Moricandia arvensis extracts protect against DNA damage, mutagenesis in bacteria system and scavenge the superoxide anion

Toxicology in Vitro 23 (2009) 166–175

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Moricandia arvensis extracts protect against DNA damage, mutagenesis in bacteria system and scavenge the superoxide anion Ines Skandrani a, Ines Bouhlel a, Ilef Limem a, Jihed Boubaker a, Wissem Bhouri a, Aicha Neffati a, Mohamed Ben Sghaier a, Soumaya Kilani a, Kamel Ghedira a, Leila Ghedira-Chekir a,b,* a b

Unité de Pharmacognosie/Biologie Moléculaire 99/UR/07-03, Faculté de Pharmacie de Monastir, Rue Avicenne, Monastir 5000, Tunisia Laboratoire de Biologie Moléculaire et Cellulaire, Faculté de Médecine Dentaire de Monastir, Rue Avicenne, Monastir 5000, Tunisia

a r t i c l e

i n f o

Article history: Received 4 July 2008 Accepted 22 October 2008 Available online 30 October 2008 Keywords: Ames test Antioxidant activity DNA cleavage Moricandia arvensis

a b s t r a c t The mutagenic potential of total aqueous, total oligomers flavonoids (TOF), ethyl acetate (EA), chloroform (Chl), petroleum ether (PE) and methanol (MeOH) extracts from aerial parts of Moricandia arvensis was assessed using Ames Salmonella tester strains TA100 and TA1535 with and without metabolic activation (S9), and using plasmid pBluescript DNA assay. None of the different extracts produced a mutagenic effect, except aqueous extract when incubated with Salmonella typhimurium TA100 after metabolic activation. Likewise, the antimutagenicity of the same extracts was tested using the ‘‘Ames test”. Our results showed that M. arvensis extracts possess antimutagenic effects against sodium azide (SA) in the two tested Salmonella assay systems, except metabolized aqueous and PE extracts when tested with S. typhimurium TA100 assay system. Different extracts were also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals, except PE and aqueous extracts. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non enzymatic (NBT/Riboflavine assay) systems. TOF extract was the more effective one in inhibiting both xanthine oxidase activity and NBT reduction. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction There has been increasing realization in recent years that several plant-derived polyphenolic compounds may possess anticancer and apoptosis inducing properties (Mukhtar et al., 1998). Therefore, the role of plant-derived polyphenols in chemoprevention of cancer has emerged as an interesting area of research. Plant polyphenols are natural antioxidants and most of their pharmacological properties are considered to be due to their antioxidant action (Ames et al., 1995). This is generally considered to reflect their ability to scavenge endogenously generated oxygen radicals or those radicals formed by various xenobiotics, radiation etc. However, some data in the literature suggest that the antioxidant properties of the polyphenolic compounds may not fully account for their chemopreventive effects (Gali et al., 1992). Most plant polyphenols possess both antioxidant as well as pro-oxidant properties (Inoue et al., 1994). The pro-oxidant action of polyphenolics may be an important mechanism of their anticancer and apoptosis inducing properties (Hadi et al., 2000). In addition, the genotoxic

* Corresponding author. Address: Laboratoire de Biologie Moléculaire et Cellulaire, Faculté de Médecine Dentaire de Monastir, Rue Avicenne, Monastir 5000, Tunisia. Tel.: +216 97316282; fax: +216 73461150. E-mail address: [email protected] (L. Ghedira-Chekir). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.10.010

and mutagenic effects of vegetable extracts have been studied over the years by researchers concerned with indiscriminate consumption of such products. Damage to the users’ genetic material could lead to mutagenesis and carcinogenesis as well as other toxic effects (Umbuzeiro-Valent et al., 1999; Ramos et al., 2001). For this reason, antimutagenic properties elicited by plant species have a full range of prospective applications in human healthcare. In addition, in the recent years there has been increasing interest in antimutagenesis (Calomme et al., 1996) of plant origin compounds. Even for populations which use herbs traditionally, encouraging the use of species with chemopreventive actions could be helpful as part of life expectancy improvement strategies: costs are significantly low, herbs have usually little or no toxicity during longterm oral administration and are available at large scale. Moricandia arvensis (Cruciferae) includes five species distributed in northern Africa, southern Europe, and western Asia (Pottier-Alapetite, 1979). In Tunisia, the leaves of M. arvensis are used in traditional cooking. Decoctions of leaves and stems were employed in the treatment of syphilis (Le Floch, 1983) and scorbut (Cheieb and Boukhris, 1998). Moricandia arvensis Ssp suffruticosa collected from the Est of Tunisia showed an important antioxidant activity and also serves as a source of various products, including polyphenols (Braham et al., 2005). Owing to their frequent use in traditional medicine, we investigated the mutagenicity,

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antimutagenicity, oxidative DNA damage-protecting activity and antioxidant potential of M. arvensis subsp eu-arvensis collected from the southern region of Tunisia.

2. Materials and methods 2.1. Chemicals Dimethylsulfoxide USP grade (DMSO), aflatoxin B1 (AFB 1) and 2-aminoanthracene (2-AA), sodium dodecyl sulfate (SDS) (St Louis, MO, USA), sodium azide (SA), L-histidine, D-biotine, hydroxylamine, glucose-6-phosphate (G-6-P), b-nicotinamide adenine dinucleotide phosphate sodium salt (NADP), ampicilline, xanthine (X), hydrogen peroxide (H2O2), magnesium dichloride (MgCl2), sodium acetate and xanthine oxidase (XOD) were purchased from Sigma–Aldrich (St Louis, USA). Nutrient Broth No. 2 (Oxoid) and agar–agar were procured from Difco (Paris, France). Aroclor 1254 from Supelco (USA). Potassium chlorure (KCl) from Chemipharma (Le Bardo, Tunisia). Potassium phosphate (KH2PO4), 3-nitrotetrazolium blue chloride (NBT), agarose and ethidium bromure (BET) were obtained from Fluka (Steinheim, Germany), Riboflavine from Merck (Darmstadt, Germany), chloridric acid (HCL) from Panreac (Barcelone, Espagne), ethylene diamine-tetraacetic acid (EDTA) from Sigma–Aldrich (Steinheim, Germany). Tris was procured from Promega (Madison, USA).

