Acacia salicina extracts protect against DNA damage and mutagenesis in bacteria and human lymphoblast cell K562 cultures

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Nutrition Research 28 (2008) 190 – 197 www.elsevier.com/locate/nutres

Acacia salicina extracts protect against DNA damage and mutagenesis in bacteria and human lymphoblast cell K562 cultures Ines Bouhlel a , Soumaya Kilani a , Ines Skandrani a , Rebaii Ben Amar a , Aicha Nefatti a , François Laporte c , Isabelle Hininger-Favier d , Kamel Ghedira a , Leila Chekir-Ghedira a, b,⁎ a

Unité de Pharmacognosie/Biologie Moléculaire 99/UR/07-03. Faculté de Pharmacie de Monastir, Rue Avicenne 5000 Monastir, Tunisie b Laboratoire de Biochimie et de Biologie Moléculaire, Faculté de Médecine dentaire de Monastir, Rue Avicenne, 5000, Tunisie c Département de Biologie Intégrée, Laboratoire des Lipides et Biologie Moléculaire, Centre Hospitalier Universitaire, BP 217, 38043 Grenoble Cedex 09, France d Laboratoire de Nutrition, Vieillissement et Maladies Cardiovasculaires, Université Joseph Fourier-Grenoble1, 38043 Grenoble Cedex 09, France Received 7 September 2007; revised 2 December 2007; accepted 11 December 2007

Abstract Three extracts were prepared from the leaves of Acacia salicina: aqueous, methanol, and ethyl acetate extracts. The antigenotoxic properties of these extracts were investigated by assessing the inhibition of mutagenicity of the indirect-acting mutagen benzo[a]pyrene using the Ames assay and the genotoxicity of the direct-acting mutagen, hydrogen peroxide, using the “Comet assay.” Aqueous, methanol, and ethyl acetate extracts at doses of 500, 50, and 500 μg per plate reduced benzo[a]pyrene mutagenicity by 95%, 82%, and 40%, respectively, in Salmonella typhimurium TA98 strain and by 91%, 66% and 63%, respectively, at the same doses with a TA97 assay system. Human lymphoblast cells K562 were pretreated with 50% inhibition concentration of each extracts and then treated by H2O2, for the Comet assay. The Comet assay results showed that ethyl acetate and methanol extracts decreased the DNA damage caused by H2O2 by, respectively, 34.8% and 31.3%. We envisaged also the study of the antioxidant effect of these extracts by the enzymatic xanthine/xanthine oxidase assay. Results indicated that methanol and ethyl acetate extracts were potent inhibitors of xanthine oxidase and superoxide anion scavengers. We conclude that these integrated approaches to antigenotoxicity and antioxidant assessment may be useful to help compare the beneficial effects associated with using A salicina as medicinal and dietary plant. © 2008 Elsevier Inc. All rights reserved. Keywords: Abbreviations:

Acacia salicina; Ames test; Comet assay; Antigenotoxic activity; Antioxidant activity; Human lymphoblast cells K562 B[a]P, benzo [a] pyrene; BET, ethidium bromide; CML, human chronic myelogenous leukemia; DMSO, dimethylsulfoxide; EDTA, ethylene-diamine tetra-acetic acid; G6P, glucose-6-phosphate; IC50, fifty percent inhibition; NADP, nicotinamide-adenine dinucleotide phosphate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; X, xanthine; XOD, xanthine oxidase

1. Introduction Cancer, cardiovascular disease, brain dysfunction, and a diminished immune system are leading causes of death in the ⁎ Corresponding author. Unité de Pharmacognosie/Biologie Moléculaire 99/UR/07-03. Faculté de Pharmacie de Monastir, Rue Avicenne 5000 Monastir, Tunisie. Tel.: +216 97 3162 82; fax: +216 73 461 150. E-mail addresses: [email protected], [email protected] (L. Chekir-Ghedira). 0271-5317/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2007.12.011

world, and oxidative stress is thought to be an important contributing factor in their development. Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses favoring the former, leading to oxidative damage [1-3]. The process can result from a deficiency in antioxidant defense mechanisms, or from an increase in ROS, because of exposure to elevated ROS levels, the presence of toxins metabolized to ROS, or excessive activation of ROS systems, such as those mediated

