versión impresa ISSN 0102-695X
Rev. bras. farmacogn. vol.22 no.3 Curitiba mayo/jun. 2012 Epub 2012
Recep Liman*; Uğur Gürol Gökçe; Dilek Akyıl; Yasin Eren; Muhsin Konuk
Biology Department, Faculty of Science and Literatures, Afyon Kocatepe University, Afyonkarahisar, Turkey
Genotoxic and mutagenic effects of aqueous extract from aerial parts Linaria genistifolia (L.) Mill. subsp. genistifolia, Plantaginaceae (Lg-ext) were investigated by using both Allium cepa root meristematic cells and bacterial reverse mutation assay in Salmonella typhimurium TA98 and TA100 with or without metabolic activation system (S9), respectively. In Allium root growth inhibition test, EC50 value was determined approximately 15 g/L and 0.5xEC50, EC50 and 2xEC50 concentrations of Lg-ext were introduced to onion tuber roots and distilled water and methyl methane sulfonate (MMS, 10 ppm) used as a negative and positive control, respectively. The characteristic effect caused by tested preparations was an increase of mitotic index (MI) in 7.5 g/L and 15 g/L (except 24 and 96 h) and simultaneous decrease of MI in 30 g/L and in MMS. While stickiness, bridges, chromosome laggards and disturbed anaphase-telophase were observed in anaphase-telophase cells, c-metaphase, pro-metaphase, polyploidy and binuclear cells were observed in other cells. Lg-ext was not found to be mutagenic on S. typhimurium TA 98 and TA100 with or without S9. The results were also analyzed statistically by using SPSS for Windows, and Duncan's multiple range tests were performed respectively. These results indicate that Lg-ext exhibits genotoxic activity in A. cepa root meristematic cells but not mutagenic activity in Ames test system.
Keywords: Allium, Ames chromosome aberration, Linaria genistifolia, Plantaginaceae
The genus Linaria, a well known genus of the family Plantaginaceae, is widely distributed throughout the northern hemisphere with its centre of distribution in the Mediterranean basin and eastern Asia. It comprises currently around 200 species, twenty of which are in Turkey popularly known as "nevruz otu" and nine of them endemics (Davis, 1978; Seçmen et al., 2000). Some Linaria species have been used in folk remedies for various purposes such as hemorrhoid, skin eruptions, sores, ulcers, diuretic, laxative, tonic, antiscorbutic, antidiabetic and to treat some vascular disorders. Dosage is critical and it should not be given to pregnant women, since the plant might be slightly toxic (San Feliciano et al., 1993; Baytop, 1999). Linaria species are reported to contain alkaloids, iridoid glucosides, flavonoids, aurones and diterpenoids (Otsuka, 1992; Ilieva et al., 1993; Bianco et al., 1996; Hua et al., 2002; Ahmad et al., 2006; Tundis et al., 2008; Ferhat et al., 2010). They contains a poisonous glucoside that is reported to be mildly poisonous to cattle (Moroshita, 1991).
Vicia faba, Tradescantia paludosa, Pisum sativum, Hordeum vulgare, Crepis capillaris and A. cepa are used to study of cytogenetic and genotoxic effect of plant extract or chemical(s). Among them, Allium test is one of the best-established test systems in order to determine the toxicity in the laboratories (Fiskesjö, 1985; Grant, 1992; Rank, 2003; Saxena et al., 2005; Konuk et al., 2007; Liman et. al., 2011). Because onions are easy to store and to handle, and also macroscopic and microscopic parameters can be observed easily. Moreover this system is well correlated with the data obtained from eukaryotic and prokaryotic systems (Fiskesjö, 1988).
Examining the mutagenicity of plant extract or chemical(s), Ames test, or the so-called Salmonella/microsome test, is widely used (Konuk et al., 2008; Uysal et al., 2010; Liman et al., 2010). This test can be carried out rapidly and cheaply and it is also one of the most reliable short-term bacterial test systems (Maron & Ames, 1983; Mortelmans & Zeiger, 2000). In this system, S. typhimurium mutant strains, obtained from S. typhimurium LT2 parental line in vitro, are employed (Ames et al., 1975).