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extracts were dissolved in sterile distilled water whereas PE, Chl and EA extracts were resuspended in dimethylsulfoxide (DMSO). The doses of extracts we tested in the Ames assay were 500, 250 and 100 lg/assay. Whereas the tested doses in the plasmid DNA test were 250, 50 and 10 lg/assay for all extracts except with TOF extract (50, 10 and 5 lg/assay). In the enzymatic system assay (X/XOD), the tested doses were 300, 150, and 50 lg/assay, except with aqueous extract (1000, 800, 600, 300, 150, 50, 10 lg/assay). But in the non enzymatic system assay (NBT/Riboflavine), concentrations tested were 10, 3, 1, 0.3, 0.1 mg/ml. All tested doses and concentrations are in accordance with our previous investigations (Hayder et al., 2003, 2005; Abdelwahed et al., 2007; Ben Mansour et al., 2007; Ben Ammar et al., 2008) where a number of preliminary dose-finding tests involving a number of plant extracts were conducted. This means that the doses were suitable for testing the majority of the extracts, however, not necessarily all. 2.4. Bacterial strains Salmonella typhimurium TA100 (hisG46/rfa/DuvrB/pKM101) and S. typhimurium TA1535 (hisG46/rfa/DuvrB) strains were kindly supplied by Dr Felzen, (UERJ, Brazil). For all assays, an inoculum (100 ll) of a thawed permanent culture was added to 5 ml of Nutrient Broth No. 2 and incubated at 37 °C with shaking until a concentration of approximately 2–5  108 bacteria per milliliter was obtained. Plasmid DNA pBluescript KS was purchased from STRATAGENE.

2.2. Plant material 2.5. S9 fraction Moricandia arvensis subsp. eu-arvensis was collected from Oued Ghezran at Gafsa, a region situated in the southern Tunisia, in December 2005. Identification was carried out by Pr. Cheieb (Department of Botany, Faculty of Sciences, University of Sfax), according to the flora of Tunisia (Pottier-Alapetite, 1979). A voucher specimen (M.a-12.05) has been kept in our laboratory for future reference. The leaves were shade-dried, powdered, and stored in a tightly closed container for further use.

The S9 microsome fraction is prepared from livers of rats treated with Aroclor 1254 (Maron and Ames, 1983). The components of S9 mix were 8 mM MgCl2, 32.5 mM KCl, 5 mM G6P, 4 mM NADP, 0.1 M sodium phosphate buffer (pH 7.4), and S9 fraction at a concentration of 0.68 mg/ml of mix. The S9 mix was prepared freshly for each assay. 2.6. Salmonella microsome assay

2.3. Preparation of plant extracts The powdered leaves were extracted with boiling water from 15 to 20 min. After filtration, the extracts were frozen and lyophilised (aqueous extract). The residue was dissolved in water. In order to obtain an extract enriched in total oligomers flavonoids (TOF), the powdered leaves were macerated in a water/acetone mixture (1:2), during 24 h with continuous stirring. The extract was filtered, and the acetone was evaporated under low pressure in order to obtain an aqueous phase. Tannins were partially removed by precipitation with an excess of NaCl during 24 h at 5 °C, and the supernatant was recovered. The latter was extracted with ethyl acetate, concentrated, and precipitated with an excess of chloroform. The precipitate was separated and yielded the TOF extract, which was dissolved in water (Ghedira et al., 1991). Petroleum ether (PE), chloroform (Chl), ethyl acetate (EA), and methanol (MeOH) extracts were obtained by Soxhlet extraction (6 h). These four types of extract, with different polarities, were concentrated to dryness and each residue was kept at 4 °C. TOF, MeOH, EA, PE, aqueous and Chl extract compositions were reported by Skandrani et al. (2007). The family compounds detected in these extracts were essentially flavonoids and tannins compounds. The percentages of tannins detected in Chl, EA and MeOH extracts were, respectively, 0.61%, 0.18% and 0.10%, and the percentages of flavonoids detected in TOF, EA and MeOH extracts were, respectively, 13.37%, 13.74% and 8.83%. Whereas sterols were detected only in PE, EA and Chl extracts with a respective percentages of 15%, 0.25% and 12.5%. Then, aqueous, MeOH and TOF

The mutagenicity assay with S. typhimurium was performed as described by Maron and Ames (1983). 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 h at 37 °C, approximate cell density (2–5)  108 cells/ml) and 500 ll of sodium phosphate buffer (0.2 M, pH 7.4 for assay without S9) or 500 ll of S9 mix were added to 2 ml aliquots of top Agar (supplemented with 0.5 mM L-histidine and 0.5 mM 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 (1983). The plates were incubated at 37 °C for 48 h and the revertant bacterial colonies of each plate were counted. Negative and positive control cultures gave numbers of revertants per plate that were within the normal limits found in the laboratory. An extract was considered mutagenic if the number of revertants per plate was at least doubled in S. typhimurium TA 100 strain and tripled in S. typhimurium TA1535 strain, over the spontaneous revertant frequency. Data were collected with a mean ± standard deviation of three plates (n = 3). 2.7. Antimutagenic assay A modified plate incorporation procedure (Ferrer et al., 2002) was employed to determine the effect of all extracts on sodium azide (SA) induced mutagenicity. In brief, 0.5 ml of phosphate buffer for direct mutagen SA was distributed in sterilized capped tubes

