In vitro exposure to thiacloprid-based insecticide formulation promotes oxidative stress, apoptosis and genetic instability in bovine lymphocytes
Introduction
Pesticides are widely used in agriculture and for veterinary and public health purposes against various kinds of pests. Apart from their beneficial effects such as crop protection, increasing yields and treatment of diseases, they are often detected in foods (Skretteberg et al., 2015), water (Kwon et al., 2017) and in agricultural land (Sankoh et al., 2016) as pollution, and they affect non-target organisms (Saladin et al., 2003) including animals and humans. Occupational and environmental exposures together with poisonings are the most common ways in which humans may be affected. Pesticides are able to form a covalent bond with the nucleophilic centres of cellular molecules. Long-term pesticide exposure can disturb the function of various organs and systems in the body, and has been linked with human chronic diseases such as cancer, neurodegenerative diseases, multiple sclerosis and diabetes (Gangemi et al., 2016; Mostafalou and Abdollahi, 2013; Saravi and Dehpour, 2016).
Neonicotinoids, a class of insecticides acting selectively on the nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS) of insects (Han et al., 2018), have replaced classical insecticides due to their lower resistance, higher effectiveness and less danger to mammals (Jeschke et al., 2013). Thiacloprid, a chloronicotinyl neonicotinoid, known under the commercial name Calypso® 480SC (480 g.L−1), is used to protect fruit, vines, vegetables and ornamental plants against sucking and voracious insects. Thiacloprid is extensively metabolized and excreted through urine within 24 h after oral administration. The organ primarily affected in rats, mice and dogs is the liver; prostate toxicity has also been detected in dogs (EFSA, 2019; EPA, 2013). Thiacloprid has been shown to cause fetal resorptions, skeletal retardations and changes in motor and locomotor activity in offspring and adult rats respectively (EPA, 2013). Because its mutagenic and clastogenic effects long remained unnoticed, the active substance was classified as “unlikely to be genotoxic to humans”. On the other hand, carcinogenic changes in rats (thyroid adenomas and uterine adenocarcinomas) and mice (ovarian luteoma) have been observed, and thiacloprid is marked as “likely to be carcinogenic to humans” (EPA, 2013; FAO, 2010). Moreover, it was found that thiacloprid exhibits a high level of acute toxicity to fish (Tomizawa and Casida, 2005).
But its adverse effects on non-target organisms have caught people's attention. Previous studies have found changes in honey bees´ immune response (Brandt et al., 2016) and behaviour (Tison et al., 2016) after pure thiacloprid treatment. Then experiments on greenhouse whitefly Trialeurodes vaporariorum (Safavi and Bakhshaei, 2017), predacious coccinellids (Katsarou et al., 2009) and Daphnia magna (Takács et al., 2017) have shown increased toxicity, lower survival and high mortality (up to 100%) respectively after Calypso insecticide formulation treatment. In fish, namely the early stages of Cyprinus carpio (Velisek and Stara, 2018) growth decrease, development delay and changes in antioxidant enzyme levels were found after exposure to pure thiacloprid. Other researchers have observed micronucleus induction in rats (Hendawi et al., 2016; Sekeroglu et al., 2013) and a decrease in cell proliferation associated with higher levels of chromosomal aberrations in bovine lymphocytes (Galdikova et al., 2015) after treatment with this commercial product. Kocaman et al. (2014) tested pure thiacloprid on human lymphocytes; both genotoxic and cytotoxic effects were detected.
Since there is a lack of data regarding thiacloprid formulation effects on animal/bovine lymphocytes, we decided to analyze the potential cytotoxic and genotoxic effects of thiacloprid-based insecticide formulation (Calypso®) on bovine peripheral lymphocytes in vitro in more detail, especially upon short-term exposure. Cattle represent a suitable model for monitoring of pesticide exposure because of their high ingestion of contaminated feed or polluted soil during grazing (Rhind, 2005). Moreover, milk and other tissues, which could contain accumulated pesticides during their life, may have harmful effects on their offspring, and on humans (Pardio et al., 2012; Pardio et al., 2003). In addition, lymphocytes are widely used to evaluate potential cytotoxic and genotoxic pesticide effects (Ferri et al., 2018; Jacobsen-Pereira et al., 2018; Schwarzbacherova et al., 2017).
Section snippets
Sample collection and cell cultivation
A one-year-old bull (Holstein crossbreed) was the source of whole blood samples, from which peripheral lymphocytes were isolated using HISTOPAQUE®-1077 (Sigma Aldrich, Poznan, Poland) according to the manufacturer's instructions. Then they were cultured at a concentration of 2 × 105 per mL of culture (total volume of culture medium per concentration was 2 mL) in cell culture plates at 37 °C for 24 h. The only exception was the micronucleus test, where the 72 h cultivation is required. Separated
Results
Results of cell viability assessment after 4-h treatment with insecticide concentrations are shown on Fig. 1A. Acridine orange-ethidium bromide staining was used. Gradual decrease in cell viability was seen; the maximum level of cells in late apoptosis (up to 27%; p <0.01) was found at the highest concentration tested (240 μg.mL−1).
For detection of apoptotic events in insecticide-treated cells, Annexin V staining (Fig. 1B, C) and TUNEL assays (Fig. 1D, E) were performed. Apoptotic cell death
Discussion
Neonicotinoids, a new class of insecticides, act preferably on insect nicotinic acetylcholine receptors (nAChRs) as their agonists. These receptors belong in the group of ligand gated ion channels, whose role is to enable rapid excitatory cholinergic transmission (Tomizawa and Casida, 2005; Tomizawa and Yamamoto, 1993). However, various side effects on non-target animals have been observed after exposure to neonicotinoids, such as hepatotoxicity (Toor et al., 2013), neurotoxicity (Lonare et
Conclusions
To conclude, we detected lower cell viability accompanied by DNA damage in bovine lymphocytes after their exposure to thiacloprid-based formulation. Genetic instability was probably mediated by increased levels of oxidative damage. Moreover, apoptosis as a consequence of oxidative damage was detected. Despite the fact that neonicotinoids are already under supervision because of their harmful effects both in insects and mammals, further research should be conducted using different in vitro and
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This study was supported by the Slovak Scientific Agency VEGA [project 1/0242/19] and IGA 01/2019: Assessment of genotoxic and cytotoxic effect of acetamipride insecticide in cell cultures.
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