The objective of this study was to investigate the genotoxic and mutagenic effects of Lg-ext by employing both A. cepa anaphase-telophase test and bacterial reverse mutation assay in S. typhimurium TA98 and TA100 strains with or without S9, respectively.
Materials and Methods
The S. typhimurium test strains TA98 and TA 100 were obtained from Nuran Diril, Hacettepe University, Turkey. While TA98 were used for determining the frame shift, TA100 was used to determine the base pair exchange type of mutations. Allium cepa (2n=16) onion bulbs, 25-30 mm diameter, without any treatment, were purchased from a local supermarket.
S9 from Liver of rat (Sprague-Dawley), Bacto agar, nutrient broth no.2 (Oxoid), 2-aminoanthracene (2AA), β-nicotinamide-adenine dinucleotide phosphate (β-NADP), glucose-6-phosphate (G6P), mitomycin-C (MMC), ampicillin and histidine were obtained from Sigma-Aldrich. Sodium azide (SA), citric acid monohydrate, sodium hydroxide, potassium chloride, sodium chloride, and dimethyl sulphoxide (DMSO) were purchased from Riedel. 4-Nitro-O-phenylenediamine (NPD) and 2-aminofluorene (2AF) were purchased from Fluka. Magnesium chloride, crystal violet, potassium phosphate and sodium ammonium phosphate were obtained from Merck.
Plant collection and extraction
The aerial parts of Linaria genistifolia (L.) Mill. subsp. genistifolia, Plantaginaceae, were collected from Başören Village, Afyonkarahisar, Turkey, in late-May 2010. The taxonomic identification of plant materials was confirmed by Dr Mehmet Temel, Department of Biology, Afyon Kocatepe University, Turkey. A voucher specimen was deposited at the Herbarium of Afyon Kocatepe University (number Kala 1410). Air dried and powdered aerial parts of the L. genistifolia subsp. genistifolia (80 g) were extracted with 1 L boiling water for 10 min (Sofowora, 1999). Both the decoctions and squeezed extracts were filtered with a 2.5-µm filter (Whatman® no. 42) to remove the suspended particles and stored at 4 ºC until usage. These were considered as the stock solutions and freshly prepared extracts were applied daily.
Allium cepa anaphase-telophase test
Prior to initiating the test, the outer scales of the bulbs and the dry bottom plate were removed without destroying the root primordia. In order to determine effective concentration (EC50), for each water sample, a series of six bulbs were placed in distilled water for 24h and afterwards the best growing five bulbs exposed for four days (d) to the Lg-ext solutions (10, 20, 40, 60 and 80 g/L, respectively) at room temperature (~21±4 ºC). The test concentrations were renewed at every 24 h during the experiments. On the 5th day, root lengths (lengths of ten roots from each bulb) were measured from both Lgext exposed bulbs, and control group. EC50 value was considered as the concentration which retards the growth of root 50% when compared to the control.
In the determination of application doses, 2xEC50, EC50 and 0.5x EC5, positive (MMS, 10 ppm) and negative control group were used for 12, 24, 48, 72 and 96 h. Fixation and staining of the root tip cells were carried out as reported earlier (Yıldız et al., 2009; Liman et al., 2010). The MI and the frequencies of chromosomal aberrations (CA) were carried out according to Saxena et al. (2005). For each test group, five slides (1 root tip/slide) were prepared by squashing root tips with 45% acetic acid. Slides were randomly coded and scored blindly. For MI, the different stages of mitosis were counted in a total of 5000-6000 cells (1000 cells/slide) per concentration, and expressed as a percentage. In chromosome aberration test, 100 cells in anaphase or telophase were examined for aberrations per slide if it is possible.
Ames plate incorporation test
Cytotoxic doses of Lg-ext (3000, 1500, 750, 375, 187.5 and 93.75 mg/plate) were determined by following the method of Dean et al. (1985).
