naringin + bleomycin treatment was evaluated by correlating the frequency of MNBNC with surviving fraction, where the surviving fraction was plotted on the x-axis whereas the MNBNC frequency on y-axis. The surviving fraction of V79 cells declined with increasing frequency of MNBNC in the bleomycin treated group and the lowest survival was recorded for the concentration that induced the greatest number of MNBNC. The correlation between the cell survival and micronuclei formation was linear quadratic (Fig. 4a). Treatment of V79 cells with 1 mM naringin before exposure to various concentrations of bleomycin also showed a similar correlation between surviving fraction and MNBNC induction except that the correlation between surviving fraction and MNBNC formation was linear for the cells treated with 1 mM naringin before bleomycin treatment (Fig. 4b). Discussion Chemotherapy has been used successfully to treat several neoplastic disorders in man. The use of single or combination of chemotherapeutic agents has no doubt increased the survival of patients receiving these treatments. However, one of the important clinical consequences of their use has been the induction of secondary malignancies in patients receiving chemotherapy (Pedersen-Bjergaard and Rowley, 1994). Bleomycin is one of the cytotoxic agents used either alone or in combination with other drugs to treat neoplastic disorders. The aim of the present investigation was to evaluate the protective potential of naringin, a bioflavonoid on cell survival and DNA damage in V79 cells treated with bleomycin. The cytotoxic effect of antitumor agents in a biological system must be defined by its impairment of the reproductive integrity of individual cells. In in vitro Copyright © 2006 John Wiley & Sons, Ltd. Figure 4. Correlation between cell survival and micronuclei formation in V79 cells treated or not with 1 mM NIN before exposure to different concentrations of bleomycin. a: Bleomycin alone; b: Naringin + Bleomycin J. Appl. Toxicol. 2007; 27: 122–132 DOI: 10.1002/jat 128 A. JAGETIA ET AL. systems, this impairment can be assessed by the inability of cells to proliferate indefinitely and form colonies under the appropriate experimental conditions. Bleomycin treatment resulted in a concentration dependent decline in the survival of V79 cells. An identical effect has been reported earlier in cultured fibrosarcoma (SA-1) and Ehrlich lettre ascites carcinoma cells receiving bleomycin (Cemazar et al., 1998). Other chemotherapeutic agents such as paclitaxel, vindesine, doxorubicin and teniposide have been reported to cause a concentration dependent decline in the cell survival earlier (Babudri et al., 1984; Rownisky et al., 1988; Hanuaske et al., 1992; Jagetia and Adiga, 1995, 1998; Jagetia and Nayak, 1996; Adiga and Jagetia, 1999). Pretreatment of V79 cells with 1 mM naringin inhibited the bleomycin-induced decline in the cell survival and this inhibition was greater at the highest concentration of bleomycin (see Fig. 2). Reports regarding the use of naringin to protect against the bleomycin-induced cytotoxic effects are unavailable. However, naringin has been reported to protect V79 cells against iron-induced cytotoxicity (Jagetia et al., 2004). 2-[(Aminopropyl)amino] ethanethiol (WR-1065) has been reported to protect against the bleomycininduced cytotoxicity in V79 cells (Nagy and Grdina, 1986). Amifostine, an aminothiol, has also been reported to protect against the melphalan-induced genotoxicity (Buschini et al., 2000). Micronuclei are acentric fragments or a complete chromosome that fails to attach to the mitotic spindle during cytokinesis and are excluded from the main nuclei. Different mechanisms may be involved in the formation of micronuclei, including chromosome breakage (clastogenesis) and spindle disruption (aneugens) (Heddle et al., 1983; Majer et al., 2001). Yet micronuclei are among the most extensively used cytogenetic markers that indicate early biological effects associated with DNA-damaging agents. Among various techniques used to detect genetic and genotoxic effects, the micronucleus test is simple, cheap, less cumbersome and allows convenient and easy application. The protection afforded by naringin against bleomycin-induced cytotoxicity was