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Cytoprotective and antigenotoxic potential of Mangiferin, a glucosylxanthone against cadmium chloride induced toxicity in HepG2 cells
Food and Chemical Toxicology 47 (2009) 592–600
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Cytoprotective and antigenotoxic potential of Mangiferin, a glucosylxanthone against cadmium chloride induced toxicity in HepG2 cells B.S. Satish Rao a,*, M.V. Sreedevi b, B. Nageshwar Rao a a b
Division of Radiobiology and Toxicology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, Karnataka, India Division of Biotechnology, Manipal Life Sciences Centre, Manipal, Manipal University, Manipal 576104, Karnataka, India
a r t i c l e
i n f o
Article history: Received 3 October 2008 Accepted 15 December 2008
Keywords: Apoptosis Cadmium Comet assay Cytotoxicity Mangiferin Micronuclei
a b s t r a c t Mangiferin (MGN), a glucosylxanthone present in large amounts in the leaves and edible mango fruits of Mangifera indica. Here, we report about MGN’s potential for mitigating cadmium chloride (CdCl2) induced cytotoxic and genotoxic effects in HepG2 cells growing in vitro. The cytoprotective potential was assessed by MTT, clonogenic and apoptotic assays, while antigenotoxic effect by micronucleus and comet assay. The established cytotoxic and genotoxic effects were well indicated after CdCl2 treatment and was mitigated by pretreatment with MGN. MGN prior to CdCl2 treatment increased the cell survival (MTT), surviving fraction (clonogenic assay) and inhibited sub-G1 population (flow cytometric analysis). Further, inhibition of CdCl2 induced apoptotic cell death by MGN was confirmed by microscopic and DNA fragmentation assays. A significant (p < 0.01) reduction in the micronuclei frequency and comet parameters after MGN pretreatment to CdCl2 clearly indicated the antigenotoxic potential. Similarly, the reactive oxygen species generated by the CdCl2 treatment were inhibited significantly (p < 0.001) by MGN. Taken together, our study revealed that MGN has potent cytoprotective and antigenotoxic effect against CdCl2 induced toxicity in HepG2 cell line and which may be attributed to decrease in CdCl2 induced reactive oxygen species levels and resultant oxidative stress. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Metals due to their natural abundance and by virtue of their universal usage in all spheres of life they are recognized as an integral part of our life today. Heavy metals, as a group, include both metals essential for normal biological functioning (e.g. Zn, Cu, Mn, Fe) and non-essential metals (e.g. Cd, Pb, Hg). Acute and chronic exposure of cadmium occurs through food, air, water, exposure to industrial products, occupational exposure and cigarette smoking (Elinder et al., 1976; Bertin and Averbeck, 2006). It accumulates in the body tissues, such as lung, bone, reproductive organs, the immune system and most extensive accumulation occurring in kidney and liver (Jarup et al., 1998). Liver and kidney cadmium concentrations are comparable after short-term exposure and the kidney concentration exceeds the liver concentration following prolonged exposure. Most important toxicity dysfuncAbbreviations: CdCl2, cadmium chloride; DCFH-DA, dicholorofluorescein diacetate; DMSO, dimethyl sulphoxide; EDTA, ethylene diamine tetraacetic acid; FCS, fetal calf serum; MEM, Eagle’s minimum essential medium; MGN, Mangiferin; MNBNC, micronucleated binucleate cells; OTM, olive tail moment; ROS, reactive oxygen species. * Corresponding author. Tel.: +91 820 2922122; fax: +91 820 2571919. E-mail addresses: [email protected], [email protected] (B.S. Satish Rao). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.12.017
tions resulting from cadmium ingestion include renal injuries (including tubular and glomerular dysfunctions), immune deficiencies, apathies, bone injuries (Osteomalacia and Osteoporosis), femoral pain, lumbago and skeleton deformations (Satarug et al., 2002; Jin et al., 2004). It causes a syndrome called ‘‘Itai–itai” characterized by bone deformities (Kasuya et al., 2000). These negative effects on human’s health are due to its low excretion rate (halflife as long as 15–20 years) (Elinder et al., 1976; Jin et al., 2004), and its accumulation in the organism. Fortunately, mammalian cells have evolved with strategies to detect and to manage with different forms of stress to adapt or to overcome cell death by inherent mechanisms such as expression of metallothioneins and heat shock proteins. Metallothioneins, low molecular weight cysteine-rich proteins play an important role in homeostasis of metals including cadmium. Cadmium intoxication and resultant hazards for human health has been reviewed recently by Godt et al. (2006). In humans, cadmium intoxification is strongly associated with induction of cancer of the lungs and a weaker association with cancers of prostate, kidney, and testes (Waalkes, 2003). Although, the cadmium induced mutagenicity is poorly understood, it is believed that it is mediated by mechanisms such as induction of reactive oxygen species and inhibition of DNA repair (Fotakis and Timbrell, 2006). Therefore, mitigation of cadmium induced toxicity is of primary importance
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in the field of toxicological research and public health. Several of the nutrients were known to play an important role in assisting the body’s natural processes of detoxification and elimination of cadmium. Elements like calcium, selenium and zinc have shown protective effect against cadmium induced toxicity (Hurna et al., 1997; Ciesielska et al., 2000). Besides, many of the anti-oxidants like glutathione (GSH), Vitamin C, E, selenium, glycine, cysteine, and salicylic acid have also shown their roles in mitigating cadmium toxicity (Singhal et al., 1987; Bolkent et al., 2007). Xanthones the biologically active plant phenols are found in some of the tropical plants such as Mangifera indica, Swertia mussotii and Swertia franchetiana. Like flavonoids, natural xanthones and their derivatives were known to have health promoting effects (Pinto et al., 2005). Mangiferin (1,3,6,7-tetrahydroxyxanthone-C2-b-D-glucoside, MGN), a naturally occurring glucosylxanthone, is present in M. indica, in large amounts in the leaves, and edible mango fruits. MGN has exhibited several beneficial pharmacological properties such as anti-viral, anti-inflammatory, hypoglycemic effects, antidiabetic analgesic and anti-oxidant activities (Zheng and Lu, 1990; Ojewole, 2005). As liver being the primary target organ for cadmium induced toxicity, in the study herein, we used human hepatoma cells (HepG2) having phase I and phase II xenobiotic metabolising systems similar to that of normal liver cells (Sundermann et al., 2004). Therefore, the objective of this study was to investigate the cytoprotective and antigenotoxic potential of MGN, on Cadmium chloride (CdCl2) induced toxicity in HepG2 cell line. 2. Materials and methods 2.1. Chemicals Cadmium chloride (CdCl2) (CAS 10108-64-2), Mangiferin (MGN) (CAS 4773-960), Eagle’s Minimum Essential Medium (MEM), Trypsin (0.1%), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], Trypan blue (0.1%), Cytochalasin-B, Propidium iodide, Ethidium bromide, RNAse, Proteinase K, Agarose, 20 ,70 dicholorofluorescein diacetate (DCFH-DA) and Fetal calf serum (FCS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acridine orange (AO) was purchased from BDH Chemicals Ltd, Poole, England. Other chemicals such as Absolute alcohol, Dimethyl sulphoxide (DMSO), Ethylene diamine tetraacetic acid (EDTA), Sodium bicarbonate and Hydrochloric acid were purchased from Qualigens Fine Chemicals (A division of GlaxoSmithKline Pharmaceuticals), Mumbai, India. 2.2. Cell line and culture HepG2 cells originally derived from human hepatocellular carcinoma were procured from National Centre for Cell Sciences, Pune, India. The cells were grown in 25-cm2 T-flasks (Falcon, Becton Dickinson, USA) with loosened caps, containing MEM supplemented with 10% FCS, 1% L-glutamine and 50 lg/ml gentamycin sulfate, at 37 °C in a CO2 incubator (NuAire, Plymouth, MN, USA) in an atmosphere of humidified 5% CO2 in 95% air. 2.3. Preparation of CdCl2/MGN solutions CdCl2 was dissolved in MEM and prepared freshly whenever required, whereas MGN was dissolved in 0.02% DMSO at a concentration of 1 mM and diluted with MEM immediately before use. 2.4. Cytotoxic and cytoprotective studies using MTT assay A fixed number (5 105) of exponentially growing cells were seeded into several individual 25 cm2 culture T-flasks and allowed to grow. Twenty four hours after culture initiation, several of such individual cell cultures were used for the experiments and they were divided into following groups: Group 1 (control): The cells of this group were not treated with MGN/CdCl2. Group 2 (CdCl2 alone): The cultures of this group were treated with 2.5, 5, 10, 25, 50, 100, 200, 300, 400, 500 and 600 lM of CdCl2 alone for 3, 6, 12, 24 and 48 h. Group 3 (MGN alone): The cells of this group were treated with different concentrations (10, 25, 50, 100, 200, 400 and 600 lM) of MGN alone for 3, 6, 12, 24 and 48 h. Group 4 (MGN + CdCl2): This group of cells was treated with 50 lM of MGN for 2 h, before treating with 100 lM of CdCl2 for 3, 6, 12 and 24 h.
