Turk J Biol 36 (2012) 622-629 © TÜBİTAK doi:10.3906/biy-1110-18

Genoprotective potential of Brassica juncea (L.) Czern. against mercury-induced genotoxicity in Allium cepa L.

Sonia SHARMA, Avinash NAGPAL, Adarsh Pal VIG Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar-143005, Punjab - INDIA

Received: 21.10.2011 ● Accepted: 03.04.2012

Abstract: Brassica juncea (L.) Czern. is an important species of the family Brassicaceae with immense medicinal value. The main goal of this study was to investigate the antigenotoxic effects of different concentrations (0.1%-1.0%) of chloroform extract of B. juncea seeds on mercury-induced (0.75 ppm) genotoxic effects in root cells of Allium cepa. Three different modes of treatment were performed: pretreatment, posttreatment, and simultaneous treatment. For pretreatment, freshly emerged root tips were first treated with different concentrations of seed extract for 3 h and then treated with Hg for 3 h. For posttreatment, the roots were first treated with Hg and then with extract concentrations. For simultaneous treatment, different concentrations of extract were added along with the Hg. The study revealed that chloroform extract was neither toxic nor in possession of genotoxic activity. Treatment with Hg caused a decrease in mitotic index and induction of chromosomal aberrations as compared to the negative control. This effect was reversed by treatment with different concentrations of chloroform extract of seeds of B. juncea. Maximum percentage inhibition (95.3%) of genotoxic effects was observed with 1% chloroform extract (post- and simultaneous treatment). This study clearly indicates that the chloroform extract of B. juncea seeds has antigenotoxic potential against mercury-induced genotoxicity in a dose-dependent manner by evaluating A. cepa root chromosomal aberration assay.

Key words: Antigenotoxicity, chloroform extract, Allium cepa root chromosomal aberration assay

Introduction consistent effect of mercurials is the induction of Heavy metal contamination of the biosphere has c-mitosis through disturbance of the spindle activity, increased for the past few decades (1-3). The majority resulting in the formation of polyploid and aneuploid of metals have the potential to cause mutations and cells and c-tumors (5-7). Exposure to inorganic salts can be referred to as environmental mutagens. Among of mercury in A. cepa reduced the mitotic index heavy metals, mercury is a major environmental in the root tip cells and increased the frequency genotoxin required in various items like barometers, of chromosomal aberrations, which was directly thermometers, pesticides, preservatives, paints, proportional to the concentrations used and to the dental fillings, etc. and is also consumed in agricultural duration of exposure (8). industries for seed dressing, etc. (4,5). Hg is also Plants have been utilized as medicines for known to induce various clastogenic and mutagenic thousands of years. According to World Health effects in higher plants. The genotoxic effects of Hg Organization estimates, up to 80% of the world’s depend on its oxidation state, its concentration, and population depends on traditional medicinal systems the duration of exposure. The most noticeable and for some aspects of primary health care. The use of

