Muhammad Mudassir Ali, Zainab Farhad, Muhammad Wasim, Sohail Raza, Mikhlid H. Almutairi, Kainat Zahra, Muhammad Usman Saleem, Khalid Mehmood
Abstract
Ivermectin (IVM) is an anti-parasitic drug which is used for treating parasitic infestations. It has been used in humans for treating intestinal strongyloidiasis and onchocerciasis however, currently researchers are investigating its potential for treating coronavirus SARS-CoV-2. Due to its broad-spectrum activities, IVM is being used excessively in animals which has generated an interest for researchers to investigate its toxic effects. Cytotoxic and genotoxic effects have been reported in animals due to excessive usage of IVM. Therefore, this study aims to evaluate the cytotoxic and genotoxic effects of IVM on the Madin-Darby-Bovine-Kidney (MDBK) cell line by examining the expression of a DNA damage-responsive gene (OGG1). Cytotoxicity of IVM was tested using an assay (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereas the genotoxicity was evaluated using comet assay along with micronucleus assay. Moreover, the gene expression of DNA damage response gene (OGG1) was measured by qRT-PCR, after extraction of RNA from the MDBK cell line using the TRIzol method and its conversion to cDNA by reverse-transcriptase PCR. During the experiment, cell viability percentage was measured at different doses of IVM i.e., 25%, 50%, 75%, along with LC50/2, LC50 and LC50*2. It was observed that the gene expression of OGG1 increased as the concentration of IVM increased. It was concluded that IVM has both cytotoxic and genotoxic effects on the MDBK cell line.
Introduction
Ivermectin (IVM) is a semi-synthetic macrocyclic compound which is effective against numerous parasitic diseases [1] including onchocerciasis. It has been reported to be effective against symptomatic alleviation caused by Onchocerca volvulus along with having the potential to reduce transmission of the aforementioned parasite [1]. The IVM exhibits systemic anti-parasitic activities against helminths, arachnids, insects [2] and is being used to combat parasitic issues related to animal and human health. It also provides positive effects to humans, livestock and pets however, its progressive impacts are still being investigated [3,4]. The IVM combat infections caused by many parasites and offers significant results for treating intestinal strongyloidiasis in humans. Currently being investigated as a potential treatment for COVID-19 (SARS-CoV-2) [5]. Moreover, due to its broad-spectrum effects IVM is being excessively used in both humans and animals [3]. Recently, IVM has gained significant attention due to the toxic effects caused by its excessive use. Slow metabolism of IVM in animals leads to its accumulation in tissues [6] and can be toxic to humans if they consume the meat of an animal that has been excessively treated with IVM [7].
Materials and methods
Cell line
For this study MDBK cell line was obtained from Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan. The DMEM (Dulbecco’s Modified Eagle Medium) media (Gibco DMEM, Thermo Fisher Scientific, USA) was used for MDBK cells.
Growth and sub-culturing of cell line
The MDBK cell line was sub-cultured by using already present cell line and DMEM media. Sub-culturing of the MDBK cell line was done by using frozen and fresh cell line stock. Frozen stock was thawed at 37°C in a water bath after which media was added and growth was obtained. Culture media was prepared using 3 mL antibiotic (Streptomycin) with 10 ml FBS (Fetal Bovine Serum) and DMEM was added up to 100 ml volume. Preceding media was removed from the flask and 3 mL for t25 and 6 ml for t75 trypsin was added for trypsinization (detachment of cells).
Results
MTT assay
During this experiment metabolically active cells reduced tetrazolium to formazan by the action of dehydrogenase enzymes. Viable cells were indicated by purple color whereas, dead cells were not stained. The percentage of cell viability decreased as the doses of IVM increased as shown in “Fig 1”. At LC50 dose of IVM the percentage of cell viability decreased up to 50%.
Discussion
The IVM is derived from avermectin B which is an antiparasitic drug that comes from bacterium Streptomyces. It is effective against worms and arthropods and is useful in treating parasitic infestation related to both humans and domestic animals. However, its toxic nature can cause development of micronuclei [14]. The IVM works by binding to ligand-gated ion channel receptors such as glutamate, GABA and glycine. These receptors are responsible for motility, reproduction and feeding actions in the parasite. In vitro studies have shown that IVM has antiviral properties at very high doses [15]. The IVM is a proven antimicrobial and antiviral agent known for prophylaxis or treatment that prevents re-occurring of disease [16,17] and is used widely for treatment of endoparasites and ectoparasitic infections in animals as well as in humans [18]. Many nematodes have become resistant to IVM due to its excessive use, under dosage genetic defense of pests and many other factors [19].
Conclusion
Nexus to the above results of this study suggest that Ivermectin (IVM) has significant cytotoxic and genotoxic effects on the Madin-Darby-Bovine-Kidney (MDBK) cell line particularly at higher concentrations. The genotoxic effects were evident from the increased expression level of the OGG1 gene in the MDBK cell line which concludes that IVM cause DNA damage in MDBK cells. Furthermore, it is recommended that studies related to the toxic effects of IVM at molecular level should be conducted to combat the hazardous effects caused by IVM.
Citation: Ali MM, Farhad Z, Wasim M, Raza S, Almutairi MH, Zahra K, et al. (2024) Evaluation of genotoxic effect via expression of DNA damage responsive gene induced by ivermectin on MDBK cell line. PLoS ONE 19(5): e0296255. https://doi.org/10.1371/journal.pone.0296255
Editor: Adekunle Akeem Bakare, University of Ibadan Faculty of Science, NIGERIA
Received: August 15, 2023; Accepted: December 4, 2023; Published: May 3, 2024
Copyright: © 2024 Ali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript.
Funding: The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R191), King Saud University, Riyadh, Saudi Arabia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0296255#abstract0