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in an ice bath, then 0.1 ml of test compounds (50 ll of mutagen and/or 50 ll of test compound) and 0.1 ml of bacterial culture (prepared as described in mutagenicity test) were added. After vortexing gently and preincubating at 45 °C for 30 min, 2 ml of top agar supplemented with 0.5 mM L-histidine and D-biotine were added to each tube and vortexed for 3 s. The resulting entire was overlaid on the minimal agar plate. The plates were incubated at 37 °C for 48 h 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 the mutagen by the following formula: Inhibition rate (%) = [1  (number of revertants on test plates  number of spontaneous revertants)/(number of revertants on mutagen control plates  number of spontaneous revertants)]  100. Each dose was tested in triplicate.

was prepared in the same manner as the assay mixture to measure the total uric acid production. The uric acid production was calculated from the differential absorbance. To detect the superoxide scavenging activity, 2 ml of the colouring reagent consisting of sulphanilic acid solution (300 lg/ml), N(1-naphtyl) ethylenediamine dihydrochloride (5 lg/ml) and acetic acid (16.7%, v/v) were added. This mixture was allowed to stand for 30 min at room temperature and the absorbance was measured at 550 nm on a Helios Alpha, Spectronic Unicam (Cambridge, England) spectrophotometer. The dose-effect curve for each tested extract was linearized by regression analysis and used to derive the IC50 values. Values are presented as mean ± standard deviation of three determinations. 2.10. Superoxide radical scavenging activity

2.8. Reaction with plasmid pBluescript DNA DNA damage and DNA protecting activities of extracts were prospected on pBluescript KS DNA vector (purchased from STRATAGENE). Plasmid DNA was oxidized with H2O2 + UV treatment in the presence or absence of extracts and checked on 0.7% agarose in 1xTAE buffer (2 M Tris, 1 M sodium acetate, 50 mM EDTA, pH 8) according to Russo et al. (2000) after modifications. In brief, the experiments were performed in a volume of 9 ll in an Eppendorf tube containing 2.34 lg of pBluescript plasmid DNA, H2O2 was added to a final concentration of 147 mM with and without 4 ll of extract at various doses. The reaction was initiated by UV irradiation and continued at ambient temperature for 5 min on the surface of UV transilluminator (Bioblock Scientific, TF35 C, France) with intensity of 180 W, at k of 254 nm. After irradiation, the mixture was incubated at room temperature during 15 min. Finally, the reaction mixture along with gel loading dye was placed on 0.7% agarose gel for electrophoresis. Untreated pBluescript KS DNA was used as a control in each run of gel electrophoresis along with UV and H2O2 treatment. Gel was stained with BET and photographed with bio-print (Vilbert lourmat, France). 2.9. Xanthine/xanthine oxidase assay The enzyme xanthine oxidase catalyzes the oxidation of xanthine to uric acid. During this reaction, molecular oxygen acts as an electron acceptor, producing superoxide radicals according to the following equation:

Xanthine þ O2

!

Xanthine oxidase

uric acid þ O 2 þ H 2 O2 :

Xanthine oxidase activity was evaluated under aerobic condition (Kong et al., 2000), by the spectrophotometric measurement of the production of uric acid from xanthine. The inhibition of xanthine oxidase activity was measured according to the increase in absorbance at 290 nm as proposed by Cimanga et al. (2001), while the superoxide anion scavenging activity was detected spectrophotometrically with the nitrite method described by Oyangagui (1984) with some modifications introduced by Hu et al. (1995) and Russo et al. (2005). The assay mixture consisted of 100 ll of compound test solution, 200 ll xanthine (final concentration 0.1 mM) as the substrate, hydroxylamine (final concentration 0.2 mM), 200 ll EDTA (0.1 mM) and 300 ll distilled water. The reaction was initiated by adding 200 ll xanthine oxidase (11 mU) dissolved in phosphate buffer (KH2PO4, 0.2 M, pH 7.5). The assay mixture was incubated at 37 °C for 30 min. Before measuring the uric acid production at 290 nm, the reaction was stopped by adding 100 ll of 0.58 mM HCL. The absorbance was measured spectrophotometrically against a blank solution prepared as described above, but replacing xanthine oxidase with buffer solution (no production of uric acid). A control solution without test compound

The assay was based on the capacity of the samples to enhance the aerobic photochemical reduction of nitroblue tetrazolium (NBT) in the presence of riboflavine (Beauchamp and Fridovich, 1971). For all assays, the reaction mixture contained EDTA (6.5 mM), riboflavine (4 lM), NBT (96 lM) and phosphate buffer (51.5 mM, pH 7.4). The volume of tested sample was of 100 ll/assay. The occurrence of superoxide and/or free radicals was indirectly evaluated by the increase in absorbance of formazan at 560 nm, after 30 min of incubation at 30 °C from the beginning of illumination (Banerjee et al., 2005). The assay run without any test compound (containing only NBT–riboflavine) was used as the reference. All assays were realized in triplicate.

3. Results 3.1. Mutagenic activities of extracts The results of mutagenicity with and without metabolic activation are reported in Table 1. Experiments showed no mutagenicity of the different tested doses using the S. typhimurium TA100 assay system, except the aqueous extract which increases the number of revertants when incubated in the presence of the S9 metabolic system at a concentration of 100 lg/plate by comparison to the spontaneous mutation frequency. Results obtained with the Ames test showed that none of the tested extract induced any mutagenic effect within the tested dose range, in the S. typhimurium TA1535 assay system in the absence of the S9 system. Whereas in the presence of S9, all the tested extracts induce mutagenic effect except the aqueous and PE extracts. 3.2. Antimutagenic activities of extracts The possible antimutagenic potential of M. arvensis extracts was examined against the direct-acting mutagens SA (1.5 lg/plate), with the same tester strains, using the plate incorporation assay. Antimutagenic effects usually appear as an attenuation of the increase in the number of his+ revertant colonies induced by mutagenic effect of SA. The results of antimutagenic effects of extracts are presented in Figs. 1 and 2 as plots of the percentage of the remaining mutagenicity. MeOH, TOF, EA and Chl extracts showed the most important antimutagenic effect against SA in TA100 assay system (Fig. 1). The highest antimutagenic activity was observed at the same dose 100 lg/plate with MeOH (87.77%) and TOF (85.83%) extracts (Fig. 1). But EA and Chl extracts reduced the SA induced mutagenicity by, respectively, 82.93% (at a dose of 500 lg/assay) and 72.95% (at a dose of 250 lg/assay). Whereas PE and aqueous extracts increased the mutagenicity induced by positif control SA. MeOH and PE extracts showed the most important antimutagenic effect against SA in TA1535 assay system, in a dose