622 S. SHARMA, A. NAGPAL, A. P. VIG

antimutagens/anticarcinogens in everyday life is Loba Chemic Pvt. Ltd, Mumbai, India; and S D Fine- the most effective procedure for preventing various Chem Limited, Mumbai, India. genetic diseases. Antimutagens/antigenotoxins are Preparation of the chloroform extract of seed the chemical agents that interfere with mutagen- induced DNA damage to protect the cells/organisms Seeds of Indian mustard were washed with water, air- from the harmful consequences of exposure to dried, and thoroughly ground to a fine powder. The mutagens (9). seed powder (250 g) was first extracted with hexane (500 mL) using a Soxhlet extractor for approximately A number of bioassays are being used to study the 110-115 h to remove oil. The seed meal was air-dried genotoxicity of different environmental chemicals/ overnight and extracted with 500 mL of chloroform mixtures (10-16). The same assays have also been by placing the mixture on a shaker for about 2 nights. used to determine the antigenotoxicity of different Chloroform was distilled off to concentrate the chemicals/plant extracts. Among different assays chloroform extract. Chloroform extract (2 g) was available, the Allium cepa root chromosomal first solubilized in DMSO (1 mL), and then distilled aberration assay is widely used to determine water was added to make a total volume of 200 mL genotoxic and antigenotoxic effects of different plant (stock). extracts (17-23). Toxicity assay B. juncea (Indian mustard), a member of the Healthy onion bulbs were allowed to root in tap water. family Brassicaceae, is a multipurpose plant with After 2-3 days, the onion bulbs with freshly emerged immense medicinal value. Seeds of B. juncea are used roots were placed on coupling jars filled with different in the treatment of tumors and act as antibiotic agents. concentrations of B. juncea seed extract. Among Brassica vegetables appear to be protective against different concentrations (0.1, 0.25, 0.5, 0.75, 1, and 2 cancer and heart diseases (24-26). Glucosinolates ppm) of mercury, we found a concentration of 0.75 and other sulfur-containing metabolites present in ppm to be the EC value, and so this concentration Brassica act as anticancer agents due to their ability 50 was used as a negative control in the study. The to induce detoxification enzymes in mammalian experiment was done in triplicate. On the fourth day cells and to reduce the rate of tumor development of treatment, onion bulbs with roots were thoroughly (27,28). Currently, there are no published data on the washed, and the root length and root number of each antigenotoxic effects of Indian mustard seed extract. onion bulb were measured and compared with the Therefore, the present investigation was planned to controls. evaluate the antigenotoxic effects of the chloroform extract of seeds of Indian mustard against mercury- Allium cepa root chromosomal aberration assay induced genotoxicity in A. cepa root chromosomal Healthy onion bulbs were allowed to root in tap aberration assay. water contained in coupling jars kept in the dark. Freshly emerged roots (0.5-1.0 cm) were treated with different concentrations of chloroform extract of Materials and methods seeds of B. juncea and Hg (0.75 ppm) for 3 h. Three Brassica juncea (L.) Czern. kinds of treatments, pretreatment, posttreatment, Certified and disease-free seeds of Brassica and simultaneous treatment, were applied. For juncea (Indian mustard) were procured from the pretreatment, the roots were first treated with Department of Plant Breeding, Punjab Agriculture different concentrations (1.0%, 0.75%, 0.5%, 0.25%, University, Ludhiana, India. and 0.1%) of seed extract for 3 h followed by Hg treatment (0.75 ppm). For posttreatment, onion Chemicals bulbs were first treated with Hg (0.75 ppm) and then Mercury was purchased from Qualigens Fine with different concentrations of the extract for 3 h. Chemicals, Mumbai, India. HCl, orcein, glacial acetic For simultaneous treatment, the freshly emerged acid, and other chemicals were bought from Thomas root tips were exposed to 0.75 ppm Hg and different Baker (Chemicals) Pvt. Limited, Mumbai, India; concentrations of the extract simultaneously.

623 Genoprotective potential of Brassica juncea (L.) Czern. against mercury-induced genotoxicity in Allium cepa L.

Table 1. Effect of different concentrations of chloroform extract of Brassica juncea seeds on root number and root length of Allium cepa.

Root number Roots length (cm) Concentrations (Mean ± SE) (Mean ± SE) NC 26.33 ± 2.603 4.06 ± 0.636 PC 12.67 ± 1.764* 1.46 ± 0.521 0.1% 27.67 ± 1.202 3.06 ± 0.353 0.25% 28.33 ± 2.603 3.06 ± 0.437 0.50% 29.67 ± 1.856 3.23 ± 0.498 0.75% 29.33 ± 1.764 3.33 ± 0.811 1.0% 31.00 ± 1.155 3.90 ± 0.569

NC: negative control (distilled water); PC: positive control (0.75 ppm Hg). *P < 0.05 in one-way ANOVA. Root number: F-ratio = 10.517*; honestly significant difference (HSD) = 9.3086. Root length: F-ratio = 2.254; HSD = 2.7200.

