Regular Article
Evaluation of Cytotoxicity Induced by the Anti-Cancerous Drugs Doxorubicin and Erlotinib in Allium cepa Assay for Eco-Safety Monitoring
2021 年 86 巻 3 号 p. 195-199
詳細
2021 年 86 巻 3 号 p. 195-199
Allium cepa assay is performed to evaluate the cytotoxicity of anti-cancerous drugs namely, Doxorubicin and Erlotinib. The doses administered are much lower than the pharmaceutical concentrations. The objective of the work is to determine the safe dose(s) of the drugs after disposal to the environment for eco-safety monitoring. Assessments of mitotic indices and cytological aberrations observed in dividing and resting cells to untreated control suggest that the doses employed are sub-lethal to lethal. Types of cytological abnormalities encountered in treated cells indicate that both the drugs are clastogenic (fragments, rings, bridges, and cells with micronucleus) apart from affecting the spindle apparatus (vagrant chromosomes and multipolarity) and cellular metabolism (stickiness of chromosomes and giant cells formation). The present results can be used as an index for safe dose(s) monitoring of the drugs for ecological safety.
Today, the disease of cancer is one of the major concerns for human beings due to its increasing tendency of occurrence worldwide (Bernard and Christopher 2014), and therefore the consumption of anti-cancerous drugs is expected to be higher over the years (Besse et al. 2012). Most of the anti-cancerous drugs instigate immense systemic side effects in the host because of their non-selective mode of action by targeting both cancerous and non-cancerous cells (Allwood et al. 2002). Heath et al. (2020) reported that the anti-cancerous drugs are hazardous compounds causing toxication to the reproductive system of test organisms, and so they bear a potential threat to the ecosystem following their release in the environment through feces and urine of patients. Furthermore, due to negligence and unawareness of common people (including patients and their relatives, unskilled staff members of the medical clinics and pharmaceutical industries), the soil and water bodies can also be polluted by the hazardous anti-cancerous drugs (Schmidt and Redshaw 2015) through improper disposal of hospital waste and municipal wastewater (Zhang et al. 2013). Thus, it is of serious concern that residual pharmaco-chemical deposits in soil and water can be accumulated into the human system through direct and/or indirect (through a rooted system of edible plants) integration into the food chain (Tanoue et al. 2014). As such, considering the above facts, there exists utmost necessity to determine the ‘Cytothreat’ of toxic anti-cancerous drugs (parental compounds/residual forms/degradable products) for assessment of environmental risk.
Allium cepa (2n=16) bioassay (root tip mitotic cells) is used to ascertain chromosome damages and disturbances in the mitotic cycle (Leme and Marin-Morales 2009) exposed to environmental pollutants (Basu et al. 2019) including anti-cancerous drugs (Mercykutty and Stephen 1980, Lutterbeck et al. 2015, Fernández-Casañas and Chan 2018) due to its simplicity, cost-effectivity and efficiency (Barberio et al. 2011, Verma and Srivastava 2018). Results of the Allium test corroborate with the data obtained in the mammalian test system (El-Shahaby et al. 2003, Teixeira et al. 2003). The present study describes the cytotoxicity of two anti-cancerous drugs namely, Doxorubicin (Rivankar 2014) and Erlotinib (Wang et al. 2011) using A. cepa assay system. The root tip cells of A. cepa are subjected to lower doses than the pharmaceutical concentrations of the drugs to foresee whether they are cytotoxic or not. The objective of the work is to specify the safe dose(s) of the compounds after their disposal to the environment for use as an index in ecological safety.
Doxorubicin (Pfizer-Perth Pvt. Ltd., Australia) and Erlotinib (Radha Kishan Pharmaceuticals, India) were the drugs evaluated for their toxicity using the Allium test. Working concentrations (Doxorubicin—0.0100, 0.0125, 0.0150 and 0.0200 g L−1; Erlotinib—0.001, 0.002, 0.004 and 0.006 g L−1) of the test chemicals were prepared using the stock (Doxorubicin—2% injectable solution; Erlotinib—150 mg tablet) following serial dilutions by ddH2O.
TreatmentsA. cepa var. aggregatum bulbs were sprouted in sand-saw dust (1 : 1) trays and dipped in test concentrations of Doxorubicin and Erlotinib for 24 h duration (half-life: Doxorubicin 20 to 48 h—https://www.rxlist.com; Erlotinib 36 h—https://www.accessdata.fda.gov/drugsatfda_docs/label/2008/021743s010lbl.pdf). Treatments were performed in Petri plates. Three sprouted onion bulbs were treated in each concentration. Following treatments, six roots (two roots for each bulb) from each dose were cut, fixed in acetic acid–ethanol (1 : 1) for 30 min, and preserved in 70% ethanol under the refrigerator for further use. Control sets using ddH2O were maintained at 18±1°C under similar laboratory conditions.
