2019 Volume 84 Issue 3 Pages 263-269
Allium cepa (2n=16) assay is used to determine cytotoxicity of environmental pollutants like heavy metal arsenic (in the form of arsenic trioxide—concentration used: 0.010, 0.050, 0.075 and 0.100 mg L−1 for 24 h duration) and azo-dye metanil yellow (concentration used: 100, 150, 200 and 400 mg L−1 for 24 h duration) with an objective to understand the toxic effects of the test materials on cells and chromosomes of a plant-based system. Assessment of cytotoxicity reveals that arsenic trioxide can induce chromosomal breakages, affects spindle organization and causes cellular metabolic defects; whereas, metanil yellow predominantly affects cellular metabolism. Cytological disturbances are mostly dose-dependent, and arsenic trioxide depicts pronounced effectivity in inducing mitotic aberrations in root tip cells of A. cepa than metanil yellow (in relation to employed doses). Furthermore, aqueous plant extracts (used due to its operational simplicity and cost-effectivity) of the leaf (Coriandrum sativum L., Ocimum tenuiflorum L., and Pteris vittata L.) and seed (Nigella sativa L.) samples are used to ascertain their amelioration potentiality against the environmental toxicants. The ameliorative study (decrement in their observed values) involves attributes like mitotic index, total abnormal dividing cell frequency and frequency of giant and anucleate cells in resting stages. Results suggest that all the employed extracts are ameliorative, and can be explored further for their role in bioremediation.
Heavy metals and azo-dyes released in the environmental inter-collegium mostly through industrial (Sleiman et al. 2007, Abdel-Tawwab et al. 2017, Pirkarami and Olya 2017) wastes, and can percolate and accumulate in the soil as end-point deposition following atmospheric rainwater and transportation. Due to their prolonged self-life (Fazio et al. 2014, Bankole et al. 2017), they cause cellular toxicity to different components of the ecosystem. The rooted plant species, principal producer of the food chain, can easily be intoxicated by the industrial effluents (like heavy metals and azo-dyes) deposited within the soil (primary site of interaction between the components). Cytotoxicity encompassing alterations in the dividing cell frequency and chromosome architecture is induced by the heavy metals (Kiran and Şahin 2006, Ünyayar et al. 2006, Pandey and Upadhyay 2010, Bhat et al. 2011, Kwankua et al. 2012, Tommonaro et al. 2015, Gupta et al. 2018, Kumbhakar et al. 2018, Pramanik et al. 2019) and azo-dyes (Dwivedi and Kumar 2015, Kumbhakar et al. 2018) in plant species providing insight to subcellular mode of interaction (cellular damages caused). Accumulated toxicants in plant species can be transmitted to consumers, especially human beings, through the edible product(s) causing serious health hazards (Chandra and Nagaraja 1987, Dees et al. 1997, Miller et al. 2002, Soucy et al. 2003, Banerjee and Giri 2014).
Arsenic, a toxic metalloid, can exist in soil and water in the form of arsenic trioxide (As2O3, inorganic arsenic). Elementary arsenic accumulates in humans through drinking water (Nordstrom 2002, Meharg et al. 2009) and food grain crops, and induces cellular toxicity and diseases. Spectroscopic analyses reveal that As2O3 binds to the DNA and RNA (Nafisi et al. 2005) and is reported to be a clastogenic/genotoxic compound (Kumar et al. 2014). Metanil yellow, non-permitted (prevention of Food Adulteration Act 1954, India) synthetic azo-dye, is a toxic chemical used as an adulterant (adulteration) in some food items in India (Nath et al. 2015). The chemical is reported to possess toxic effects on different vital organs like stomach, liver, kidney, abdomen, and testis of humans (Chandra et al. 1987). Thus, the two environmental pollutants namely, arsenic trioxide and metanil yellow can be uptaken from sources and accumulated in humans causing health hazards.
The use of plant species and their value added products for treatment of diseases is an age-old practice in Ayurvedic medicine, which is greatly enhanced in recent years (Oyeyemi and Bakare 2013) due to their perceived efficacy, operational simplicity, cost effectivity and very low to no incidences of adverse side effects (Bhattacharya and Haldar 2013). Bhattacharya (2017) reviewed plants and their products in amelioration of arsenic toxicity in experimental models like mice, rabbits, rats, and cats. Leaf extracts (methanolic/ethanolic) of Coriandrum sativum are reported to possess protective effect against metanil yellow induced lipid peroxidation in the liver (Hazra et al. 2016) and cardiac (Dome et al. 2017) tissues of a goat. Kumar et al. (2010) suggest that bioactive compounds of leaf methanolic extract of Amaranthus spinosus exhibit hepatoprotective and antioxidant effects against paracetamol-induced acute liver damage in rats.
