Evaluation of Synthesised heterocyclic compounds for their Antimitotic activity by Onion Root Tip assay

Ojaswi Ghadge^1,2*, Supriya Mahajan¹

¹C.U. Shah College of Pharmacy, Juhu, Santacruz (W), Mumbai, Maharashtra, India 400049.

²SVB’s College of Pharmacy, Dombivli (E), Maharashtra, India 421204.

*Corresponding Author E-mail: ojaswi.ghadge@svbpharmacy.edu.in, drojaswighadge@gmail.com

ABSTRACT:

Cancer is still one of the leading causes of death worldwide. Considering the benefits of small molecules for treating malignancies, novel derivatives of benzothiazole were synthesised (1-8). These compounds were subjected to Allium cepa assay for evaluating cytotoxic, antimitotic and genotoxic potential. Cytotoxicity was evaluated by observing root growth inhibition. Antimitotic effect was evaluated by calculating mitotic index (MI). Genotoxicity was studied by observing chromosomal aberrations (CA). The studies were carried out in three different concentrations of tests and controls. As compared to negative control, lesser root growth was observed at all concentration of the test compounds. Among all tested compounds, Compound 1 showed maximum root growth inhibition for all the three concentrations. Dose-dependent inhibitory effect was exhibited on root meristems. During the determination of antimitotic activity, Compound 1 showed least MI value and Compound 3 showed highest MI value. Compounds 1, 2 and 4 exhibited insignificant chromosomal aberrations (CA). Among these, Compound 2 had shown the least per cent chromosomal aberration at all the three concentrations. The statistical analysis was carried out using MAX-STAT-LITE Software, 3.60. The one-way ANOVA at 95% confidence level was used for statistical analysis. Values of p<0.05 were considered to be significant.

KEYWORDS: Allium assay, Benzothiazole, Antimitotic, Mitotic index, Chromosomal aberrations

INTRODUCTION:

As per WHO, cancer is still one of the leading causes of death worldwide that accounts for more than 10 million deaths by 2022. The available anticancer drugs have lot of limitations with respect to toxicity, adverse effects, resistance. Hence, synthesis of new molecules for various types of cancers requires due attention^1-3,13. Synthesis of new molecules to fulfil the constant need of small heterocyclics for anticancer therapy and their faster screening at preliminary level is an important approach with respect to cost and labour saving^4-6,9.

Small heterocyclic molecules have gained attention due to their broad pharmacological activities^7-9. Benzothiazole is one of the most important heterocyclic compounds, receiving overwhelming attention due to its diverse molecular design and remarkable biological importance^8-12. This heterocyclic scaffold is readily substituted at the unique methylene centre of the thiazole ring and it is weakly basic in nature^14-15. The present research work aims to test the synthesized benzothiazole derivatives shown in Figure 1 (Compounds 1-8) as potential antimitotic agents.

Mitosis has been an attractive target for anticancer therapies since fast proliferation was identified as one of the hallmarks of cancer cells. Thus, an assessment of cytotoxic and antimutagenic activity is necessary to understand their antiproliferative activity^16-18.

Allium cepa root tip meristems have been widely used for the evaluation of cytotoxicity, anti-mitotic activity, and genotoxicity^16,19,20. Characterized by rather homogenous meristematic cells, very large chromosomes and only sixteen chromosome numbers (2n = 16), the Allium cepa species (common onion) is ideal for use in bioassays. Onion root meristem cells are very sensitive to genetic damage by chemicals. Allium test could be useful in correlating the antimitotic effect of the plant in Allium cepa with that of mammalian cells as it is reported that Allium test shows good correlation with mammalian test systems. Allium assay has been shown to have correlation with tests in other living systems and serve as an indicator of toxicity of the tested material^21,22. The assay also helps to study genotoxicity by observing chromosomal aberrations. Chromosomal aberrations refer to any irregularity or abnormality of chromosome distribution, number, structure, or arrangement. A chromosomal aberration is defined as any abnormality in the structure or the number of chromosomes. Chromosomal aberrations refer to genotoxicity^23-28.