Data assessmentTreated and control roots were cytologically analyzed following 2% aceto-orcein:1 N HCl (9 : 1) staining mixture and subsequently squashed in 45% acetic acid. The prepared slides (triplicate for each dose of the treatment) were studied under Leitz Laborlux S compound microscope attached with Leica E3 Scientific camera. The suitable cells were photographed. Mitotic index (MI: number of dividing cells into mitosis/total cells scored ×100) and chromosomal aberrations (CA) in dividing and resting cells were studied to assess the cytotoxicity of the drugs in the test system to untreated controls.
Statistical analysisData obtained for MI and total CA (both in dividing and resting cells) in treatments in relation to respective control were statistically analyzed using one-way ANOVA (analysis of variance) and computation of CD (critical difference) at a 0.05 probability level.
MI and types and frequencies of CA studied in Doxorubicin and Erlotinib treated cells to respective controls are depicted in Table 1 and Fig. 1. To controls, MI values are significantly (p＜0.05) decreased in treatments (Doxorubicin: 6.64 to 3.71; Erlotinib: 5.88 to 0.78) and it is mostly dose-dependent (excepting Doxorubicin—0.0150 g L−1). The total cessation of dividing cells is noted in both the chemicals at higher administered doses. In relation to respective control, MI decreases 29.22%, 52.00%, 44.11% and 59.90% in Doxorubicin and 38.50%, 50.95%, 65.17% and 91.85% in Erlotinib treatments as per doses administered from lower to higher concentrations.
| Chemotherapeutic drugs | Dose (g L−1) | Total cells scored | Total no. of dividing cells | Mitotic index | Abnormal diving cells (%) | Abnormal resting cells (%) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Stickiness | Fragment(s) | Ring configuration of chromosomes | Vagrant chromosome | Bridge(s) | Multipolarity | Total | Micronuclei | Giant cells | Other abnormalities | Total | |||||
| Doxorubicin | 0 | 2412 | 223 | 9.25 | 1.79 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.79 | 0.00 | 0.00 | 0.00 | 0.00 |
| 0.0100 | 3524 | 234 | 6.64 | 11.11 | 1.71 | 0.85 | 0.00 | 5.13 | 0.00 | 18.80 | 0.36 | 0.73 | 0.18 | 1.27 | |
| 0.0125 | 2252 | 100 | 4.44 | 8.00 | 2.00 | 0.00 | 2.00 | 4.00 | 0.00 | 16.00 | 0.19 | 1.21 | 0.18 | 1.58 | |
| 0.0150 | 4355 | 225 | 5.17 | 11.11 | 0.00 | 4.44 | 0.00 | 4.44 | 0.00 | 19.99 | 0.24 | 2.42 | 0.36 | 3.02 | |
| 0.0200 | 3910 | 145 | 3.71 | 13.79 | 0.00 | 0.00 | 3.45 | 3.45 | 0.00 | 20.69 | 0.13 | 4.78 | 0.00 | 4.91 | |
| CD at 5% | 0.130 | 1.533 | 0.121 | ||||||||||||
| Erlotinib | 0 | 2656 | 254 | 9.56 | 1.28 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 1.28 | 0.00 | 0.00 | 0.00 | 0.00 |
| 0.001 | 3129 | 184 | 5.88 | 3.26 | 0.54 | 1.09 | 1.09 | 1.63 | 0.00 | 7.61 | 0.34 | 3.40 | 0.03 | 3.77 | |
| 0.002 | 3820 | 179 | 4.69 | 2.23 | 0.56 | 0.56 | 1.12 | 1.68 | 0.00 | 6.15 | 0.05 | 2.09 | 0.49 | 2.63 | |
| 0.004 | 2611 | 87 | 3.33 | 1.15 | 0.00 | 0.00 | 8.05 | 0.00 | 0.00 | 9.20 | 0.36 | 1.62 | 0.56 | 2.54 | |
| 0.006 | 3065 | 24 | 0.78 | 8.33 | 0.00 | 0.00 | 3.74 | 0.00 | 0.43 | 12.50 | 0.40 | 2.63 | 0.43 | 3.46 | |
| CD at 5% | 0.160 | 0.716 | 0.382 | ||||||||||||
Abnormal diving cell—Diving cells possessing chromosomal aberrations; Abnormal resting cell—Interphase cells consisting of various nuclear and cellular morpho-physiological deformities; CD—Critical difference
Untreated control cells show 2n=16 chromosomes at metaphase (Fig. 1a). The only type of aberration studied in control cells is the sticky and clumped configuration of chromosomes (1.79% and 1.28%). CA observed in treated cells are stickiness and clumping of chromosomes (Fig. 1b), fragments (Fig. 1c), rings (Fig. 1d), bridges with or without an associated fragment (Fig. 1e–j), and vagrant chromosomes (Fig. 1j) in dividing cells and micronucleus (Fig. 1m) and giant cells (Fig. 1n–o) in resting cells. Abnormalities like asynchronous division (Erlotinib—0.004 g L−1, 1.15%; Fig. 1e), multipolarity (Erlotinib—0.006 g L−1, 4.17%; Fig. 1k) and binucleate cells (Erlotinib—0.002 g L−1, 0.49%, Fig. 1p) are also observed in treatments. In comparison to controls, CA in dividing cells is enhanced significantly (p＜0.05) in treatments and is mostly dose-dependent (excepting: 0.0125 g L−1 Doxorubicin). Total aberration frequency in resting cells is found to increase significantly (p＜0.05) with higher doses of Doxorubicin, however, in Erlotinib treatments, it enhances from 2.54% (0.004 g L−1) to 3.53% (0.001 g L−1).