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I. Skandrani et al. / Toxicology in Vitro 23 (2009) 166–175 Table 1 Mutagenic effect of different extracts from M. arvensis in S. typhimurium TA100 and TA1535 assay systems in the presence and absence of exogenous metabolic activation system (S9). Strain Extracts Aqueous

TA 100 Dose lg/plate

500 250 100 TOF 500 250 100 MeOH 500 250 100 EA 500 250 100 Chl 500 250 100 PE 500 250 100 Spontaneous revertants PC

TA 1535

S9

+S9

S9

+S9

64 ± 0 71 ± 1 88 ± 2 83 ± 6.5 73 ± 2 69 ± 2 89 ± 5 82 ± 4 77 ± 9 96 ± 4 82 ± 4 67 ± 1 66 ± 3.5 72 ± 6 69 ± 7 65.5 ± 9.5 101 ± 10 66.5 ± 2.5 92 ± 2 427 ± 4

91 ± 2 147 ± 6 226 ± 7 101 ± 3 97 ± 1 90 ± 5 108 ± 7 105 ± 3 103 ± 4 101 ± 0 102 ± 2 101 ± 6 99 ± 7 82 ± 2 80 ± 8 104 ± 5 103 ± 0 100 ± 2 100 ± 10 319 ± 10

11 ± 1 12.5 ± 0.5 9.5 ± 0.5 13.5 ± 1 16.5 ± 2 20 ± 1 18 ± 3 23 ± 1 13.5 ± 1 17 ± 5 15 ± 7 14 ± 2 22 ± 0.5 19 ± 8 17.5 ± 8 20 ± 3 17.5 ± 1.5 19 ± 2 17 ± 6 360 ± 7

16 ± 2 28 ± 4 24 ± 0 84 ± 0 89 ± 2 93.5 ± 3 69 ± 5 21.5 ± 8 20 ± 4 183 ± 4 55 ± 1 52 ± 2 120 ± 9 80 ± 6 72 ± 7 47 ± 11 23.5 ± 9 18 ± 3 22 ± 1 97 ± 8

Positive control (PC): TA100/S9 and TA1535/S9, SA (1.5 lg/plate); TA100/+S9, AFB1 (10 lg/plate) and TA1535/+S9, 2-AA (1 lg/plate). PE extract (petroleum ether extract), MeOH extract (methanol extract), TOF extract (total oligomers flavonoids), Chl extract (chloroform extract), EA extract (ethyl acetate extract).

dependent manner (respectively, 70.8% and 64.91% at the same dose 500 lg/plate) (Fig. 2). Whereas TOF extract decreased the mutagenicity induced by SA in a reverse dose dependent manner, and aqueous extract gave the same range of antimutagenicity with all tested concentrations (respectively, 59.23% and 49.25% at the same dose 100 lg/plate) (Fig. 2). All the tested extracts showed a lower antimutagenic activity in the TA1535 assay system when compared to the TA100 assay system except aqueous and PE extracts. Likewise, Chl and EA extracts exhibited a weak inhibition activity against SA induced mutagenicity in the S. typhimurium TA1535 assay system. Thus, the highest inhibition rates obtained with Chl and EA extracts were, respectively, 34.93% and 9.87%, when 500 lg of each extract was added to the TA1535 assay system.

IP (%) of SA induced mutagenicity

100

3.3. Reaction with plasmid pBluescript DNA The free radicals and the damages resulting from the oxidization of the DNA are implied in several pathologies such as cancers (Olinski et al., 2002). Cells possess generally efficient DNA repair mechanisms for oxidative DNA damage (Lindhal and Wood, 1999), but damaged forms of DNA oxidation is persistent during replication of DNA, leading to mutation. In order to evaluate the ability of the extracts to generate breaks in the phosphodiester bands of DNA, or unlike to protect it against the genotoxic effect of hydroxyls radicals generated by photolysis of the hydrogen peroxide after exhibition to the UV light, the plasmid DNA was treated with different concentration of each extract. Fig. 3 shows the electrophoretic pattern of DNA after UV-photolysis of H2O2 in the presence or absence of M. arvensis extracts. pKS plasmid DNA showed two bands on agarose gel electrophoresis (lane A) the faster moving prominent band corresponded to the native supercoiled circular DNA (Sc DNA) (I) and the slower moving band was the open circular form (Oc DNA) (II). The UV irradiation of DNA in the presence of H2O2 (lane B) resulting the cleavage of Sc DNA to give prominent Oc DNA and a faint linear (Lin) DNA (III) indicating that OH generated from UV-photolysis of H2O2 produced DNA strand scission. Although both O 2 and H2O2 are potentially cytotoxic, most of the oxidative damage in biological systems is caused by the OH, which is generated by the reaction between O 2 and H2O2 in the presence of metals ions (Guitteridge, 1984). The results showed that the treatment with all extracts doses did not result in changes in plasmid DNA conformation (Figs. 3– 5). These observations suggest that if the extracts cause DNA damage, it is not through direct DNA chain breakage. In the same way, protective effect of the extracts in inhibiting OH induced DNA cleavage, was also studied (Figs. 6 and 7). A protective effect was observed when DNA was exposed to UV irradiation and treated with 250 and 50 lg/assay doses of MeOH and TOF extracts (Fig. 6, lanes C–D; Fig. 7, lanes C–D). However at the lowest tested dose (10 lg/assay) of these two extracts, we observed a weak regeneration of Sc DNA (Fig. 6, lane E; Fig. 7, lane E). Whereas Chl extract effectively inhibited OH induced DNA cleavage at the different tested doses (Fig. 7, lanes K–L–M). By contrast with the inhibitory activity of Chl extract, the addition of EA extract (Fig. 6, lanes, F–G–H) at various concentrations to the reaction mixture of H2O2, induced no protection effect towards the damage of native Sc DNA. A similar effect was observed in the pres-

87 77

80 60 40 20 0 eous

-20 -40

H

-60 -80 -100

EA

Dose( µg/assay)

Fig. 1. Effect of M. arvensis extracts on the mutagenicity induced by SA (sodium azide) (1.5 lg/plate) in the S. typhimurium TA100 assay system. PE extract (petroleum ether extract), MeOH extract (methanol extract), TOF extract (total oligomers flavonoids), Chl extract (chloroform extract), EA extract (ethyl acetate extract).