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The MTT assay was carried out according to the protocol described by Mossman (1983). Preliminary experiments were conducted to titrate the number of cells to be seeded onto the 96-well plates and finally optimized to be 5 103 cells per well. The cell viability was assessed at different post exposure time intervals. Briefly, following the treatment, at the stipulated time as mentioned above, medium was aspirated; 100 ll of the MTT stock (1 mg/ml) was added to each of the 96 wells. After 4 h of incubation at 37 °C in a 5% CO2 incubator, the medium was removed and purple colored precipitate of formazan was solubilized in 100 ll of DMSO. After 5–10 min of incubation at 37 °C, the optical densities (OD) were read on multi-well spectrophotometer (Tecan, Austria) at 540 nm wavelength. IC50 (Inhibitory concentration of the drug causing 50% cell death) of CdCl2 for different time intervals was calculated from this experiment by calculating the viability (%) as follows:
Percent viability ¼
Average of TestðODÞ Average of BlankðODÞ 100: Average of ControlðODÞ Average of BlankðODÞ
2.5. Evaluation of cytotoxic and antigenotoxic effects of MGN A fixed number (5 105) of cells were inoculated into several individual culture flasks and were allowed to grow for 24 h. Exponentially cultures were then divided into following groups: Group 1 (control): This group of cells was treated with neither MGN/CdCl2. Group 2 (MGN alone): This group of cells was treated with different concentrations (0, 2.5, 5, 10, 25, 50, 75 and100 lM) of MGN for 2 h. Group 3 (CdCl2 alone): The cells of this group were treated with various concentrations of CdCl2 (0, 2.5, 5, 10, 25, 50, 100 and 200 lM) for 3 h. Group 4 (MGN + CdCl2): This group of cells was treated with different concentrations (0, 2.5, 5, 10, 25, 50, 75 and 100 lM) of MGN for 2 h, before treating with 10 lM of CdCl2 for 3 h. After the treatment with the MGN alone or with CdCl2, media was removed and cells were dislodged by trypsin EDTA (0.1%) treatment. The cytoprotective effect and the antigenotoxic effect of the MGN were analyzed by clonogenic, micronucleus and comet assays. 2.5.1. Clonogenic assay To assess the clonogenicity of the cells treated with CdCl2 alone or in combination with MGN colony-forming assay was performed according to the method of Puck and Marcus (1955). The cells from the stock culture were trypsinized by a mild 2 min exposure to trypsin EDTA and. counted using a hemocytometer An appropriate number of cells were plated into 60 mm petridishes (Nunc, Denmark) in triplicate containing 5 ml of growth medium. After various treatments, the drug containing growth medium was replaced with fresh medium and cells were allowed to grow for 10–12 days to form colonies. At the end of the last day, the medium was removed and the petridishes were washed with Phosphate Buffered Saline (PBS). The colonies were stained with Crystal violet (1%) for 30 min, rinsed with water and viable colonies containing more than 50 cells were counted. The plating efficiency (PE) and the surviving faction (SF) were calculated as follows:
Number of colonies counted 100; Number of cells seeded Number of colonies counted : Surviving fraction ðSFÞ ¼ Number of cells seeded ðPE=100Þ Plating efficiency ðPEÞ ¼
2.5.2. Micronucleus assay A separate experiment was conducted to assess the effect of MGN on CdCl2 induced micronucleus formation. This assay was performed according to the original method of Fenech and Morley (1985) with slight modifications of Rao et al. (2006). The cells were treated with CdCl2 alone or in combination with MGN. After various treatments, the cells from each group were dislodged by a mild 2 min exposure to trypsin EDTA. Briefly, the cells were re seeded, allowed to attach for 6 h and incubated with 3lg/ml of cytochalasin-B to inhibit cytokinesis. The cells were left undisturbed and allowed to grow for another 29 h. Thereafter, the medium containing cytochalasin-B was removed and cells were washed once with PBS. Finally, cells were dislodged with trypsin EDTA treatment, centrifuged, subjected to mild hypotonic treatment (0.75% ammonium oxalate) for 3 min, 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 onto precleaned coded slides to avoid observer’s bias. Triplicate cultures were used for each concentration for each group and the results were confirmed by repeating the experiments three times. The slides containing cells were stained with 0.01% acridine orange in Sorensen’s buffer (pH 6.8), and washed two times in the buffer. The buffer mounted slides were observed under a fluorescence microscope (Carl Zeiss Photomicroscope III, Germany) for the presence of micronuclei in the binucleate cells (BNC). A minimum of thousand BNC was scored from each culture and the frequency of micronucleated binucleate cells (MNBNC) was determined. The
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micronuclei identification was done according to the criteria of Fenech and Morley (1985) and the values were expressed as mean ± SEM (standard error of the mean) from three independent experiments. 2.5.3. Comet assay (single cell gel electrophoresis) The changes in the CdCl2 induced DNA damage by MGN was evaluated by single cell gel electrophoresis (comet assay). This assay was performed under alkaline conditions according to the procedure of Singh et al. (1988) with minor modifications of Collins et al. (1997). Exponentially growing cells were treated with CdCl2 alone or in combination with MGN. After the various treatments the comet slides were prepared. Briefly, slides frosted at one end were covered with 100 ll of 1.5% normal melting agarose prepared in Ca and Mg free PBS at 37 °C. The aliquots containing 1 105 harvested cells in 1 ml from the culture medium were centrifuged at 1500 rpm for 5 min. The cells pelleted were resuspended in 200 ll of 0.75% low melting agarose layered on to the first layer and allowed to solidify under a cover slip on ice. A third layer of 0.75% low melting agarose without the cells was made in a similar manner. Subsequently slides were exposed to lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base, 1% Triton-X 100, 10% DMSO) for 1 h at 4 °C, rinsed with distilled water and placed in electrophoresis buffer (10 N NaOH pH 13, 200 mM EDTA) for 20 min to allow DNA unwinding and electrophoresed at 20 V further for 20 min. The slides were then neutralized using neutralization buffer (0.4 M Tris pH 7.5). All the steps were conducted under a reduced light level to prevent additional DNA damage and slides were stained with 50 ll of ethidium bromide (20 lg/ml). The slides were visualized at 40 magnification using fluorescence microscope (Olympus BX51, Olympus Microscopes, Tokyo, Japan) equipped with a 515–535 nm excitation filter, a 590 nm barrier filter and a CCD camera (Cool SNAP-Procf Digital Color Camera Kit Version 4.1, Media Cybergenetics, Silver Spring, Maryland, USA). A total of 100 cells per sample were analyzed. The comets thus captured were analyzed using Komet software (Version 5.5, Kinetic Imaging Ltd., Bromborough, UK). The mean olive tail moment (OTM) was selected as the parameter that best reflects DNA damage, and calculated as follows:
% OTM ¼ ðHead MeanÞ Tail % DNA=100:
2.5.4. Cell death by apoptosis 2.5.4.1. Analysis of sub-G1 cell population by flow cytometry. Flow cytometry was performed in order to determine the apoptotic sub-G1 hypodiploid cells. The cells (1 106) were seeded in culture flask and allowed to attach and grow overnight. After 24 h, cells were treated with various concentrations of MGN (10–100 lM) for 2 h followed by treatment with 50 lM of CdCl2 for 24 h. At the end of treatment, both floating as well as attached cells were collected, washed with PBS two times and fixed with 70% ethanol. Cells were then washed three times with PBS to remove the traces of ethanol. Hypotonic rinse solution (0.1% Triton-X, 0.1% sodium citrate, 100 lg/ml RNAse) was added and incubated for a period of 30 min at 37 °C. This was followed by addition of 25 ll of Propidium iodide (50 lg/ml) and incubation at 4 °C for 15 min in dark. After appropriate gating, 10,000 events per sample were acquired on FACSCalibur (Becton Dickinson, USA) using CellQuest software. At least 10,000 events were acquired, cell debris was excluded by gating and the percentage of sub-G1 cell population (hypo-diploid fraction) was determined using Win MDI version 2.9 software (USA).
2.5.4.4. Intracellular reactive oxygen species (ROS) estimation. Fluorescence spectrophotometry was used to measure intracellular ROS using 20 ,70 - DCFH-DA fluorescence assay as described by Bai and Cederbaum (2003). In brief, exponentially growing HepG2 cells were treated with various concentrations of MGN (10– 100 lM) for 2 h followed by treatment with 10 lM of CdCl2 for 3 h. This was followed by incubation of the cells with 5lM DCFH-DA in MEM for 30 min at 37 °C in the dark. The cells were washed in PBS, trypsinized, and resuspended in 3 ml of PBS, and the intensity of fluorescence was immediately read in a fluorescence spectrophotometer (RF-5301PC, Shimadzu) at 488 nm for excitation and at 525 nm for emission. The results were expressed as arbitrary units of the fluorescence intensity.
3. Statistical analysis All the data were expressed as mean ± SEM. The statistical significance between the treatments was evaluated by One-way ANOVA and with Bonforroni’s post-hoc test using GraphPAD InStat, Software, USA.
4. Results 4.1. Cytotoxicity studies using MTT assay Treatment of cells with various concentrations of CdCl2 (2.5– 600 lM) showed a time and concentration dependent decline in the cell survival. IC50 values of CdCl2 alone treatment were 550, 525, 350, 27 and 5 lM for 3, 6, 12, 24, 48 h post-CdCl2 treatment times, respectively (Fig. 1). Exposure of HepG2 cells to different concentrations of MGN alone for 2 h did not alter the cell viability significantly. When the cells were treated with increasing concentrations of MGN (10–100 lM) for 2 h before exposure to IC50 (lM) concentration of CdCl2 for 3 h, increased cell survival was observed. Maximum elevation in cell survival was significantly (p < 0.01) greater in 50 lM MGN pretreated group when compared with other groups (Fig. 2A). Cytoprotective activity of MGN was evaluated by treating HepG2 cells with 50 lM of MGN for 2 h before treating with 100 lM of CdCl2 for various time periods. A significant (p < 0.001) elevation in cell survival of 1.4-fold was observed in MGN pretreated group in comparison with 24 h CdCl2 alone treated group (Fig. 2B).
2.5.4.2. Fluorescence microscopic analysis of apoptotic cells. The morphological changes that occur in a cell during apoptosis/necrosis were also analyzed by the differential uptake of fluorescent DNA binding ethidium bromide and acridine orange (AO/EtBr) stains. Cells were treated with 25 and 50 lM concentrations of MGN for 2 h followed by treatment with 50 lM of CdCl2 for 24 h. Briefly, at the end of the treatment times, cells were washed with cold PBS and stained with AO/EtBr (1:1 v/v) at a final concentration of 100 lg/ml and observed under a fluorescent microscope (Olympus BX51, Japan). According to this staining method, the live cells have normal green nuclei, early apoptotic cells have bright green nuclei with condensed or fragmented chromatin, late apoptotic cells display condensed and fragmented orange chromatin, and cells that have died from direct necrosis have structurally normal orange/red nuclei (Renvoize et al., 1998).
2.5.4.3. Detection of DNA fragmentation by agarose electrophoresis. The formation of ladder pattern from the DNA fragmentation indicating apoptosis was performed according to the protocol described by Giri et al. (2003) with minor modifications. Exponentially growing cells were treated with various concentrations of MGN (10–100 lM) for 2 h followed by treatment with 50 lM of CdCl2 for 24 h. After the various treatments, the floating and adherent cells were pooled and lysed overnight at 37 °C in a lysis buffer (0.02M EDTA, 0.05 M Tris HCl, 1% Nonidet P-40). After centrifugation at 800 rpm for 10 min, the supernatant was collected and incubated with 10ll of RNAse (10 mg/ml) and 20 ll of 20% Sodium dodecyl sulfate at 56 °C for 2 h. This was followed by treatment with 5 ll of Proteinase K (20 mg/ml) at 56 °C for 8 h. DNA was precipitated overnight with ammonium acetate and ethanol and the resultant pellet was dissolved in 20 ll of TE buffer. Later, 10 lg of DNA was separated on a 1.5% agarose gel and the fragmented ladder pattern was visualized under a UV illuminator after ethidium bromide staining.
Fig. 1. Changes in the cell survival after treating HepG2 cells with CdCl2 as assessed by MTT assay. (}) 3 h (IC50 550 lM); (5) 6 h (IC50 525 lM); (D) 12 h (IC50 350 lM); (s) 24 h (IC50 27 lM) and (h) 48 h (IC50 5 lM).
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Fig. 2. (A) Changes in viability of CdCl2 exposed cells with or without prior treatment with MGN. (HepG2 cells were treated with different doses of Mangiferin for 2 h followed by treatment with 550 lM of cadmium for 3 h). The significant levels *p < 0.05, **p < 0.01 compared to CdCl2 group and ***p < 0.001 compared with control HepG2 group. (B) Effect of MGN (50 lM for 2 h) on HepG2 cells treated with 100 lM of CdCl2 for various time periods. The significant levels *p < 0.05, **p < 0.001 and no symbol = non significant when compared with respective CdCl2 treated group.
4.2. Clonogenic assay
4.3. Micronucleus assay
The clonogenic assay is the gold standard for screening cytotoxic activity of any test agent, it was used to study the effect MGN on cytotoxic effect of CdCl2. Treatment of HepG2 cells with increasing concentrations of CdCl2 resulted in a concentration dependent decline in the cell survival as evidenced by the reduction in surviving fraction, which was lowest at 200 lM CdCl2 (Fig. 3). Treatment of the cells with 25 lM MGN for 2 h before exposure to different concentrations of CdCl2 resulted in an elevation in the cell survival when compared with the CdCl2 alone treatment. The cytoprotective effect of MGN increased at all concentration of CdCl2 used. The surviving fraction increased by 0.22, 0.19 and 0.16 for 25, 50 and 75 lM CdCl2 in MGN pretreated group when compared with the CdCl2 alone treated group.
The protection against CdCl2 induced genotoxicity by MGN was studied by micronucelus assay. Exposure of HepG2 cells to various concentrations of CdCl2 resulted in a concentration dependent elevation in the frequency of MNBNC at all the post-treatment times. MGN treatment alone did not alter the spontaneous frequency of micronuclei in binucleate cells up to 100lM. Treatment of HepG2 cells with 10 lM of CdCl2 resulted in a significant (p < 0.001) elevation in the MNBNC, and this increase in MNBNC was 4.15-folds higher than that of control MNBNC frequency. Exposure of the cells to different concentrations of MGN before treatment with 10 lM CdCl2 caused a decline in the CdCl2 induced micronuclei formation and a maximum decrease in MNBNC’s was observed for 25 lM MGN (Table 1) and this dose was considered as the optimum protective concentration. Therefore, further studies were carried out using this MGN concentration. Pretreatment of HepG2 cells with 25 lM MGN before exposure to different concentrations of CdCl2 resulted in a significant (p < 0.01) decline in the frequency of MNBNC when compared with the CdCl2 alone treatment (Fig. 4).
Table 1 Effect of various concentrations of MGN on the micronuclei induction in HepG2 cells exposed to 10 lM of CdCl2. MGN (lM)
0 2.5 5.0 10 25 50 75 100
Fig. 3. Effect of MGN (25 lM, 2 h) on the cell survival of HepG2 cells treated with different concentrations of CdCl2 for 3 h as assessed by clonogenic assay.
Frequency of micronucleated binucleate cells (MNBNC) ± SEM MGN alone
MGN + CdCl2
26.00 ± 0.64 26.75 ± 1.66 28.00 ± 1.21 28.50 ± 1.78 29.25 ± 1.54 31.50 ± 1.27 31.75 ± 2.15 32.25 ± 2.56
108.25 ± 1.15 89.00 ± 1.21 81.75 ± 1.54a 76.50 ± 2.07a 71.75 ± 1.21b 78.25 ± 2.72a 83.25 ± 2.56a 88.75 ± 2.89
MGN = Mangiferin, CdCl2 = cadmium chloride, SEM = standard error of the mean. The significant levels: a = p < 0.05, b = p < 0.01, and no symbol = non-significant, when compared with the respective control group.
