Study of the Methylation of Bovine GSTP1 Gene under the Influence of Pesticide Mospilan 20SP Alone and in Combination with Pesticide Orius 25EW.

DNA methylation, one of the most studied epigenetic mechanisms, when present in the promoter region of genes, causes inhibition of gene expression, and conversely, hypomethylation of these regions enables gene expression. DNA methylation is susceptible to nutritional and environmental influences, and undesirable alterations in methylation patterns manifested in changes in the expression of relevant genes can lead to pathological consequences. In the present work, we studied the methylation status of the bovine GSTP1 gene under the influence of pesticide Mospilan 20SP alone and in combination with pesticide Orius 25EW in in vitro proliferating bovine lymphocytes. We employed methylation-specific PCR, and when studying the effect of pesticide combinations, we also used its real-time version followed by a melting procedure. Our results showed that Mospilan 20SP alone at 5, 25, 50, and 100 µg.ml-1 and 5, 10, 25, and 50 µg.ml-1 for the last 4 and 24 hours of culture with in vitro proliferating bovine lymphocytes, respectively, did not induce methylation of the bovine GSTP1 gene. The same results were revealed when studying the effect of the combination of the pesticides added to the lymphocyte cultures for the last 24 hours of cultivation in the following amounts: 1.25, 2.5, 5, 10, and 25 µg.ml-1 of Mospilan 20SP and 1.5, 3, 6, 15, and 30 µg.ml-1 of Orius 25EW. We have also revealed that the less laborious real-time MSP followed by a melting procedure may replace MSP for studying the methylation status of the GSTP1 gene.

Evaluation of GSTP1, GSTA4 and AChE Gene Methylation in Bovine Lymphocytes Cultured In Vitro with Miconazole Alone and in Combination with Mospilan 20SP.

5-methylcytosine (5mC) is one of the most important epigenetic modifications. Its increased occurrence in regulatory sequences of genes, such as promoters and enhancers, is associated with the inhibition of their expression. Methylation patterns are not stable but are sensitive to factors such as the environment, diet, and age. In the present study, we investigated the effects of fungicide miconazole, both alone and in combination with the insecticide Mospilan 20SP, on the methylation status of bovine GSTP1, GSTA4, and AChE genes in bovine lymphocytes cultured in vitro. The methylation-specific PCR technique was used for the objectives of this study. We found that miconazole alone at concentrations of 1.25, 2.5, 5, 10, 25, and 50 µg/mL after 24 h exposure probably did not induce changes in methylation for all three genes analysed. The same results were found for the combination of pesticides at 24 h exposure and the following concentrations for each of them: 0.625, 1.25, 2.5, 5, and 12.5 µg/mL. Thus, we can conclude that the fungicide miconazole alone, as well as in combination with the insecticide Mospilan 20SP, was unlikely to cause changes to the methylation of bovine GSTP1, GSTA4, and AChE genes.

Detection of the T1640C RYR1 mutation indicating malignant hyperthermia in dogs.

Malignant hyperthermia (MH) is a clinical syndrome exhibiting elevation of expired carbon dioxide, hyperthermia, muscle rigidity, rhabdomyolysis, acidosis and hyperkalaemia, as well as cardiac dysrhythmia and renal failure. The syndrome manifests itself as a response to anaesthetic agents, such as e.g., halothane, desflurane, and succinylcholine. Depending on the animal species, MH is characterised by autosomal dominant or recessive inheritance, and so far two genes have been identified whose mutations can be linked to MH: RYR1 and CACNA1S. In different species, various mutations of the RYR1 gene have been described which may underlie MH. One of these mutations in dogs is T1640C, which results in the substitution of alanine for valine of the amino acid 547 (V547A) in the RYR1 protein. In our work, we aimed to investigate MH at the DNA level by identifying the T1640C mutation in a group of 50 dogs. For this purpose we used the PCR-RFLP technique, and in six dogs also direct sequencing of PCR products and subsequent comparison of their sequences with the RYR1 gene sequence in an online database. The results of our study show that none of the dogs analysed had any mutant allele of the RYR1 gene, indicating that none should be affected by MH.

The adverse effects of synthetic acaricide tau-fluvalinate (tech.) on winter adult honey bees.

