Research Articles

2021  |  Vol: 6(1)  |  Issue: 1(January-February) | https://doi.org/10.31024/apj.2021.6.1.2
Genotoxic potential of Bohadschia vitiensis aqueous extract: A sea cucumber species, using Allium cepa genotoxicity model

Nishani J. Jayathilake1, Uthpala A. Jayewardena2

1Department of Zoology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Sri Lanka

2Department of Zoology, Faculty of Natural Sciences, The Open University of Sri Lanka, Sri Lanka

*Address for Correspondening Author

Nishani J. Jayathilake

Department of Zoology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Sri Lanka

 

Abstract

Objective: Bohadschia vitiensis is historically proven for potential therapeutic values with anti-hypertension, anti-inflammatory, antimicrobial, antioxidant, antithrombotic, wound healing properties. Current study investigates the potential genotoxic effect of aqueous extract of Bohadschia vitiensis using Allium cepa genotoxicity model in order to validate the therapeutic usage. Materials and methods: Root grown onion bulbs were exposed to several concentrations of the extract; 62.5, 125, 250, 500 µg/ml, positive control (Dimethyl sulfoxide) and negative control (aged tap water) for 2 days. Immediately after the exposure period, root tips were harvested and observed in order to identify the existing stage of the cells and chromosomal aberrations. Results: The Mitotic Index was negatively correlated with the exposed concentrations and only the mitotic index of 500µg/ml was significantly different compared to the negative control. The extract showed no significant lethal genotoxic effect in comparison to the negative control within the tested range.  Microscopic observations of the root tips exposed to the extract, revealed 0.1-0.2% of chromosomal aberrations including chromosomal bridges, chromosomal breaks, c-mitosis and vagrants. Conclusion: Moreover, the tested concentrations showed no significant effect on percentage chromosomal aberrations except 500µg/ml that showed only a sub lethal effect. Further, evaluation for toxicity with animal models is warranted for validating the toxicity of the extract prior to therapeutic applications.

Keyword: Genotoxicity; Allium cepa model, Sri Lankan Sea Cucumber species, Bohadschia vitiensis


Introduction

Sea cucumbers are soft bodied, marine echinoderms, usually harvestable from sea floor worldwide (Anderson et al., 2011) and have long been appreciated in East Asia for its nutritional and therapeutic value (Bordbar et al., 2011; Kiew and Don, 2012). Sea cucumbers are a delicacy for Chinese, Korean and Japanese and available at international markets in fresh, dry, frozen and processed forms (Dhinakaran and Lipton, 2014) as well as a well-known remedy for hypertension, asthma, rheumatism, cuts, burns, impotence and constipation in traditional medicines (Bordbar et al., 2011).

Promising researches pointed out that sea cucumbers contain several bioactive compounds such as triterpene glycosides, glycosaminoglycans, gangliosides, collagen, branched-chain fatty acid and lectins (Kiew and Don, 2012), with a high biochemical and structural diversity(Kijjoa and Sawangwong, 2004) which are probably responsible for anticancer/antiproliferative, antiangiogenic, anticoagulant, anti-hypertension, anti-inflammatory, anti-microbial, antioxidant, antithrombotic, antiviral, wound healing, anti-leishmanial  activities etc.( Bordbar et al., 2011). Owing to this, there is an increasing focus for producing commercial products; nutraceuticals, beauty products and many commercialized items around the world (Kijjoa and Sawangwong, 2004). Up to date, nutritional supplements, tablets for joint problems, skin tonners, variety of creams are popular in the market globally.

Bohadschia vitiensis (Family Holothuriidae) is a relatively abundant species in North West and East coast and a low value sea cucumber species in Sri Lanka (Dissanayake and Stefansson, 2010). Extracts and few isolated compounds from Bohadschia vitiensis have been reported to possess several biological activities including antifungal activity, Spermicidal activity (Lakshmi et al., 2010), antioxidant activity (Nursiol et al., 2019), antinociceptive effects (Ridzwan et al., 2003) and anti-inflammatory activity (Jayathilake and Gunathilake, 2020). Particularly, local people in Sri Lanka believe that water extracts of Bohadschia sp. have a healing effect on inflammatory conditions and arthritic pain, but more scientific evidences are yet to be discovered. Despite the profound therapeutic value and the widespread usage of sea cucumbers, some of them may contain potentially toxic, carcinogenic and teratogenic compounds.