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(OTM) in a concentration dependent manner at all the post-treatment times (Fig. 5A). Treatment of cells with different concentrations of MGN (10–100 lM) alone did not alter baseline DNA damage significantly. Pretreatment of HepG2 cells with different concentrations (2.5–100 lM) of MGN before exposure to 50 lM CdCl2 caused decline in the CdCl2 induced percentage of tail DNA and OTM and a maximum decrease was observed for 25 lM MGN (Table 2), and was considered as the optimum protective concentration. A separate experiment was conducted to study the effect of 25 lM MGN on the DNA damage induced by increasing doses of CdCl2 (10–100 lM). Treatment of HepG2 cells with different concentrations of CdCl2 alone caused a concentration dependent elevation in the DNA damage (Fig. 5B). Pretreatment with 25 lM MGN showed a dose-dependent reduction in the DNA damage when compared with CdCl2 alone treated group. The reduction in the DNA damage by MGN was statistically significant (p < 0.01) at all concentrations of CdCl2, when compared with CdCl2 alone treatment. Fig. 4. Effect of various concentrations of CdCl2 for 3 h on the micronuclei induction in HepG2 cells pretreated with 25 lM for MGN for 2 h. The significant levels * p < 0.05, **p < 0.01, and no symbol = non significant, when compared with respective control group. MNBNC = micronucleated binucleate cells.
4.4. Comet assay Alkaline single cell gel electrophoresis (comet assay) was used to detect the cellular DNA damage induced by CdCl2. Exposure of HepG2 cells to CdCl2 increased the comet parameters like percentage Tail DNA resultant of damaged DNA, and olive tail moment
4.5. Analysis of apoptosis using flow cytometry In order to evaluate the effect of MGN on apoptosis induced by CdCl2 in HepG2 cells, sub-G1 cell population was analyzed. Increase in the population of cells with sub-G1 DNA content indicates increase in the apoptotic cells. The sub-G1 fraction was approximately 6% both in the untreated HepG2 cells as well as after 100 lM MGN treatment. Cells treated with different concentration of MGN (10, 25, 50 and 100 lM) prior exposure to 10 lM CdCl2, showed sub-G1 fraction of 15.30%, 10.44%, 13.42% and 16.5%,
Fig. 5. (A) Genotoxic effect of various concentrations of CdCl2 on HepG2 cells. (HepG2 cells were treated with various concentrations of cadmium for 3 h). The significant levels *p < 0.01, **p < 0.001, and no symbol = non significant when compared to control. (B) Changes in CdCl2 induced DNA damage influenced by MGN (25 lM) treatment assessed by comet assay (OTM = olive tail moment). All the other explanations are as in Fig. 4.
Table 2 Effect of various concentrations of MGN on the DNA damage induced by 50 lM of CdCl2 in HepG2 cells. MGN (lM/ml)
0 10 25 50 100
Head DNA
Tail DNA
OTM
MGN alone
MGN + CdCl2
MGN alone
MGN + CdCl2
MGN alone
MGN + CdCl2
85.78 ± 1.15 84.87 ± 1.14 83.46 ± 1.08 82.92 ± 1.37 82.35 ± 1.24
64.30 ± 1.13 68.87 ± 1.19 73.80 ± 0.94a 70.53 ± 1.02 67.53 ± 1.07
14.22 ± 0.56 15.23 ± 0.71 16.64 ± 0.78 17.18 ± 1.21 17.75 ± 2.56
35.70 ± 1.13 31.13 ± 1.19 26.20 ± 1.09a 29.47 ± 1.07 32.47 ± 1.02
12.16 ± 0.72 12.52 ± 0.56 14.25 ± 0.28 15.12 ± 0.31 15.54 ± 0.42a
40.40 ± 1.30 36.91 ± 1.23 29.54 ± 1.10a 32.45 ± 1.01 33.96 ± 1.14
The HepG2 cells were exposed to MGN for 2 h + CdCl2 for 3 + 2 h post incubation. MGN = Mangiferin; CdCl2 = cadmium chloride; OTM = olive tail moment; SEM = standard error of the mean. The significant levels: a = p < 0.05 and no symbol = non-significant, when compared with the respective control group.
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respectively. The maximum decline in the sub-G1 fraction was observed at 25 lM of MGN pretreated group (Fig. 6).
Table 3 Apoptotic index of HepG2 cells treated with CdCl2 with or without MGN. MGN (lM)
Apoptotic index ± SEM (%) MGN alone
MGN + CdCl2
0 10 25 50 100
4.2 ± 0.01 4.4 ± 0.08 4.5 ± 0.04 4.8 ± 0.12 5.0 ± 0.06
37.2 ± 0.25 14.8 ± 0.37c 13.2 ± 0.11c 15.8 ± 0.22c 16.6 ± 0.29c
4.6. Morphological analysis of apoptotic bodies Apoptosis detection was performed by morphological analysis by double staining with AO/EtBr. HepG2 cells when treated with 50 lM of CdCl2 alone resulted in appearance of cells in early as well as late apoptotic stages with a minimal number of necrotic cells. Membrane blebbing and alterations in nuclear morphology such as chromatin condensation and DNA fragmentation were observed in apoptotic cells. MGN treatment alone even at a dose of 100 lM did not increase the proportion of apoptotic cells indicating its non toxic nature. When the cells treated with 25 lM of MGN for 2 h before treatment with 50 lM of CdCl2 for 24 h, the proportion of apoptotic cells decreased significantly (p < 0.01) as compared to the cells treated with CdCl2 alone (Table 3). 4.7. Detection of DNA fragmentation To confirm further, the effect of MGN on CdCl2 induced apoptosis, DNA was isolated from the treated and untreated HepG2 cells and electrophoresed on 1.2% agarose gel. Cells treated with 2 lM doxorubicin served as a positive control showed a clear visible typical ladder formation. A similar ladder formation after CdCl2 treatment was observed even at a low dose of 10 lM and became more prominent at higher concentrations (Fig. 7). The nucleosomal ladder formation after CdCl2 treatment pretreated with MGN showed decrease in the intensity of the ladder indicating the inhibition of apoptosis when compared to the CdCl2 alone. Further, it was observed that the intensity of the ladder formed in 25 lM of MGN pretreated group was much less in comparison to other pretreated groups indicating that apoptosis induction is less in 25 lM MGN pretreated group (Fig. 7, Lane 14).
The HepG2 cells were exposed to MGN for 2 h following CdCl2 at 50 lM for 3 h and cells were processed immediately for microscopic analysis. The other details are as in Table 2 and the significant levels: c = p < 0.001 compared to the control.
4.8. Intracellular ROS estimations The changes in the intracellular ROS generation induced by CdCl2 as influenced by MGN were estimated. The HepG2 cells treated with CdCl2 showed dose dependent increase in DCF fluorescence levels (indicative of intracellular ROS levels) (Fig. 8A). Pretreatment with MGN decreased DCF fluorescence levels in a dose dependent manner, a significant (p < 0.001) decrease was observed at 25 lM MGN pretreated group in comparison with CdCl2 alone group (Fig. 8B). 5. Discussion Cadmium being one of the toxic heavy metal and due to its inherent nature it gets accumulated in the environment, may pose severe risk for health problems (Godt et al., 2006). Exposure to cadmium has been associated with several specific clinical complications such as renal dysfunction, liver abnormalities and bone diseases (Goyer, 1997; Kasuya et al., 2000; Satarug et al., 2002; Jin et al., 2004). Recently, there is an increasing interest on the use of dietary components such as flavonoids, polyphenols,
Fig. 6. Flow cytometric analysis of changes in CdCl2 induced sub-G1 cell population indicating apoptotic cells influenced by MGN treatment. (A) Control HepG2; (B) MGN 100 lM alone; (C) CdCl2 50 lM alone; (D) MGN (10 lM) + CdCl2; (E) MGN (25 lM) + CdCl2; (F) MGN (50 lM) + CdCl2; (G) MGN (100 lM) + CdCl2. In the graph, DNA content (PI area – X-axis) is plotted against number of cells (Y-axis). The gates on the DNA histogram: M1 represent the total number cells and M2 indicates cells which fall in the subG1 region (apoptotic cells).
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Fig. 7. Effect of Mangiferin on the CdCl2 induced DNA fragmentation in the HepG2 cells. [Lane 1 and 10 – control; Lane 2 and 11 – positive control, doxorubicin (2 lM); Lane 3–8 cells treated with CdCl2 (5, 10, 25, 50, 100 and 200 lM); Lane 12 – CdCl2, 10 lM alone; Lane 13 – MGN (10 lM) + CdCl2; Lane 14 – MGN (25 lM) + CdCl2; Lane 15 – MGN (50 lM) + CdCl2; Lane 16 – MGN (100 lM) + CdCl2; Lane 17 – MGN (100 lM) alone; Lane 9 and 18 – 3 Kb ladder marker].
Fig. 8. (A) Induction of ROS levels after 3 h treatment with CdCl2 in HepG2 cells. (B) Effect of different concentrations of MGN treatment (2 h) on CdCl2 (10 lM, 3 h) induced ROS levels in HepG2 cells. The significant levels *p < 0.05, **p < 0.001, ***p < 0.001, when compared with respective control group.