Respectively, 0.75 ppm Hg and water served as the of dividing cells among the total number of cells positive and negative control. The onion bulbs were scored per slide. The percentage inhibition (PI) of washed under tap water after each treatment. The chromosomal aberrations was calculated as follows: root tips from each treated onion bulb were fixed in PI = a – b / a – c × 100, where a is the number of Farmer’s fluid (glacial acetic acid and ethyl alcohol, aberrant cells induced by the positive control (0.75 1:3) for 24 h and stored at 4 °C. For chromosomal ppm Hg), b is the number of aberrant cells induced analysis, the root tips were hydrolyzed in 1 N HCl by seed extract and mercury (0.75 ppm), and c is the for 1 min at a fixed temperature of about 60 °C. They number of aberrant cells induced by the negative were sub­sequently processed for cytological study control (distilled water). by the aceto-orcein squash technique (1 N HCl and aceto-orcein, 1:9). The squash preparation was Statistical analysis of data examined under the microscope for different types of The mitotic index was calculated by scoring dividing chromosomal aberration assay. cells. The experimental data are presented as means Three slides were examined per onion and 6 ± standard errors (SEs) of triplicate experiments. onions were used for each treatment. The mitotic For the determination of the significance among index (MI) was determined by counting the number the mean values, one-way ANOVA was applied (P <

Table 2. Effect of different concentrations of chloroform extract of seeds of B. juncea on mitotic index observed in root tip cells of Allium cepa.

Total number of Mitotic index Concentration Number of dividing cells cells analyzed (% ± SE) NC 10,118 1200 11.86 ± 0.054 PC 11,040 1200 10.86 ± 0.657 0.10% 9843 1200 12.19 ± 0.015 0.25% 9790 1200 12.25 ± 0.02 0.50% 9717 1200 12.34 ± 0.009 0.75% 9641 1200 12.44 ± 0.01 1.00% 9246 1200 12.97 ± 0.167

NC: negative control (distilled water); PC: positive control (0.75 ppm Hg). *P < 0.05 in one-way ANOVA. Mitotic index: F-ratio = 2.8821*; HSD = 1.708931.

624 S. SHARMA, A. NAGPAL, A. P. VIG

0.05). The linear relationship between the dose and root number of Allium bulbs. The results of the root effect of chloroform extract was obtained by simple length and root number for the controls (positive and regression and correlation analysis. negative) and the treatment groups are shown in Table 1. A dose-dependent increase in root growth and root number was observed in the extract treatment Results groups compared to untreated control samples. In this study, the effects of different concentrations Mitotic index of the chloroform extract of B. juncea seeds were observed on the genotoxicity of mercury employing Table 2 shows the effect of different concentrations the Allium cepa root chromosomal aberration assay. of the chloroform extract of B. juncea on the mitotic In our earlier study, mercury was observed to cause index in root tip cells of A. cepa. A slight but significant a dose-dependent decrease in the mitotic index and increase in the mitotic index was observed following trigger different types of chromosomal aberrations treatment with different concentrations of the extract (29). with respect to the control value. This indicates that Toxicity assay the extract was not cytotoxic. The effect of the chloroform extract of B. juncea Allium cepa root chromosomal aberration assay seed extract was observed on the root length and The results on the effects of the chloroform extract

a b c

d e f

g h

Figure 1. Different types of aberrations induced by mercury (0.75 ppm) in Allium cepa root tips: a) c-mitosis, b) delayed anaphase, c) stickiness, d) vagrant chromosomes, e) chromatin bridge, f) chromatin bridge, g) chromosomal breaks, h) chromosomal breaks.

