Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(4): 99-109, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-984 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: S. Koleničová, B. Holečková, M. Galdíková, V. Schwarzbacherová, M. Drážovská (2020) Genotoxicity test- ing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay. Caryologia 73(4): 99-109. doi: 10.13128/caryologia-984 Received: June 25, 2020 Accepted: September 24, 2020 Published: May 19, 2021 Copyright: © 2020 S. Koleničová, B. Holečková, M. Galdíková, V. Schwar- zbacherová, M. Drážovská. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Genotoxicity testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay Simona Koleničová1, Beáta Holečková1,*, Martina Galdíková1, Viera Schwarzbacherová1, Monika Drážovská2 1 University of Veterinary Medicine and Pharmacy in Košice, Department of Biology and Genetics, Slovak Republic 2 University of Veterinary Medicine and Pharmacy in Košice, Department of Epizootiol- ogy and Parasitology, Slovak Republic *Corresponding author. E-mail: beata.holeckova@uvlf.sk Abstract. Epoxiconazole belongs in the class of azoles which have been devel- oped to protect crops from fungal diseases. The mechanism of action of these fungi- cides is to inhibit the specific cytochrome P450 enzyme (CYP), CYP51 (lanosterol 14α-demethylase) which contributes to ergosterol biosynthesis. Since ruminants and cattle are exposed to contaminants during grazing, they are a suitable experimental model for genotoxicity testing. In our experiment, epoxiconazole (EPX) (active agent, 99% of purity) was tested in vitro for its potential genotoxic and cytotoxic effects on bovine lymphocytes, isolated from whole peripheral blood. We exposed the lympho- cytes to EPX at concentrations of 2.5, 5, 10, 25, 50 and 100 μg/mL by two different ways: immediately after isolation of lymphocytes during 2 h in RPMI 1640 medium (without phytohaemagglutinin, PHA) as well as on the last 2 h of 48-h culture (with PHA). In a second case, we chose 48 h culture because the lymphocytes usually start DNA replication 24 h after the start of the cultures; therefore, we incubated the cells longer to obtain dividing (proliferating) cells. The levels of DNA damage were measured using alkaline and neutral comet assays. The results of alkaline comet assay showed the significantly increased percentage of DNA breaks in both lymphocytes in medium with- out PHA (2 h of exposure; non-proliferating cells) and lymphocytes cultured during 48 h in medium with PHA (exposure for the last 2 h of cultivation; proliferating cells). Similarly, neutral comet assay showed dose-dependent elevation of the DNA migration induced in both non-proliferating and proliferating lymphocytes treated with EPX when compared with negative controls. Our results suggest that epoxiconazole fungicide is capable of causing damage to the genetic material of the bovine cells. Keywords: epoxiconazole, genotoxicity, cattle, comet assay. INTRODUCTION Pesticides are a significant source of environmental pollution due to their wide-ranging application in agriculture and forestry. Exposure to these pollutants can have both acute and chronic effects on target and non-target 100 Simona Koleničová et al. organisms (Berenzen et al. 2005). Long-term exposure and chronic poisoning with pesticides can trigger geno- toxic and epigenetic processes through various path- ways, interactions and doses resulting from the intensive use of pesticides and can cumulatively lead to genetic change in humans, covertly and without clinical evi- dence (Bull et al. 2006). As later indicated by Kaur and Kaur (2018), occupational exposure to pesticides in agri- cultural workers has been associated with an increased incidence of various diseases such as cancer, Parkinson’s disease, Alzheimer’s disease, reproductive disorders, and birth defects. Conazoles are a class of azole-based fungicides which are widely used as pesticides in the cultivation of crops despite their suspected endocrine disrupting prop- erties (Roelofs et al. 2014) but also as human and vet- erinary pharmaceuticals for the treatment of oropharyn- geal, vaginal as well as systemic candida