xanthones to mitigate the effects of toxic environmental pollutants including certain heavy metals such as Cd, Hg, Zn and Fe. Many of these flavonoids were well known anti-oxidants and protect against damage caused by reactive oxygen species (Pietta, 2000). Further, few of these dietary flavonoids are also efficient modulators of enzymes involved in biotransformation reactions (Rodeiro et al., 2008a). Such dietary agents may have great utility in counteracting the effect of toxic heavy metals as they are consumed daily and have wide acceptability than other agents that have similar properties. The well known beneficial properties of such natural products motivated us to carry out this study on MGN for its cytoprotective and antigenotoxic potential. Here, we report for the first time the ameliorating effect of MGN against CdCl2 induced cytotoxic and genotoxic effects using cultured HepG2 cells. MGN, a naturally occurring glucosylxanthone, has catechol moiety, a pharmacophore with well established anti-oxidant activity, this moiety was also known to present in numerous other flavonoids. The hepatoprotective property due to its anti-oxidant poten-
tial has been established earlier (Rodeiro et al., 2008b). Apart from its well documented anti-oxidant property, MGN has many other pharmacological activities including anti-inflammatory, anti-viral, anti-bacterial and hypoglycemic effects (Zheng and Lu, 1990; Ojewole, 2005). In our study, treatment of HepG2 cells with CdCl2 resulted in a concentration dependent elevation in cytotoxicity as evidenced by the continuous decline of viable cells with increasing doses of CdCl2. Interestingly, in the presence of MGN, CdCl2 treated HepG2 cells showed reduction in cytotoxicity compared to cells treated with CdCl2 alone. Although, the protective potential of MGN against the cytotoxicity induced by several of the hepatotoxins such as tert-butyl hydroperoxide, ethanol, carbon tetrachloride and lipopolysaccharide (Rodeiro et al., 2008b) has been well documented, here we report its beneficial effect against heavy metal toxicity. Furthermore, our study has also demonstrated protective effect of MGN against CdCl2 induced genotoxicity, as evaluated by micronucleus and comet assay. A plethora of earlier reports convincingly
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demonstrated the genotoxic and co genotoxic potential of cadmium in plants, animals and mammalian cells (Waisberg et al., 2003; Deckert, 2005; Bertin and Averbeck, 2006). Moreover, earlier in vitro studies have indicated cadmium induced effects such as single and double strand breaks in DNA, DNA–protein cross links, oxidative DNA damages, mutations and chromosomal damage (Misra et al., 1998; Mouron et al., 2004). The mechanistic insights into the genotoxic effect of cadmium revealed both the direct and indirect pathways. Among all the DNA lesions, DNA double strand breaks (DSB) were considered as critical if unrepaired or misrepaired they contribute to cell killing or cell transformation (Bertin and Averbeck, 2006). In our study, we demonstrate the non-genotoxic property of MGN as it failed to induce any micronuclei and comet tail length by itself. Our results, further confirms the antimutagenic and antigenotoxic effect of MGN as indicated in an earlier experimental findings of Rodeiro et al., 2006 using bacterial and rodent mutagenic tests. Although, the exact mechanism involved in the protection of cadmium induced DNA damage is not clear, Mangiferin was shown to be protective against oxidative damage and mutagenesis due to its free radical scavenging property (Sato et al., 1992). Cadmium induces oxidative stress arising from indirect processes like decrease in cellular anti-oxidants and exhalation of ROS by mitochondria (Waisberg et al., 2003), leading to DNA damage as well as misfolding of cellular proteins (Bertin and Averbeck, 2006). Protective effects of natural compounds against cadmium toxicity are usually found at a particular optimum concentration. In our study, the effect of MGN on CdCl2 induced ROS was studied using DCFH-DA assay. CdCl2 alone elevated the intracellular ROS levels in a concentration dependent manner. In the presence of MGN, ROS levels were found to be declining and the maximum decline was found at 25 lM of an optimal concentration. This decrease in the CdCl2 induced ROS at 25 lM of MGN may account for the decline in the observed genotoxic as well as cytotoxic effect. However, MGN at higher concentrations was known to form oxidized products like quinoids as a result of its anti-oxidant activities, which accumulates in the mitochondria and reacts with membrane thiol groups. This shifts its anti-oxidant property to thiol arylation (Andreu et al., 2005) which might be the possible reason for the observed ineffectiveness in lowering the ROS levels observed at the doses more than 25 lM MGN in our studies. In the present study, CdCl2 induced apoptotic cells in a dose dependent manner as indicated in DNA ladder pattern after gel electrophoresis. This is in accordance with the earlier studies on the production of reactive oxygen species, which was implicated in CdCl2 induced apoptosis (Oh and Lim, 2006; Oh et al., 2006). Further, the CdCl2 induced cell death by apoptosis was attributed to the activation of caspase-8 via FAS receptor followed by BID cleavage and induction of mitochondrial caspase cascade after release of cytochrome C in HepG2 cells and betulin, a triterpene inhibited cadmium induced apoptotic cell death by its anti-oxidant effect (Oh and Lim, 2006). In the present study, although MGN alone did not induce any apoptotic HepG2 cells up to a concentration of 100 lM, pretreatment of cells with 25 lM of MGN reduced the percentage of apoptotic cells. On the basis of the earlier reports and present findings it is clear that CdCl2 induced generation of ROS must have resulted in increased apoptotic cells, while the inhibiting action of MGN on the production of ROS as well as scavenging of free radicals generated must have resulted in decreased apoptotic cells. Mammalian cells have complex strategies to detect and manage heavy metal induced stress on cells. Metallothionein (MT), low molecular weight cysteine-rich proteins are on the forefront in the homeostasis of these essential metals. These metal binding proteins were known to protect against cadmium toxicity and oxidative stress (Andrews, 2000). Besides, it is also understood that
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GSH also has a role in the protection of cadmium induced cytotoxicty. This was substantiated by the fact that GSH supplementation protected against cadmium induced hepatotoxicity and nephrotoxicity (Singhal et al., 1987). Furthermore, it was reported that Mangiferin increased GSH levels and also increased catalase activity (Sarkar et al., 2004). Therefore, the role of MT and GSH in ameliorating cadmium induced cytotoxicity cannot be ruled out in the present study. The studies are underway to understand the role of MT as well as GSH for the observed cytoprotective action of MGN. The present study revealed that MGN, a glucosylxanthone, an active component of M. indica has potent cytoprotective and antigenotoxic effect against CdCl2 induced toxicity in HepG2 cell line. The cytoprotective and antigenotoxic property of MGN may be attributed to decrease in CdCl2 induced ROS levels and resultant oxidative stress. Taken together, the earlier health promoting pharmacological properties of MGN and with a yet another implication from the present study it may be concluded that MGN may be a good agent for counteracting the negative effects of several other heavy metals. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgement This research work was carried out under the ITREOH program activity with the support of Manipal University, Manipal. The authors are thankful to Dr. K. Satyamoorthy, The Director, Manipal Life Sciences Centre, Manipal University, Manipal for his help and encouragement during this study. References Andreu, G.L., René, D., Velho, A.J., Curti, C., Vercesi, A.E., 2005. Mangiferin, a natural occurring glucosylxanthone, increases susceptibility of rat liver mitochondria to calcium-induced permeability transition. Arch. Biochem. Biophys. 439, 184– 193. Andrews, G.K., 2000. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 59, 95–104. Bai, J., Cederbaum, A.I., 2003. Catalase protects HepG2 cells from apoptosis induced by DNA damaging agents by accelerating the degradation of p53. J. Biol. Chem. 278, 4660–4667. Bertin, G., Averbeck, D., 2006. Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review). Biochimie 88, 1549–1559. Bolkent, S., Koyuturk, M., Bulan, O.K., Tunali, S., Yanardag, R., Tabakoglu, A.O., 2007. The effects of combined alpha-tocopherol, ascorbic acid, and selenium against cadmium toxicity in rat intestine. J. Environ. Pathol. Toxicol. Oncol. 26, 21–27. Ciesielska, S.A., Stachura, A., Slotwinska, M., Kaminska, T., Sniezko, R., Paduch, R., Abramczyk, D., Filar, J., Kandefer-Szerszen, M., 2000. The inhibitory effect of zinc on cadmium-induced cell apoptosis and reactive oxygen species (ROS) production in cell cultures. Toxicology 145, 159–171. Collins, A., Dusinska, M., Franklin, M., Somorovska, M., Petrovská, H., Duthie, S., Fillion, L., Panayiotidis, M., Raslova, K., Vaughan, N., 1997. Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ. Mol. Mutagen. 30, 139–146. Deckert, J., 2005. Cadmium toxicity in plants: is there any analogy to its carcinogenic effect in mammalian cells? Biometals 18, 475–481. Elinder, C.G., Lind, B., Kjellstorm, T., Linnman, L., Friberg, L., 1976. Cadmium in kidney cortex, liver and pancreas from Swedish autopsies. Estimation of biological half time in kidney cortex, considering calories intake and smoking habits. Arch. Environ. Health 31, 292–302. Fenech, M., Morley, A.A., 1985. Measurement of micronuclei in lymphocytes. Mutat. Res. 147, 29–36. Fotakis, G., Timbrell, J.A., 2006. Modulation of cadmium chloride toxicity by sulphur amino acids in hepatoma cells. Toxicol. In Vitro 20, 641–648. Giri, K., Ghosh, U., Bhattacharyya, N.P., Basak, S., 2003. Caspase 8 mediated apoptotic cell death induced by b-sheet forming polyamine peptides. FEBS Lett. 555, 380–384. Godt, J., Scheidig, F., Grosse, S.C., Esche, V., Brandenburg, P., Reich, A., Groneberg, D.A., 2006. The toxicity of cadmium and resulting hazards for human health. J. Occup. Med. Toxicol. 1, 22. Goyer, R.A., 1997. Toxic and essential metal interactions. Annu. Rev. Nutr. 17, 37– 50.
600
B.S. Satish Rao et al. / Food and Chemical Toxicology 47 (2009) 592–600
Hurna, E., Siklenka, P., Hurna, S., 1997. Effect of selenium on cadmium genotoxicity investigated by micronucleus assay. Vet. Med.-(Praha) 42, 339–342. Jarup, L., Berglund, M., Elinder, C.G., Nordberg, G., Vahter, M., 1998. Health effects of cadmium exposure – a review of literature and a risk estimate. Scand. J. Work Environ. Health 24, 1–51. Jin, T., Kong, Q., Ye, T., Wu, X., Nordberg, G.F., 2004. Renal dysfunction of cadmiumexposed workers residing in a cadmium-polluted environment. Biometals 17, 513–518. Kasuya, M., Teranishi, H., Aoshima, K., Katoh, T., Horiguchi, H., Morikawa, Y., Nishijo, M., Iwata, K., 2000. Water pollution by cadmium and the onset of Itai–itai disease. Water Sci. Technol. 25, 149–156. Misra, R.R., Smith, G.T., Waalkes, M.P., 1998. Evaluation of the direct genotoxic potential of cadmium in four different rodent cell lines. Toxicology 126, 103– 114. Mossman, T., 1983. Rapid colorimetric assay for cell growth and survival: application to proliferation and cytotoxicity assay. J. Immunol. Methods 65, 55–63. Mouron, S.A., Grillo, C.A., Dulout, F.N., Golijow, C.D., 2004. A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells. Mutat. Res. 568, 221–231. Oh, S.H., Lim, S.C., 2006. A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation. Toxicol. Appl. Pharmacol. 212, 212–223. Oh, S.H., Choi, J.E., Lim, S.C., 2006. Protection of betulin against cadmium-induced apoptosis in hepatoma cells. Toxicology 1, 1–12. Ojewole, J.A.O., 2005. Anti-inflammatory, analgesic and hypoglycemic effects of Mangifera indica Linn. (Anacardiaceae) stem-bark aqueous extract. Methods Find. Exp. Clin. Pharmacol. 27, 547–554. Pinto, M.M., Sousa, M.E., Nascimento, M.S., 2005. Xanthone derivatives: new insights in biological activities. Curr. Med. Chem. 12, 2517–2538. Pietta, P.G., 2000. Flavonoids as antioxidants. J. Nat. Prod. 63, 1035–1042. Puck, T.T., Marcus, P.I., 1955. A rapid method for viable cell titration and clone production with HeLa cells in tissue culture: the use of x-irradiated cells to supply conditioning factors. Proc. Natl. Acad. Sci. USA 41, 432–437. Rao, B.S.S., Shanbhoge, R., Upadhya, D., Jagetia, G.C., Adiga, S.K., Kumar, P., Guruprasad, K., Gayathri, P., 2006. Antioxidant, anti-clastogenetic and