The present work describes the cytotoxic effects induced by arsenic trioxide and metanil yellow (considering recommended doses as well as higher concentrations) in root tip meristematic cells of Allium cepa L. and subsequently protective roles against toxicity are studied using aqueous (due to simplicity and cost effectivity in operational mode) extracts of Coriandrum sativum L. (leaf), Nigella sativa L. (seed), Ocimum tenuiflorum L. (leaf) and Pteris vittata L. (leaf). The species are reported to possess significant therapeutic uses (C. sativum—Mahendra and Bisht 2011, Laribi et al. 2015; N. sativa—Yarnell and Abascal 2011, Datta et al. 2012; O. tenuiflorum—Puri 2002, Prakash and Gupta 2005; P. vittata—Wahid et al. 2015). The objective of the work is to determine the nature of cytotoxicity induced by the pollutants in a plant-based system as well as to foresee whether or not the employed plant extracts possess ameliorative potentiality against the cytotoxic effects. An Allium test performed as it is a classical test system for determination of chromosomal aberrations (Barberio et al. 2011, Fatma et al. 2018, Verma and Srivastava 2018), and this test is reported to exhibit identical results as that of mammalian test systems (El-Shahaby et al. 2003, Teixeira et al. 2003).
Amount of 0.01 mg of arsenic trioxide (As2O3, Merck-AR grade) was dissolved in 100 mL of deionized water to make the concentration of 0.1 mg L−1 and subsequently, different concentrations like 0.05, 0.075 and 0.01 mg L−1 were prepared following dilution with deionized water.
Powdered metanil yellow was procured in packets from the local market (No. A9512). A stock solution of 400 mg L−1 was prepared by dissolving 40 mg of metanil yellow dye powder in 100 mL of deionized water. From the stock, 200, 150 and 100 mg L−1 concentrations were prepared following dilutions with deionized water.
Preparation of plant extractsLeaf (Coriandrum sativum L., Ocimum tenuiflorum L., Pteris vittata L.) and seed (Nigella sativa L.) samples of the experimental materials were procured from medicinal plant garden, Narendrapur Ramkrishna Mission, West Bengal, India.
Leaf samples (10 g newly formed leaves in each case) of the plant species were taken from fully grown healthy plants prior to reproductive stage. Leaf and seed (10 g of seeds) samples were crushed to powder form with the help of liquid nitrogen in a cold chamber at 4°C. Each of the powdered samples was thoroughly dissolved in 100 mL deionized water with the help of a magnetic stirrer for 1 h and subsequently filtered through a Whatman No. 1 filter paper. The final volumes of the aqueous extracts were made up to 100 mL.
TreatmentsSprouted (root length 1 to 2 mm; sprouted in sand-saw dust tray) bulbs (4 in each concentration) of Allium cepa cv. aggregatum (obtained from farmers of North 24 Paraganas, West Bengal) were dipped in different concentrations of As2O3 (0.01, 0.05, 0.075 and 0.1 mg L−1) and metanil yellow (100, 150, 200 and 400 mg L−1) in Petri plates for 24 h duration. After 24 h treatment, 1 to 2 roots (total of 6 roots from 4 bulbs in each set of treatment) from each bulb were cut, fixed in acetic ethanol (1 : 1) for 30 min and preserved in 70% ethanol at 16°C under refrigeration for assessment of cytotoxicity. Each of the four bulbs of each treatment was dipped in aqueous extracts of C. sativum, N. sativa, O. tenuiflorum, and P. vittata in Petri plate for 24 h duration. Following 24 h exposure in aqueous extracts, 6 roots from each set were cut, fixed in acetic–ethanol (1 : 1) for 30 min and preserved in 70% ethanol for further studies.
Determination of cytotoxicityAs2O3 and metanil yellow treated roots and roots dipped in aqueous extracts were cytologically assessed. Six roots (3 slides) from each set of treatment were selected, and root tips were squashed in 45% acetic acid following staining in aceto-orcein–1M HCl (9 : 1) for overnight. The slides were observed under a Leitz Laborlux S Compound Microscope and suitable cells were photographed using a Leica E3 scientific camera attached to it. Mitotic index (number of dividing cells/total cells estimated×100) and, aberration types both in dividing and resting stages and their frequencies were analyzed. Untreated control roots were also studied under similar laboratory conditions (21±1°C). Ameliorative effects of the aqueous extracts were studied with reference to fold increase (+) or decrease (−) in relation to respective values.
Statistical analysisData obtained from treatments (including control) for dividing cell frequencies and total aberration frequencies in dividing cells were statistically analyzed to assess significant variations between/among treatments using one way ANOVA (analysis of variance) test and computation of CD (critical difference) at 0.05 probability level.
