Genotoxicity and anti-genotoxicity of fennel plant (Foeniculum vulgare Mill) fruit extracts using the somatic mutation and recombination test (SMART)

The antigenotoxic action of fennel (Foeniculum vulgare) were assessed using the eye (w/w + ) somatic mutation and recombination test (SMART) assay of Drosophila melanogaster. Fennel is used for various medicinal purposes. Methyl methanesulfonate (MMS) was used as the positive control. Fruit extracts of fennel did not show genotoxicity at the doses used. In the co-treatment, a dose-dependent decrease in mutation frequency was observed for this plant. The fennel at 8% (w/v) demonstrated a marked decrease in MMS with an inhibition rate of 41.16%. The results demonstrate that the fennel exerted a significant and potent anti-mutagenic activity in eyes spot of D. melanogaster.


INTRODUCTION
Recently, there has been considerable interest in the mutagenicity and antimutagenicity of medicinal plants (Romero-Jimenez et al., 2005;Patenkovic et al., 2009;Ribeiro and Salvadori, 2003).It is sometimes argued that frequently used plants in traditional medicine are safe, due to their long-term use and are considered to have no side effects because they are natural (Van den Berg et al., 2011).This concept is largely circumstantial and it is important to determine toxicology of plant extracts, especially those that are used frequently over long periods.
Foeniculum vulgare Mill.(Apiaceae family) is commonly known as fennel.The plant is native to the Mediterranean region, and temperate regions of Asia.It was introduced and distributed to subtropical regions, Europe and North America and also cultivated worldwide (He and Huang, 2011).The fruits, leaves and roots can be used, but the fruits are most active medicinally and are the part normally used (EFSA, 2009).
this test, such as food additives, beverages and insecticides (Osaba et al., 2002).These one generation tests make use of the wing or eye imaginal disc cells in larvae and have proven to be very efficient and sensitive.They are based on the principle that the loss of heterozygosity of suitable recessive markers can lead to the formation of mutant clones of cells that are then expressed as spots on the wings or eyes of the adult flies (Graf et al., 1998).
The purpose of this work was to study the genotoxic activity of fennel and to evaluate its potential anti-genotoxic effect using methyl methanesulfonate (MMS) as a mutagen

Preparation of the aqueous extract
The fresh fruit of fennel was purchased at the local market in the city of Tetouan, Morocco.Plant material was prepared according to the traditional method used in Morocco.Ten grams of dried, ground plant material were extracted in 100 ml of distilled water and allowed for over-night shaking.After filtering, solutions were used immediately in treatments.

Negative and positive controls
Pure distilled water served as a negative control.A 1 mM aqueous solution of MMS [66-27-3] (Sigma) were used as the positive control.MMS was purchased from Sigma and was reagent grade.

Markets and strains
The following Drosophila melanogaster strains were used: Okwhite, a strain carrying the X-linked eye color marker white (w) and Ok-yellow.

Treatment procedure
Plant extract and mutagen were administered by chronic cotreatment feeding exposure from the egg stage.The Oregon k (Ok) was used.Twenty Oregon K-yellow-virgin female flies were mated with w males (20) flies for three days and then transferred to bottles (200 mL) containing 3 g of instant Drosophila medium (Carolina Biological Supply, Burlington, USA) dissolved with equal volumes of the test plant extract and MMS.
Drosophila larvae were exposed to fennel extract at three concentrations: 2, 4 and 8%.This was done by adding the solutions to the flies' medium.The solutions were always freshly prepared, immediately before use.The flies were permitted to lay eggs for three days.Newly hatched flies were counted and the females were transferred to fresh medium; two to six days later, their eyes were inspected for the presence of white spots.All experiments were carried out at 25°C and 65% relative humidity.

Scoring of eyes
The scoring of the etherized flies was carried out in a liquid consistin g of 90 parts ethanol, one part Tween-80 and nine parts water (Smijs et al., 2004).Because of the fast evaporation of this solution, a second solution (90% water + 10% first solution) was added when necessary (Gaivao and Comendador, 1996).
Inspection of the eyes for the presence of white spots was performed using stereomicroscope at a magnification of 80x using a light source that was provided by a glass fiber illumination of 20 W. Large white spots are seen as white parts in the red eyes and small white spots are dark ommatidia in red eye.Spots separated from each other by at least four nonmutated ommatidia were counted as independent events, and the smallest size of white clone expected to be counted accurately is 2 (Vogel and Nivard, 1993).An increase in white clone frequency was only accepted as a positive response if it was significantly higher than that of both the concurrent and pooled experimental controls.

Statistical test
The statistical significance of the differences between spot frequencies in the experimental groups and control was calculated using the Chi-square test for proportions as described by Frei and Wurgler (1988).The percentage of genotoxicity inhibition was calculated based on the spot frequencies according to the formula: [(mutagen alone-mutagen + plant)/mutagen alone] x 100 (Abraham, 1994).