radioprotective effect of Coleus aromaticus on Chinese hamster fibroblast cells (V79) exposed to gamma radiation. Mutagenesis 21, 237–242. Renvoize, C., Biola, A., Pallardy, M., Breard, J., 1998. Apoptosis: identification of dying cells. Cell Biol. Toxicol. 14, 111–120. Rodeiro, I., Cancino, L., González, J.E., Morffi, J., Garrido, G., González, R.M., Nuñez, A., Delgado, R., 2006. Evaluation of the genotoxic potential of Mangifera indica L. extract (Vimang), a new natural product with antioxidant activity. Food Chem Toxicol. 10, 1707–1713. Rodeiro, I., Donato, M.T., Lahoz, A., González-Lavaut, J.A., Laguna, A., Castell, J.V., Delgado, R., Gomez-Lechón, M.J., 2008a. Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes. Chem. Biol. Interact. 17, 1–10. Rodeiro, I., Donato, M.T., Martínez, I., Hernández, I., Garrido, G., González-Lavaut, J.A., Menéndez, R., Laguna, A., Castell, J.V., Gómez-Lechón, M.J., 2008b. Potential hepatoprotective effects of new Cuban natural products in rat hepatocytes culture. Toxicol. In Vitro 22, 1242–1249. Sarkar, A., Sreenivasan, Y., Ramesh, G.T., Manna, S.K., 2004. Beta-D-Glucoside suppresses tumor necrosis factor-induced activation of nuclear transcription factor kappaB but potentiates apoptosis. J. Biol. Chem. 279, 33768–33781. Satarug, S., Baker, J.R., Reilly, P.E., Moore, M.R., Williams, D.J., 2002. Cadmium levels in the lung, liver, kidney cortex and urine samples from Australians without occupational exposure to metals. Arch. Environ. Health 57, 69–77. Sato, T., Kawamoto, A., Tamura, A., Tatsumi, Y., Fujii, T., 1992. Mechanism of antioxidant action of pueraria glycoside (PG)-1 (an isoflavonoid) and Mangiferin (a xanthonoid). Chem. Pharm. Bull. 40, 721–724. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184–191. Singhal, R.K., Anderson, M.E., Meister, A., 1987. Glutathione, a first line of defense against cadmium toxicity. FASEB J. 1, 220–223. Sundermann, M.V., Knasmüller, S., Wu, X.J., Darroudi, F., Kassie, F., 2004. Use of a human-derived liver cell line for the detection of cytoprotective, antigenotoxic and cogenotoxic agents. Toxicology 198, 329–340. Waalkes, M.P., 2003. Cadmium carcinogenesis. Mutat. Res. 533, 107–120. Waisberg, M., Joseph, P., Hale, B., Beyersmann, D., 2003. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192, 95–117. Zheng, M.S., Lu, Z.Y., 1990. Antiviral effect of mangiferin and isomangiferin on herpes simplex virus. Chin. Med. J. (Engl.) 103, 160–165.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Cytoprotective and antigenotoxic potential of Mangiferin, a glucosylxanthone against cadmium chloride induced toxicity in HepG2 cells B.S. Satish Rao a,*, M.V. Sreedevi b, B. Nageshwar Rao a a b
Division of Radiobiology and Toxicology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, Karnataka, India Division of Biotechnology, Manipal Life Sciences Centre, Manipal, Manipal University, Manipal 576104, Karnataka, India
a r t i c l e
i n f o
Article history: Received 3 October 2008 Accepted 15 December 2008
Keywords: Apoptosis Cadmium Comet assay Cytotoxicity Mangiferin Micronuclei
a b s t r a c t Mangiferin (MGN), a glucosylxanthone present in large amounts in the leaves and edible mango fruits of Mangifera indica. Here, we report about MGN’s potential for mitigating cadmium chloride (CdCl2) induced cytotoxic and genotoxic effects in HepG2 cells growing in vitro. The cytoprotective potential was assessed by MTT, clonogenic and apoptotic assays, while antigenotoxic effect by micronucleus and comet assay. The established cytotoxic and genotoxic effects were well indicated after CdCl2 treatment and was mitigated by pretreatment with MGN. MGN prior to CdCl2 treatment increased the cell survival (MTT), surviving fraction (clonogenic assay) and inhibited sub-G1 population (flow cytometric analysis). Further, inhibition of CdCl2 induced apoptotic cell death by MGN was confirmed by microscopic and DNA fragmentation assays. A significant (p < 0.01) reduction in the micronuclei frequency and comet parameters after MGN pretreatment to CdCl2 clearly indicated the antigenotoxic potential. Similarly, the reactive oxygen species generated by the CdCl2 treatment were inhibited significantly (p < 0.001) by MGN. Taken together, our study revealed that MGN has potent cytoprotective and antigenotoxic effect against CdCl2 induced toxicity in HepG2 cell line and which may be attributed to decrease in CdCl2 induced reactive oxygen species levels and resultant oxidative stress. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Metals due to their natural abundance and by virtue of their universal usage in all spheres of life they are recognized as an integral part of our life today. Heavy metals, as a group, include both metals essential for normal biological functioning (e.g. Zn, Cu, Mn, Fe) and non-essential metals (e.g. Cd, Pb, Hg). Acute and chronic exposure of cadmium occurs through food, air, water, exposure to industrial products, occupational exposure and cigarette smoking (Elinder et al., 1976; Bertin and Averbeck, 2006). It accumulates in the body tissues, such as lung, bone, reproductive organs, the immune system and most extensive accumulation occurring in kidney and liver (Jarup et al., 1998). Liver and kidney cadmium concentrations are comparable after short-term exposure and the kidney concentration exceeds the liver concentration following prolonged exposure. Most important toxicity dysfuncAbbreviations: CdCl2, cadmium chloride; DCFH-DA, dicholorofluorescein diacetate; DMSO, dimethyl sulphoxide; EDTA, ethylene diamine tetraacetic acid; FCS, fetal calf serum; MEM, Eagle’s minimum essential medium; MGN, Mangiferin; MNBNC, micronucleated binucleate cells; OTM, olive tail moment; ROS, reactive oxygen species. * Corresponding author. Tel.: +91 820 2922122; fax: +91 820 2571919. E-mail addresses: [email protected], [email protected] (B.S. Satish Rao). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.12.017
tions resulting from cadmium ingestion include renal injuries (including tubular and glomerular dysfunctions), immune deficiencies, apathies, bone injuries (Osteomalacia and Osteoporosis), femoral pain, lumbago and skeleton deformations (Satarug et al., 2002; Jin et al., 2004). It causes a syndrome called ‘‘Itai–itai” characterized by bone deformities (Kasuya et al., 2000). These negative effects on human’s health are due to its low excretion rate (halflife as long as 15–20 years) (Elinder et al., 1976; Jin et al., 2004), and its accumulation in the organism. Fortunately, mammalian cells have evolved with strategies to detect and to manage with different forms of stress to adapt or to overcome cell death by inherent mechanisms such as expression of metallothioneins and heat shock proteins. Metallothioneins, low molecular weight cysteine-rich proteins play an important role in homeostasis of metals including cadmium. Cadmium intoxication and resultant hazards for human health has been reviewed recently by Godt et al. (2006). In humans, cadmium intoxification is strongly associated with induction of cancer of the lungs and a weaker association with cancers of prostate, kidney, and testes (Waalkes, 2003). Although, the cadmium induced mutagenicity is poorly understood, it is believed that it is mediated by mechanisms such as induction of reactive oxygen species and inhibition of DNA repair (Fotakis and Timbrell, 2006). Therefore, mitigation of cadmium induced toxicity is of primary importance
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in the field of toxicological research and public health. Several of the nutrients were known to play an important role in assisting the body’s natural processes of detoxification and elimination of cadmium. Elements like calcium, selenium and zinc have shown protective effect against cadmium induced toxicity (Hurna et al., 1997; Ciesielska et al., 2000). Besides, many of the anti-oxidants like glutathione (GSH), Vitamin C, E, selenium, glycine, cysteine, and salicylic acid have also shown their roles in mitigating cadmium toxicity (Singhal et al., 1987; Bolkent et al., 2007). Xanthones the biologically active plant phenols are found in some of the tropical plants such as Mangifera indica, Swertia mussotii and Swertia franchetiana. Like flavonoids, natural xanthones and their derivatives were known to have health promoting effects (Pinto et al., 2005). Mangiferin (1,3,6,7-tetrahydroxyxanthone-C2-b-D-glucoside, MGN), a naturally occurring glucosylxanthone, is present in M. indica, in large amounts in the leaves, and edible mango fruits. MGN has exhibited several beneficial pharmacological properties such as anti-viral, anti-inflammatory, hypoglycemic effects, antidiabetic analgesic and anti-oxidant activities (Zheng and Lu, 1990; Ojewole, 2005). As liver being the primary target organ for cadmium induced toxicity, in the study herein, we used human hepatoma cells (HepG2) having phase I and phase II xenobiotic metabolising systems similar to that of normal liver cells (Sundermann et al., 2004). Therefore, the objective of this study was to investigate the cytoprotective and antigenotoxic potential of MGN, on Cadmium chloride (CdCl2) induced toxicity in HepG2 cell line. 2. Materials and methods 2.1. Chemicals Cadmium chloride (CdCl2) (CAS 10108-64-2), Mangiferin (MGN) (CAS 4773-960), Eagle’s Minimum Essential Medium (MEM), Trypsin (0.1%), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], Trypan blue (0.1%), Cytochalasin-B, Propidium iodide, Ethidium bromide, RNAse, Proteinase K, Agarose, 20 ,70 dicholorofluorescein diacetate (DCFH-DA) and Fetal calf serum (FCS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acridine orange (AO) was purchased from BDH Chemicals Ltd, Poole, England. Other chemicals such as Absolute alcohol, Dimethyl sulphoxide (DMSO), Ethylene diamine tetraacetic acid (EDTA), Sodium bicarbonate and Hydrochloric acid were purchased from Qualigens Fine Chemicals (A division of GlaxoSmithKline Pharmaceuticals), Mumbai, India. 2.2. Cell line and culture HepG2 cells originally derived from human hepatocellular carcinoma were procured from National Centre for Cell Sciences, Pune, India. The cells were grown in 25-cm2 T-flasks (Falcon, Becton Dickinson, USA) with loosened caps, containing MEM supplemented with 10% FCS, 1% L-glutamine and 50 lg/ml gentamycin sulfate, at 37 °C in a CO2 incubator (NuAire, Plymouth, MN, USA) in an atmosphere of humidified 5% CO2 in 95% air. 2.3. Preparation of CdCl2/MGN solutions CdCl2 was dissolved in MEM and prepared freshly whenever required, whereas MGN was dissolved in 0.02% DMSO at a concentration of 1 mM and diluted with MEM immediately before use. 2.4. Cytotoxic and cytoprotective studies using MTT assay A fixed number (5 105) of exponentially growing cells were seeded into several individual 25 cm2 culture T-flasks and allowed to grow. Twenty four hours after culture initiation, several of such individual cell cultures were used for the experiments and they were divided into following groups: Group 1 (control): The cells of this group were not treated with MGN/CdCl2. Group 2 (CdCl2 alone): The cultures of this group were treated with 2.5, 5, 10, 25, 50, 100, 200, 300, 400, 500 and 600 lM of CdCl2 alone for 3, 6, 12, 24 and 48 h. Group 3 (MGN alone): The cells of this group were treated with different concentrations (10, 25, 50, 100, 200, 400 and 600 lM) of MGN alone for 3, 6, 12, 24 and 48 h. Group 4 (MGN + CdCl2): This group of cells was treated with 50 lM of MGN for 2 h, before treating with 100 lM of CdCl2 for 3, 6, 12 and 24 h.