Mitotic index and, types and frequency of cytological abnormalities recorded in untreated control and in test materials are documented in Table 1 and Fig. 1a–x. In relation to control, mitotic index is reduced significantly (p＜0.05) in treatments (As2O3: 14.50 to 7.82%; metanil yellow: 12.61 to 5.36%) and the reduction is dose-dependent. The mitodepressive effect due to treatments suggests that both As2O3 and metanil yellow affect normal cell cycle progress leading to suppression of DNA synthesis (Sudhakar et al. 2001, Patlolla et al. 2012) and microtubule organization (Butt and Vahidy 1994) as the possible consequence of reduced ATP production (Jain and Sarbhoy 1988). Reduction in dividing cell frequency in mitotic cells following exposure to heavy metals (Barbosa et al. 2010, Pandey and Upadhyay 2010, Aslam et al. 2014, Abubacker and Sathya 2017, Kumbhakar et al. 2018) and azo-dyes (Dwivedi and Kumar 2015, Kumbhakar et al. 2018) is reported earlier in plant species.
| Treatments | Concentration (mg L−1) | Total no. of cells scored | No. of dividing cells | Mitotic index (%) | Abnormality in dividing cells (%) | Frequency of abnormal dividing cells (%) | Cells with micronuclei (%) | Giant cells (%) | Anucleated cells (%) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Stickiness | Diplochromosomes | Fragments | Polyploid | Bridge(s) | Laggard(s) | Multipolarity | |||||||||
| As2O3 | 0 | 2890 | 427 | 14.78 | 0.47 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.47 | 0.00 | 0.00 | 0.00 |
| 0.010 | 1310 | 190 | 14.50 | 1.58 | 1.05 | 1.05 | 2.11 | 1.05 | 1.58 | 1.05 | 9.47 | 0.54 | 2.77 | 0.36 | |
| 0.050 | 1223 | 157 | 12.84 | 1.91 | 0.64 | 1.27 | 3.18 | 1.91 | 1.27 | 0.64 | 10.83 | 0.19 | 3.94 | 0.66 | |
| 0.075 | 1114 | 120 | 10.77 | 1.67 | 0.00 | 2.50 | 4.17 | 1.67 | 0.83 | 1.67 | 12.50 | 0.10 | 3.72 | 0.63 | |
| 0.100 | 1291 | 101 | 7.82 | 1.98 | 0.99 | 1.98 | 3.96 | 1.98 | 1.98 | 0.99 | 14.85 | 0.00 | 4.03 | 0.76 | |
| CD at 5% level | 0.69 | 0.76 | |||||||||||||
| Metanil yellow | 0 | 2890 | 427 | 14.78 | 0.47 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.47 | 0.00 | 0.00 | 0.00 |
| 100 | 1729 | 218 | 12.61 | 4.13 | 0.92 | 0.00 | 0.00 | 1.38 | 0.92 | 0.00 | 7.34 | 0.26 | 2.85 | 0.73 | |
| 150 | 1480 | 197 | 13.31 | 5.08 | 1.02 | 0.00 | 0.00 | 1.52 | 0.00 | 1.02 | 8.63 | 0.16 | 2.34 | 0.70 | |
| 200 | 1233 | 117 | 9.49 | 5.13 | 0.85 | 0.00 | 0.00 | 1.71 | 0.85 | 1.71 | 10.26 | 0.09 | 1.97 | 1.70 | |
| 400 | 1324 | 71 | 5.36 | 7.04 | 1.41 | 0.00 | 0.00 | 4.23 | 0.00 | 0.00 | 12.68 | 0.00 | 2.08 | 1.92 | |
| CD at 5% level | 0.54 | 0.82 | |||||||||||||
Mitotic cells of untreated control show 2n=16 chromosomes (Fig. 1a–b). Clumped and sticky configuration of chromosomes is the only aberration recorded in few (0.47%) control cells. As2O3 and metanil yellow treated cells depict abnormalities like stickiness of chromosomes (Fig. 1c, q), diplochromosomes (Fig. 1g), chromosomal fragments (Fig. 1f), polyploid cell formation (Fig. 1d–f), laggards (Fig. 1h), bridges (Fig. 1i, r) and multipolarity (Fig. 1j) in dividing cells; micronuclei (condensed type—Fig. 1l, s; uniformly one in number), uninucleate giant (Fig. 1o, v–w), binucleate (equal and unequal sized nuclei) giant (Fig. 1m–n) and anucleate cells (Fig. 1p) in resting stages. As2O3 treatment (0.10 mg L−1) shows deformity in cellular architecture at anaphase (Fig. 1k) in 0.99% cell. It is significant to mention that fragments and polyploid cells are only noted in As2O3 treatments, and their occurrences are mostly dose-dependent. Metanil yellow treatments predominantly show the cellular and nuclear shape and size deformities, chromatin fragmentation (Fig. 1x) and disintegration (Fig. 1t), chromatin connection between adjacent cells (Fig. 1u) and bi-nucleate cell formation.
