INTRODUCTION

The use of chemical products to keep cattle healthy and with high yields is based on the need to meet the growing demand for animal products worldwide. Drugs are routinely and prophylactically applied to animals for the treatment of parasitic diseases. There is little knowledge of studies on the genotoxicity (defined as DNA damage) and/or cytotoxicity of IVM in individuals (vectors, mammals and humans), which is available for parasite control (1,2 ). However, there is a lot of in vivo literature associated with the evaluation of forms of administration, therapeutic doses (3), sensitivity of invertebrates to the drug (4) and resistance (5,6).

To detect the genotoxic damage caused by various agents, different tests can be used that determine alterations caused to genetic material, in both somatic and germ cells. The Comet or Single Cell Gel Electrophoresis (SCGE) assay detects single and double-stranded DNA damage, allowing for the evaluation of DNA fractionation and repair levels in cell populations without the need to work with proliferating cells. The name derives from the appearance of cells after the test: an intensely bright head and a tail whose length and intensity are related to the amount of chain breaks in the DNA. In contrast, undamaged cells have intact nuclei without tails (7).

All living things are directly or indirectly exposed, not only to the active components of pesticides, but also to their commercial formulation. They include mixtures of one or more active substances with toxic activity and a theoretically biologically inert excipient. However, numerous in vitro and in vivo studies have shown that, in most cases, the potential risks to which living organisms are exposed from commercial formulations are more damaging than the active compounds. The objective of this study was to evaluate the genetic damage caused by the use of Ivermectin (IVM) in zebu cattle (Bos taurus indicus).

MATERIALS AND METHODS

Population study. Fifteen Cebu Brahman cows were used, between three and four years of age, with an average weight of 350 kg, body condition of between 3.0 and 3.5 and a good health status. The livestock belonged to the cattle on the Cielo Lindo Hacienda, located in the rural area of the municipality of Corozal, in the northeast region of the Department of Sucre (Colombia). This hacienda is located at an altitude of 174 masl, with an average temperature of 30ºC and precipitation of 1,300 mm. The selected animals were kept in quarantine for more than seventy days, with no supply of medication before the start of the study.

Experiment group. A unifactorial experiment design was used, with three experiment groups, each with five animals. The first group was exposed to 1% IVM concentrations. The second group was exposed to 3.15% IVM concentrations, and the third group was the control (without an IVM application). The IVM doses corresponded to those formulated by the commercial products and their application followed the biosecurity protocols established on the hacienda. To evaluate the possible difference between the treatments and control, a one-way variance (ANOVA) analysis and multiple comparison tests of n-treatment means were performed with a Dunnett control to compare the treatments with the control, without IVM. Statistical analyzes were performed using free access software R (8).

Collection of samples. The blood samples were collected at 9, 18 and 27 days post-treatment with jugular vein punctures, caudal or medial, collected in Vacutainer® tubes with EDTA (anticoagulant). 8 ml of whole blood was drawn.

The samples were processed in the microbiology laboratory of the Universidad de Sucre using the comet test technique, according to the protocol of Singh et al (7), as modified by Ramírez and Cuenca (9). The parameters validated to determine the damage assessed by the comet test included: DNA migration, DNA concentration in the tail, tail moment, and Olive moment (10). For this study, the tail length (μm) was used as the measurement parameter (11,12). The criterion for determining genetic damage was the mean of the control plus a standard deviation plus one (X ± SD + 1) and, according to this value, the type of damage was classified into five categories: 0: -18 μm); 1: cells with low damage (19-37 μm); 2: cells with medium damage (38-56 μm): 3, cells with high damage (57-75 μm) and 4: cells with total damage (greater than 75 μm). (13).

The total damage count was calculated using the Damage Index (DI) technique, calculated as: DI = Σ PDD x DD, where DD is the degree of damage (from 0 to 4) and P is the percentage of cells that show damage. For each individual, 100 cells per plate (total two plates) were analyzed. The images of the comets were photographed and analyzed with the Open Comet program (14).

Complementary material - Comet test. Cells were suspended in 0.5% low melting agarose (PML) in PBS and pipetted onto previously coated slides with a 1% normal melting point (NMP) agarose layer. Cell suspension slides were placed on cover-slips and brought to 4°C for ten minutes, then submerged in lysis buffer (2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Tris-HCl pH 10, 1% Triton X-100 and 10 % DMSO) for one hour at 4°C in the dark to allow lysis of the embedded cells and unwinding of the DNA. After incubation of the cells in the lysis solution, the slides were exposed to an alkaline buffer solution (1mM Na 2 EDTA, 300 mM NaOH buffer), pH> 13, for twenty minutes to degrade the DNA. Finally, the slides were electrophoresed for twenty minutes at 25v and 300mA in the same alkaline buffer (pH> 13), after which the slides were washed with 0.4 Tris-HCl buffer (pH 7.5) to neutralize the excess alkali and remove detergents. Finally, the plates were fixed with ethanol, stained with ethidium bromide (1ug/ml) and examined under a fluorescent microscope at a 40X magnification.

RESULTS

Regardless of the IVM concentration, the presence of nuclei with DNA migration (Figure 1a and b) was observed at a percentage greater than 75% in all cells observed per plaque, demonstrating the ability of the IVM compound to produce simple chain breaks in the DNA molecule. The comet classification values denoted cells with high levels of damage (Grade 3-cells with high damage).

DISCUSSION

For many years, IVM has been the best-selling antiparasitic drug in the world, but little is known about the genotoxic potential. The results of this research demonstrate the ability of IVM to induce single-strand breaks in DNA, which were evaluated using the comet assay methodology (Figure 1). The concentrations of IVM significantly increased the frequency of damaged cells, together with an increase in the amount of migrated DNA, as compared to negative control values (Table 1). In vitro, González et al (15) analyzed the deleterious capacity of IVM and one of its commercial formulations with a genotoxic assay. The results showed that both compounds exert a genotoxic and cytotoxic effect when exposed to equimolar concentrations of the active compound in a range of 5.0 to 50 μg/ml, inducing single strand breaks in the DNA molecule. On the other hand, in situ assays, as reported by Montes et al (16), demonstrated that concentrations IVM of 3.15% produced nuclei with DNA migration, corroborating the results of this study.

The results found in the present study constitute concrete evidence for the induction of genomic damage as exerted by IVM, using the comet assay methodology, which has been widely used to detect genomic lesions that are possibly corrected, once DNA damage is common in specific cells and tissues (17,18,19).

In conclusion, the results suggest that the genetic damage found in the studied livestock may be associated with the use of IVM; however, a single bioassay is insufficient as an indicator to characterize the toxicity of a studied pesticide study.

REFERENCES

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Author notes

donicer.montes@unisucre.edu.co