I. Bouhlel et al. / Nutrition Research 28 (2008) 190–197

by chronic infection and inflammation [4]. In addition to endogenously produced antioxidants and enzymes that catalyze the metabolism of ROS, the ROS can be scavenged by exogenously obtained antioxidants, such as polyphenolic compounds, flavonoids, ascorbic acid, and other biologically active components that have a positive influence on health [5,6]. Because antioxidants from dietary and medicinal plant sources, particularly those that contain phenolic compounds, have a significant antioxidant activity and are able to help combat degenerative diseases, such as cancer, cardiovascular diseases, immune system decline, and brain dysfunction [7], many investigators have searched for chemopreventive and antioxidant agents from natural products to identify dietary treatments for humans. Acacia is an evergreen tree that is native to Australia but is now widely distributed in the Mediterranean area. Acacia species are frequently used for the treatment of various illnesses because of their reputed pharmacologic effects. The published actions for Acacia include hypoglycemic [8], antiinflammatory [9], cestodial [10], antiplatelet aggregation [11], spasmogenic and vasoconstrictor [12], antihypertensive [13], and inhibitory against hepatitis C virus [14]. In Tunisian traditional medicine, the use of Acacia differs according to the species and according to the region of the country. Based on information gathered from traditional healers, herbalists, and inhabitants from rural regions, Acacia salicina is frequently used in such diverse applications as the treatment of inflammatory diseases, as febrifuge to treat cancer, and to promote human fertility. In Tunisia, infusions made from fresh or dried leaves are taken orally, or alternatively, chopped fresh leaves are applied directly to inflamed sores. Traditional medicinal uses of Acacia in north Tunisia are somewhat different; in this region, Acacia is used for the treatment of diarrhea and rheumatism. Recently we reported the presence of flavonoids and tannins in A salicina extracts [15,16], and based on this information, we examined the ability of A salicina extracts to protect against oxidative and genotoxic damage caused by carcinogens and ROS in vitro. Therefore, we investigated the genotoxic, antigenotoxic, and antioxidant properties of extracts from A. salicina collected from Monastir in the center of Tunisia. 2. Methods and materials 2.1. Plant materials The leaves of A salicina were collected in the Monastir region of Tunisia in November 2003. Botanical identification was carried out by Pr M Chaieb (Department of Botany, Faculty of Sciences, University of Sfax, Sfax, Tunisia), according to the flora of Tunisia [17]. A voucher specimen (ASm-11.03) has been deposited in the laboratory of Pharmacognosy, Faculty of Pharmacy of Monastir, Tunisia. The leaves were shade-dried, powdered, and stored at room temperature in a tightly closed container for analysis and use.