I. Skandrani et al. / Toxicology in Vitro 23 (2009) 166–175

IP (%) of SA induced mutagenicity

170

80 70 60 50 40 30 20 10 0 queous E eOH OF Hl EA

-10

PE 50µg/assay (-UV)

PE 50µg/assay + UV

PE 10µg/assay (-UV)

PE 10µg/assay + UV

MeOH 250µg/assay (–UV)

MeOH 250µg/assay + UV

E

F G

H

K

L

M N

O

MeOH 10µg/assay + UV

PE 250µg/assay + UV

D

MeOH 10µg/assay (-UV)

PE 250µg/assay (-UV)

B C

MeOH 50µg/assay + UV

H2O2 + UV

A

MeOH 50µg/assay (-UV)

Control

Fig. 2. Effect of M. arvensis extracts on the mutagenicity induced by SA (sodium azide) (1.5 lg/plate) in the S. typhimurium TA1535 assay system. PE extract (petroleum ether extract), MeOH extract (methanol extract), TOF extract (total oligomers flavonoids), Chl extract (chloroform extract), EA extract (ethyl acetate extract).

P

III II I Fig. 3. Agarose gel electrophoresis of plasmid DNA treated with petroleum ether (PE) and methanol (MeOH) extracts. 2.34 lg/4 ll of pBluescript DNA was incubated with 250, 50 and 10 lg/assay of different extracts and irradiated with UV for 5 min, the reaction products were electrophoresed in 0.7% agarose gel. A: DNA, B: DNA + hydrogen peroxide (H2O2) + UV, C: DNA + PE (250 lg/assay), D: DNA + PE (250 lg/assay) + UV, E: DNA + PE (50 lg/assay), F: DNA + PE (50 lg/assay) + UV, G: DNA + PE (10 lg/assay), H: DNA + PE (10 lg/assay) + UV, K: DNA + M (250 lg/assay), L: DNA + M (250 lg/assay) + UV, M: DNA + MeOH (50 lg/assay), N: DNA + MeOH (50 lg/assay) + UV, O: DNA + MeOH (10 lg/assay), P: DNA + MeOH (10 lg/assay) + UV. I: supercoiled form, II: circular-relaxed form, III: linear form.

ence of the highest dose of (500 lg/assay) of PE extract. At the lower tested concentration (50 lg/assay), PE extract showed a pro-oxidant effect provoking DNA degradation at the dose of 10 lg/assay (Fig. 6, lane M). When 250 lg/assay of aqueous extract was added to the reaction mixture, we obtained a weak regeneration of the Sc DNA form (Fig. 7, lane F) which disappeared at the 50 and 10 lg/ assay tested doses (Fig. 7, lane G and H). 3.4. Xanthine oxidase inhibition and superoxide scavenging activity The superoxide radical (O 2 ) is a highly toxic species that is generated by numerous biological 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 reacts with nitric oxide to generate peroxynitrite, which is known to be toxic towards DNA, lipids and proteins. The antioxi-

dant activity of M. arvensis extract leaves was evaluated by the X/XOD enzymatic system. The influence of the M. arvensis leaf extracts on XOD activity evaluated by uric acid formation as the final product, and their effect on the superoxide anions (O 2 ) enzymatically generated by this system, were evaluated in vitro. The IC50 (50% inhibitory concentration) values of the tested extracts for the inhibition of XOD and as scavengers of superoxide anions (O 2 ) are given in Table 2. Fifty percentage inhibition of uric acid production was obtained at IC50s of 180, 950, 140, 27, 235 and 210 lg/assay with, respectively, MeOH, aqueous, EA, TOF, Chl and PE extracts. Indeed, TOF extract is the best inhibitor of XOD. It was concluded that M. arvensis extracts were effective inhibitors of XOD. Whereas, it appears from the IC50s values of superoxide anions measured in the presence of MeOH, aqueous and TOF extracts (respectively, 145, 95 and 180 lg/assay) that aqueous extract is the most potent superoxide scavenger. Comparaison of the IC50s values of XOD activity and O 2 scavenging activity showed that aqueous extracts have better superoxide scavenging activity than inhibitory effect of XOD. Whereas TOF, EA and Chl extracts were more

171

Aqueous 50µg/assay (-UV)

Aqueous 50µg/assay + UV

Aqueous 10µg/assay (-UV)

Aqueous 10µg/assay + UV

MeOH 250µg/assay (-UV)

MeOH 250µg/assay + UV

C

D

E

F

G

H

K

L M

MeOH 50µg/assay + UV

MeOH 10µg/assay + UV

Aqueous 250µg/assay + UV

B

MeOH 10µg/assay (-UV)

Aqueous 250µg/assay (-UV)

A

MeOH 50µg/assay (-UV)

H2O2 + UV

III

Control

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N O

P

II I

TOF 50µg/assay (-UV)

TOF 50µg/assay + UV

TOF 10µg/assay (-UV)

TOF 10µg/assay + UV

TOF 5µg/assay (-UV)

TOF 5µg/assay + UV

EA 250µg/assay (-UV)

EA 250µg/assay + UV

EA 50µg/assay (-UV)