and mycosis infections (Kjaerstad et al. 2010). These fungicides act by inhibiting a specific cytochrome P450 (CYP) enzyme, CYP51 (lanosterol 14α-demethylase), which mediates a critical step in the biosynthesis of ergosterol, a steroid required for the synthesis of the fungal cell wall (Zarn et al. 2003). For this reason they are called demethylation inhibitor (DMI) or ergosterol-biosynthesis-inhibiting (EBI) fungicides. Besides their effects on fungal CYP51, triazole-based conazoles have the potential to interact with the mammalian cytochrome P450 (CYP) system, e.g. via inhibition of aromatase (CYP19) which can lead to numerous toxicological effects (Chambers et al. 2014). As reported by Roelofs et al. (2013) conazoles also cause catalytic inhibition of the CYP17 enzyme, responsible for the conversion of pregnenolone and progesterone to androgen precursors. Exposure to these compounds from multiple environmental matrices can cause many negative effects including carcinogenicity hepatotoxicity, reproductive and developmental toxicities (Goetz and Dix 2009; Hester et al. 2012; Heise et al. 2015; Mu et al. 2016; Heise et al. 2018). In spite of the large production and extensive usage of many conazoles, accurate data on human exposure levels are scarce. Besides occupational and pharmaceutical exposure, individuals can also be exposed to conazoles through environmental, food, resi- dent or bystander exposure. This is confirmed by the increasing concentrations of conazole pesticides found in surface and waste waters (Kahle et al. 2008). Epoxiconazole (EPX) belongs in the triazole class of pesticides and is used worldwide as a fungicide for plant protection. It is known to combat various target fungal diseases in cereals, rice, sugar beets, bananas, coffee, and soybeans (Passeport et al. 2011). This DMI fungicide was effectively used for the control of Fusarium head blight of wheat in China (Chen et al. 2012). In Europe and Australia, the epoxiconazole is part of several commer- cially successful one-compound fungicide formulations (Epic, Opus) or two-compound formulations composed from combinations of epoxiconazole with different pes- ticide (Splice, Swing Gold, Tango Super, Venture etc.). Glyphosate, DDTs and the broad-spectrum fungicides boscalid, epoxiconazole and tebuconazole were the most frequently found in agricultural soil samples of 11 member states of the European Union (EU) (Silva et al. 2019). These findings confirmed the previous study of Hvĕzdová et al. (2018), where conazoles showed the second most frequent occurrence among currently used pesticides (CUPs) in Central European arable soils. In the Czech Republic, Vašíčková et al. (2019) determined that epoxiconazole was one of the main contributors to the overall pesticide mixture toxicity: the measured lev- els and its frequent presence in soils represented a risk for the agroecosystems. This contribution might be a result of low biodegradability and photochemical sta- bility of the EPX molecule that makes it very persistent in soil and aquatic sediment (Passeport et al. 2011) and allows entering multiple environmental media through spray drift or surface runoff (Potter et al. 2014). Bovine farm animals are exposed to chemical agents through grazing, so they are the first in which adverse effects of pesticides might occur (Drážovská et al. 2016). For this reason, in this study, we would like to present new data from an experiment where DNA damage was investigated after exposure of bovine peripheral lympho- cytes to epoxiconazole. Both alkaline and neutral comet assays were used as the methods of choice for detection of single-strand and double-strand DNA breaks. MATERIALS AND METHODS Blood samples were collected by means of jugular venipuncture from two healthy bulls (Slovak indigenous cattle, 6 month old). The animals were kept in healthy conditions, not treated with any drugs and fed with clean feed. The study was conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. Lymphocytes iso- lated from whole blood were used for the comet assays. Epoxiconazole (CAS registry number 133855-98-8, 99% purity, Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) and