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The MTT assay was carried out according to the protocol described by Mossman (1983). Preliminary experiments were conducted to titrate the number of cells to be seeded onto the 96-well plates and finally optimized to be 5 103 cells per well. The cell viability was assessed at different post exposure time intervals. Briefly, following the treatment, at the stipulated time as mentioned above, medium was aspirated; 100 ll of the MTT stock (1 mg/ml) was added to each of the 96 wells. After 4 h of incubation at 37 °C in a 5% CO2 incubator, the medium was removed and purple colored precipitate of formazan was solubilized in 100 ll of DMSO. After 5–10 min of incubation at 37 °C, the optical densities (OD) were read on multi-well spectrophotometer (Tecan, Austria) at 540 nm wavelength. IC50 (Inhibitory concentration of the drug causing 50% cell death) of CdCl2 for different time intervals was calculated from this experiment by calculating the viability (%) as follows:
Percent viability ¼
Average of TestðODÞ Average of BlankðODÞ 100: Average of ControlðODÞ Average of BlankðODÞ
2.5. Evaluation of cytotoxic and antigenotoxic effects of MGN A fixed number (5 105) of cells were inoculated into several individual culture flasks and were allowed to grow for 24 h. Exponentially cultures were then divided into following groups: Group 1 (control): This group of cells was treated with neither MGN/CdCl2. Group 2 (MGN alone): This group of cells was treated with different concentrations (0, 2.5, 5, 10, 25, 50, 75 and100 lM) of MGN for 2 h. Group 3 (CdCl2 alone): The cells of this group were treated with various concentrations of CdCl2 (0, 2.5, 5, 10, 25, 50, 100 and 200 lM) for 3 h. Group 4 (MGN + CdCl2): This group of cells was treated with different concentrations (0, 2.5, 5, 10, 25, 50, 75 and 100 lM) of MGN for 2 h, before treating with 10 lM of CdCl2 for 3 h. After the treatment with the MGN alone or with CdCl2, media was removed and cells were dislodged by trypsin EDTA (0.1%) treatment. The cytoprotective effect and the antigenotoxic effect of the MGN were analyzed by clonogenic, micronucleus and comet assays. 2.5.1. Clonogenic assay To assess the clonogenicity of the cells treated with CdCl2 alone or in combination with MGN colony-forming assay was performed according to the method of Puck and Marcus (1955). The cells from the stock culture were trypsinized by a mild 2 min exposure to trypsin EDTA and. counted using a hemocytometer An appropriate number of cells were plated into 60 mm petridishes (Nunc, Denmark) in triplicate containing 5 ml of growth medium. After various treatments, the drug containing growth medium was replaced with fresh medium and cells were allowed to grow for 10–12 days to form colonies. At the end of the last day, the medium was removed and the petridishes were washed with Phosphate Buffered Saline (PBS). The colonies were stained with Crystal violet (1%) for 30 min, rinsed with water and viable colonies containing more than 50 cells were counted. The plating efficiency (PE) and the surviving faction (SF) were calculated as follows:
Number of colonies counted 100; Number of cells seeded Number of colonies counted : Surviving fraction ðSFÞ ¼ Number of cells seeded ðPE=100Þ Plating efficiency ðPEÞ ¼
2.5.2. Micronucleus assay A separate experiment was conducted to assess the effect of MGN on CdCl2 induced micronucleus formation. This assay was performed according to the original method of Fenech and Morley (1985) with slight modifications of Rao et al. (2006). The cells were treated with CdCl2 alone or in combination with MGN. After various treatments, the cells from each group were dislodged by a mild 2 min exposure to trypsin EDTA. Briefly, the cells were re seeded, allowed to attach for 6 h and incubated with 3lg/ml of cytochalasin-B to inhibit cytokinesis. The cells were left undisturbed and allowed to grow for another 29 h. Thereafter, the medium containing cytochalasin-B was removed and cells were washed once with PBS. Finally, cells were dislodged with trypsin EDTA treatment, centrifuged, subjected to mild hypotonic treatment (0.75% ammonium oxalate) for 3 min, 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 onto precleaned coded slides to avoid observer’s bias. Triplicate cultures were used for each concentration for each group and the results were confirmed by repeating the experiments three times. The slides containing cells were stained with 0.01% acridine orange in Sorensen’s buffer (pH 6.8), and washed two times in the buffer. The buffer mounted slides were observed under a fluorescence microscope (Carl Zeiss Photomicroscope III, Germany) for the presence of micronuclei in the binucleate cells (BNC). A minimum of thousand BNC was scored from each culture and the frequency of micronucleated binucleate cells (MNBNC) was determined. The
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micronuclei identification was done according to the criteria of Fenech and Morley (1985) and the values were expressed as mean ± SEM (standard error of the mean) from three independent experiments. 2.5.3. Comet assay (single cell gel electrophoresis) The changes in the CdCl2 induced DNA damage by MGN was evaluated by single cell gel electrophoresis (comet assay). This assay was performed under alkaline conditions according to the procedure of Singh et al. (1988) with minor modifications of Collins et al. (1997). Exponentially growing cells were treated with CdCl2 alone or in combination with MGN. After the various treatments the comet slides were prepared. Briefly, slides frosted at one end were covered with 100 ll of 1.5% normal melting agarose prepared in Ca and Mg free PBS at 37 °C. The aliquots containing 1 105 harvested cells in 1 ml from the culture medium were centrifuged at 1500 rpm for 5 min. The cells pelleted were resuspended in 200 ll of 0.75% low melting agarose layered on to the first layer and allowed to solidify under a cover slip on ice. A third layer of 0.75% low melting agarose without the cells was made in a similar manner. Subsequently slides were exposed to lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base, 1% Triton-X 100, 10% DMSO) for 1 h at 4 °C, rinsed with distilled water and placed in electrophoresis buffer (10 N NaOH pH 13, 200 mM EDTA) for 20 min to allow DNA unwinding and electrophoresed at 20 V further for 20 min. The slides were then neutralized using neutralization buffer (0.4 M Tris pH 7.5). All the steps were conducted under a reduced light level to prevent additional DNA damage and slides were stained with 50 ll of ethidium bromide (20 lg/ml). The slides were visualized at 40 magnification using fluorescence microscope (Olympus BX51, Olympus Microscopes, Tokyo, Japan) equipped with a 515–535 nm excitation filter, a 590 nm barrier filter and a CCD camera (Cool SNAP-Procf Digital Color Camera Kit Version 4.1, Media Cybergenetics, Silver Spring, Maryland, USA). A total of 100 cells per sample were analyzed. The comets thus captured were analyzed using Komet software (Version 5.5, Kinetic Imaging Ltd., Bromborough, UK). The mean olive tail moment (OTM) was selected as the parameter that best reflects DNA damage, and calculated as follows:
% OTM ¼ ðHead MeanÞ Tail % DNA=100:
2.5.4. Cell death by apoptosis 2.5.4.1. Analysis of sub-G1 cell population by flow cytometry. Flow cytometry was performed in order to determine the apoptotic sub-G1 hypodiploid cells. The cells (1 106) were seeded in culture flask and allowed to attach and grow overnight. After 24 h, cells were treated with various concentrations of MGN (10–100 lM) for 2 h followed by treatment with 50 lM of CdCl2 for 24 h. At the end of treatment, both floating as well as attached cells were collected, washed with PBS two times and fixed with 70% ethanol. Cells were then washed three times with PBS to remove the traces of ethanol. Hypotonic rinse solution (0.1% Triton-X, 0.1% sodium citrate, 100 lg/ml RNAse) was added and incubated for a period of 30 min at 37 °C. This was followed by addition of 25 ll of Propidium iodide (50 lg/ml) and incubation at 4 °C for 15 min in dark. After appropriate gating, 10,000 events per sample were acquired on FACSCalibur (Becton Dickinson, USA) using CellQuest software. At least 10,000 events were acquired, cell debris was excluded by gating and the percentage of sub-G1 cell population (hypo-diploid fraction) was determined using Win MDI version 2.9 software (USA).
2.5.4.4. Intracellular reactive oxygen species (ROS) estimation. Fluorescence spectrophotometry was used to measure intracellular ROS using 20 ,70 - DCFH-DA fluorescence assay as described by Bai and Cederbaum (2003). In brief, exponentially growing HepG2 cells were treated with various concentrations of MGN (10– 100 lM) for 2 h followed by treatment with 10 lM of CdCl2 for 3 h. This was followed by incubation of the cells with 5lM DCFH-DA in MEM for 30 min at 37 °C in the dark. The cells were washed in PBS, trypsinized, and resuspended in 3 ml of PBS, and the intensity of fluorescence was immediately read in a fluorescence spectrophotometer (RF-5301PC, Shimadzu) at 488 nm for excitation and at 525 nm for emission. The results were expressed as arbitrary units of the fluorescence intensity.
3. Statistical analysis All the data were expressed as mean ± SEM. The statistical significance between the treatments was evaluated by One-way ANOVA and with Bonforroni’s post-hoc test using GraphPAD InStat, Software, USA.
4. Results 4.1. Cytotoxicity studies using MTT assay Treatment of cells with various concentrations of CdCl2 (2.5– 600 lM) showed a time and concentration dependent decline in the cell survival. IC50 values of CdCl2 alone treatment were 550, 525, 350, 27 and 5 lM for 3, 6, 12, 24, 48 h post-CdCl2 treatment times, respectively (Fig. 1). Exposure of HepG2 cells to different concentrations of MGN alone for 2 h did not alter the cell viability significantly. When the cells were treated with increasing concentrations of MGN (10–100 lM) for 2 h before exposure to IC50 (lM) concentration of CdCl2 for 3 h, increased cell survival was observed. Maximum elevation in cell survival was significantly (p < 0.01) greater in 50 lM MGN pretreated group when compared with other groups (Fig. 2A). Cytoprotective activity of MGN was evaluated by treating HepG2 cells with 50 lM of MGN for 2 h before treating with 100 lM of CdCl2 for various time periods. A significant (p < 0.001) elevation in cell survival of 1.4-fold was observed in MGN pretreated group in comparison with 24 h CdCl2 alone treated group (Fig. 2B).
2.5.4.2. Fluorescence microscopic analysis of apoptotic cells. The morphological changes that occur in a cell during apoptosis/necrosis were also analyzed by the differential uptake of fluorescent DNA binding ethidium bromide and acridine orange (AO/EtBr) stains. Cells were treated with 25 and 50 lM concentrations of MGN for 2 h followed by treatment with 50 lM of CdCl2 for 24 h. Briefly, at the end of the treatment times, cells were washed with cold PBS and stained with AO/EtBr (1:1 v/v) at a final concentration of 100 lg/ml and observed under a fluorescent microscope (Olympus BX51, Japan). According to this staining method, the live cells have normal green nuclei, early apoptotic cells have bright green nuclei with condensed or fragmented chromatin, late apoptotic cells display condensed and fragmented orange chromatin, and cells that have died from direct necrosis have structurally normal orange/red nuclei (Renvoize et al., 1998).
2.5.4.3. Detection of DNA fragmentation by agarose electrophoresis. The formation of ladder pattern from the DNA fragmentation indicating apoptosis was performed according to the protocol described by Giri et al. (2003) with minor modifications. Exponentially growing cells were treated with various concentrations of MGN (10–100 lM) for 2 h followed by treatment with 50 lM of CdCl2 for 24 h. After the various treatments, the floating and adherent cells were pooled and lysed overnight at 37 °C in a lysis buffer (0.02M EDTA, 0.05 M Tris HCl, 1% Nonidet P-40). After centrifugation at 800 rpm for 10 min, the supernatant was collected and incubated with 10ll of RNAse (10 mg/ml) and 20 ll of 20% Sodium dodecyl sulfate at 56 °C for 2 h. This was followed by treatment with 5 ll of Proteinase K (20 mg/ml) at 56 °C for 8 h. DNA was precipitated overnight with ammonium acetate and ethanol and the resultant pellet was dissolved in 20 ll of TE buffer. Later, 10 lg of DNA was separated on a 1.5% agarose gel and the fragmented ladder pattern was visualized under a UV illuminator after ethidium bromide staining.
Fig. 1. Changes in the cell survival after treating HepG2 cells with CdCl2 as assessed by MTT assay. (}) 3 h (IC50 550 lM); (5) 6 h (IC50 525 lM); (D) 12 h (IC50 350 lM); (s) 24 h (IC50 27 lM) and (h) 48 h (IC50 5 lM).
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Fig. 2. (A) Changes in viability of CdCl2 exposed cells with or without prior treatment with MGN. (HepG2 cells were treated with different doses of Mangiferin for 2 h followed by treatment with 550 lM of cadmium for 3 h). The significant levels *p < 0.05, **p < 0.01 compared to CdCl2 group and ***p < 0.001 compared with control HepG2 group. (B) Effect of MGN (50 lM for 2 h) on HepG2 cells treated with 100 lM of CdCl2 for various time periods. The significant levels *p < 0.05, **p < 0.001 and no symbol = non significant when compared with respective CdCl2 treated group.
4.2. Clonogenic assay
4.3. Micronucleus assay
The clonogenic assay is the gold standard for screening cytotoxic activity of any test agent, it was used to study the effect MGN on cytotoxic effect of CdCl2. Treatment of HepG2 cells with increasing concentrations of CdCl2 resulted in a concentration dependent decline in the cell survival as evidenced by the reduction in surviving fraction, which was lowest at 200 lM CdCl2 (Fig. 3). Treatment of the cells with 25 lM MGN for 2 h before exposure to different concentrations of CdCl2 resulted in an elevation in the cell survival when compared with the CdCl2 alone treatment. The cytoprotective effect of MGN increased at all concentration of CdCl2 used. The surviving fraction increased by 0.22, 0.19 and 0.16 for 25, 50 and 75 lM CdCl2 in MGN pretreated group when compared with the CdCl2 alone treated group.
The protection against CdCl2 induced genotoxicity by MGN was studied by micronucelus assay. Exposure of HepG2 cells to various concentrations of CdCl2 resulted in a concentration dependent elevation in the frequency of MNBNC at all the post-treatment times. MGN treatment alone did not alter the spontaneous frequency of micronuclei in binucleate cells up to 100lM. Treatment of HepG2 cells with 10 lM of CdCl2 resulted in a significant (p < 0.001) elevation in the MNBNC, and this increase in MNBNC was 4.15-folds higher than that of control MNBNC frequency. Exposure of the cells to different concentrations of MGN before treatment with 10 lM CdCl2 caused a decline in the CdCl2 induced micronuclei formation and a maximum decrease in MNBNC’s was observed for 25 lM MGN (Table 1) and this dose was considered as the optimum protective concentration. Therefore, further studies were carried out using this MGN concentration. Pretreatment of HepG2 cells with 25 lM MGN before exposure to different concentrations of CdCl2 resulted in a significant (p < 0.01) decline in the frequency of MNBNC when compared with the CdCl2 alone treatment (Fig. 4).
Table 1 Effect of various concentrations of MGN on the micronuclei induction in HepG2 cells exposed to 10 lM of CdCl2. MGN (lM)
0 2.5 5.0 10 25 50 75 100
Fig. 3. Effect of MGN (25 lM, 2 h) on the cell survival of HepG2 cells treated with different concentrations of CdCl2 for 3 h as assessed by clonogenic assay.
Frequency of micronucleated binucleate cells (MNBNC) ± SEM MGN alone
MGN + CdCl2
26.00 ± 0.64 26.75 ± 1.66 28.00 ± 1.21 28.50 ± 1.78 29.25 ± 1.54 31.50 ± 1.27 31.75 ± 2.15 32.25 ± 2.56
108.25 ± 1.15 89.00 ± 1.21 81.75 ± 1.54a 76.50 ± 2.07a 71.75 ± 1.21b 78.25 ± 2.72a 83.25 ± 2.56a 88.75 ± 2.89
MGN = Mangiferin, CdCl2 = cadmium chloride, SEM = standard error of the mean. The significant levels: a = p < 0.05, b = p < 0.01, and no symbol = non-significant, when compared with the respective control group.