MATERIALS AND METHODS:

Procedure for Allium assay:

Healthy and nearly equal sized bulbs, weighing in the range of 30-35 g, of common onion (Allium cepa L.) were chosen for the experiments. For each test compound, the onion bulbs were randomly divided into eight groups, with three onion bulbs in each group. Each onion bulb was kept on a container containing tap water so that only the stem portion is dipped into water. After three days, when most of the roots attained the length of about 20–30 mm, the unhealthy smaller roots were discarded and the three groups of onion root bulb were transferred to the beakers having three different concentrations of the synthesized compounds. Onions of the fourth group were kept on the containers containing distilled water (control 1). Onions from fifth group were kept on the containers containing the solvent used for preparing dilutions of the synthesized compounds (methanol: water, 1:2, control 2 for compounds 1, 3, 5 and 8 and DMSO: water, 1:3, control 3 for compounds 2, 4. 6 and 7). Onions from the sixth, seventh and eighth groups were kept on the containers containing solutions of a well-known mutagenic agent⁹, ethyl methane sulphonate (EMS)^19,20, positive controls.

To study the effect of synthesized compounds on root growth inhibition, The length of onion roots in different groups were measured daily for seven days. To study the effect of synthesized compounds on mitotic index (MI), the mitotic index (MI)^19-20 was calculated by counting 1,000 cells per root.

Preparation of squashed root meristem samples:

After 24 h of the treatment with control 1, control 2, control 3, positive control and test compounds (1-8), 3-4 roots were cut from the onions in each group and stored in Carnoy’s fixative (glacial acetic acid: absolute ethanol, 3:1) at room temperature and were used within 24 h for preparing squashed root meristem samples. Fixed roots were boiled gently for 1-2 min in a watch glass containing Orcein acetic stain and then placed on clean slides. The darkly stained meristem portions were cut with a razor blade and pressed with thumb using a cover slip. For determining Mitotic Index, these squash preparations of 3-4 roots were thoroughly examined through compound microscope, under 40X magnification for counting number of the cells at division phase. Only normal cells were counted for determining Mitotic Index. Abnormal cells and chromosomal aberrations were not taken into consideration^18,21,23-30.

Figure 1: Synthesized benzothiazole derivatives (1-8) under experiment.

To study the effect of synthesized compounds on chromosomal aberrations, the squashed root meristem samples were used. The squashed root meristem samples were observed for the presence of various chromosomal aberrations including chromosomal fragments, distorted shapes and elongated nuclei^26-33.

Results and discussion for onion root tip (Allium) assay:

Effect of synthesized compounds on root growth inhibition: The values of average root growth were reported in terms of length in millimeter (mm). Root growth was measured for three onions in each group and reported as Mean ± SEM. Tables 1 presents the values of average root growth in Allium cepa treated with methanol: water (1:2) and various concentrations of positive control and test compounds 1, 3, 5 and 8. Table 2 presents the values of average root growth in Allium cepa treated with DMSO: water (1:3) and various concentrations of positive control and test compounds 2, 4, 6 and 7.

As observed in Table 1 and 2, it can be noted that the difference between the mean of root growth for control 1 -control 2 and control 1 -control 3 was not statistically significant. This means that the control 2 and 3 did not inhibit the root growth significantly. It was observed that inhibition of root growth by all the test compounds (1, 3, 5 and 8) was statistically significant from day 4 to day 7, when compared with control 1 and control 2. At each concentration of compounds 1, 3, 5 and 8, the root growth was found to be lesser than that of the control 1 and control 2 from day 4 to day 7. However, at each concentration the root growth in all the test compounds was more than that observed in the positive control. Inhibition of root growth by the positive control (EMS) was statistically significant at all the three concentrations as shown in Tables 1 and 2. Inhibition of root growth for compound 2 was not statistically significant at 50 μg/mL for day 4 and day 5. Inhibition of root growth for compounds 7 was not statistically significant at 50 μg/mL for day 4. For all other compounds statistically significant inhibition in root growth was observed for day 4 to day 7. At each concentration of compounds 2, 4, 6 and 7, the root growth was found to be lesser than that of control 1 and control 2 from day 4 to day 7. As compared to the positive control, the higher root growth was observed at all the concentrations of test compounds.