Ames test was performed as a standard plate incorporation assay with S. typhimurium strains TA98 and TA100 with or without S9 (Maron & Ames, 1983). Selection of the strains was based on the testing and strain selection strategies of Mortelmans & Zeiger (2000). These strains were tested on the basis of associated genetic markers. For each tester strain, a specific positive control was always used to test the experimental flaws, if any. While 4-nitro-O-phenylenediamine (NPD) for TA 98 and sodium azide (SA) for TA100 were used as positive controls without S9, 2-aminofluorene (2AF) and 2-aminoanthracene (2AA) were used as positive controls with S9, respectively.
S9 mix (500 µL) (or 500 µL phosphate buffer), the test solution (100 µL) for each concentration and a cell suspension (100 µL) from an overnight culture (1-2x109 cells/mL) were added to 2 mL top agar (kept at 45 ºC) and vortexed for 3 s. The entire mixture was overlaid on the minimal agar plate. The plates were incubated at 37 ºC for 72 h and then the revertant bacterial colonies on each plate were counted. Both the positive and negative controls (distilled water) were maintained concurrently. Samples were tested on triplicate plates in two independent parallel experiments.
The data of root length, MI, mitotic phases, CA expressed as percentages, and the levels of significance in different treatment groups were analyzed statistically. For these, Duncan multiple range tests were performed by using one-way analysis of variance (ANOVA) on SPSS 15.0 version for Windows software both A. cepa anaphase-telophase and Ames test.
Results and Discussion
Herbal medicine is still the mainstay of about 75-80% of the whole population, mainly in developing countries, for primary health care because of better cultural acceptability, better compatibility with the human body and fewer side effects. However, an increase in the usage of these plants in the developed world has been observed for a few years (Parekh et al., 2005). Therefore, knowledge of mutagenic and toxic effects of these plants become very important. Because many plants synthesize toxic substances for defense against viruses, bacteria and fungi etc. and these compounds could have potentially deleterious effects in humans. Neither phytochemical nor biological studies of L. genistifolia subsp. genistifolia have been previously reported on the topic studied.
Test systems to determine the genotoxicity and/ or mutagenicity can be divided into groups based on the biological systems employed and their genetic endpoint detected. Bioassays with prokaryotes enable the detection of agents that induce gene mutation and primary DNA damages. On the other hand, analyses with eukaryotes enable the detection of a greater damage extent, varying from gene mutations to chromosome damages and aneuploidies (Houk, 1992; Leme & Marin-Morales, 2009). Using both pro- and eukaryotic test system make the results both strengthen and correlate to verify if the chemical(s) has/have really any bad effects on the genes.
Allium root growth test results are shown in Table 1. The EC50 was found to be approximately 15 g/L. It can be easily seen that the effect of Lg-ext in Allium root growth was dose-dependent. Root growth decreased at all concentrations tested and yielded with statistically significant results (p<0.05). In the meantime over 40 g/L concentration, roots became dark colored, thicker and gel like formations. The inhibition of root growth generally related to apical meristematic activity (Webster & Macleod, 1996), and to cell elongation during differentiation (Fusconi et al., 2006) and the appearance of stunted roots indicate the retardation of growth and cytotoxicity (Yıldız et al., 2009). As reported earlier, neoclerodane diterpenoids and flavonoids were isolated from Linaria saxatilis var. glutinosa by showing cytotoxic activity in different neoplastic cell cultures (Gordaliza et al., 1997). Tundis et al. (2005) determined the antiproliferative action of several flavones isolated from Linaria reflexa Desf., Plantaginaceae, against the large cell lung carcinoma cell line COR-L23, hepatocellular carcinoma cell line HepG-2, renal adenocarcinoma cell line ACHN, amelanotic melanoma cell line C32 and colorectal adenocarcinoma cell line Caco-2, and reported that pectolinarigenin and some flavonoid glycosides like pectolinarin exhibited strong cytotoxic activity on COR-L23 cell line with an IC50 value of 5.03 and 4.07 µM, respectively. Akkol & Elçi (2009) suggested that the extracts of Linaria species (L. grandiflora, L. genistifolia subsp. confertiflora and L. aucheri) had analgesic and anti-inflammatory effects without toxicity.