EA 50µg/assay + UV

EA 10µg/assay (-UV)

EA 10µg/assay + UV

A B

C

D

E

F

G

H

K

L

M

N O

P

Control

H2O2 + UV

Fig. 4. Agarose gel electrophoresis of plasmid DNA treated with aqueous and chloroform (Chl) extracts. 2.34 lg/4 ll of pBluescript DNA was incubated with 250, 50 and 10 lg/assay of different extracts and irradiated with UV for 5 min, the reaction products were electrophoresed in 0.7% agarose gel. A: DNA, B: DNA + hydrogen peroxide (H2O2) + UV, C: DNA + aqueous (250 lg/assay), D: DNA + aqueous (250 lg/assay) + UV, E: DNA + aqueous (50 lg/assay), F: DNA + aqueous (50 lg/assay) + UV, G: DNA + aqueous (10 lg/assay), H: DNA + aqueous (10 lg/assay) + UV, K: DNA + Chl (250 lg/assay), L: DNA + Chl (250 lg/assay) + UV, M: DNA + Chl (50 lg/assay), N: DNA + Chl (50 lg/assay) + UV, O: DNA + Chl (10 lg/assay), P: DNA + Chl (10 lg/assay) + UV. I: supercoiled form, II: circular-relaxed form, III: linear form.

III II I

H2O2 + UV

MeOH 250µg/assay + H2O2 + UV

MeOH 50µg/assay + H2O2+ UV

MeOH 10µg/assay + H2O2+ UV

EA 250µg/assay + H2O2+ UV

EA 50µg/assay + H2O2+ UV

EA 10µg/assay + H2O2+ UV

PE 250µg/assay + H2O2+ UV

PE 50µg/assay + H2O2+ UV

PE 10µg/assay + H2O2+ UV

III II I

Control

Fig. 5. Agarose gel electrophoresis of plasmid DNA treated with TOF and ethyl acetate (EA) extracts. 2.34 lg/4 ll of pBluescript DNA was incubated with 250, 50 and 10 lg/ assay of EA extract and 50, 10 and 5 lg/assay of TOF extract and irradiated with UV for 5 min, the reaction products were electrophoresed in 0.7% agarose gel. A: DNA, B: DNA + hydrogen peroxide (H2O2) + UV, C: DNA + TOF (50 lg/assay), D: DNA + TOF (50 lg/assay) + UV, E: DNA + TOF (10 lg/assay), F: DNA + TOF (10 lg/assay) + UV, G: DNA + TOF (5 lg/assay), H: DNA + TOF (5 lg/assay) + UV, K: DNA + EA (250 lg/assay), L: DNA + EA (250 lg/assay) + UV, M: DNA + EA (50 lg/assay), N: DNA + EA (50 lg/ assay) + UV, O: DNA + EA (10 lg/assay), P: DNA + EA (10 lg/assay) + UV. I: supercoiled form, II: circular-relaxed form, III: linear form.

A

B

C

D

E

F

G

H

K

L

M

Fig. 6. Inhibition of hydrogen peroxide (H2O2) induced DNA strand breakage by MeOH, EA and PE extracts. 2.34 lg/4 ll of pBluescript DNA was incubated with 250, 50 and 10 lg/assay of different extracts and irradiated with UV for 5 min, the reaction products were electrophoresed in 0.7% agarose gel. A: DNA, B: DNA + hydrogen peroxide (H2O2) + UV, C: DNA + MeOH extract (250 lg/assay) + H2O2 + UV, D: DNA + MeOH extract (50 lg/assay) + H2O2 + UV, E: DNA + MeOH extract (10 lg/assay) + H2O2 + UV, F: DNA + EA extract (250 lg/assay) + H2O2 + UV, G: DNA + EA extract (50 lg/assay) + H2O2 + UV, H:DNA + EA extract (10 lg/assay) + H2O2 + UV, K: DNA + PE extract (250 lg/ assay) + H2O2 + UV, L: DNA + PE extract (50 lg/assay) + H2O2 + UV, M: DNA + PE extract (10 lg/assay) + H2O2 + UV. I: supercoiled form, II: circular-relaxed form, III: linear form.

TOF 50µg/assay + H2O2 + UV

TOF 10µg/assay + H2O2+ UV

TOF 5µg/assay + H2O2+ UV

Aqueous 250µg/assay + H2O2+ UV

Aqueous 50µg/assay + H2O2+ UV

Aqueous 10µg/assay + H2O2+ UV

Chl 250µg/assay + H2O2+ UV

Chl 50µg/assay + H2O2+ UV

Chl 10µg/assay + H2O2+ UV

III

H2O2 + UV

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Control

172

A

B

C

D

E

F

G

H

K

L

M

II I Fig. 7. Inhibition of hydrogen peroxide (H2O2) induced DNA strand breakage by TOF, aqueous and Chl extracts. 2.34 lg/4 ll of pBluescript DNA was incubated with 250, 50 and 10 lg/assay of aqueous and Chl extracts and 50, 10 and 5 lg/assay of TOF extract and irradiated with UV for 5 min, the reaction products were electrophoresed in 0.7% agarose gel. A: DNA, B: DNA + hydrogen peroxide (H2O2), C: DNA + TOF (50 lg/assay) + H2O2 + UV, D: DNA + TOF (10 lg/assay) + H2O2 + UV, E: DNA + TOF (5 lg/ assay) + H2O2 + UV, F: DNA + aqueous (250 lg/assay) + H2O2 + UV, G: DNA + aqueous (50 lg/assay) + H2O2 + UV, H: DNA + aqueous (10 lg/assay) + H2O2 + UV, K: DNA + Chl (250 lg/assay) + H2O2 + UV, L: DNA + Chl (50 lg/assay) + H2O2 + UV, M: DNA + Chl (10 lg/assay) + H2O2 + UV I: supercoiled form, II: circular-relaxed form, III: linear form.