RESULTS
The fennel fruits extract was first analyzed for the evaluation of its mutagenic activity.The numbers of small spots, large spots with the total number of spots are given in Table 1.Fennel extract was not toxic in the chronic feeding.At the concentrations tested, it did not show any effect on the frequencies of somatic mutations when compared with their respective negative controls.The data obtained with this approach showed that the fennel extracts does not induce mutagenicity at the selected doses.
MMS showed to be clearly mutagenic (Figure 1).A high value of genotoxic activity with a high significant response (p < 0.001) was detected for all the spot categories: small spot, large spots as well as total spots for all the concentration used when compared with the negative control.MMS at 1 mM showed a highly significant increase in induced somatic mutations (53.03 spot per 100 eyes).
The anti-genotoxic effect was established by means of a chronic co-administration of fruits water extract of fennel with MMS to larvae of D. melanogaster.Our result showed a dose-dependent inhibition of genotoxicity in larvae (Table 2).The overall inhibition activity is 31.38,34.83 and 41.16% for 2, 4 and 8% concentration of fennel, respectively.The chronic co-administration of fennel was effective in significantly reducing the frequencies of large and totals spots induced by MMS.

DISCUSSION
The use of D. melanogaster in the evaluation of genotoxicity has been well established as a test-system.Due   to a genome similarity as compared to mammals and easy maintenance in the laboratory, these flies represent an appropriate organism to run in vivo short-term tests (Graf et al., 1996;Vogel et al., 1999).SMART is a simple and fast short-term assay when compared with other in vivo tests.Through the use of these test-systems, it is possible to evaluate the genotoxic activity of a single compound as well as complex mixtures (Patenkovic et al., 2009: Schneider et al., 2009;Lehmann et al., 2010).
The somatic assays take advantage of the possibility to expose such large populations of mitotically growing cells in the imaginal discs of larvae.If a genetic alteration occurs in one of these imaginal disc cells, this alteration will be present in all the descendant cells and will form a clone of mutant cells.If the alteration causes a visible change in the phenotype, the mutant cell clone can be detected as a spot of mutant cells on the body surface of the adult flies.Thus, due to its capabilities, SMART was chosen to evaluate the anti-genotoxic effects of the aqueous fennel extract.
The fennel was first analyzed for the evaluation of its genotoxic/mutagenic activity.Three concentrations of the fennel were tested.The concentrations chosen are not toxic according to progeny mortality relative to the control.Fennel, at the concentrations tested, did not show any effect on the frequencies of somatic mutations when compared with their respective negative controls.
The evaluation of the antimutagenic potential of the aqueous extract from fennel was established by means of a co-treatment protocol in which the extract was administered simultaneously with the mutagens used, MMS a direct-acting genotoxin.MMS is a monofunctional alkylating agent known for its ability to interact directly with DNA, and produces genotoxic damage in different models in vitro and in vivo (Arnaiz et al., 1996).Under our experimental conditions, MMS showed to be clearly mutagenic.The aqueous extract from fennel displayed significant antimutagenic effect on the somatic mutations induced by MMS.
The mutational spectra induced in Drosophila by MMS suggest the involvement of apurinic sites as mutagenic lesions (Vogel et al., 1990).In addition, a clear relationship exists between the extent of the DNA Nalkylation and the efficiency of the MMS to induce mitotic recombination in the Drosophila wing-spot test (Arnaiz et al., 1996).Against this direct acting mutagen, the fennel used in this study showed a significant antimutagenic activity.Considering the genotoxin co-administered with the plant, there is possibility that the antimutagens from fennel exert their protective effect by interacting with MMS in desmutagenic manner without affecting the genetic material directly (Kuroda et al., 1992).MMS does not require metabolic activation; therefore, the natural compounds present in this plant may interact directly with the methyl radical groups of MMS and inactivate them by chemical reaction.It is also possible that these compounds compete to interact with the nucleophilic sites in DNA, thus altering the binding of the mutagen to these sites.
The mutagenicity, antimutagenicity and anticarcinogenicity of fennel and some of its components were subjected to several investigations (Ebeed et al., 2010;Hassan et al., 2011) and indicated that trans-anethole, the main component of fennel oil, does not increase the mutant frequency in the Salmonella/microsome test and it did not induce chromosome aberrations in Chinese hamster ovary cells.In addition, pretreatment with transanethole and eugenol led to significant antigenotoxic effects against ethyl methane sulfonate (EMS), cyclophosphamide (CPH), procarbazine (PCB), N-methyl-N'nitro-N-nitrosoguanidine (MNNG) and urethane (URE).Both trans-anethole and eugenol exerted dose-related antigenotoxic effects against PCB and URE.There was no significant increase in genotoxicity of trans-anethole and eugenol even when administered at high doses (Abraham, 2001).

Conclusion
This research indicates that fennel may serve as potential dietary sources of natural anti-mutagen for improving human nutrition and health.Purification and identification of the active compounds in these herbs is required for a better understanding of the protective mechanisms involved and for the possible application in the food industry and in medicine.

Figure 1 .
Figure 1.Mutagenic effect of MMS in eye spot test of D. melanogaster.

Table 1 .
Mutagenic effect of fennel fruit extract in Drosophila eye spot test (w/w + ).

Table 2 .
Antimutagenic effect of fennel fruit extract on MMS that induced mutagenicity in Drosophila eye spot test (w/w + ).