Table 2 summarizes the effect of Lg-ext on MI and mitotic phase in the root meristematic cells of A. cepa treated for 12, 24, 48, 72 and 96 h. While at all concentrations used in the incubations of root in 7.5 g/L and 15 g/L (except 24 and 96 h) increased MI, applications of 30 g/L and MMS decreased MI compared to negative control at each exposure time. The highest values were obtained from 12 h examination of 7.5 g/L concentrations of Lg-ext (45.18±0.75), and the lowest one in 96 h applications of MMS (20.14±0.62). The increased and decreased MI showed statistically significant results (p<0.05). As stated by Fernandes et al. (2007), the cytotoxicity levels of an agent can be determined by the increase or decrease in the MI. MI lower than the negative control may indicate that the growth and development of exposed organisms have been affected by test compounds. On the other hand, MIs above those of the negative control are result of the induction of increased cell division, which may characterize an event detrimental to cells, leading to uncontrolled proliferation and even tumor formation (Hoshina et al., 2002). The increased cell proliferation activity can be the consequence of a reduction of the time necessary for DNA repair (Evseeva et al., 2003).
All concentrations of Lg-ext used in the experiment caused changes in the percentage of particular phases' distribution in comparison to the control. The characteristic effect caused by tested preparations was an increase of prophase index except in 12 h at 30 g/L and simultaneous decrease of anaphase index except in 72 and 96 h at 7.5 g/L and telophase index except in 12 h at 30 g/L. This might be an indication of the blockage of chfr point (control point between prophase/metaphase). Scolnic & Halazonetis (2000) reported that chrf defines a checkpoint that delays entry into metaphase in response to mitotic stress. Most of the increased and decreased phase index showed statistically significant results (p<0.05).
In the A. cepa anaphase-telophase chromosome aberration test conducted with root meristematic cells of A. cepa as shown in Table 3. The most frequent abnormalities were stickiness, anaphase bridges, chromosome laggards and disturbed anaphase-telophase in anaphase-telophase cells. The effect of Lg-ext concentration on CA was significantly different (p<0.05) except in 12 h at 7.5 g/L compared to the negative control. Analysis of the chromosomes showed that 7.5 g/L of Lg-ext concentration except in 12 and 24 h and 15 g/L of Lg-ext concentration in 12 and 96 h induced chromosomal aberrations but other Lg-ext concentrations decreased chromosomal aberrations. No aberration was recorded in the chromosome of A. cepa exposed to the 30 g/L of Lg-ext concentration in 72 and 96 h. Stickiness (especially at 7.5 g/L in 48 h) indicates highly irreversible type toxic effect of Lg-ext, and its occurrences during the study could be because of sub-chromatid linkage between chromosomes (Mc-Gill et al., 1974; Chauhan et al., 1986; Kovalchuk et al., 1998; Ajay & Sarbhoy, 1988). Anaphase bridges could happen during the translocation of the unequal chromatid exchange or due to dicentric chromosome presence or due to the breakage and fusion of chromosomes and chromatids. This bridges cause structural chromosome mutations (El-Ghamery et al., 2000; Luo et al., 2004). Disturbed anaphase-telophase (especially at 15 g/L in 12 h and at 7.5 g/L in 72 h) and chromosome laggards could occur by the effect of Lg-ext on microtubule formations (Amer & Ali, 1986; Kumari et al., 2009). Such spindle malfunctioning may arise due to inhibition of tubulin polymerization (Kuriyama & Sakai, 1974). The occurrence of chromosome laggards at anaphase was due to the failure of the chromosomes or acentric chromosome fragments to move to either of the pole.