Table 2 IC50 of M. arvensis extracts for inhibition of xanthine oxidase activity and reduction of superoxide level Extracts

Inhibition of xanthine oxidase activity IC50 (lg/assay)

Superoxide anion scavenging activity IC50 (lg/assay)

TOF Aqueous MeOH EA Chl PE

27 950 180 140 235 210

180 95 145 – – Pro-oxidant

PE extract (petroleum ether extract), MeOH extract (methanol extract), TOF extract (total oligomers flavonoids), Chl extract (chloroform extract), EA extract (ethyl acetate extract).

effective inhibitors of uric acid formation than O 2 scavengers. While PE extract exhibits XOD inhibitory activity, and at the same time increases the generation of O 2 (Fig. 8). 3.5. Generation of superoxide anion detected by the non enzymatic NBT/Riboflavine system The test implements two principal reactions (Liochev and Fridovich, 1995):

ðaÞ 2NBTH ! NBT þ NBTH2 ðFormazanÞ ðbÞ NBTH þ O2 $ NBT þ O 2 ða quasi-equilibriumÞ: When the riboflavine is photochemically activated, it reacts with the NBT to give NBTH (Beauchamp and Fridovich, 1971) that leads to formazan according to the reaction (a). In presence of oxygen, concentrations of radical species are controlled by the quasi equilibrium (b). Thus, superoxide anions appear indirectly when the test is performed under aerobic conditions. In the presence of an antioxidant that can donate an electron to NBT, the purple color typical of the formazan decays, a change that can be followed spectrophotometrically at 560 nm. TOF extract was the most potent superoxide scavenger in this assay (Fig. 9). This extract produced a 76.4% decrease of NBT photoreduction at a concentration of 10 mg/ml and an IC50 value of 0.75 mg/ml. Whereas MeOH and EA extracts exhibited lower scavenging activity, respectively, 39.47 and 55.96%, at a concentration of 10 mg/ml than TOF extract. This antioxidant activity of the

Fig. 8. Activity of petroleum ether extract (PE) from M. arvensis as scavenger of superoxide anion generated by the enzymatic system xanthine/xanthine oxidase (X/XOD) assay system.

tested extracts may be correlated with their phenolic contents found in our previous study (Skandrani et al., 2007). It is Known that generation of the O 2 anion may lead to the formation of H2O2. However, at neutral pH the peroxide ion immediately protonates to give hydrogen peroxide (H2O2). Alternatively, in aqueous solution the superoxide anion undergoes dismutation to form H2O2 and O2 (Halliwell and Gutteridge, 1984). Therefore the rate of generation of superoxide anion by PE, Chl and aqueous extracts was compared. The increase in absorption at 560 nm is observed on reduction of NBT by superoxide anion. Chl, PE and aqueous extracts showed a pro-oxidant effect and increased the generation of O 2 . PE seems to be the most important producer of superoxide radical as compared to Chl and aqueous extracts. 4. Discussion The absence of mutagenicity for MeOH, EA, Chl and TOF extracts of M. arvensis in two Salmonella tested strains in the absence of the S9 activation system, as well as the absence of phosphodiester band breaks in plasmid DNA at any tested concentration of different extracts, indicates that DNA does not seem to be revelant target for these extracts. Results obtained with S. typhimurium TA100

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Fig. 9. Antioxidant activity of M. arvensis extracts towards superoxide anion generated by the non enzymatic system NBT/Riboflavine. TOF extract (total oligomers flavonoids), PE extract (petroleum ether), chloroform extract (Chl), ethyl acetate extract (EA), methanol extract (MeOH).

strain in the presence of the S9 activation system, showed that all extracts even at high concentration, did not induce increase in number of revertants when compared to the number of spontaneous revertants, except the aqueous extract which have been evaluated as mutagenic by doubling the number of revertants at the lowest tested concentration (100 lg/assay). The absence of mutagenicity when a large excess of the aqueous extract was added to the assay system, could be explained by the inhibition of the penetration through the cell membrane at high doses of extract components. Whereas in the presence of TA1535 strain, after metabolic activation, all extracts induce a mutagenic effect, except aqueous and PE extracts. This suggests that several metabolic intermediates and reactive oxygen species (ROS) derived from Chl, EA, MeOH and TOF extracts during the process of microsomal enzyme activation are capable of damaging DNA strands. Both strains TA1535 and TA100 contain the same hisG46 mutation and differ only by the presence of plasmid pKM101 in strain TA100. When the plasmid PKM101 was introduced into strain TA1535, the resulting strain, TA100, became the most sensitive of all of the S. typhimurium tester strains (Zeiger et al., 1985). However, the original base pair substitution strain, TA1535, continued to be part of the test battery because it was thought that some mutagenic chemicals would be detected in this strain but not detected when strain TA100 was used. In fact to explain the results stipulating that EA, TOF, MeOH and Chl extracts are mutagenic in S. typhimurium TA1535 strain but not in S. typhimurium TA100 strain, we suppose that extracts may induce more mutations in strain TA1535 than they do in strain TA100, though it is difficult to imagine a molecular mechanism for such differential activity (Prival and Zeiger, 1998). Cases in which strain TA1535 is more sensitive than strain TA100 to the mutagenic effects of chemicals have been reported previously by other authors (McCann et al., 1975; Maragos et al., 1993). Results of antimutagenic activity showed that TOF, MeOH, EA and Chl extracts inhibited strongly SA mutagenicity in the presence of TA100 strain. This important antimutagenic effect may be ascribed to the composition of the tested extracts. In fact, MeOH and TOF extracts were enriched in phenolic compounds, whereas EA and Chl extracts were enriched with both phenolic and steroid molecules as described in our previous study (Skandrani et al., 2007). These families of compounds are known to have antioxidant