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(OTM) in a concentration dependent manner at all the post-treatment times (Fig. 5A). Treatment of cells with different concentrations of MGN (10–100 lM) alone did not alter baseline DNA damage significantly. Pretreatment of HepG2 cells with different concentrations (2.5–100 lM) of MGN before exposure to 50 lM CdCl2 caused decline in the CdCl2 induced percentage of tail DNA and OTM and a maximum decrease was observed for 25 lM MGN (Table 2), and was considered as the optimum protective concentration. A separate experiment was conducted to study the effect of 25 lM MGN on the DNA damage induced by increasing doses of CdCl2 (10–100 lM). Treatment of HepG2 cells with different concentrations of CdCl2 alone caused a concentration dependent elevation in the DNA damage (Fig. 5B). Pretreatment with 25 lM MGN showed a dose-dependent reduction in the DNA damage when compared with CdCl2 alone treated group. The reduction in the DNA damage by MGN was statistically significant (p < 0.01) at all concentrations of CdCl2, when compared with CdCl2 alone treatment. Fig. 4. Effect of various concentrations of CdCl2 for 3 h on the micronuclei induction in HepG2 cells pretreated with 25 lM for MGN for 2 h. The significant levels * p < 0.05, **p < 0.01, and no symbol = non significant, when compared with respective control group. MNBNC = micronucleated binucleate cells.
4.4. Comet assay Alkaline single cell gel electrophoresis (comet assay) was used to detect the cellular DNA damage induced by CdCl2. Exposure of HepG2 cells to CdCl2 increased the comet parameters like percentage Tail DNA resultant of damaged DNA, and olive tail moment
4.5. Analysis of apoptosis using flow cytometry In order to evaluate the effect of MGN on apoptosis induced by CdCl2 in HepG2 cells, sub-G1 cell population was analyzed. Increase in the population of cells with sub-G1 DNA content indicates increase in the apoptotic cells. The sub-G1 fraction was approximately 6% both in the untreated HepG2 cells as well as after 100 lM MGN treatment. Cells treated with different concentration of MGN (10, 25, 50 and 100 lM) prior exposure to 10 lM CdCl2, showed sub-G1 fraction of 15.30%, 10.44%, 13.42% and 16.5%,
Fig. 5. (A) Genotoxic effect of various concentrations of CdCl2 on HepG2 cells. (HepG2 cells were treated with various concentrations of cadmium for 3 h). The significant levels *p < 0.01, **p < 0.001, and no symbol = non significant when compared to control. (B) Changes in CdCl2 induced DNA damage influenced by MGN (25 lM) treatment assessed by comet assay (OTM = olive tail moment). All the other explanations are as in Fig. 4.
Table 2 Effect of various concentrations of MGN on the DNA damage induced by 50 lM of CdCl2 in HepG2 cells. MGN (lM/ml)
0 10 25 50 100
Head DNA
Tail DNA
OTM
MGN alone
MGN + CdCl2
MGN alone
MGN + CdCl2
MGN alone
MGN + CdCl2
85.78 ± 1.15 84.87 ± 1.14 83.46 ± 1.08 82.92 ± 1.37 82.35 ± 1.24
64.30 ± 1.13 68.87 ± 1.19 73.80 ± 0.94a 70.53 ± 1.02 67.53 ± 1.07
14.22 ± 0.56 15.23 ± 0.71 16.64 ± 0.78 17.18 ± 1.21 17.75 ± 2.56
35.70 ± 1.13 31.13 ± 1.19 26.20 ± 1.09a 29.47 ± 1.07 32.47 ± 1.02
12.16 ± 0.72 12.52 ± 0.56 14.25 ± 0.28 15.12 ± 0.31 15.54 ± 0.42a
40.40 ± 1.30 36.91 ± 1.23 29.54 ± 1.10a 32.45 ± 1.01 33.96 ± 1.14
The HepG2 cells were exposed to MGN for 2 h + CdCl2 for 3 + 2 h post incubation. MGN = Mangiferin; CdCl2 = cadmium chloride; OTM = olive tail moment; SEM = standard error of the mean. The significant levels: a = p < 0.05 and no symbol = non-significant, when compared with the respective control group.
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respectively. The maximum decline in the sub-G1 fraction was observed at 25 lM of MGN pretreated group (Fig. 6).
Table 3 Apoptotic index of HepG2 cells treated with CdCl2 with or without MGN. MGN (lM)
Apoptotic index ± SEM (%) MGN alone
MGN + CdCl2
0 10 25 50 100
4.2 ± 0.01 4.4 ± 0.08 4.5 ± 0.04 4.8 ± 0.12 5.0 ± 0.06
37.2 ± 0.25 14.8 ± 0.37c 13.2 ± 0.11c 15.8 ± 0.22c 16.6 ± 0.29c
4.6. Morphological analysis of apoptotic bodies Apoptosis detection was performed by morphological analysis by double staining with AO/EtBr. HepG2 cells when treated with 50 lM of CdCl2 alone resulted in appearance of cells in early as well as late apoptotic stages with a minimal number of necrotic cells. Membrane blebbing and alterations in nuclear morphology such as chromatin condensation and DNA fragmentation were observed in apoptotic cells. MGN treatment alone even at a dose of 100 lM did not increase the proportion of apoptotic cells indicating its non toxic nature. When the cells treated with 25 lM of MGN for 2 h before treatment with 50 lM of CdCl2 for 24 h, the proportion of apoptotic cells decreased significantly (p < 0.01) as compared to the cells treated with CdCl2 alone (Table 3). 4.7. Detection of DNA fragmentation To confirm further, the effect of MGN on CdCl2 induced apoptosis, DNA was isolated from the treated and untreated HepG2 cells and electrophoresed on 1.2% agarose gel. Cells treated with 2 lM doxorubicin served as a positive control showed a clear visible typical ladder formation. A similar ladder formation after CdCl2 treatment was observed even at a low dose of 10 lM and became more prominent at higher concentrations (Fig. 7). The nucleosomal ladder formation after CdCl2 treatment pretreated with MGN showed decrease in the intensity of the ladder indicating the inhibition of apoptosis when compared to the CdCl2 alone. Further, it was observed that the intensity of the ladder formed in 25 lM of MGN pretreated group was much less in comparison to other pretreated groups indicating that apoptosis induction is less in 25 lM MGN pretreated group (Fig. 7, Lane 14).
The HepG2 cells were exposed to MGN for 2 h following CdCl2 at 50 lM for 3 h and cells were processed immediately for microscopic analysis. The other details are as in Table 2 and the significant levels: c = p < 0.001 compared to the control.
4.8. Intracellular ROS estimations The changes in the intracellular ROS generation induced by CdCl2 as influenced by MGN were estimated. The HepG2 cells treated with CdCl2 showed dose dependent increase in DCF fluorescence levels (indicative of intracellular ROS levels) (Fig. 8A). Pretreatment with MGN decreased DCF fluorescence levels in a dose dependent manner, a significant (p < 0.001) decrease was observed at 25 lM MGN pretreated group in comparison with CdCl2 alone group (Fig. 8B). 5. Discussion Cadmium being one of the toxic heavy metal and due to its inherent nature it gets accumulated in the environment, may pose severe risk for health problems (Godt et al., 2006). Exposure to cadmium has been associated with several specific clinical complications such as renal dysfunction, liver abnormalities and bone diseases (Goyer, 1997; Kasuya et al., 2000; Satarug et al., 2002; Jin et al., 2004). Recently, there is an increasing interest on the use of dietary components such as flavonoids, polyphenols,
Fig. 6. Flow cytometric analysis of changes in CdCl2 induced sub-G1 cell population indicating apoptotic cells influenced by MGN treatment. (A) Control HepG2; (B) MGN 100 lM alone; (C) CdCl2 50 lM alone; (D) MGN (10 lM) + CdCl2; (E) MGN (25 lM) + CdCl2; (F) MGN (50 lM) + CdCl2; (G) MGN (100 lM) + CdCl2. In the graph, DNA content (PI area – X-axis) is plotted against number of cells (Y-axis). The gates on the DNA histogram: M1 represent the total number cells and M2 indicates cells which fall in the subG1 region (apoptotic cells).
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Fig. 7. Effect of Mangiferin on the CdCl2 induced DNA fragmentation in the HepG2 cells. [Lane 1 and 10 – control; Lane 2 and 11 – positive control, doxorubicin (2 lM); Lane 3–8 cells treated with CdCl2 (5, 10, 25, 50, 100 and 200 lM); Lane 12 – CdCl2, 10 lM alone; Lane 13 – MGN (10 lM) + CdCl2; Lane 14 – MGN (25 lM) + CdCl2; Lane 15 – MGN (50 lM) + CdCl2; Lane 16 – MGN (100 lM) + CdCl2; Lane 17 – MGN (100 lM) alone; Lane 9 and 18 – 3 Kb ladder marker].
Fig. 8. (A) Induction of ROS levels after 3 h treatment with CdCl2 in HepG2 cells. (B) Effect of different concentrations of MGN treatment (2 h) on CdCl2 (10 lM, 3 h) induced ROS levels in HepG2 cells. The significant levels *p < 0.05, **p < 0.001, ***p < 0.001, when compared with respective control group.