Table 1: Average growth (in mm) in roots of Allium cepa treated with methanol: water as control 2 and various concentrations of positive control and test compounds 1, 3, 5 and 8

Control code	Concentration (μg/mL)	Root length (Mean ± SEM) was measured daily for seven days
		Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
Control 1 (Water)	-	3.6±0.8	7.3±0.7	17±1.6	24.6±0.8	31.7±1.4	39.3±2.9	44.7±2.6
Control 2 (methanol: water, 1:2)	-	2.0±0.5	5.7±0.9	14.7±1.3	22.3±1.4^ns	29±0^ns	37±1.5^ns	43.3±1.6^ns
Positive control (EMS)	50	-	-	-	8.66±0.8***	10±0.5***	11±0.5***	11.33±0.8***
	100	-	-	-	7.66±0.3***	9.66±0.8***	10±0.5***	11±0.5***
	200	-	-	-	3.33±0.8***	4±1***	5.33±0.8***	6.33±0.3***
1	50	-	-	-	20.7±0.6*	22±0.5*	22±1*	23±0.6*
	100	-	-	-	17.3±1.2**	18.7±0.6**	19.3±03***	20±0***
	200	-	-	-	12±1***	13±0.5***	13±0***	14.3±0.3***
3	50	-	-	-	19±0.57*	20.33±0.88*	23±0.57*	27.6±0.88*
	100	-	-	-	18±0.33**	19.33±0.33**	20.3±0.33**	21.33±0.66**
	200	-	-	-	14±1.73***	13±1***	14.66±0.88***	15±0.57***
5	50	-	-	-	18.6±1.33*	20.6±0.88*	22±1.52*	27.6±1.33*
	100	-	-	-	14.33±0.33***	16.33±0.33***	19±0.57***	23.33±0.33***
	200	-	-	-	11.33±1.85***	13.33±2.02***	14±1.73***	15.66±0.66***
8	50	-	-	-	19.3±0.8*	22.3±0.3*	25.3±0.8*	28.7±0.3*
	100	-	-	-	14±0**	18±0.5**	22.7±0.8**	26.3±0.3**
	200	-	-	-	9.6±0.3***	14.3±0.8***	19.7±0.8***	23±1***

The statistical analysis was carried out using MAX STAT LITE software, Version 3.60. The one-way ANOVA at 95 % confidence level was used for statistical analysis. Values of p <0.05 were considered to be significant.

*Indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001, is statistically significant when compared to control 1, while ns= not significant when compared to control 1

Table 2: Average growth (in mm) in roots of Allium cepa treated with DMSO: water as control 3 and various concentrations of positive control and test compounds 2, 4, 6 and 7

Compound code	Concentration (μg/mL)	Root length (Mean ± SEM) was measured daily for seven days
		Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
Control 1 (Water)		3.6±0.8	7.3±0.7	17±1.6	24.6±0.8	31.7±1.4	39.3±2.9	44.7±2.6
Control 3 (DMSO:water 1:3)		1±0.57	3.66±0.88	9±1.52	19±2.08^ns	25.33±1.76^ns	30±2.08^ns	36.33±3.84^ns
2	50				22.3±0.6^ns	24.3±0.6^ns	26.7±0.8*	30±1.1*
	100				18.6±0.6	21.6±0.3*	23.7±0.3*	25.7±0.3*
	200				16±0.5**	18.3±0.6**	21.7±0.8**	24.7±0.3**
4	50	-	-	-	18.3±0.3*	21.7±1.2*	24.7±0.3*	26.3±0.3*
	100	-	-	-	14.6±0.3**	18±0.5**	22.7±0.6**	25±0.5**
	200	-	-	-	12±0.5***	16.3±1.2***	20±0.5***	22.3±0.8***
6	50	-	-	-	17.66±0.33**	19±0.57**	21.6±0.33**	27±0.57**
	100	-	-	-	14.33±0.88***	15.3±1.45***	18.3±2.72***	20.66±1.85***
	200	-	-	-	13.66±2.33***	14.6±1.76***	18±0.57***	18.66±0.66***
7	50	-	-	-	19±2.08^ns	21.6±1.45*	27±1.52*	30±0.57*
	100	-	-	-	17.66±0.66**	19.6±1.20**	22±0**	24.33±0.88**
	200	-	-	-	14±0***	15±0.57***	18±0.57***	20.33±0.88***

*Indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001, is statistically significant when compared to control 1, while ns= not significant when compared to control

From Tables 1 and 2, it was observed that A. cepa bulbs which were exposed to various concentrations of compounds 1-8, the root growth was retarded significantly from 4^th to 7^th day when compared with the root growth in control 1, control 2 and control 3. Of the three concentrations (50 μg/mL, 100 μg/mL and 200 μg/mL) used, root growth was maximally inhibited in A. cepa bulbs treated with 200 μg/mL of test compounds, thus, exhibiting a dose-dependent inhibitory effect on the root meristems. Among all the tested compounds, compound 1 showed maximum inhibition in root growth for all the three concentrations from day 4 to day 7.

Effect of synthesized compounds on Mitotic index (MI): It has been observed from Table 3 that the MI values for onions kept in contact with control 1, control 2 and control 3 were found to be higher than those treated with various concentrations of compounds 1- 8. The mitotic index (MI) for the onion root tips kept in contact with control 2 and control 3, which was not significantly different than that of control 1. This indicated that the solvents did not reduce the MI significantly. For higher anticancer potential the compound is expected to possess higher antimitotic activity thus lower MI values. Since compound 1 has lowest MI value, it is said to possess higher anticancer potential as compared to the other test compounds. MI values for onion root tips kept in contact with positive control (EMS) were found to be significant when compared with control 1.

It was noted that a significant decrease in the mitotic index from 50.08±2.58 (for control 1, Table 7.15) to 11.16±1.04 (Table 7.15) for compound 1 at the concentration of 200 μg/mL was recorded. Therefore, it can be concluded that compound 1 showed superior antimitotic activity among all the tested compounds. Among all the synthesized compounds, compound 3 showed the lowest activity since it showed the highest MI at all the three concentrations as compared to the other compounds. Significant difference was observed between the MI values of controls (control 1, 2 and 3) and positive control. It was also observed that the cell division was arrested at the initial stage, a prophase of mitotic division, so most of the observed cells at the concentrations of 50, 100 and 200 μg/mL showed cells dominantly in a prophase, while the remaining division stages were negligible.

Table 3: Effect of controls and the synthesized compounds 1, 2, 3, 4, 5, 6, 7 and 8 on % chromosomal aberrations

Compound code	Mitotic (MI) (Mean±SEM)	% CA	Mitotic Index (MI) (Mean±SEM)	% CA	Mitotic Index (MI) (Mean±SEM )	% CA
Concentrations	50 μg/mL		100 μg/mL		200 μg/mL
Control 1	50.08±2.58	0.1	50.08±2.58	0.1	50.08	0.1
Positive control	11.7±0.26	2.03	10.36±0.37	2.7	9.3±0.416	3.26
Control 2	46.1±1.28	0.3	46.1±1.28	0.3	46.1±1.28	0.3
Compound 1	35.63±2.18	0.26	20.96±0.39	0.26	11.16±1.04	0.36
Compound 3	42.7±2.10	0.3	41.93±1.28	0.46	39.53±1.04	0.56
Compound 5	37.4±2.40	0.26	31.93±1.03	0.36	30.33±0.43	0.5
Compound 8	36.9±0.75	0.46	33.8±0.94	0.53	21.6±0.92	0.63
Control 3	45.83±1.35	0.1	45.83±1.35	0.1	45.83±1.35	0.1
Compound 2	39.33±0.81	0.1	34.4±1.26	0.16	31.83±0.49	0.16
Compound 4	38.63±0.97	0.1	31.70±1.30	0.4	27.36±1.03	0.43
Compound 6	40.86±1.64	0.46	38.4±0.96	0.63	32.93±0.93	0.7
Compound 7	40.60±1.47	0.6	37.06±2.56	0.6	31.63±1.52	0.73

The inhibition of cell division by the synthesized compounds could be due to interaction between the compound and DNA of cells. Experiment was conducted in triplicate, and data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey multiple comparison post-test. The means, with 95 % confidence limits, and the standard errors for results of the mitotic index (MI) for each concentration of the synthesized compounds were calculated.