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2.2. Extraction method The powdered leaves were extracted with boiling water for 15 to 20 minutes. After filtration, the extracts were freeze-dried and yielded the aqueous extract. The residues were dissolved in water. Ethyl acetate and methanol extracts were obtained by soxhlet extraction (6 hours). These organic phases were concentrated to dryness. The residues were kept at 4°C. Before use, the extracts were resuspended in dimethylsulfoxide (DMSO). In the present study, 3 extracts were investigated. The extracts were used at the concentrations of 50, 250, and 500 μg per plate in Ames test; 10, 50, 150, 300, and 600 μg/ mL in xanthine (X)/X oxidase (XOD) assay; and 200 μg/mL for the methanol extract, 250 μg/mL for the ethyl acetate extract, and 120 μg/mL for the aqueous extract in cell culture. These concentrations were in accordance with our previous investigations where several preliminary dose-testing involving several plant extracts were conducted [18-20]. 2.3. Inhibition of xanthine oxidase activity and superoxide radical scavenging effect Both inhibition of XOD activity and the superoxide anion scavenging activity, were assessed in vitro in 1 assay. The inhibition of XOD activity was measured according to the increase of uric acid ultraviolet absorbance at 290 nm as proposed by Cimanga et al [21], whereas the superoxide anion scavenging activity was detected spectrophotometrically with the nitrite method described by Oyangagui [22], with some modifications introduced by Russo et al [23]. Briefly, the assay mixture consisted of 100 μL of the tested compound solution, 200 μL (X) (Sigma, Aldrich, St Louis, MO) (final concentration, 50 μmol/L) as the substrate, hydroxylamine (final concentration, 0.2 mmol/L), 200 μL EDTA (0.1 mmol/L), and 300 μL distilled water. The reaction was initiated by adding 200 μL XOD (Sigma) (11 mU mL −1) dissolved in phosphate buffer (KH2PO4 20.8 mmol/L, pH 7.5). The assay mixture was incubated at 37°C for 30 minutes. Before uric acid production measurement at 290 nm, the reaction was stopped by adding 0.1 mL of HCl 0.5 mol/L. The absorbance was measured spectrophotometrically against a blank solution, prepared as described above, but replacing XOD with buffer solution. Another control solution without the tested compound was prepared in the same manner as the assay mixture to measure the total uric acid production (100%). The uric acid production was calculated from the differential absorbance. To detect the superoxide scavenging activity, 2 mL of the coloring reagent consisting of sulphanilic acid solution (final concentration, 300 μg/mL), N-(1-naphtyl) ethylenediamine dihydrochloride (final concentration, 5 μg/mL) and acetic acid (16.7%, vol/vol) were added. This mixture was left for 30 minutes at room temperature and the absorbance was measured at 550 nm on a Helios α-spectrophotometer. The dose-response curve for each test compound was linearized by regression analysis and used to derive the 50% inhibition (IC50) values.

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2.4. Bacterial tester strains Salmonella typhimurium strains TA98 and TA97, which are histidine-requiring mutants, were kindly provided by Pr. Felzen (Universidade do Estado do Rio de Janeiro [UERJ], Rio de Janeiro, Brazil) and maintained as described by Maron and Ames [24]. The genotypes of the test strains were checked routinely for their histidine requirement, deep rough (rfa) character, ultraviolet sensitivity (uvrB mutation), and presence of the R factor. They were stored at −80°C. S typhimurium TA98 and TA97 strains are known to be more responsive to benzo[a]pyrene (B[a]P) mutagen [24]. 2.5. Activation mixture The S9 microsome fraction was prepared from livers of rats treated with Aroclor 1254 (Supelco, Bellefonte, Pa) [24]. The composition of S9 mix was 8 mmol/L MgCl2, 32.5 mmol/L KCl, 5 mmol/L glucose-6-phosphate, 4 mmol/L nicotinamide-adenine dinucleotide phosphate, 0.1 mol/L sodium phosphate buffer (pH 7.4), and S9 fraction at a concentration of 0.04 mL/mL (S9 mix). The S9 fraction was stored at −80°C. 2.6. Salmonella microsome assay The mutagenicity assay with S typhimurium was performed as described by Maron and Ames [24]. The experiments were performed with and without an exogenous metabolic system, the S9 fraction in S9 mix. One hundred microliters of an overnight culture of bacteria [cultivated for 16 hours at 37°C, approximate cell density (2-5) × 10 8 cells/ mL] and 500 μL of sodium phosphate buffer (0.2 mol/L, pH 7.4 for assay without S9) or 500 μL of S9 mix were added to 2-mL aliquots of top Agar (supplemented with 0.5 mmol/L L-histidine and 0.5 mmol/L D-biotine) containing different concentrations of each extract. The resulting complete mixture was poured on minimal agar plates prepared as described by Maron and Ames [24]. The plates were incubated at 37°C for 48 hours and the revertant bacterial colonies of each plate were counted. Negative control without the tested compound was prepared in the same manner as the assay mixture; it gave the number of spontaneous revertants, whereas the positive control was prepared as described above with adding the toxicant B[a]P. An extract was considered as mutagenic if the number of revertants per plate was at least doubled in TA98 and TA97 strains over the spontaneous revertant frequency [24]. Data were collected with a mean ± SD of 3 plates (n = 3). 2.7. Antimutagenicity testing To evaluate the antimutagenic ability of A salicina extracts, we analyzed their effect on stable mutations inferred by the B[a]P. Therefore, a modified plate incorporation procedure [24] was used to determine the effect of all isolates on B[a]P induced mutagenicity. Briefly, 0.5 mL of the S9 mixture for indirect mutagen B[a]P (7.5 or 10 μg per plate) was distributed in sterilized capped