The giant cells (cells with larger dimension as compared to other regular cells in mitotic squash preparations) are possibly the outcome of the accumulation of cellular defects due to the abnormal mitotic activity under physiological stress induced by the treating agents. As2O3 shows polyploid cells and giant cells; whereas, metanil yellow documents no polyploid cells but with variable shaped giant cells suggesting that endoreduplication may not be the cause of giant cell formation. Hayashi et al. (2013) opined that giant cells are the outcome of chaotic mitotic activity due to physiological stresses. Furthermore, such cells are not observed in any squash preparation of untreated control. The occurrence of anucleate cells in metanil yellow treatment may be the possible consequences of cytomictic behavior of chromosomes.
Frequency of abnormal dividing cells is found to enhance significantly among/between the doses of As2O3 and metanil yellow. As2O3 is found effective to induce mitotic aberrations in root tip cells of A. cepa at a considerably lower concentration than those of employed for metanil yellow. Result suggests that As2O3 can induce clastogenic effect as well as can affect spindle dysfunctioning causing a high frequency of cells with polyploid chromosome number. However, both As2O3 and metanil yellow induce defects in cellular metabolism resulting in cellular and nuclear aberrations. On the contrary, Roychoudhary and Giri (1989) opined that metanil yellow can induce polyploid cell formation and can bring about clastogenic activity at higher doses in the root tip of A. cepa. Kumbhakar et al. (2018) reported mitotic aberrations in root tip cells of Nigella sativa following treatments with methyl orange and malachite green. Cytotoxic effects of heavy metals (copper, cadmium, lead, mercury and arsenic) are also reported in plant species (Ünyayar et al. 2006, Bhat et al. 2011, Kwankua et al. 2012, Tommonaro et al. 2015, Gupta et al. 2018, Kumbhakar et al. 2018, Pramanik et al. 2019).
The ameliorative potentiality of aqueous plant extractsData (dividing cell frequency, total aberration frequency in dividing cells, giant and anucleate cell frequencies in resting cells) relating to the aqueous plant extracts inducing a reduction in cytological aberrations caused due to As2O3 and metanil yellow treatments is presented in Fig. 2. As compared to treatments, there is a profound decrement in mitotic index and cytotoxicity in dividing cells following amelioration by the aqueous plant extracts. All the studied plant extracts possess ameliorative potentiality as evinced from several folds of decrement in cytotoxicity in dividing cells. Such decrement in mitotic aberrations is mostly inversely proportional to doses of treatments. Results, however, suggest that the effectivities due to ameliorations of all the employed plant extracts are nearly similar as observed by CD values computed through ANOVA tool performed among extracts for each dose of treatment. Mitotic index is found to reduce considerably in extracts, and such results can be exploited to suppress cell proliferation and contribute to the significance in cancer research.
Furthermore, it is noteworthy to mention that in comparison to As2O3 and metanil yellow treatments (Table 1), reduction in the frequency of giant and anucleate (Fig. 2A–H) cells following plant extract treatments suggesting some sort of reversal of cellular and nuclear disintegration which was caused by the studied cytotoxicants. Prajitha and Thoppil (2016) evaluated the protective effect of aqueous leaf extracts of A. spinosus on A. cepa meristematic cells against H2O2 induced damage. The authors opined that active compounds present in the extracts possibly scavenged the free radicals generated by H2O2 reducing genetic damage imposed by the toxicant. The antigenotoxic potentiality of plant extracts is due to its total phenolic content (Maurich et al. 2004) which forms strong ligand complexes with metal ions (Ferguson 2001, Hale et al. 2002). Plant extracts can also induce DNA glycosylase enzymes which are capable to repair DNA bases by alkylation (Steele and Kelloff 2005).
The present study highlights that the bioactive compound(s) present in administered aqueous extracts can be explored, and effectively utilized to control cytotoxic as well as genotoxic effects of toxic chemicals in the living system leading to bioremediation.
The authors are thankful to University of Kalyani for providing necessary facilities. Authors SB and SG thankfully acknowledge the financial assistance received from DST PURSE II, University of Kalyani and WBHESTB, Govt. of West Bengal India (Award No. SR/PURSE Phase 2/37 (G) dated 2nd November 2017). The authors are greatly thankful to the honorable reviewers for their valuable suggestion for upgrading the article.