xanthones to mitigate the effects of toxic environmental pollutants including certain heavy metals such as Cd, Hg, Zn and Fe. Many of these flavonoids were well known anti-oxidants and protect against damage caused by reactive oxygen species (Pietta, 2000). Further, few of these dietary flavonoids are also efficient modulators of enzymes involved in biotransformation reactions (Rodeiro et al., 2008a). Such dietary agents may have great utility in counteracting the effect of toxic heavy metals as they are consumed daily and have wide acceptability than other agents that have similar properties. The well known beneficial properties of such natural products motivated us to carry out this study on MGN for its cytoprotective and antigenotoxic potential. Here, we report for the first time the ameliorating effect of MGN against CdCl2 induced cytotoxic and genotoxic effects using cultured HepG2 cells. MGN, a naturally occurring glucosylxanthone, has catechol moiety, a pharmacophore with well established anti-oxidant activity, this moiety was also known to present in numerous other flavonoids. The hepatoprotective property due to its anti-oxidant poten-
tial has been established earlier (Rodeiro et al., 2008b). Apart from its well documented anti-oxidant property, MGN has many other pharmacological activities including anti-inflammatory, anti-viral, anti-bacterial and hypoglycemic effects (Zheng and Lu, 1990; Ojewole, 2005). In our study, treatment of HepG2 cells with CdCl2 resulted in a concentration dependent elevation in cytotoxicity as evidenced by the continuous decline of viable cells with increasing doses of CdCl2. Interestingly, in the presence of MGN, CdCl2 treated HepG2 cells showed reduction in cytotoxicity compared to cells treated with CdCl2 alone. Although, the protective potential of MGN against the cytotoxicity induced by several of the hepatotoxins such as tert-butyl hydroperoxide, ethanol, carbon tetrachloride and lipopolysaccharide (Rodeiro et al., 2008b) has been well documented, here we report its beneficial effect against heavy metal toxicity. Furthermore, our study has also demonstrated protective effect of MGN against CdCl2 induced genotoxicity, as evaluated by micronucleus and comet assay. A plethora of earlier reports convincingly
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demonstrated the genotoxic and co genotoxic potential of cadmium in plants, animals and mammalian cells (Waisberg et al., 2003; Deckert, 2005; Bertin and Averbeck, 2006). Moreover, earlier in vitro studies have indicated cadmium induced effects such as single and double strand breaks in DNA, DNA–protein cross links, oxidative DNA damages, mutations and chromosomal damage (Misra et al., 1998; Mouron et al., 2004). The mechanistic insights into the genotoxic effect of cadmium revealed both the direct and indirect pathways. Among all the DNA lesions, DNA double strand breaks (DSB) were considered as critical if unrepaired or misrepaired they contribute to cell killing or cell transformation (Bertin and Averbeck, 2006). In our study, we demonstrate the non-genotoxic property of MGN as it failed to induce any micronuclei and comet tail length by itself. Our results, further confirms the antimutagenic and antigenotoxic effect of MGN as indicated in an earlier experimental findings of Rodeiro et al., 2006 using bacterial and rodent mutagenic tests. Although, the exact mechanism involved in the protection of cadmium induced DNA damage is not clear, Mangiferin was shown to be protective against oxidative damage and mutagenesis due to its free radical scavenging property (Sato et al., 1992). Cadmium induces oxidative stress arising from indirect processes like decrease in cellular anti-oxidants and exhalation of ROS by mitochondria (Waisberg et al., 2003), leading to DNA damage as well as misfolding of cellular proteins (Bertin and Averbeck, 2006). Protective effects of natural compounds against cadmium toxicity are usually found at a particular optimum concentration. In our study, the effect of MGN on CdCl2 induced ROS was studied using DCFH-DA assay. CdCl2 alone elevated the intracellular ROS levels in a concentration dependent manner. In the presence of MGN, ROS levels were found to be declining and the maximum decline was found at 25 lM of an optimal concentration. This decrease in the CdCl2 induced ROS at 25 lM of MGN may account for the decline in the observed genotoxic as well as cytotoxic effect. However, MGN at higher concentrations was known to form oxidized products like quinoids as a result of its anti-oxidant activities, which accumulates in the mitochondria and reacts with membrane thiol groups. This shifts its anti-oxidant property to thiol arylation (Andreu et al., 2005) which might be the possible reason for the observed ineffectiveness in lowering the ROS levels observed at the doses more than 25 lM MGN in our studies. In the present study, CdCl2 induced apoptotic cells in a dose dependent manner as indicated in DNA ladder pattern after gel electrophoresis. This is in accordance with the earlier studies on the production of reactive oxygen species, which was implicated in CdCl2 induced apoptosis (Oh and Lim, 2006; Oh et al., 2006). Further, the CdCl2 induced cell death by apoptosis was attributed to the activation of caspase-8 via FAS receptor followed by BID cleavage and induction of mitochondrial caspase cascade after release of cytochrome C in HepG2 cells and betulin, a triterpene inhibited cadmium induced apoptotic cell death by its anti-oxidant effect (Oh and Lim, 2006). In the present study, although MGN alone did not induce any apoptotic HepG2 cells up to a concentration of 100 lM, pretreatment of cells with 25 lM of MGN reduced the percentage of apoptotic cells. On the basis of the earlier reports and present findings it is clear that CdCl2 induced generation of ROS must have resulted in increased apoptotic cells, while the inhibiting action of MGN on the production of ROS as well as scavenging of free radicals generated must have resulted in decreased apoptotic cells. Mammalian cells have complex strategies to detect and manage heavy metal induced stress on cells. Metallothionein (MT), low molecular weight cysteine-rich proteins are on the forefront in the homeostasis of these essential metals. These metal binding proteins were known to protect against cadmium toxicity and oxidative stress (Andrews, 2000). Besides, it is also understood that
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GSH also has a role in the protection of cadmium induced cytotoxicty. This was substantiated by the fact that GSH supplementation protected against cadmium induced hepatotoxicity and nephrotoxicity (Singhal et al., 1987). Furthermore, it was reported that Mangiferin increased GSH levels and also increased catalase activity (Sarkar et al., 2004). Therefore, the role of MT and GSH in ameliorating cadmium induced cytotoxicity cannot be ruled out in the present study. The studies are underway to understand the role of MT as well as GSH for the observed cytoprotective action of MGN. The present study revealed that MGN, a glucosylxanthone, an active component of M. indica has potent cytoprotective and antigenotoxic effect against CdCl2 induced toxicity in HepG2 cell line. The cytoprotective and antigenotoxic property of MGN may be attributed to decrease in CdCl2 induced ROS levels and resultant oxidative stress. Taken together, the earlier health promoting pharmacological properties of MGN and with a yet another implication from the present study it may be concluded that MGN may be a good agent for counteracting the negative effects of several other heavy metals. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgement This research work was carried out under the ITREOH program activity with the support of Manipal University, Manipal. The authors are thankful to Dr. K. Satyamoorthy, The Director, Manipal Life Sciences Centre, Manipal University, Manipal for his help and encouragement during this study. References Andreu, G.L., René, D., Velho, A.J., Curti, C., Vercesi, A.E., 2005. Mangiferin, a natural occurring glucosylxanthone, increases susceptibility of rat liver mitochondria to calcium-induced permeability transition. Arch. Biochem. Biophys. 439, 184– 193. Andrews, G.K., 2000. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 59, 95–104. Bai, J., Cederbaum, A.I., 2003. Catalase protects HepG2 cells from apoptosis induced by DNA damaging agents by accelerating the degradation of p53. J. Biol. Chem. 278, 4660–4667. Bertin, G., Averbeck, D., 2006. Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review). Biochimie 88, 1549–1559. Bolkent, S., Koyuturk, M., Bulan, O.K., Tunali, S., Yanardag, R., Tabakoglu, A.O., 2007. The effects of combined alpha-tocopherol, ascorbic acid, and selenium against cadmium toxicity in rat intestine. J. Environ. Pathol. Toxicol. Oncol. 26, 21–27. Ciesielska, S.A., Stachura, A., Slotwinska, M., Kaminska, T., Sniezko, R., Paduch, R., Abramczyk, D., Filar, J., Kandefer-Szerszen, M., 2000. The inhibitory effect of zinc on cadmium-induced cell apoptosis and reactive oxygen species (ROS) production in cell cultures. Toxicology 145, 159–171. Collins, A., Dusinska, M., Franklin, M., Somorovska, M., Petrovská, H., Duthie, S., Fillion, L., Panayiotidis, M., Raslova, K., Vaughan, N., 1997. Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ. Mol. Mutagen. 30, 139–146. Deckert, J., 2005. Cadmium toxicity in plants: is there any analogy to its carcinogenic effect in mammalian cells? Biometals 18, 475–481. Elinder, C.G., Lind, B., Kjellstorm, T., Linnman, L., Friberg, L., 1976. Cadmium in kidney cortex, liver and pancreas from Swedish autopsies. Estimation of biological half time in kidney cortex, considering calories intake and smoking habits. Arch. Environ. Health 31, 292–302. Fenech, M., Morley, A.A., 1985. Measurement of micronuclei in lymphocytes. Mutat. Res. 147, 29–36. Fotakis, G., Timbrell, J.A., 2006. Modulation of cadmium chloride toxicity by sulphur amino acids in hepatoma cells. Toxicol. In Vitro 20, 641–648. Giri, K., Ghosh, U., Bhattacharyya, N.P., Basak, S., 2003. Caspase 8 mediated apoptotic cell death induced by b-sheet forming polyamine peptides. FEBS Lett. 555, 380–384. Godt, J., Scheidig, F., Grosse, S.C., Esche, V., Brandenburg, P., Reich, A., Groneberg, D.A., 2006. The toxicity of cadmium and resulting hazards for human health. J. Occup. Med. Toxicol. 1, 22. Goyer, R.A., 1997. Toxic and essential metal interactions. Annu. Rev. Nutr. 17, 37– 50.
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Hurna, E., Siklenka, P., Hurna, S., 1997. Effect of selenium on cadmium genotoxicity investigated by micronucleus assay. Vet. Med.-(Praha) 42, 339–342. Jarup, L., Berglund, M., Elinder, C.G., Nordberg, G., Vahter, M., 1998. Health effects of cadmium exposure – a review of literature and a risk estimate. Scand. J. Work Environ. Health 24, 1–51. Jin, T., Kong, Q., Ye, T., Wu, X., Nordberg, G.F., 2004. Renal dysfunction of cadmiumexposed workers residing in a cadmium-polluted environment. Biometals 17, 513–518. Kasuya, M., Teranishi, H., Aoshima, K., Katoh, T., Horiguchi, H., Morikawa, Y., Nishijo, M., Iwata, K., 2000. Water pollution by cadmium and the onset of Itai–itai disease. Water Sci. Technol. 25, 149–156. Misra, R.R., Smith, G.T., Waalkes, M.P., 1998. Evaluation of the direct genotoxic potential of cadmium in four different rodent cell lines. Toxicology 126, 103– 114. Mossman, T., 1983. Rapid colorimetric assay for cell growth and survival: application to proliferation and cytotoxicity assay. J. Immunol. Methods 65, 55–63. Mouron, S.A., Grillo, C.A., Dulout, F.N., Golijow, C.D., 2004. A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells. Mutat. Res. 568, 221–231. Oh, S.H., Lim, S.C., 2006. A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation. Toxicol. Appl. Pharmacol. 212, 212–223. Oh, S.H., Choi, J.E., Lim, S.C., 2006. Protection of betulin against cadmium-induced apoptosis in hepatoma cells. Toxicology 1, 1–12. Ojewole, J.A.O., 2005. Anti-inflammatory, analgesic and hypoglycemic effects of Mangifera indica Linn. (Anacardiaceae) stem-bark aqueous extract. Methods Find. Exp. Clin. Pharmacol. 27, 547–554. Pinto, M.M., Sousa, M.E., Nascimento, M.S., 2005. Xanthone derivatives: new insights in biological activities. Curr. Med. Chem. 12, 2517–2538. Pietta, P.G., 2000. Flavonoids as antioxidants. J. Nat. Prod. 63, 1035–1042. Puck, T.T., Marcus, P.I., 1955. A rapid method for viable cell titration and clone production with HeLa cells in tissue culture: the use of x-irradiated cells to supply conditioning factors. Proc. Natl. Acad. Sci. USA 41, 432–437. Rao, B.S.S., Shanbhoge, R., Upadhya, D., Jagetia, G.C., Adiga, S.K., Kumar, P., Guruprasad, K., Gayathri, P., 2006. Antioxidant, anti-clastogenetic and
radioprotective effect of Coleus aromaticus on Chinese hamster fibroblast cells (V79) exposed to gamma radiation. Mutagenesis 21, 237–242. Renvoize, C., Biola, A., Pallardy, M., Breard, J., 1998. Apoptosis: identification of dying cells. Cell Biol. Toxicol. 14, 111–120. Rodeiro, I., Cancino, L., González, J.E., Morffi, J., Garrido, G., González, R.M., Nuñez, A., Delgado, R., 2006. Evaluation of the genotoxic potential of Mangifera indica L. extract (Vimang), a new natural product with antioxidant activity. Food Chem Toxicol. 10, 1707–1713. Rodeiro, I., Donato, M.T., Lahoz, A., González-Lavaut, J.A., Laguna, A., Castell, J.V., Delgado, R., Gomez-Lechón, M.J., 2008a. Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes. Chem. Biol. Interact. 17, 1–10. Rodeiro, I., Donato, M.T., Martínez, I., Hernández, I., Garrido, G., González-Lavaut, J.A., Menéndez, R., Laguna, A., Castell, J.V., Gómez-Lechón, M.J., 2008b. Potential hepatoprotective effects of new Cuban natural products in rat hepatocytes culture. Toxicol. In Vitro 22, 1242–1249. Sarkar, A., Sreenivasan, Y., Ramesh, G.T., Manna, S.K., 2004. Beta-D-Glucoside suppresses tumor necrosis factor-induced activation of nuclear transcription factor kappaB but potentiates apoptosis. J. Biol. Chem. 279, 33768–33781. Satarug, S., Baker, J.R., Reilly, P.E., Moore, M.R., Williams, D.J., 2002. Cadmium levels in the lung, liver, kidney cortex and urine samples from Australians without occupational exposure to metals. Arch. Environ. Health 57, 69–77. Sato, T., Kawamoto, A., Tamura, A., Tatsumi, Y., Fujii, T., 1992. Mechanism of antioxidant action of pueraria glycoside (PG)-1 (an isoflavonoid) and Mangiferin (a xanthonoid). Chem. Pharm. Bull. 40, 721–724. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184–191. Singhal, R.K., Anderson, M.E., Meister, A., 1987. Glutathione, a first line of defense against cadmium toxicity. FASEB J. 1, 220–223. Sundermann, M.V., Knasmüller, S., Wu, X.J., Darroudi, F., Kassie, F., 2004. Use of a human-derived liver cell line for the detection of cytoprotective, antigenotoxic and cogenotoxic agents. Toxicology 198, 329–340. Waalkes, M.P., 2003. Cadmium carcinogenesis. Mutat. Res. 533, 107–120. Waisberg, M., Joseph, P., Hale, B., Beyersmann, D., 2003. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192, 95–117. Zheng, M.S., Lu, Z.Y., 1990. Antiviral effect of mangiferin and isomangiferin on herpes simplex virus. Chin. Med. J. (Engl.) 103, 160–165.