tubes in an ice bath, then 0.1 mL of test compounds (50 μL of mutagen and/or 50 μL of test compound) and 0.1 mL of bacterial culture (2-5) × 10 8 cells/mL were added. After vortexing gently and preincubating at 37°C for 30 minutes, 2 mL of top agar supplemented with 0.05 mol/L L-histidine and D-biotine (Sigma-Aldrich, St Louis, MO) were added to each tube and vortexed for 3 seconds. The resulting entire was overlaid on the minimal agar plate. The plates were incubated at 37°C for 48 hours, and the revertant bacterial colonies on each plate were counted. The inhibition rate of mutagenicity (%) was calculated relative to those in the control group with only the mutagen by the following formula: Percent inhibition ð%Þ ¼ f1  ½ðnumber of revertants on test plates  number of negative controlÞ =ðnumber of revertants on positive control plates  number of negative controlÞg 100:

2.8. Cell culture The human chronic myelogenous leukemia (CML) cell line K562 was maintained in RPMI 1640 (Invitrogen Life Technology, Cergy Pontoise, France) medium supplemented with 10% fetal bovine serum, L-glutamine (4%), and gentamycine (1%) (all from Sigma Cell Culture, Courtaboeuf, France), in humidified incubator at 37°C and an atmosphere enriched with 5% CO2. The culture medium was renewed every 2 to 3 days. 2.9. Assay for cytotoxic activity To control if the tested concentrations of plant extracts impacted cell viability, we estimated viability of K562 cells by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, which is based on the cleavage of a tetrazolium salt by mitochondrial dehydrogenase in viable cells. The in vitro test against the human CML cell line K562 was performed essentially according to the method described previously [25]. Cells were seeded into 96-μL plates and incubated overnight. The test samples were dissolved in DMSO and were added in serial dilution (the final DMSO concentrations in all assays did not exceed 0.01%). Twenty 4 hours after seeding, 100 μL of new media or test compounds was added before incubating the plates for 48 hours. Cells were washed once before adding 50 μL fetal bovine serum–free medium containing 5 mg/mL MTT. After 4 hours of incubation at 37°C, the medium was discarded, and the formazan blue formed in the cells was replaced by adding 50 μL DMSO. Negative control without the tested compound was prepared in the same manner. Optical density (OD) was measured at 540 nm. The cytotoxicity index (%) was calculated according to the following equation [26]: The % of cell viability ¼ ð1 ðOD of treated cells=OD of negative controlÞÞ 100:

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Table 1 IC50 of A. salicina extracts for inhibition of xanthine oxidase and reduction of superoxide anion levels Extracts

Concentration (μg/mL)

Inhibition of xanthine oxidase activity (%)

Methanol extract

300 150 50 10 300 150 50 600 300 150 50

76.4 ⁎ ± 0.15 73.1 ⁎ ± 0.6 11.08 ± 1.7 – 82.1 ⁎ ± 1.5 56.8 ⁎ ± 2.1 29.6 ⁎ ± 0.33 – 51.5 ⁎ ± 0.98 36.5 ⁎ ± 0.75 7.4 ± 3.33

Ethyl acetate extract

Aqueous extract

IC50 (μg/mL) a, b 87.5

112

275

Inhibition of superoxide anion (%) 95.6 ⁎ ± 1.45 92.7 ⁎ ± 0 81 ⁎ ± 1.45 43.5 ⁎ ± 2.11 75.8 ⁎ ± 0.35 62.9 ⁎ ± 2.2 47.1 ⁎ ± 1.7 54.7 ⁎ ± 2.9 35.7 ⁎ ±1.4 20.4 ± 3.6 9.4 ± 4.3

IC50 (μg/mL) a, b 12

70

550

–, not tested. a Values are means ± SD (n = 3). b Values obtained from regression lines with 95% of confidence level. IC50 is defined as the concentration sufficient to obtain 50% of maximum inhibition. ⁎ P b .05 compared with the negative control (without the tested compound) using Dunett test.