Variety of cells and species are used to test genotoxic effects of testing agents ranging from pharmaceuticals, industrial chemicals, pesticides, biocides, food additives, cosmetics ingredients, to veterinary drugs (Corvi and Madia, 2017), relevant in the context of international legislations aiming at the protection of human and animal health. Mainly, categories of microorganisms, cell cultures, insects, plants, crustaceans, mollusks, fish and mammals (Sponchiado et al., 2016) are used while setting several biomarkers for bio monitoring (Barras and Nadal, 2004).

Allium cepa test is a frequently used model which is satisfactorily sensitive for innumerous compounds present in natural extracts (Yuet et al., 2012; Timothy, 2014; Debnath et al., 2018) providing cytotoxicity and genotoxicity end points with chromosomal alterations (Kumari et al., 2011; Hemachandra and Pathirathne, 2016). Evidently, mutagens can be detected cytologically by cellular inhibition; disruption in metaphase; induction of chromosomal aberrations, numerical and structural changes ranging from chromosomal fragmentation to the disorganization of the mitotic spindle, and consequently of all subsequent dependent mitotic phases (Tedesco et al., 2015). A. cepa bioassay is more promising due to the availability of both cytotoxicity and genotoxicity end points and the cost effectiveness (Wijeyaratne et al., 2019). High sensitivity, clearly visible mitotic phases in Allium meristematic cells, possessing a stable chromosome number, diversity in the chromosome morphology, stable karyotype (2n=16), clear and fast response to the genotoxic substances, lack of spontaneous chromosomal damages are added benefits (Fiskesjo, 1985). Further, according to validated information, results of Allium cepa assay shows no difference from in vivo tests when comparing the results.  Toxicity evaluation of sea cucumber extracts is crucial for developing therapeutic products, effective for human use. Aim of present study was to evaluate the genotoxic potential of Bohadschia vitiensis water extract using Allium cepa bioassay.

Materials and methods

Collection and identification of plant material

Specimen of sea cucumber, Bohadschia vitiensis (560g) were collected from commercial catches of fishermen, from coastal areas of Pallimunei, Mannar, Sri Lanka (8° 03′, 8° 35′N: 77° 15′, 77° 36′E) in the month of January 2018. Precise identification of species was carried out (Purcell et al., 2012) by morphology, and ossicle analysis based on Purcell, Samyn, and Conand (2012). Samples were packed in plastic bags with ice during transportation. After removing the internal organs, samples were kept at -20ºC until extractions.

Preparation of extract

Water extract of Bohadschia vitiensis (WE) was prepared according to Ridzwan, Leong and Idid (2003) with slight modifications. Briefly, thawed samples of B. vitiensis were thoroughly washed with distilled water. The visceral organs were removed, and the body wall was diced, followed by homogenization with mortar and pestle. The homogenized sample was incubated in distilled water (1:2 w/v) and occasionally shaken for 4 hrs. The extract was then centrifuged at 3000rpm for 20 min (HERMLE Labortechnik GmbH, D-78564, Wehingen, Germany). The supernatant was carefully collected, freeze-dried, and used for the toxicity assay.

Allium cepa genotoxicity assay

The genotoxicity assay for the WE was performed as previously described procedure by Hemachandra and Pathiratne (2016). Commercially available equal sized (5-6g) Allium cepa onion bulbs which are in good condition were used for the assay. After the bottom plates and the dead scales were removed, bulbs(n=5) were placed on glass vials filled with aged tap water (dechlorinated) without exposing to direct sunlight. The temperature was maintained at 28±2°C during the period 48h and provided with renewed water supply every 24 h. Later, they were treated with different concentrations (500, 250, 125, 62.5µg/mL) for another 48h. Dimethyl sulfoxide (DMSO) was used as the positive control and aged tap water was used as the negative control. Distilled water was used as dilution water for preparing concentration series. The exposure solutions were renewed daily. At the end of exposure period, 3 bulbs were randomly selected (n=3) and 5-6 tips (1-2mm) were obtained from each bulb. Immediately, the tips were dipped in 1:3 aceto alcohol solution incubating at 60°C for 10 min. Then the tips were transferred in to 1N HCl solution and placed it again in the incubator at 60°C for 10 min. Then the root tips were transferred and exposed to acetocarmine solution for 10 min or until the root tips were properly stained with deeply stained edges of the root tips. The roots were placed on a glass slide with a drop of 5% acetocarmine and cover slip was placed on the glass slide providing a slight pressure to make the cells squash on the surface of glass slide. One slide was prepared for each bulb. The slides were coded randomly and examined blindly under light microscope (×400). Microscopic analysis included counting the number of cells undergoing mitotic stages and chromosomal aberrations in 1000 dividing cells.