confirmed by micronucleus assay at the genomic level and by comet assay at the molecular level. Bleomycin treatment increased the frequency of micronuclei in V79 cells in a concentration dependent manner. Several chemotherapeutic agents such as vincristine, vinblastine, vindesine, taxol, doxorubicin and teniposide have been reported to induce micronuclei (Jagetia and Jacob, 1992; Jagetia and Nayak, 1996; Jagetia and Adiga, 1995, 1998; Adiga and Jagetia, 1999; Jagetia and Baliga, 2002). Similarly, ionizing radiations have been reported to increase the frequency of micronuclei in a dose dependent manner in vitro and in vivo (Jagetia and Ganapathi, 1994; Jagetia and Adiga, 2000; Jagetia and Reddy, 2002). This increase in micronucleus formation after bleomycin treatment was two to three fold higher at a concentration of 10 µg ml−1 and more. Copyright © 2006 John Wiley & Sons, Ltd. Bleomycin induces single- and double-strand breaks through the formation of kinetically competent activated bleomycin in the presence of Fe2+ and O2 (Sam and Peisach, 1993; Absalon et al., 1995; Harsch et al., 2000) and these DNA strand breaks are susequently converted in to chromosome fragments and finally to micronuclei after one cell division. This may be the reason for micronuclei formation in V79 cells after bleomycin treatment. Naringin protected cellular genome against bleomycin-induced insult as evidenced by a significant reduction in the micronuclei frequency. A similar effect has been observed against radiation-induced micronuclei and chromosome damage in mice bone marrow cells treated with naringin (Jagetia and Reddy, 2002; Jagetia et al., 2003). Naringin has also been reported to reduce the iron-induced DNA damage in V79 cells (Jagetia et al., 2004). Cysteine, an amino acid, has been reported to protect against bleomycin-induced genomic damage (Chatterjee and Raman, 1993). Single cell gel electrophoresis (SCGE) or the comet assay has been considered as a rapid, simple and sensitive technique for measuring DNA damage (Fairbairn et al., 1995). Thus, the comet assay has been shown to be a very sensitive method for detecting genetic damage induced by different genotoxic agents (Olive and Durand, 1992; Malyapa et al., 1998) as well as for examining DNA repair under a variety of experimental conditions (Fairbairn et al., 1995). The comet assay determines the amount of DNA damage (both single- and double-strand breaks and conformational changes), alkali labile sites and cross links in a cell exposed to DNA damaging agents after removing most of the non-DNA material and applying a weak electric field to the remaining DNA embedded in an agarose gel (Bauch et al., 1999). The whole cells are embedded in an agarose gel, lysed and treated in situ with alkali to render the DNA singlestranded prior to running the gel. In an appropriate electrical field, the genomic DNA migrates out of the nucleus into the agarose and is then stained with the intercalating fluorescent dye, ethidium bromide, allowing visualization of the DNA. Viewed microscopically the combination of the DNA that has stayed within the confines of the nucleus and the ‘tail’ of DNA that has migrated makes individual cells look like comets. Quantitative microscopic evaluation is done by measuring the length and intensity of the comet in relation to the signal of the nonmigrating nuclear DNA in comparison with standards (Singh et al., 1988; Collins et al., 1997; Olive, 1999). The present study investigated the ability of naringin to protect against bleomycin-induced DNA strand break induction and its repair kinetics in V79 cells by comet assay. Exposure of V79 cells to 10 µg ml−1 bleomycin caused a significant increase in DNA damage as evidenced by an increase in olive tail moment and greater migration of DNA into the comet tails. Migration of DNA into comet tails (% tail DNA) and increase in OTM J. Appl. Toxicol. 