2.10. Comet assay As eukaryotic cells compared with prokaryotic cells have more complicated morphological and biochemical structures, the Comet assay with human lymphocytes was used to detect DNA damage. Before each experiment, frosted microscope slides were precoated with 2 layers (100 μL) of normal agarose (1% in milli-Q water) and left at room temperature to allow agarose to dry. The cells were treated during 2 hours with the previously detected IC50 cell viability concentration of the different extracts. Cells were harvested in phosphate-buffered saline (PBS) after a 2-hour stress with 50 μmol/L H2O2. The cell dilution (20 000 cells in 60 μL) was mixed with an equal volume of low-melting-point agarose (1.2% in PBS). This agarose cell suspension (120 μL) was spread onto each precoated slide and covered with a cover slip. After 10 minutes on ice, the cover slip was gently removed, and the slides were placed in a tank filled with the lysate buffer (2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L acid Tris, 1% sodium sarcosinate pH 10, 1% of Triton × 100, and 10% DMSO). They were immerged for 1 hour in this buffer (4°C, in the dark). The slides were then transferred into the electrophoresis buffer (NaOH 10 N, EDTA 200 mmol/L, pH 13 in deionized water) during 20 minutes at room temperature in the dark. Electrophoresis was carried out for 15 minutes at 25 V, 300 mA. Finally, the slides were gently rinsed with neutralization solution (0.4 mol/L Tris MA Base, Sigma, pH 7.5) 3 times for 5 minutes each time. Staining of DNA was accomplished using 50 μL of ethidium bromide solution at 20 μg/mL in PBS per slide. The slides were examined using an epifluorescence microscope (Zeiss Axioskop 20; Carl Zeiss, Microscope Division, Oberkochen, Germany) equipped with a mercury lamp HBO (50 W, 516-560 nm, Zeiss) at 20× magnification and linked to a pulnix TM 765 camera (Kinetic Imaging, Liverpool, UK). Fifty randomly selected cells per slide and 3 slides per condition were analyzed with the image analysis system Komet 3.1 (Kinetic Imaging) [27]. The

genotoxicity parameter retained was the “Tail DNA,” that is, percentage of DNA in the Comet tail relative to the total amount of DNA in the entire Comet. A DNA repair result (%) was calculated relative to DNA damage in the control group cells treated with H2O2 only by the following formula: DNA repair ð%Þ ¼ ð1  ðtail DNA of H2 O2 stressed cells treated by each extract =tail DNA of H2 O2 stressed cells onlyÞÞ  100:

2.11. Statistical analysis All experiments were done in triplicate, and data were expressed as means ± SD of 3 independent experiments. A 1-way analysis of variance and the Dunett test were used to determine significant differences of multiple comparisons (SPSS program, ver 11.5; SPSS, Chicago, Ill) [28]. 3. Results 3.1. Xanthine oxidase inhibition and superoxide scavenging activity The IC50 values of the tested extracts for the inhibition of xanthine oxidase and as scavengers of superoxide anions (O2· −) are given in Table 1. Both inhibition of xanthine oxidase and scavenging effect on superoxide anions were measured in one assay. Inhibition of xanthine oxidase involves a decrease in the production of uric acid and in superoxide anions, which can be followed spectrophotometrically. For each tested extract, 2 IC50 values (50% inhibitory concentration) were calculated by linear regression analysis: 50% inhibition of XOD activity and 50% reduction of the superoxide level. The half-maximal inhibitory concentrations of the extracts are listed in Table 1. Fifty percent inhibition of uric acid production was obtained at IC50s of 87.5, 112, and 275 μg/mL, respectively, for methanol, ethyl acetate, and aqueous extracts. Likewise, it appears from the IC50s values of superoxide anions measured in the presence of methanol, ethyl acetate, and aqueous extracts (respectively,