The mitotic index (MI), Percentage mitotic inhibition (PMI), Percentage chromosomal aberrations (PCA) were calculated using following formulae:

Statistical analysis

Minitab 18 statistical software was used to analyses the results. The means with 95% confidence limit and standard errors for each set of data were calculated. Correlation and One way-ANOVA were applied with 95 CI (significance at the p<0.05) for results prediction.

Results

Cytogenotoxic effects of Bohadschia vitienis aqueous extract on Allium cepa root cells were tabulated in Table 1. Mitotic index (MI) declined gradually exhibiting negative correlation (r = 0.935) along the concentration series.  This was significant at the highest dose, reporting a low MI of 17.7 compared to the 26.8 at the negative control (One-Way ANOVA, DF=3, F=11.86, p=0.003).

Table 1. Cytogenotoxic effects of aqueous extract of Bohadschia vitiensis on Allium cepa root    cells

Treatment group

Concentration

(µg/ml)

MI+SEM (%)

PMI± SEM

(%)

C-mitosis

Stickiness

Vagrants

Chromosomal bridges

Chromosomal breaks

PCA (%)

Negative control

Tap water

26.8±2.0

0

1

-

1

-

-

0.2

Positive control

(DMSO)

0.1M

7.8±1.8*

70.9±6.5

48

65

37

25

34

20.9

Extract

(WE)

500

17.7±1.7*

33.9±6.3

-

-

1

1

-

0.2

250

21.7±1.3

19.1±4.9

1

-

-

-

1

0.2

125

24.1±1.8

9.9±6.6

2

-

-

-

-

0.2

62.5

29.2±0.5

9.3±1.8

-

-

1

-

-

 0.1

MI-Mitotic index, PMI-Percentage Mitotic Inhibition, PCA-Percentage Chromosomal aberrations, SEM-Standard Error Mean; 1000 cells/concentration of extract and controls, n = 3; MI values are means ± SEM; *p < 0.05 significantly different when compared to negative control (tap water)

Percentage inhibition of mitotic index (PMI) as a percentage of the control (Figure 1) revealed a significant decline (nearly 34%) at the highest dose, 500 µg/ml (DF=4, F=12.75, p=0.001). Nevertheless, 50% inhibition; the boundary level of genotoxicity, was not exceeded by any of the exposed concentrations.

Figure 1. Alterations of the mitotic index of Allium cepa by the WE (water extract) of B. vitiensis, positive control (DMSO) and negative control (Tap water)

 

Prophase, Metaphase, Anaphase and Telophase of A cepa meristematic cell division were clearly observed under a light microscope (×400). Meanwhile, extract exposed cells exhibited variety of chromosomal aberrations; c-mitosis, vagrants, stickiness, chromosomal bridges and breaks in low in quantity.

Figure 2. Percentage chromosomal aberrations in the Allium cepa cells induced by the WE (water extract) of B. vitiensis

 

Percentage chromosomal aberrations of each exposed concentration of the extract (Figure 2) remained approximately similar to the negative control and showed no significant correlation with the concentration level (p<0.05). Table 1 presents frequencies and types of chromosomal aberrations induced by the extract. The lowest percentage of chromosomal aberrations (0.1%) was exhibited by 62.5g/ml concentration of the extract. Besides, the highest tested concentration (500µg/ml) showed lower percentage chromosomal aberration (0.2%) compared to the positive control (20.9%) and it was as same as that of the negative control (0.2%). The highest percentage chromosomal aberration (0.2%) among four doses was recorded in higher doses (500, 250, 125 µg/ml) while lowest PCA (0.1%) was reported in the lowest dose (62.5 µg/ml). However, the PCA of positive control was the highest 20.9% of all the treatments while 0.2% chromosomal aberrations were reported in the negative control.

