This study investigated the potential genotoxic effects of the fungicide Tango® Super using methods of conventional cytogenetic analysis, fluorescence in situ hybridization (FISH) and detection of DNA fragmentation in bovine lymphocytes. After exposure of two donor cell cultures to several concentrations of fungicide (0.5, 3.0 and 15.0 mg.ml-1 for conventional cytogenetic analysis; 0.5 and 3.0 mg.ml-1 for FISH) we detected the insignificant occurrence of chromosome and chromatid breakages. In both donors we observed a significant decrease in mitotic index (MI) percentage with increasing concentrations of fungicide (P < 0.01; P < 0.001), which indicated a cytotoxic effect of the preparation. Electrophoretic analysis of DNA fragmentation in lymphocytes exposed to increasing concentrations (0.5; 1.5; 3.0; 6.0 and 15.0 mg.ml-1) of this preparation showed its ability to induce formation of fragments, which is a characteristic manifestation of the last stage of apoptosis.
Different chemicals can have genotoxic effects on the body, as confirmed by chromosome damage detection. Using conventional cytogenetic analysis and fluorescence in situ hybridization, we tested the extent of chromosome damage caused by the acetamiprid-based insecticide Mospilan 20SP on bovine peripheral blood lymphocytes at concentrations of, 2.5, 5, 25 and 50 µg.ml−1 after a 24 h incubation period. During the experiment, the presence of unstable aberrations—chromosomal and chromatid breaks and gaps—were detected by conventional cyto-genetic analysis. With increasing insecticide concentrations, we observed a statistically significant increase in chromosome damage frequency after 24 hours of exposure. Fluorescence in situ hybridization was used to detect stable structural aberrations; whole-chromosome painting probes for bovine chromosomes 1 and 7 (BTA 1 and BTA 7) were used for this purpose. As a result of exposure to the insecticide, neither BTA 1/BTA 7 translocations nor other types of translocations were observed.
The interactions between the fungicide tebuconazole and human serum albumin were investigated using fluorescence and circular dichroism spectroscopies. The experimental results showed that the fluorescence quenching of the protein by the tebuconazole molecule was a result of the formation of a ligand-protein complex with a binding constant of 8.51×103 l.mol−1 and the number of binding sites in the macromolecule was close to 1. These findings demonstrated the fact that although the binding affinity of tebuconazole to the protein may be slight, it was very similar to other triazole fungicides. In addition, tebuconazole stabilized the α-helical secondary structure of the human serum albumin due to the increase of the α-content in the protein macromolecule.
Acetamiprid, that is known as the commercial formulation Mospilan® 20SP is the part of the neonicotinoid insecticide group and is widely used against various pests. In our study we assessed the potential clastogenic effects of Mospilan® in human peripheral blood lymphocytes in vitro using a chromosome aberration test. The lymphocytes were treated with acetamiprid in the concentration range of 5, 10, 25 and 50 µg.ml−1 for 24 and 48 h. After 24 h exposure, the insecticide induced statistically significant higher levels of chromosome aberrations from the concentration of 10 µg.ml−1 (P < 0.05 and P < 0.001) and a significant decrease in mitotic index (MI) at the concentrations of 25 and 50 µg.ml−1 (P < 0.05 and P < 0.01), respectively. After a 48 h exposure, we found a dose dependent increase in the percentage of chromosome aberrations at all concentrations (P < 0.05; P < 0.01 and P < 0.001) and a decrease in MI at concentrations of 25 and 50 µg.ml−1 (P < 0.05 and P < 0.01). Our results indicated that neonicotinoid insecticide formulations containing acetamiprid may have potential cytotoxic and genotoxic effects.
The epigenetic mechanisms represent a dynamic, reversible and heritable manner modulating gene expression during the life cycle of an animal organism. They generate the specific epigenetic marks which constitute so-called epigenome. One of the most studied epigenetic mechanisms/marks is DNA methylation which is, similarly as the whole epigenome, susceptible to environmental and nutritional influences. The aberrations of the DNA methylation profile may alter gene expression leading to pathologic consequences. Pesticides along with their pest-reducing effects may also negatively affect non-target organisms. In our preliminary study, we investigated an effect of the pesticide Mospilan on the DNA methylation of the bovine GSTP1 gene which plays an important role in the cell detoxification processes. The specific primers for the GSTP1 Methylation-specific PCR (MSP) analysis were proposed and tested with the DNA from the Mospilan-treated bovine lymphocytes. It seems that the pesticide with the concentration of 100 µg.ml−1 did not induce DNA methylation changes in GSTP1 gene in bovine lymphocytes.
Nowadays many chemicals are widely used in agriculture to ensure high crop yields or in veterinary/human medicine to cure diseases. After their improper usage they may contaminate the environment, persist in it and adversely affect both the target and/or the non-target organisms. One of the ways to detect the occurrence of chemicals in the environment is to assess their impact on aquatic and farm animals; both are directly or indirectly exposed via their feed and water. The micronucleus assay is a standardly used cytogenetic test for the simultaneous detection of clastogenic and aneugenic agents. Additionally, cytotoxic effects are also assessed by analysing the proliferation changes using the cytokinesis-blocked proliferation index. The occurrence of micronuclei is analysed in many types of cells like the peripheral blood cells, bone marrow or cell lines according to standards for micronuclei detection. The analysis of published results has shown that the micronucleus assay is, together with the chromosomal aberration test, one of the most often used test in genotoxicity assessment. Its results have contributed to reassessing the use of multiple chemicals available on the market. Moreover, it is a compulsory test before approving the chemical/ pesticide for the market.