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Table 2 Mutagenic effect of different extracts from A salicina in S typhimurium TA98 and TA97 assay systems in the presence and absence of exogenous metabolic activation system (S9) Extracts

Dose (μg/plate)

– – 50 250 500 50 250 500 50 250 500

NC PC Aqueous extract

Methanol extract

Ethyl acetate extract

His + revertants/plate

His + revertants/plate

TA98

TA97

−S9

+S9

−S9

+S9

30 ± 4 422 ± 8 32 ± 5 35.5 ± 5.5 34 ± 7 40 ± 9 36.5 ± 4.5 38.5 ± 6.5 45.5 ± 3.5 47.5 ± 6.5 43.5 ± 7.5

32.5 ± 2.5 152 ± 8 31.5 ± 4.5 34 ± 6 32 ± 6 37 ± 3 36.5 ± 4.5 41 ± 6 43.5 ± 4.5 36.5 ± 1.5 39 ± 6

70 ± 5 211 ± 9 73 ± 4 76 ± 10 69 ± 11 79 ± 8 71 ± 10 86 ± 7 73 ± 4 85 ± 6 84 ± 3

74.5 ± 6.5 215 ± 12 73 ± 4 81 ± 3 79 ± 7 80.5 ± 2.5 84.5 ± 3.5 79 ± 6 90 ± 8 88.5 ± 4.5 93.5 ± 2.5

Values are means ± SD (n = 3). Positive control: TA98/−S9, sodium azide (1.5 μg/plate); TA97/−S9, 4-nitrophenyldiamine (10 μg/plate); TA98/+S9, B[a]P (7.5 μg/plate); TA97/+S9, B[a]P (10 μg/plate). PC, positive control; NC, negative control (without the tested compound).

12, 70, and 550 μg/mL) that methanol extract is the most potent superoxide scavenger as it was the most enriched in polyphenolic compounds [16] known to have antioxidant activity [29]. Nonetheless, ethyl acetate and aqueous extract are considered as efficacious superoxide scavengers. 3.2. Mutagenic activity of extracts In a series of experiments preceding the antimutagenicity studies, it was ascertained that the different amounts of extracts added to the indicator bacteria does not influence their viability, and mutation frequencies do not change significantly when compared with spontaneous mutation frequencies. The results of Ames test with and without metabolic activation are reported in Table 2. None of the tested extracts induced a significant increase in the revertant number in TA98 and TA97 strains even with or without the S9 metabolic system.

3.3. Antimutagenicity assay Doses of 7.5 and 10 μg/plate of B[a]P were chosen for the antimutagenicity studies because these doses were not toxic and induced, respectively, 138 ± 13 revertants in S typhimurium TA98 and 211 ± 6 revertants in S typhimurium TA97 strains. The inhibitory effect of different extracts of A. salicina on the mutagenicity of positive mutagens using the plate incorporation is illustrated in Table 3. The addition of aqueous and ethyl acetate extracts reduced significantly B[a]P-induced mutagenicity, and a dose-response effect was observed. At the concentrations of 50, 250, and 500 μg per plate, the aqueous extract reduced B [a]P mutagenicity, respectively, by 92.4%, 93.7%, and 95.5% with S typhimurium TA98 strain, and 45%, 86.9%, and 91.5% with TA97 strain. At the same concentrations, ethyl acetate extract decreased the number of revertants in TA98 strain to 29.8%, 40%, and 46.6% and those in TA97 to

Table 3 Effect of the different extracts from the leaves of A. salicina on the mutagenicity induced by B[a]P in S. typhimurium TA98 and TA97 assay systems in the presence of exogenous metabolic activation system (S9) Extracts

NC PC Aqueous extract

Methanol extract

Ethyl acetate extract

Dose (μg/ plate) – – 50 250 500 50 250 500 50 250 500

TA98

TA97

Nb revertants/plate

% Inhibition of mutagenesis

Nb revertants/plate

% Inhibition of mutagenesis

25.5 ± 1.5 138 ± 13 34 ⁎ ± 1 32.5 ⁎ ± 2.5 30.5 ⁎ ± 4.5 45.5 ⁎ ± 1.5 54 ⁎ ± 3 53 ⁎ ± 2 104.5 ⁎ ± 5.5 93 ⁎ ± 3 83.5 ⁎ ± 3.5