introduced into culture flasks at concentrations of 2.5, 5, 10, 25, 50 and 100 μg/mL. The fungicide doses were chosen according to study of Šiviková et al. (2018), where the fungicide cytotoxicity level was identified at a 101Genotoxicity testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay concentration of more than 100 μg/mL. The final DMSO concentration was 0.1% in both the treated and untreat- ed (negative control) cells. Hydrogen peroxide (H2O2, Mikrochem, SR, 250 μM) was used as a positive control agent. Cell cultivation and treatment For comet assay, lymphocytes were immediately isolated from bovine whole blood using the Histo- paque®-1077 (Sigma-Aldrich, St. Louis, MO, USA) sepa- ration medium. Isolated lymphocytes were treated with epoxiconazole for 2 h in two different ways: immedi- ately after isolation (non-proliferating lymphocytes) and for the last 2 h of 48 h cultivation (i.e. pre-cultivation of lymphocytes before 2 h treatment to obtain proliferat- ing lymphocytes). Medium for non-proliferating lym- phocytes consisted from 4 ml RPMI 1640 medium sup- plemented with L-glutamine and 15 μM HEPES, 1 ml bovine foetal serum (BOFES) and 40 µl antibiotic/anti- mycotic mixture (100 U/mL penicillin, 0.1 mg/mL strep- tomycin and 0.25 µg/mL amphotericin) (Sigma-Aldrich, St. Louis, MO, USA). Immediately after isolation lym- phocytes were added to the medium and exposed to the test fungicide for two hours (2 h) (i.e. concurrently with their addition to the medium) according the procedure of Calderón-Segura et al. (2012). In the experiment with proliferating lymphocytes phytohaemagglutinin (PHA-L, 20 µg/mL, PAN Biotech, Germany) was added to the above-described culture medium. The isolated lymphocytes were subsequently incubated at 37°C for 48 h and exposed to epoxiconazole for the last 2 h of cultivation. The cells of positive controls were treated with H2O2 (250 mM) for 5 minutes (Horváthová et al. 2006). Cytotoxicity After exposure completed, the cells were washed twice with phosphate-buffered saline (Dulbecco A, pH 7.4) and resuspended to a final volume 1mL with PBS. Cytotoxic effects on the bovine peripheral lymphocytes were evaluated using the trypan blue dye exclusion staining (0.4% trypan blue), where the number of viable (shiny) and dead (blue) cells were scored (viability test). Alkaline comet assay The alkaline comet assay procedure was the same for both non-proliferating and proliferating lympho- cytes. Each concentration tested was represented on special microscope comet slides (CometSlidesTM 2-Well, TREVIGEN, Gaithersburg, Maryland, US) treated to promote agarose adherence, in this case ready‒to‒use low melting point agarose (LMPA). The cells were mixed with 0.75% LMPA in PBS. The cell suspension was pipet- ted onto the agarose layer, fitted with a cover slip and left to set at 4°C. After removal of the cover slips, the microscope slides were immersed in cold lysing solu- tion (2.5 M NaCl, 0.1 M Na2EDTA, 10 mM Tris, plus 1% Triton X-100) for 1 h at 4°C. The slides were then transferred to a horizontal gel electrophoresis tank with electrophoresis solution (0.3 M NaOH, 1 mM Na2EDTA, pH>13) for 40 min unwinding at 4 °C, and then electro- phoresis was conducted at 25V and 300mA for 30 min. The slides were neutralized two times for 10 min with 0.4 M Tris-HCl (pH=7.4), stained with ethidium bromide (5 μg/mL ) on both sides, and fitted with cover slips. All of these steps were carried out in the dark and cold (4ºC) to prevent the occurrence of additional DNA dam- age (Collins 2002). Neutral comet assay The slides were lysed in cold lysing solution (2.5 M NaCl, 0.1M disodium ethylene diaminetetraacetic acid (EDTA disodium salt), 10 mM Tris-HCl, pH=9.5, 1% N-lauroylsarcosine sodium salt, 1% TritonX-100) for 1h at 4°C. Then the slides were moved to an electropho- retic tank with TBE buffer in which the “unwinding” was performed for 1 hour, followed by electrophoresis (20V) for 40 min. After electrophoresis, the slides were neutralized in blossom with neutralizing solution (0.4 M Tris, pH=7.4) for 2 x 10 min. After drying, the glasses were stained with ethidium bromide (5 μg/mL) (Gyori et al. 2014). DNA damage