Currently several pyrethroids (e.g., flumethrin and tau-fluvalinate) are used in apiculture worldwide as acaricides/miticides. The long half-lives of pyrethroids in synthetic acaricides applied to hive matrices, may adversely affect the health of bee colony. The potentially adverse effects of synthetic acaricide/miticide tau-fluvalinate (tech.) on winter honeybees were assessed in this study (OECD 245 2017). No dose-dependent mortality in in vitro reared winter honeybees was observed after chronic oral 10-day exposure to syrup (50% w/v) spiked with a maximum concentration of 750 µg a.i./kg diet and its 1/10 concentration. The No Observed Effect Concentration is ≥ 750 µg a.i./kg diet. Tau-fluvalinate testing for the sublethal effects on bee immune system showed up-regulated gene expression encoding abaecin, lysozyme, and defensin in both tested groups, however the expression of hymenoptaecin gene was reduced. Moreover, tau-fluvalinate significantly induced levels of DNA damage in exposed bees, which can result in adverse genotoxic effect.

Chromosomal Aberrations in Cattle.

Chromosomal aberrations and their mechanisms have been studied for many years in livestock. In cattle, chromosomal abnormalities are often associated with serious reproduction-related problems, such as infertility of carriers and early mortality of embryos. In the present work, we review the mechanisms and consequences of the most important bovine chromosomal aberrations: Robertsonian translocations and reciprocal translocations. We also discuss the application of bovine cell cultures in genotoxicity studies.

Interaction of Conazole Pesticides Epoxiconazole and Prothioconazole with Human and Bovine Serum Albumin Studied Using Spectroscopic Methods and Molecular Modeling.

The interactions of epoxiconazole and prothioconazole with human serum albumin and bovine serum albumin were investigated using spectroscopic methods complemented with molecular modeling. Spectroscopic techniques showed the formation of pesticide/serum albumin complexes with the static type as the dominant mechanism. The association constants ranged from 3.80 × 104-6.45 × 105 L/mol depending on the pesticide molecule (epoxiconazole, prothioconazole) and albumin type (human or bovine serum albumin). The calculated thermodynamic parameters revealed that the binding of pesticides into serum albumin macromolecules mainly depended on hydrogen bonds and van der Waals interactions. Synchronous fluorescence spectroscopy and the competitive experiments method showed that pesticides bind to subdomain IIA, near tryptophan; in the case of bovine serum albumin also on the macromolecule surface. Concerning prothioconazole, we observed the existence of an additional binding site at the junction of domains I and III of serum albumin macromolecules. These observations were corroborated well by molecular modeling predictions. The conformation changes in secondary structure were characterized by circular dichroism, three-dimensional fluorescence, and UV/VIS absorption methods.

DNA methylation studies in cattle.

Investigation of the role of epigenetics in cattle breeding is gaining importance. DNA methylation represents an epigenetic modification which is essential for genomic stability and maintenance of development. Recently, DNA methylation research in cattle has intensified. The studies focus on the definition of methylomes in various organs and tissues in relation to the expression of genes underlying economically important traits, and explore methylome changes under developmental, environmental, disease, and diet influences. The investigations further characterize the methylation patterns of gametes in connection with their quality, and study methylome alterations in the developing naturally or assisted produced zygotes, embryos, and fetuses, considering their viability. A wide array of technologies developed for accurate and precise analysis of DNA methylation patterns is employed for both single-gene and genome-wide studies. Overall, the research is directed towards the identification of single methylation markers or their combinations which may be useful in the selection and breeding of animals to ensure cattle improvement.

In vitro exposure to thiacloprid-based insecticide formulation promotes oxidative stress, apoptosis and genetic instability in bovine lymphocytes.

A proprietary thiacloprid-based neonicotinoid insecticide formulation is widely used in agriculture to protect vegetables and fruit against various pests. However, its effect on animal cells has not been fully elucidated. In this study, bovine peripheral lymphocytes were incubated with different concentrations of this formulation (10; 30; 60; 120 and 240 µg.mL-1) for 4 h to address the potential cytotoxic and genotoxic effects of the insecticide. Insecticide formulation treatment resulted in decreased cell viability and proliferation, p53-mediated cell cycle arrest at the G0/G1 phase, and apoptosis induction accompanied by elevated levels of mitochondrial superoxide and protein carbonylation. Oxidant-based DNA damage and DNA damage response (DDR) were also observed, namely the formation of micronuclei, DNA double-strand breaks and slightly elevated recruitment of p53 binding protein (53BP1) foci. Our results contribute to the elucidation of insecticide effects on animal lymphocyte cultures after short-term exposure. Due to increased application of neonicotinoids worldwide, resulting in both higher yields and adverse effects on non-target animals and humans, further in vivo and in vitro experiments should be performed to confirm their cytotoxic and genotoxic activities during short-term exposure.