In addition to these anomalies, others (c-metaphase, pro-metaphase, polyploidy and binuclear cell) were also observed. While the lowest anomalies were observed 0.04±0.02% at the 15 in 12 h, the highest ones were observed 0.72±0.46% at 7.5 g/L in 48 h. Statistically significant (p<0.05) frequencies of other anomalies were recorded in 12 h at 7.5 g/L, all 24 h applications, in 72 h at 15 and 30 g/L, and also in 96 h at 7.5 g/L. It was not observed any anomalies in 48 and 96 h at 30 g/L. C-metaphase, a possibly reversible effect, might occur due to disturbed microtubules by Lg-ext and could be induced aneuploidies (Fiskesjö, 1988; Shahin & El-Amodi, 1991; Odeigah et al., 1997). Polyploidy cells can occur due to the lack of fragmoplast that prevents the formation of the daughter cells (Vidakovivé-Cifrek et al., 2002; Fernandes et al., 2007). Binuclear cells are accepted as the inhibition of cytokinesis in any control points of the cellular cycle (Ateeq et al., 2002).
The results from the Ames test are shown in Table 4. The interpretation of the Ames test results for genotoxicity testing of chemicals United States Environmental Protection Agency (USEPA, 1996) methods were followed. According to the guideline, a mutagenic potential is assumed, if the revertant frequency is 2.0 or higher over the solvent control or dose-related increase in the number of revertant colonies in one or more strains (Mortelmans & Zeiger, 2000). Noncytotoxic doses of the Lg-ext tested were determined firstly. The results obtained suggested that all tested concentrations were not determined as cytotoxic. The average revertant colony numbers in negative control were 34.8±1.48 for TA98 and 147.6±12.05 for TA100 with S9 and 32±3.31 and 105±7.21 without S9 respectively. Spontaneous revertants were within the normal values for the four strains examined. While application of S9 in TA98 was decreased revertant colony numbers, application of S9 in TA100 was increased revertant colony numbers. On the contrary, the plates with the positive control mutagens (SA, 2AF, 2AA and NPD) showed significant increases relative to the spontaneous mutation rate in the two tested strains. While the highest value observed was in the TA100 with S9 at 750 µg/plate concentration of Lg-ext (216.4±11.78), and the lowest value was in the TA98 with S9 at 750 µg/plate concentration of Lg-ext (25.6±1.14). Most of the results, increasing or decreasing relative to negative control group, were not statistically significant at p<0.05 (Duncan test) in examined strains. In order to establish a dose-response relationship, 5 different concentrations of Lg-ext were tested, and no induced revertants were observed along the dose range tested in either with or without S9 with two strains. The results of the present study showed that all applications of Lg-ext were not found to be mutagenic S. typhimurium TA98 and TA100 with or without S9.
In our knowledge, there is no report on the genotoxicty and mutagenicity of Linaria except for the present paper. On the other hand, Linaria genus one of the member of Scrophulariaceae and there are some reports on the other members of this family. Pereira-Martins et al. (1998) reported that flavone cirsitakaoside extracted from the medicinal herb Scoparia dulcis was able to retard the progression of the cell cycle and showed mutagenicity in the in vitro system but not in the in vivo system at the concentrations tested. Flavones from Limnophila geoffrayi were not exhibit mutagenic activity in the Bacillus subtilis recassay (Suksamrarn et al., 2003). BacoMindTM, standardized extract of Bacopa monnieri, demonstrated no mutagenic effect in the Ames test (Dipanwita et al., 2008).
In conclusion, our results indicate that Lg-ext was found cytotoxic and genotoxic in Allium test but not found mutagenic in Ames test. Although some Linaria species have been used in folk remedies for various purposes, they should be used with caution, always following the traditional methods of preparation exactly. Further studies are necessary to determine the active ingredient or compound(s) of Linaria genistifolia subsp. genistifolia and should be tested in other biological test systems to obtain definitive conclusions on their cytotoxic, genotoxic and mutagenic potential.
The authors wish to thank Afyon Kocatepe University Scientific Research Committee for supporting this study financially (Project No: 11.FEN.BL.16) and Dr. M. Temel for the identification of the plant.
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Received 27 Jul 2011
Accepted 16 Dec 2011
* Correspondence: Recep Liman Biology Department, Faculty of Science and Literatures, Afyon Kocatepe University, ANS Campus, Gazligol Yolu, 03200-Afyonkarahisar/Turkey firstname.lastname@example.org Tel: +90 272 228 13 39 Fax: +90 272 228 12 35