evaluation Comets were analysed with a Nikon ECLIPSE Ni-U f luorescence microscope, equipped with a Texas Red single band pass filter. A total of 100 nucleoids per slide (three slides for each concentration - 300 nucleoids) were scored visually and five classes of damage were recorded, from 0 (undamaged) to 4 (maximally damaged) accord- ing to DNA fluorescence intensity in proportion compar- ing the comet tail and head. The scores 0-4 were attrib- uted according to visual analysis of nucleoids. The overall score for each slide was therefore between 0-400 (Collins 2002). The percentage of damaged cells and the extent of % DNA damage in the comet tail were calculated. 102 Simona Koleničová et al. Statistical analysis Statistical analysis was performed using simple anal- ysis of variance (ANOVA, Student’s t test), which was used to evaluate % DNA breaks comparing treated and untreated groups (controls). RESULTS The results of our analysis of DNA damage using both alkaline and neutral comet assays in non-prolifer- ating (2 h exposure to fungicide) and proliferating (48h cultivation and exposure to fungicide for the last 2h) lym- phocytes from bovine peripheral blood after exposure to epoxiconazole at concentrations of 2.5; 5; 10; 25; 50 and 100 μg/mL, are summarized in Fig. 1a, b and Fig. 2a, b. The percentage viability of non-proliferating and prolif- erating lymphocytes from bovine peripheral blood fol- lowing exposure to epoxiconazole is shown in Fig. 3a, b (alkaline comet assay) and Fig. 4a, b (neutral comet assay). Regarding the results of alkaline comet assay after 2h exposure of non-proliferating lymphocytes to epoxi- conazole, increases in DNA damage with statistical sig- nificance were found starting from concentration 5 µg/ mL in donor 1 (5 µg/mL * p<0.05; 10, 25, 50 and 100 µg/ mL ** p <0.01; ANOVA and Student’s t test; Fig. 1a) as well as donor 2 (5 µg/mL * p <0.05; 10, 25, 50 and 100 µg/mL ** p<0.01; ANOVA and Student’s t test; Fig. 1a). After 48h cultivation and exposure to epoxiconazole for the last 2 h, DNA damage was observed in proliferat- ing lymphocytes with statistical significance in donor 1 (5 µg/mL * p <0.05; 10, 25 µg/mL ** p <0.01; 50 and 100 µg/mL *** p <0.001; ANOVA and Student’s t test; Fig. 1b) as well as donor 2 (10 µg/mL * p<0.05; 25, 50 µg/mL ** p<0.01; 100 µg/mL *** p<0.001; ANOVA and Student’s t test; Fig. 1b). The viability of non-proliferating lymphocytes was greater than 95% in both donors (Fig. 3a), and for pro- liferating lymphocytes it was greater than 94.7% in both donors, too (Fig. 3b). Statistically significant increases in DNA damage with double-stranded breaks in proliferating and non- proliferating lymphocytes were detected using neutral comet assay after exposure to epoxiconazole (Fig. 2a, b) at the same concentrations as for alkaline comet assay. Lymphocyte viability is shown in Fig. 4 a, b. Figure 2. Percentages of DNA in tail estimated by means of neutral comet assay in bovine peripheral blood lymphocytes (non-proliferat- ing) treated with epoxiconazole for 2 h (a) and in bovine peripheral blood lymphocytes (proliferating 48 h) treated with epoxiconazole for the last 2 h. NC (negative control): DMSO; PC (positive control): H2O2 (250μM); a: p<0.05; b: p<0.01; c: p<0.001; mean ± SD. Figure 1. Percentages of DNA in tail estimated by means of alkaline comet assay in bovine peripheral blood lymphocytes (non-proliferat- ing) treated with epoxiconazole for 2 h (a) and in bovine peripheral blood lymphocytes ( proliferating 48 h) treated with epoxiconazole for the last 2 h (b). NC (negative control): DMSO; PC (positive con- trol): H2O2 (250μM); a: p<0.05; b: p<0.01; c: p<0.001; mean ± SD. 103Genotoxicity testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay DNA damage results detected using neutral comet analysis after exposure of non-proliferating lymphocytes to epoxiconazole indicate statistically significant DNA damage in donor 1 from the lowest concentration (2.5 and 5 µg/mL *p<0.05; 10 µg/mL ** p<0.01; 25, 50 and 100 µg/mL *** p<0.001; ANOVA and Student’s t test; Fig. 2a) and in donor 2 from concentration 5 µg/mL (5 µg/ mL * p <0.05, 10, 50 µg/mL ** p <0.01; 25 and 100 µg/ mL *** p<0.001; ANOVA and Student’s t test; Fig. 2a). Proliferating lymphocytes showed statistical signifi- cance in donor 1 from starting from concentration 10 µg/mL (25 µg/mL ** p<0.01; 10, 50 and 100 µg/mL *** p<0.001; ANOVA and Student’s t test; Fig. 2b) and in donor 2 starting from concentration 10 µg/mL (10, 50 µg/mL ** p<0.01; 25 and 100 µg/mL *** p<0.001; ANO- VA and Student’s t test; Fig. 2b). Lymphocyte viability found in both donors after epoxiconazole exposure was higher than 95.8% (Fig. 4a) in non-proliferating lymphocytes and higher than 90% in proliferating ones (Fig. 4b). DISCUSSION Comet assay (single-cell gel electrophoresis) is one of the most popular methods employed for the evaluation of DNA damage and repair in eukaryotic cells (Singh 2016; Lu et al. 2017; Moller 2018) This method is used to study processes dealing with DNA damage in various fields, such as environmental toxicology, biological pro- cess monitoring, radiation biology, nutritional studies and cancer studies (Olive 2009; Wasson et al. 2008). This test has a wide spread in genotoxicity testing mainly due to advantages such as simplicity of the test, low cost and high sensitivity (Hartmann et al. 2003; Tice et al. 2000). The comet test is a universal and sensitive method meas- uring single-stranded and / or double-stranded DNA breaks as well as photodimers (Collins et al. 2008). There are two basic variants for determining DNA damage using comet analysis under alkaline or neutral condi- tions (Östling 1984; Singh 1988). Visual classification of nucleoids and calculation of percentage DNA at the tail is commonly presented up today (Collins et al. 2002; García et al. 2004; Bruschweiler et al. 2016; Hamdi et al. 2018) as an alternative to image analysis. In the present study, the possible genotoxic and cy totoxic effects of epoxiconazole fungicide were assessed in bovine lymphocy tes using alkaline and neutral variants of the comet assay. Treatment was per- formed on non-proliferating and proliferating lym- phocytes to evaluate whether the status of cells has an impact on the DNA damage level. Therefore, we evalu- Figure 3. Viability of bovine peripheral blood lymphocytes used in alkaline comet assay. Cells were treated with fungicide epoxicona- zole for 2h (a) and for the last 2h of 48h cultivation (b). NC (nega- tive control): DMSO. Figure 4. Viability of bovine peripheral blood lymphocytes used in neutral comet assay. Cells were treated with fungicide epoxicona- zole for 2h (a) and for the last 2h of 48h cultivation (b). NC (nega- tive control): DMSO. 104 Simona Koleničová et al. Figure 5. DNAdamage was investigated after exposure of bovine peripheral lymphocytes to epoxiconazole. The cells were treated with the fungicide for 2 h (non-proliferating lymphocytes) and for the last 2 h of the 48-hour culture (proliferating lymphocytes). Positive control was H2O2 (5 min). The results of the alkaline comet assay are shown in the first column (picture a, b, c) and the neutral comet assay in the second column (d, e, f ). Negative control: a, d. Selected concentration 50 μg/mL: b, e. Positive control: c, f. 105Genotoxicity testing of bovine lymphocytes exposed to epoxiconazole using alkaline and neutral comet assay ated two different experiments. The first one was with non-dividing (non-proliferating) lymphocytes exposed to EPX immediately after isolation for 2 hours, as indi- cated by Calderón-Segura et al. (2012). The second with lymphocytes stimulated to divide by phytohaemaggluti- nin (PHA) during 48 hours taking account the results of Bausinger and Speit (2014) who revealed that DNA syn- thesis starts in T lymphocytes (similarly like in periph- eral blood mononuclear cells, PBMC) around 24 h after stimulation with PHA. Therefore we chose 48-hour (24 h plus 24 h) cultivation allowing lymphocyte proliferation in the medium at least during one cell cycle. We treat- ed the cultured lymphocytes with EPX the last 2 h for the examination of the epoxiconazole ability to induce DNA damage in proliferating cells. Using alkaline comet assay we showed that epoxiconazole induced statistically significant DNA damage in both non-dividing (without PHA) and dividing (with