Effect of synthesized compounds on different types of chromosomal aberrations: Chromosomal aberrations are the abnormalities in chromosome arrangement/ movements during mitosis, which are also called as chromosomal abnormalities. Figure 5 presents the normal mitotic phases in the onion root tip cells treated with control 1, control 2 and control 3.

Figure 5: Various normal mitotic phases in root meristems as (a) anaphase in control 1 (b) prophase in control 1 (c) telophase in control 2 (d) metaphase in control 2 (e) prophase in control 2 (f) metaphase in control 3 (g) prophase in control 3 (h) anaphase in control 3

Various types of chromosomal abnormalities or aberrations were recorded after treating the onion root meristems with the synthesized compounds 1-8. The observed chromosomal aberrations are shown in Figure 6, which are as follows- sticky anaphase with bridges, chromosome fragments, disturbed metaphase, distorted shape or elongated nuclei, distorted metaphase, sticky anaphase, distorted metaphase, disturbed prophase, disoriented anaphase with lagging chromosome, etc.

Figure 6: Effect of test compounds on mitosis and its various stages with distorted chromosomes (chromosomal aberrations) recorded as (a) Sticky anaphase with bridges by compound 1 (b) chromosome fragments by compound 2 (c) disturbed metaphase by compound 3 (d) distorted shape or elongated nuclei by compound 4 (e) distorted metaphase by compound 5 (f) sticky anaphase by compound 6 (g) distorted metaphase by compound 7 (h) disturbed prophase by compound 8 (i) disoriented anaphase with lagging chromosome by compound 8

Highest number of chromosomal aberrations were shown by all the three concentrations of positive control, EMS. Among the test compounds 1-8, per cent chromosomal aberrations by compounds 1, 2 and 4 are insignificant as compared to per cent chromosomal aberrations by control 1, control 2, control 3, at all the three concentrations. This means that these three compounds do not produce significant chromosomal aberrations. It can be noted that among compounds 1, 2 and 4, compound 2 has shown the least per cent chromosomal aberrations at all the three concentrations. Thus, it can be concluded that the compound 2 exhibits least genotoxicity among the synthesized compounds. Since, EMS is a mutagenic agent, it shows higher per cent chromosomal aberrations. Thus, the normal cells were found to be less. Therefore, the positive control EMS showed lower MI values.

CONCLUSION:

It was noted that, among all the tested compounds, compound 1 showed maximum inhibition in root growth for all the three concentrations from day 4 to day 7. The compound 1 also exhibited least mitotic index and showed least per cent chromosomal aberrations. Since, Root growth inhibition and lower mitotic index are related to antimitotic potential and chromosomal aberrations are related to genotoxicity of the compound, it can be concluded that compound 1 has antimitotic potential with lower genotoxicity. Hence, compound 1 is considered to have anticancer potential (antimitotic) with higher safety profile (least genotoxic potential) among the tested compounds.

ACKNOWLEDGEMENT:

I am grateful for C. U. Shah College of Pharmacy and H. K. College of Pharmacy for providing the facilities and chemicals required for the experiment. I am further thankful to Dr. Bindu from Mithibai College of Arts and Science and Dr. Deepa Verma from VIVA College of Arts, Commerce and Science for giving their valuable inputs while performing Allium assay.

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Received on 01.12.2023      Revised on 16.06.2024 Accepted on 17.10.2024   Published on 10.12.2024 Available online on December 17, 2024 Asian J. Res. Pharm. Sci. 2024; 14(4):333-338. DOI: 10.52711/2231-5659.2024.00053 ©Asian Pharma Press All Right Reserved
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