– – 92.44 93.77 95.55 82.23 74.66 75.55 29.8 40 46.67

58 ± 2 211 ± 6 142 ⁎ ± 10 78 ⁎ ± 4 71 ⁎ ± 5 109.5 ⁎ ± 4.5 146 ⁎ ± 6 170 ± 12 206 ± 11 142.5 ⁎ ± 3.5 114.5 ⁎ ± 7.5

– – 45.09 86.92 91.50 66.33 42.48 26.79 3.26 44.77 63.07

Values are means ± SD (n = 3). Positive control: TA98/+S9, B[a]P (7.5 μg/plate); TA97/+S9, B[a]P (10 μg/plate). ⁎ P b .05 compared with the positive control using Dunett test.

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Fig. 1. Cytotoxicity of A. salicina leaf extracts in human K562 cells. K562 cells were treated with various concentrations of aqueous, methanol, and ethyl acetate extracts from A. salicina leaves (50, 100, 200, 400, and 800 μg/ mL of each extract) for 48 hours. Cell survival was measured by the MTT assay, as described in the Methods and materials section. Data represent the means ± SD from triplicate experiments (n = 3). Asterisk indicates P b .05, significant difference from cells treated with extract vehicle.

3.2%, 44.7%, and 63%. Methanol extract showed a higher antimutagenic effect when low doses of extracts were added to the assay system. The highest inhibition rate was obtained when 50 μg per plate was added: 82.2% to the TA98 and 66.3% to the TA97 assay systems. 3.4. Effect of A. salicina extracts on cell viability The effect of A. salicina extract on CML K562 cell proliferation was evaluated by the MTT assay. As shown in Fig. 1, treatment with increasing doses of extracts induces a dose-dependant cytotoxic effect. The IC50 for cell viability of human lymphoblast cell line K562 in the presence of methanol, ethyl acetate, and aqueous extract were 200, 250, and 120 μg/mL, respectively.

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evaluated by uric acid formation as the final product and their effect on the superoxide anions (O2· −) enzymatically generated by this system were evaluated in vitro. In fact, the superoxide radical (O2· −) is a highly toxic species that is generated by numerous biologic and photochemical reactions via the Haber-Weiss reaction; it can generate the hydroxyl radical, which reacts with DNA bases, amino acids, proteins, and polyunsaturated fatty acids and produces toxic effects. The toxicity of the superoxide radical also could be due to the perhydroxyl intermediates (HO2 ·) that react with polyunsaturated fatty acids. Finally, superoxide may react with nitric oxide to generate peroxynitrite, which is known to be toxic toward DNA, lipids, and proteins. Our data show that A. salicina extracts were effective inhibitors of XOD. Comparison of the IC50 values of XOD activity and O2· − scavenging activity, showed that each of the tested extracts have better superoxide scavenging activity than inhibitory effect of XOD. This is in good accordance with previous results described by Ljubuncic et al [30], suggesting that Crataegus aronia aqueous extract is better radical scavenger than XOD inhibitors. Antigenotoxic activity of the tested A. salicina leaf extracts were evaluated using the Ames test and the Comet assay. In all of the tested extracts, a significant antigenotoxicity toward B[a]P was observed. These data suggest that these extracts inhibit microsomal activation or that they directly protect DNA strands from the electrophilic metabolite of the mutagen. They may inhibit several metabolic intermediates and reactive ROS formed during the process.