625 Genoprotective potential of Brassica juncea (L.) Czern. against mercury-induced genotoxicity in Allium cepa L.

of B. juncea seeds on different types of chromosomal simultaneous treatment of the extract was found aberrations induced by mercury in the root tip cells of to be very effective in decreasing the clastogenic A. cepa are summarized in Table 3. Different kinds of aberrations and posttreatment was found to be chromosomal aberrations induced by mercury were effective in decreasing physiological aberrations. apportioned into physiological (c-mitosis, stickiness, For posttreatment of chloroform extract of seeds of vagrant chromosome(s), delayed anaphase(s), B. juncea, the percentage inhibition of total aberrant and laggard chromosome(s)) and clastogenic cells ranged from 62.3% for 0.1% to 95.3% for 1.0% of (chromatin bridge(s), ring chromosome(s), and the extract. Similarly, for the simultaneous treatment chromosomal break(s)) aberration categories (Figure and the pretreatment, the percentage inhibition of 1). Physiological aberrations are attributable to total aberrant cells ranged from 60.8% for 0.1% to action on the spindle apparatus and clastogenic 95.3% for 1% and 61.4% for 0.1% to 91.7% for 1% aberrations are attributable to direct breaking of the extract, respectively (Table 3). The present action on chromosomes. The effect of pre-, post-, investigation is a clear indication of the presence of and simultaneous treatment of chloroform extract showed a dose-dependent decrease in chromosomal antigenotoxic compounds in B. juncea plants. aberration frequency (Table 3). All 3 types of The linear regression analysis method of treatment were found to be equally effective. The determining the P-value (R2) represents the

Table 3. Effects of pre-, post-, and simultaneous treatment of chloroform extract of B. juncea seeds on genotoxic effects induced by mercury in root tip cells of Allium cepa.

Physiological aberrations Clastogenic aberrations Concentrations Total aberrant Percentage Percentage Treatment (%) cellsa aberrant cells inhibition Cm Da Lg Vg Sc Bg Bk Rg

NC - 4 4 1 2 - - 2 1 14 3.5 - PC - 38 24 14 10 19 45 55 3 208 52 - Pre- 0.1 8 10 7 12 16 21 14 1 89 22.25 61.4 0.25 12 7 6 10 14 18 16 - 87 21.75 65.5 0.50 10 6 6 9 10 24 12 - 90 22.5 78.8 0.75 7 4 4 3 7 20 12 - 81 20.25 83.5 1.0 3 2 4 1 4 16 14 - 78 19.5 91.7 Post- 0.1 21 10 7 7 8 23 10 - 86 21.5 62.3 0.25 19 9 5 7 6 14 10 - 55 13.75 67 0.50 15 7 5 5 4 14 11 - 61 15.25 75.7 0.75 12 6 3 3 3 21 9 - 75 18.75 84.3 1.0 6 3 2 2 1 12 9 - 46 11.5 95.3 Simultaneous 0.1 10 8 8 14 14 10 7 - 44 11 60.8 0.25 11 7 7 12 16 16 6 - 55 13.75 62.8 0.50 11 5 4 11 14 10 6 - 30 7.5 68.5 0.75 9 4 2 10 8 6 3 - 23 5.75 78.8 1.0 6 3 2 2 1 6 3 - 23 5.75 95.3 a: Out of 400 cells examined. Cm: c-mitosis, Da: delayed anaphase(s), Lg: laggard chromosome(s), Vg: vagrant chromosome(s), Sc: stickiness, Bg: chromatin bridge(s), Bk: chromosomal break(s), Rg: ring chromosome(s).

Percentage inhibition = a – b / a – c × 100, where a = number of aberrant cells induced by positive control, b = number of aberrant cells induced by seed extract and mercury, and c = number of aberrant cells induced by negative control.