2007; 27: 122–132 DOI: 10.1002/jat NARINGIN, A GRAPEFRUIT FLAVANONE, PROTECTS V79 CELLS 129 has been found to be directly proportional to the DNA damage (Kumaravel and Jha, 2006). The observation of DNA damage induction and cell death in V79 cells after bleomycin treatment is in agreement with earlier reports, where it has been reported to induce DNA damage in the peripheral blood lymphocytes and leukocytes (Jaloszynski et al., 1997; Buschini et al., 2002). Similarly, doxorubicin, an anthracycline antibiotic, has been reported to induce DNA damage (Tewey et al., 1984; Sognier et al., 1991; Delvaeye et al., 1993; Baumgartner et al., 2004). Idarubicin has also been reported to cause DNA strand breaks in a concentration-dependent manner in promyelocytic leukemia cell line, HL-60, and in murine pro-B lymphoid, BaF3 cell lines as assessed by comet assay (Blasiak et al., 2002). Exposure of γ -radiation, anticancer drugs such as mitomycin C, cisplatin, camtothecin, 5fluorouracil and nucleoside analogues have also been reported to induce DNA damage in human colon carcinoma HT-29, MCF-7 and in Chinese hamster ovary cells (Yamauchi et al., 2002). Naringin pretreament protected V79 cells against the bleomycin-induced molecular DNA damage as evident by a drastic reduction in the bleomycin-induced DNA damage at 4 and 6 h posttreatment. A similar effect has been reported earlier against the iron-induced DNA damage (Jagetia et al., 2004). Similarly, WR-2721 and genstein have been reported to protect bleomycin-induced DNA damage in the normal lymphocytes but not in tumor cells (Buschini et al., 2002). An identical effect has been observed with vitamin E ((+)-alpha-tocopherol) against the DNA damage induced by bleomycin (Wozniak et al., 2004; Lee et al., 2004). Similarly (−)-epigallocatechin-3-gallate (EGCG) has been reported to protect against bleomycininduced DNA damage in human leukocytes (Glei and Pool-Zobel, 2006). Rutin, naringin, ferulic acid, caffeine, vitamin C, E and beta-carotene have also been shown to protect against UV and radiation-induced DNA damage in various mammalian cells (Yeh et al., 2005; Maurya et al., 2005; Kumar et al., 2001; Konopacka et al., 1998). The study of DNA repair kinetics at various posttreatment times showed a continuous increase in the DNA damage up to 6 h post-bleomycin treatment in the bleomycin treatment alone group, whereas naringin pre-treatment efficiently repaired the bleomycin-induced DNA damage as evident by a significant and five-fold reduction in the migration of DNA into comet tails and OTM. A maximum repair of DNA damage by naringin was observed up to 4 h and continued until 6 h postbleomycin treatment. An identical effect in acceleration of DNA repair has been observed in V79 cells receiving curcumin, resveratrol, indole-3-carbinol and ellagic acid before treatment with N-methyl-N′-nitro-Nnitrosoguanidine (Chakraborty et al., 2004). Likewise tannic, ellagic and gallic acid have been found to repair DNA damage in B14 Chinese hamster cells exposed to Copyright © 2006 John Wiley & Sons, Ltd. Cu2+ ions and H2O2 (Labieniec and Gabryelak, 2005). The accelerated repair of DNA damage by naringin may be due to the chelation of iron that may have inhibited the reactivation of bleomycin molecule at the primary cleavage site and subsequent cleavage of complementary DNA into blunt ended double-strand breaks. This may also have resulted in the higher cell survival in naringin pretreated group. The effect of loss of genome in the form of micronuclei and DNA strand breaks was clearly evident in the form of decline in the cell survival. The greater was the number of micronuclei induced by bleomycin the higher was the decline in the surviving fraction of the cells, indicating a close correlation between cell survival and micronucleus induction. The increase in DNA strand breaks as evident by a greater amount of DNA in the comet tails may have also contributed to micronuclei formation and a subsequent cell death by bleomycin. This indicates that with the increase in genome damage there is a subsequent reduction in the reproductive integrity of the cells. The relationship between lethal events (micronucleus) and surviving fraction was found to be linear quadratic. A similar relationship was reported earlier (Jagetia and Adiga, 1995; Jagetia and Adiga, 1997; Jagetia and Adiga, 2000). A close correlation between cell survival and micronuclei-induction has been observed (Midander and Revesz, 1980; Stap and Aten, 1990; Abend et al., 1995). However, there are a few reports, where no correlation between the cell survival and MN formation was found (Bush and McMillan, 1993; Champion et al., 1995; Mariya et al., 1997). Of five cell lines studied three cell lines such as SHIN-3, DU-145 and CHO-K1 cells showed an excellent correlation between the MN frequency and surviving fraction, whereas F9 and COLO 320DM cells did not show this correlation (Guo et al., 1998). The reasons for the erratic reports to establish a link between DNA damage and clonogenic survival remain largely unclear. Treatment of V79 cells with 1 mM naringin before bleomycin exposure also showed an excellent correlation between cell survival and genomic damage except that the biological response was linear, indicating its protective action. The induction of micronuclei alone may not be responsible for the greater decline in cell survival in bleomycin treated cells. The bleomycin-induced apoptosis may reasonably explain this phenomenon, since bleomycin has been found to induce apoptosis by elevating caspase-8 and the resultant caspase-9 activity and upregulation of Fas expression (Wallach-Dayan et al., 2006). The rescuing effect of naringin and a higher cell survival in the naringin + bleomycin group may be due to the repair of DNA strand breakage (Table 1) and inhibition of bleomycin-induced apoptosis in the present study. The exact mechanism by which naringin protected belomycin-induced DNA damage in the form of DNA strand breaks, base damage and micronuclei and J. Appl. Toxicol. 2007; 27: 122–132 DOI: 10.1002/jat 130 A. JAGETIA ET AL. subsequently cell death is not known. It is possible that several mechanisms are operating simultaneously. Bleomycin is a radiomimetic agent and produces peroxyl, superoxide and hydroxyl free radicals, that react with cell DNA causing single- and double-strand breaks and cell death (Hecht, 1986; Cederberg and Ramel, 1989). Scavenging of these free radicals by naringin seems to be an important mechanism against the bleomycin-induced DNA damage and cytotoxicity. Our earlier study has shown that naringin scavenged peroxyl, superoxide, hydroxyl and ABTS cation free radicals in a concentration dependent manner (Jagetia et al., 2003). Bleomycin exerts its cytotoxic and DNA damaging effect by forming a Fe+2 coordination complex that combines with oxygen to produce highly reactive species that abstract hydrogen from C-4 of the deoxyribose, leading to DNA strand breaks or abusive sites (Smith et al., 1994). The presence of naringin may have disallowed the formation of iron–bleomycin complex thus reducing the bleomycininduced DNA damage. Naringin has been reported to reduce iron-induced toxicity in vitro (Jagetia et al., 2004). Bleomycin reacts with cytochrome P-450 and induces free radicals that induce single- and double-strand breaks (Hecht, 1986) and inhibition of cytochrome P-450 by naringin may have reduced bleomycin-induced DNA strand breaks by bringing effective repair of the broken strands of DNA and thus reducing cytotoxicity. The presence of naringin enhanced the repair of DNA damage and increased the cell survival (Table 1, Fig. 2). Naringin has been reported to inhibit cytochrome P-450 (Ueng et al., 1999). Naringin may have also reduced the bleomycin-induced oxidative stress in V79 cells thereby reducing the DNA damage and increasing the cell survival. Naringin has been reported to elevate glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase and to reduce lipid peroxidation (Jagetia and Reddy, 2005). Bleomycin has been reported to activate NF–κB, a heterodimer protein (Serrano-Mollar et al., 2003; Ortiz et al., 2004) involved in the expression of many genes responsible for cell proliferation and cell death (Baichwal and Baeuerle, 1997; Perkins, 2000). Presence of naringin may have possibly inhibited the activation of NF–κB pathway thereby increasing the survival of V79 cells. Naringin may have inhibited apoptosis by down regulating the caspase activity and apoptosis related gene and thus protecting the cells against the bleomycin-induced cell death. The present study demonstrates that naringin pretreatment reduced the bleomycin induced cytotoxicity and micronuclei formation. It also help in the repair of bleomycin induced DNA damage as evident by the reduction in the percent tail DNA and OTM. 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