3.5. Comet assay The protective effect of A. salicina extracts on H2O2induced DNA damage was assessed using the Comet assay. DNA strand breaks were represented by the mean tail DNA, that is, percentage of DNA in the Comet tail relative to the total amount of DNA [27]. Untreated control and treated cells with IC50 of the different extracts had no detectable Comet tail, whereas cells treated with 50 μmol/L of H2O2 showed significant nuclear DNA fragmentation. Compared with the tail DNA of H2O2-treated cells, ethyl acetate and methanol extracts decreased the tail DNA by 34.8% and 31.3%, respectively, at doses of 250 and 200 μg/mL (Fig. 2). In contrast, the aqueous extract showed the lowest activity. This extract decreased by 16% for DNA damage at the dose of 120 μg/mL. 4. Discussion The aim of this study was to assess the antioxidant, antigenotoxic, and antimutagenic activities of A. salicina leaves. The antioxidant activity of A. salicina leaf extracts was evaluated by the X/XOD enzymatic system. The influence of the A. salicina leaf extracts on XOD activity

Fig. 2. Inhibitory effect of A. salicina extracts on the genotoxicity of H2O2 toward K562 cells. K562 cells were treated with 250 μg/mL of ethyl acetate extract, 120 μg/mL of aqueous extract, and 200 μg/mL of methanol extract for 2 hours then stressed with 50 μmol/L of H2O2 for 2 hours. Membrane cells were lysed, and nucleus DNA was submitted to electrophoresis, as described in the Methods and materials section. T+, Control cells, only stressed with 50 μmol/L of H2O2 during 2 hours; T−, control cells without any treatment. DNA damage in the nucleus was assessed by relative intensity of tail DNA as described in the Methods and materials section. Data represent the means ± SD from triplicate experiments (n = 3). The relative intensity was calculated using the image analysis system Komet 3.1 (Kinetic Imaging Ltd, Evry, France). The asterisk and triangle indicate P b .05 (significant difference, respectively, from T+ control and T− control).

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Antigenotoxic activity of the tested extracts may be ascribed to flavonoids [31,32] coumarins [25], and tannins [33,34] detected in aqueous, methanol, and ethyl acetate extracts [16] and to sterols [35] detected in ethyl acetate extract [16]. The observed protective effect of ethyl acetate, methanol, and aqueous extracts toward B[a] P may correspond to a synergic participation of several of the above-cited compounds. We cannot, however, exclude the possibility that other compounds with antigenotoxic properties participate in the antigenotoxic effect of A. salicina leaf extracts. The weak antimutagenic activity of the methanol extract when a large excess was added could be explained by inhibition of the penetration through the cell membrane at high doses of the extract or molecules which are implied in the mutagenic inhibitory effect toward B[a]P. We must consider that it would be interesting to confirm results obtained with the bacterial test by another test using eukaryotic cell. In this case, the Comet assay was chosen. A significant decrease in DNA damage after 2 hours of exposure to ethyl acetate and methanol extracts, then 2 hours of exposure to H2O2 oxidant stress, was observed when compared with DNA damage obtained after exposure to H2O2 only. These results fit partially to those obtained with the Ames test except for the aqueous extract. Moreover, the aqueous extract does not present antioxidant activity, as shown by the X/XOD system, contrary to results from the ethyl acetate and methanol extracts. We can deduce from these results that antigenotoxicity may be ascribed to the antioxidant effects, as far as the extract without antioxidant effect (aqueous extract) toward the genotoxic H2O2 radicals that did not exhibit antigenotoxic activity toward the same genotoxicant when tested with the “Comet assay.” However, the aqueous extract exhibits an important antimutagenic activity when tested with the Ames assay. Thus, we propose that antigenotoxic effect of this extract may be ascribed to other additional mechanisms such as DNA repair enzyme induction, or it may exhibit antioxidant activity against other radical types. In conclusion, the present study demonstrates that the methanol, ethyl acetate, and aqueous extracts of A. salicina possess potent antioxidant and genoprotective activities. The extracts are capable of protecting against oxidative damage to DNA and proteins and also maintain the levels of antioxidant molecules and enzymes in vitro. The preliminary study of these phytochemicals in the present investigation depicted A. salicina extracts to contain a considerable amount of polyphenolics, to which its antioxidant activity may be ascribed. Further investigations on testing their in vivo activities and on isolation and characterization of the active compounds responsible for the antioxidant capacity of A. salicina leaves are under way in our laboratory. Acknowledgments The authors are grateful to the Rhônes-Alpes region in France, the Ministère Français des Affaires Etrangères

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