activity as described by Argolo et al. (2004) and Shon et al. (2004). The low antimutagenic activity of TOF and MeOH extracts when large excess was added to the assay system, could be explained by the inhibition of the penetration through the cell membrane, at high doses, of extract components that are implied in the mutagenic inhibitory effect induced by SA. On the other hand, TOF, EA, Chl extracts showed a weak antimutagenic activity towards the directly acting mutagen SA in the presence of the TA1535 strain compared to TA100 strain. Whereas MeOH extract exhibits the same range of antimutagenicity with the three tested doses, in the presence of TA1535 strain but this activity is less important when compared to that obtained in the TA100 assay system. In fact, strain TA100 generally presents a higher sensitivity to mutagenic insult as compared to strain S. typhimurium TA1535 due to the enhancement of SOS mutagenesis facilitated by the mucAB genes on the plasmid pKM101 that is present in TA100 (Koch and Woodgate, 1998). Therefore, we can suppose in our case that strain TA100 is more sensitive than strain S. typhimurium TA1535 to the antimutagenic effect of the tested extracts. This is the same case for aqueous and PE extracts which increased the mutagenicity induced by SA in the presence of S. typhimurium TA100 strain. This mutagenicity effect is correlated with their weak antimutagenic activity showed in the presence of TA1535 strain and may be ascribed to the interaction between extract components and the direct mutagen SA, leading to compounds with high mutagenicity. In order to investigate the mechanism by which the M. arvensis extracts exert their protective effect towards the DNA strand breakage induced by an oxidant agent (H2O2), an in vitro electrophoresis test of plasmid DNA was evaluated. The results showed that in the presence of MeOH, TOF and Chl extracts, the extent of DNA damage was significantly reduced and the protective effect was dose-dependant. The addition of EA extract at various concentrations to the reaction mixture of H2O2, induced no protection to the damage of native Sc DNA except a weak regeneration of Sc DNA in the presence of the highest tested dose of EA extract (250 lg/assay). Whereas PE extract induced no protection to the damage of native Sc DNA at the different doses tested. Indeed, we showed a total degradation of the DNA since the weakest tested dose 10 lg/assay. It is the same case for the aqueous extract which has no protective effect since 10 lg/assay, showing the presence of the two bands (linear form and circular-relaxed form), except

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a weak regeneration of Sc DNA, only in the presence of the highest tested dose (250 lg/assay). At the same time these two extracts did not present any antimutagenic effect towards the direct mutagen (SA) in the TA100 assay system. The X/XOD assay demonstrated that M.arvensis extracts were effective inhibitors of XOD, whereas only TOF, aqueous and MeOH extracts reduce O2 generation. Our previous study showed that TOF, EA, MeOH and aqueous extracts are rich in flavonoids (Skandrani et al., 2007). According to the detailed study by Cos et al. (1998) which provides a very important insight to categorize flavonoids into different classes based on their structure and biological activity related to XOD inhibition and/or O 2 scavenging, flavonoids contained in MeOH, aqueous and TOF extracts could be classified into category C which possess both the O 2 scavenging activity as well as XOD inhibitory capacity. While flavonoids contained in EA and Chl extracts could be classified into category B which can effectively inhibit XOD activity, but cannot scavenge O 2 radicals. The antioxidant activity against OH radicals generated by the photolysis of the H2O2 and antimutagenic activity of Chl extract may be the result of its action through other mechanisms than O 2 scavenging effect. Antioxidant capacity of M. arvensis extracts was also evaluated by their abilities to scavenge O 2 with the non enzymatic NBT/ Riboflavine assay system. The results indicated that TOF extract was more effective in scavenging O 2 than MeOH and EA extracts. Scavenging activity towards the superoxide anions in the enzymatic and the non enzymatic systems was mainly due to the flavonoid constituents of the TOF extract. Phenolic compounds are effective hydrogen donors, which make them good antioxidants (Rice-Evans et al., 1995). In contrast, the pro-oxidant effect of the aqueous extract detected by the non enzymatic system and PE detected by both enzymatic X/XOD system and non enzymatic NBT/ Riboflavine system, may be responsible for the absence of protection towards hydroxyl radicals in the plasmid DNA experiment, and the increasing of mutagenicity observed in the presence of S. typhimurium TA100 assay system towards the direct mutagen SA. The pro-oxidant action of plant polyphenols could contribute to cancer chemotherapy as it could cause growth arrest and induce apoptosis in cancerous cells (Galati et al., 2000). Different reactivity of MeOH, Chl and aqueous extracts towards superoxide radicals generated by either an enzymatic system (X/XOD) or a non enzymatic system (NBT/Riboflavine), indicates that the generator system of superoxide anions has an influence on the structure/ activity relationship of flavonoids (Cimanga et al., 1999). In this study, we demonstrated that M. arvensis extracts inhibit the mutagenicity induced by SA in the S. typhimurium assay. Moreover, the extracts exhibited potential free radical scavenging activity against superoxide anion and hydroxyl radicals, which could eliminate oxidative stress generated by free radicals whose removal is beyond the capacity of a biological system. Therefore, we speculate that M. arvensis extracts could be used as a chemopreventive and therapeutic agent. Further investigations on testing their in vivo activities and on isolation and characterization of the active compounds responsible for the antimutagenic and antioxidant capacity of M. arvensis leaves are under way in our laboratory. References Abdelwahed, A., Bouhlel, I., Skandrani, I., Valenti, K., Kadri, M., Guiraud, P., Steiman, R., Mariotte, A.M., Ghedira, K., Laporte, F., Dijoux-Franca, M.G., Chekir-Ghedira, L., 2007. Study of antimutagenic and antioxidant activities of gallic acid and 1,2,3,4,6 pentagalloylglucose from Pistacia lentiscus confirmation by microarray expression profiling. Chemico-Biological Interactions 165, 1–13. Ames, B.N., Gold, L.S., Willett, W.C., 1995. The causes and prevention of cancer. Proceedings of National Academy of Sciences USA 92, 5258–5265. Argolo, A.C.C., Sant’Ana, A.E.F., Pletsch, M., Coelho, L.C.B.B., 2004. Antioxidant activity of leaf extracts from Bauhinia monandra. Bioresource Technology 95, 229–233.

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