626 S. SHARMA, A. NAGPAL, A. P. VIG

100 100

80 80

60 60 y = 36.32x + 58.02 y = 34.09x + 58.45 R² = 0.977 R² = 0.996 40 40

20 20

0 0 0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1 Pretreatment Posttreatment

100

80

60 y = 37.53x + 53.72 40 R² = 0.934

20

0 0 0.25 0.5 0.75 1 Smultaneous treatment Figure 2. Relationship between different concentrations of chloroform extract of seeds of B. juncea on percentage inhibition of chromosomal aberrations induced by 0.75 ppm Hg in Allium cepa root chromosomal aberration assay. significance of the null hypothesis and indicates (31,32). The most noticeable effect of mercurials that the percentage inhibition of chromosomal was the induction of c-mitosis and polyploid and aberration was dose-dependent and positively aneuploid cells (33,34). De et al. (35) exposed water correlated (Figure 2). The reduction in percentage of cabbage for 2 days to mercuric chloride and the dose chromosomal aberrations in the extract-treated groups decreased chlorophyll content, protein, RNA, and and pre-, post-, and simultaneous treatments showed dry weight. that substances in B. juncea chloroform extract have In the present study, we investigated dose- antigenotoxic effects. dependent antigenotoxic effects of the chloroform extract of B. juncea against Hg-induced chromosomal Discussion aberrations in Allium cepa using 3 types of treatments: pre-, post-, and simultaneous treatments. Our results The secondary metabolites present in plants have been indicating the antigenotoxic potential of B. juncea reported to provide a multitude of antimutagenic, are in good agreement with those of Rani et al. (18), antigenotoxic, or anticarcinogenic effects. Plants who observed the antigenotoxic potential of the have been used as an indicator organism in studies on mutagenesis in higher eukaryotes. Plant systems ether extract of Withania somnifera in root tip cells have a variety of well-defined genetic endpoints of Allium cepa. like alterations in ploidy, chromosomal aberrations, Results obtained from this study showed that and sister chromatid exchanges (30). Dietary B. juncea chloroform extract at concentrations of constituents suppress the genotoxic action or damage 0.75 ppm and 1.0 ppm increased the mitotic index of xenobiotics through various cellular mechanisms significantly. Mitotic index values of extract-treated

627 Genoprotective potential of Brassica juncea (L.) Czern. against mercury-induced genotoxicity in Allium cepa L.

groups after mercury treatment were significantly and further studies are necessary. Studying crude higher than those of the control groups. The increase plants extracts is appropri­ate because working with in mitotic index (number of dividing cells) showed crude extracts means working with com­plex mixtures that substances in the chloroform extract have no of active compounds. Some of these compounds can cytotoxic effects. These results are in accordance be genotoxic or antigenotoxic. with the literature data on Indian goose berry (36), Considering the great versatility of uses of B. Terminalia chebula (37), pomegranates and guava juncea, public awareness regarding its antigenotoxic (38), T. arjuna (39), Hippophae rhamnoides (40), etc. value is very important. For this, further studies are Allium cepa root chromosomal aberration assays necessary with different plant and animal test systems have been used to show the antigenotoxic effects of to strengthen these findings. Withania somnifera (18), Aegle marmelos (41), etc. Seeds of B. juncea have been used as a traditional herb in Chinese medicines against cough, asthma, Acknowledgments and swelling pain. Mustard has been used as food and The authors are thankful to Guru Nanak Dev medicines in Ayurveda, as the leaves are considered a University, Amritsar, India, for providing the vegetable, while seeds are used as condiments and as necessary laboratory facilities for the work. a source of mustard oil. On the basis of our results, we conclude that Corresponding author: chloroform extract of B. juncea has a protective Adarsh Pal VIG effect on Allium cepa root meristem cells against the genotoxic effects produced by mercury in a dose- Department of Botanical and Environmental Sciences, dependent manner, but it is effective at a high dose Guru Nanak Dev University, (1%). However, the mechanism by which it acts Amritsar-143005, Punjab - INDIA remains to be investigated in different test systems E-mail: [email protected]

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