The dose-dependent decreasing values of MI suggest that both the administered drugs (Doxorubicin and Erlotinib) are cytotoxic inducing disturbances in the mitotic cycle causing hindrances in normal cell cycle progression. The mito-depressive effect induced by the anti-cancerous drugs in the plant system is reported earlier (Leme and Marin-Morales 2009, Gupta et al. 2019). In relation to control, the 22% declining value of dividing cell frequency as noted in treated root tip meristems of A. cepa is considered as sub-lethal (Antosiewicz 1990) whereas the value is observed as 50% for lethal effect (Sharma 1983, Panda and Sahu 1985). The results of the present study highlight that Doxorubicin and Erlotinib can induce both sublethal as well as lethal effects on test materials depending on the doses of treatments.
From the results, it appears that the types of CA are alike in both the anti-cancerous drugs induced cells. Sticky and clumped configuration of chromosomes are the most predominant aberrations studied, and it is reported to be the outcome of excessive formation of nucleoproteins and faulty protein–protein interaction arising due to linkage of sub-chromatid chromosome strands (Nefic et al. 2013). Formation of a vagrant chromosome and multipolarity like CAs may be caused due to defect in microtubule formation as substantiated with the earlier study (Dalton et al. 2007) where chromosome abnormalities and increased multipolarity are also found to relate with affected microtubules. Anaphase bridges are possibly the consequences of DNA double-strand breaks and incorrect fusion events (Fernández-Casañas and Chan 2018). The occurrence of chromosomal fragment(s), ring configuration of chromosomes, bridges, and micronuclei in resting cells indicates that both the drugs are clastogenic apart from affecting spindle apparatus and cellular metabolism. Antonino de-Souza Jr. et al. (2017) opined that giant cells might be the outcome of accumulated mitotic defects. The formation of binucleate cells is due to the failure of cytokinesis.
Though, the CA types observed in Doxorubicin treated A. cepa cells are comparable to the earlier record (Mercykutty and Stephen 1980) but the treated concentrations were much higher (0.1%, 1.0%, and 10.0% at different durations of mitotic cycle for 24 h) than the concentrations used in the present investigation.
Lutterbeck et al. (2015) suggest that A. cepa bioassay show higher sensitivity than other standardized tests like Ames and Umu Chromotest for evaluation of cytotoxicity (alteration in the cell cycle), genotoxicity (chromosomal aberrations), and mutagenicity (from micronucleated cells). Results of the present study suggest that both the anti-cancerous drugs are cytotoxic, genotoxic, and mutagenic and therefore, need to be carefully handled during disposal to the environment following chemotherapy. The work can also provide an insight for safe dose monitoring of Doxorubicin and Erlotinib for ecological safety.
Conceptualization, designing, and materialization of the present article by Late Dr. Animesh Kumar Datta, retired Professor, Department of Botany, Kalyani University is gratefully acknowledged. The authors paid their sincere homage and deepest regard to Dr. Datta and mourned for his untimely demise. This work was supported by the [DST-FIST program, Department of Botany, Kalyani University] under Grant [number SR/FST/LSI-547/2012(G) dated 27.05.2014]; [DST-PURSE, Kalyani University] under Grant [Award No. SR/PURSE Phase II/37 (G) dated 2nd November 2017].