Discussion

Cytogenotoxic evaluation of the water extract of sea cucumber, Bohadschia vitiensis revealed no substantial toxic effects reporting very low percentage aberrations in Allium cepa cells. Inhibition of the mitotic activity was also minimal since less than 35% inhibition was reported in all the test doses.  The Allium cepa test is a sufficiently sensitive test model (Kannangara and Pathiratne, 2015) to evaluate genotoxic effects induced by substances which cause chromosomal aberrations and disturbances in the mitotic cycle (Laughinghouse, 2012). This model is frequently used for monitoring the genotoxic potential of natural extracts, (Laughinghouse, 2012; Shehu et al., 2019) consequently, this study results reflect the application of Allium cepa root tips for assessing genotoxicity of Bohadschia vitiensis water extract using different genetic endpoints; mitotic index and chromosome aberration ( Zendehboodi, 2018).

Mitotic index is a measure of the proportion of cells in the mitotic phase of the cell cycle relevant to total cell count (Timothy, 2014). In particular, arrest of cell division will be performed by decrease of MI which reflects a direct genotoxic effect. Further, alterations of chromosomes during different phases of cell cycle (prophase, metaphase, anaphase and telophase) can be used to determine the clastogenic effect of natural extracts. According to results, mitotic index decreased with increasing test concentrations over 62.5 -500 µg/ml. Moreover, MI of the highest concentration, 500µg/ml of WE exhibited significant difference (p < 0.05) when compared to tap water which was used as the negative control, while remaining concentrations of WE contained no such effects. Nevertheless, PMI values of any treatment group are not higher than 50% in comparison with negative control showing only 9-34% inhibition. The decline of MI below 22% of the negative control is considered as sub lethal impact and the decline below 50% is considered as lethal effects on the model (Prajitha and Thoppil, 2016; Debnath, 2018). Accordingly, the highest concentration (500µg/ml) revealed sub lethal effect while other concentrations, 62.5-250µg/ml showed no substantial toxic effect. Chromosomal aberrations also provide a good indication of genotoxic potential and the type/s of active components in bioactive compounds. The chromosomal abnormalities are occurred due to blockage of DNA synthesis or inhibition of spindle formation (Akinboro and Bakare, 2007). The B. vitiensis extract showed no obvious interferences on DNA synthesis or spindle formation as the percentage chromosomal aberrations of the highest concentration of treatment (500µg/ml) is similar to the value of percentage chromosomal aberrations of the negative control.

Even though, the percentage is low, different chromosomal aberrations such as chromosomal bridges, breaks, c-mitosis and vagrant chromosomes were observed in each test concentration. Chromosome bridges indicate the clastogenic effect caused by chromosome breaks, whereas vagrant chromosomes and c-mitosis increase the risk for aneuploidy and polyploidy. Chromosomal bridges are most likely formed by breakage or fusion of chromatids or sub chromatids (Yuet et al., 2015). Further, stickiness of chromosomes occurs when they stay connected in anaphase (Yuet et al., 2015). C-mitosis is caused by incomplete spindle formation and functions while vagrants are caused by unequal distribution of chromosomes which are appeared by nondisjunction of chromatids in anaphase (Yuet et al., 2015). Particularly, C-mitosis and vagrants can be observed at low concentrations in the study. These types of aberrations are considered as week genotoxic indications which may be reversible. C-mitosis can perform polyploidy cells while vagrants form aneuploidy cells if they remain as it is.

Conclusion

In toto, it was resulted that aqueous extract of Bohadschia vitiensis which is known to consumed in folk medicines in coastal areas of Sri Lanka, revealed no significant genotoxic effects within the tested range of concentration (<500µg/ml). However, further comprehensive studies with animal models should be conducted before therapeutic applications of the extract.

Conflict of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper

Funding Statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgement

We would like to extend our gratitude to Dr. D.C.T. Dissanayake, Senior Lecturer in Department of Zoology, University of Sri Jayewardenepura, Sri Lanka for facilitating sample collection during this research work.

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