PHA stimulation) lymphocytes of cattle. EPX induced DNA migration in PHA-stim- ulated cultured lymphocytes in the same range of con- centrations like in non-stimulated ones but to a different extent; less DNA migration was observed in PHA-stimu- lated cells. It is likely that these results correspond with different capability of proliferating cells to repair DNA damage. On the contrast to alkaline comet assay, neutral comet assay showed that DNA breaks were induced in different ranges of concentrations in proliferating (divid- ing) lymphocytes (from 10 µg/mL) when compared with non-proliferating cells (from 2.5 µg/mL) (Fig. 2a, b). One of explanation might be that the lower concentration did not induce DNA damage or that neutral comet assay detects mostly double-strand breaks (Lu et al. 2017), which were probably more effectively repaired in prolif- erating lymphocytes than in non-proliferating ones. The results of the comet assay can be affected by the exposure time which is one of a crucial factor. Long incubation periods may not be appropriate for the comet assay because DNA lesions may be repaired during the time that mutagens are inactivated, leading to false neg- ative results (Sekihashi et al. 2003). According to Tice et al. (2000), an appropriate exposure time for chemical in vitro genotoxicity assessment should be around 3 to 6 hours; other papers refer 1 h, 2 h, 4 h or 24 h exposure times (Lebaily et al. 1997; Calderón-Segura et al. 2012; Želježić et al. 2016). It is known that cattle can accumulate foreign substances not only in the liver but also in the muscle (García-Repetto et al. 1997), milk (Pokorná et al. 1996) and fat (Ferré et al. 2018) thereby increasing the genet- ic risk to humans through the food chain. Guitart et al. (2010) reported that as a result of the application of fungicides in agricultural production, livestock poison- ing may occur, the clinical manifestations of which are only rarely addressed. Exposure of livestock to genotoxic substances may also induce mutations, lead to metabolic disorders, immunosuppression and decreased fertil- ity. Cattle are exposed to chemicals during grazing, so adverse effects may occur primarily in them. Besides, some of the chemical agents have a long-term cumula- tive effect and can contribute to cancer through chronic exposure. Genotoxicity assessment is an essential component of the safety analysis of all types of substances, ranging from pharmaceuticals, industrial chemicals, pesticides, biocides, food additives, cosmetics ingredients, to veteri- nary drugs, relevant in the context of international leg- islation aiming at the protection of human and animal health (ECVAM 2013). As reported by Bolognesi and Morasso (2000) pesticides have been considered poten- tial chemical mutagens. The genotoxicity of pesticides is generally considered to be the most serious of the pos- sible side effects of their usage. The formation of highly reactive substances during oxidation processes, coupled with the ability to interact with DNA, leads to a series of measurable changes, for example point mutations, chro- mosomal rearrangements, DNA adducts, DNA strand fragments and increased number of micronuclei (Medi- na et al. 2007). There are several studies testing the gen- otoxicity of pesticides and mycotoxins on bovine lym- phocytes (Lioi et al. 2004; Holečková et al. 2013; Schwar- zbacherová et al. 2017; Ferré et al. 2020). Šiviková et al. (2018) tested epoxiconazole in vitro in cultured bovine peripheral lymphocytes using chromosome aberrations, sister chromatid exchanges and micronucleus test. She found that epoxiconazole was not related to genotoxic and / or clastogenic / aneugenic effects, but had the abil- ity to significantly affect cell-cycle kinetics and induce apoptosis. Our results show that epoxiconazole can induce significant levels of DNA damage in bovine lympho- cytes, as revealed in both alkaline and neutral variants of the comet assay. On the other hand, no statistically significant DNA damage was detected by Drážovská et al. (2016), who investigated DNA damage using alkaline comet assay after 2 h exposure of bovine lymphocytes to Tango® Super fungicide (epoxiconazole/fenpropimorph). Potential genotoxic/cytotoxic effects of the epoxicona- zole/fenpropimorph-based fungicide were also investi- gated by cytogenetic assays: chromosomal aberrations, sister chromatid exchanges, micronuclei and f luores- cence in situ hybridization. The final results indicated that the tested fungicide was capable of evoking cyto- toxic effect / cell-cycle delay in peripheral cattle lympho- cytes. On the contrary Schwarzbacherová et al. (2017) 106 Simona Koleničová et al. reported stimulation od DNA-double strand breaks after 4 h exposure to epoxiconazole / fenpropimorph-based fungicide (Tango Super) using neutral comet assay. When compared with pure EPX, the results of both studies mentioned above were probably affected by the presence of fenpropimorph and inert ingredients in the tested pesticide formulation, as well as by different expo- sure times and variants of comet assay. Similarly to our observations, significantly increased percentages of comets and tail lengths were obtained after epoxiconazole treatment in the human colon car- cinoma cell line (HCT116) (Hamdi et al. 2018). Epoxi- conazole was able to induce a range of cell damage in HCT116 cells by generating ROS, which in turn induc- es mitochondrial DNA dysfunction and fragmenta- tion leading to cell death, as confirmed by the attenu- ated death of cells treated with the antioxidant N-acetyl- cysteine (NAC) prior to treatment with epoxiconazole. In the later study with F98 glioma cells the same author (Hamdi et al. 2019) showed that EPX induced cytotoxic effects, cell cycle arrest, cytoskeleton disruption, DNA damage and apoptosis via caspases dependent signal- ling. In addition Akram et al. (2019) confirmed that epoxiconazole was the potent inhibitor of 11-bhydroxy- lase (CYP11B1) and aldosterone synthase (CYP11B2) in hamster and human adrenal H295R cells; these enzymes catalyse the formation of cortisol and aldosterone in the adrenal cortex therefore in this study epoxiconazole seems to be an endocrine disruptor. Similarly, Taxvig (2007) concluded that disruption of a crucial enzyme such as CYP17, which is involved in steroid synthe- sis hormone, is one of the main endocrine-disrupting mechanisms of azole fungicides like tebuconazole and epoxiconazole. In our experiment, bovine lymphocytes were tested under in vitro conditions to obtain significant results, preceded by the testing of several methods and proce- dures to create optimal experimental conditions. Treat- ment of cells with epoxiconazole was followed by deter- mination of cell viability for each test concentration using the trypan blue exclusion method, where the per- centage of viability represents the number of viable cells compared to the total number of cells surviving after treatment. The cell viability was greater than 90% at all concentrations tested. Tice et al. (2000) recommended that in vitro treatment with chemicals should not reduce cell viability by more than 30%, and extended this simi- larly to in vivo experiments. Other researcher maintain that the allowed cell viability after exposure should be at least 70-75% (Želježić et al. 2018), or above 85 % (Evans et al. 2016), and some also report up to 95% (Lebailly et al. 2015) upon performing comet analysis. In general, the question of determining the sen- sitivity of both alkaline and neutral comet analysis is frequently discussed, so the comparison of sensitivity of individual methods is interesting from the practical point of view (Afanasieva and Sivolob 2018; Azqueta and Collins 2013; Peycheva et al. 2009; Collins et al. 2008). The alkaline variant demonstrates increased sensitiv- ity in the investigation of agents causing DNA strand breaks or inducing alkaline labile lesions of DNA. Cur- rently, the first choice is to detect low levels of DNA damage, either in lymphocyte samples or in genotoxicity testing in vitro and in vivo. According to other authors, the neutral variant is more sensitive than the alkaline one. For instance, Afanasieva et al. (2009) indicate that the neutral variant is the more sensitive method for assessing small numbers of DNA breaks. 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