Fungicide Tebuconazole Influences the Structure of Human Serum Albumin Molecule.

Studies of interactions between pesticides and target mammalian proteins are important steps toward understanding the pesticide's toxicity. Using calorimetric and spectroscopic methods, the interaction between triazole fungicide tebuconazole and human serum albumin has been investigated. The spectroscopic techniques showed that fluorescence quenching of human serum albumin by tebuconazole was the result of the formation of tebuconazole/human serum albumin complex with the static type as the dominant mechanism. The association constant was found to be 8.51 × 103 L/mol. The thermodynamic parameters were obtained as ΔH = -56.964 kJ/mol, ΔS = -115.98 J/mol·K. The main active interactions forming the tebuconazole/human serum albumin complex were identified as the interplay between hydrogen bonds and/or van der Waals forces, based on thermodynamic experiments. These binding modes were corroborated well by the predictions of molecular modeling. Hydrogen bonding of tebuconazole with Arg222, Ala215 and Ala291 of human serum albumin played a relevant role in binding. The conformation changes in secondary structure were characterized by circular dichroism and 3D fluorescence spectra.

Evaluating the genotoxic damage in bovine whole blood cells in vitro after exposure to thiacloprid.

Possible genotoxic effect of thiacloprid on bovine cultures of whole blood was investigated using chromosomal aberrations (CAs), micronuclei (MN), sister chromatid exchanges (SCEs), DNA damage and apoptotic DNA fragmentation assays. The cells of whole blood were exposed to thiacloprid (30, 60, 120, 240 and 480 µg mL-1) for the last 24 and 48 h of cultivation. Thiacloprid did not induce significant increase in CAs after 24 and 48 h; only the concentration of 120 µg mL-1 caused elevation of CAs (p < 0.05) after 24 h treatment. No clastogenic/aneugenic effect was observed by scoring of micronuclei. Considering replication damage reflected in SCEs, significant elevations were observed in both donors for 24 h (120-480 µg mL-1; p < 0.01 or p < 0.05). In comet assay, statistically significant DNA damage was observed after 2 h exposure (240 and 480 µg mL-1; p < 0.05, p < 0.01). DNA electrophoretic separation did not confirm the late apoptotic effect of thiacloprid. The decrease in additional variables such as mitotic index, cytochalasin-blocked proliferation and proliferation indices indicates the possible ability of thiacloprid to induce cytotoxic/cytostatic effects by affecting and/or inhibiting cell proliferation and to influence the cell cycle respectively.

A comprehensive study of disorder of sex development in Staffordshire bull terrier dog.

An 8-month-old female Staffordshire bull terrier was clinically examined because of external sexual organs abnormality-clitoral hypertrophy. As stated by the owner, the female dog had not been in heat yet. Serum profile of testosterone (3.39 ng/ml), as well as an anti-Mullerian hormone (24.0 ng/ml), suggested the presence of testicular tissue. On the contrary, the estimated level of 17ß-oestradiol (24.6 pg/ml) was approximately two times higher when compared with the normal anoestrus values (5-10 pg/ml). A midline laparotomy was performed to detect the cranial parts of the genital system. Gonads resembling testicle or ovotestis (left) and hypoplastic testicle (right) was visible. Cranial portion of gonads was attached to structures indicative of bilateral epididymidis. The next tubular structures-oviducts were resected along with adherent parts of a hypoplastic uterus. Histological evaluation confirmed that the examined gonad samples were testicles with modified interstitial testicular tissue. Hypertrophy of interstitial space was predominantly formed by Leydig cells. Examination of a cross-section through the head of suspected epididymidis confirmed their characteristic structures. In addition, the characteristic configuration of the oviducts was presented. The uterus consisted of three walls, in which the endometrium was hypoplastic with the presence of endometrial glands. No Y chromosome was detected by chromosomal analysis using CFA Y probe and the amplification of SRY-gene coding region (813 bp) indicated genotype 78, XX; SRY-negative. Sequencing of SOX9 gene exons 1-3 did not reveal any differences in exon 1 and 3. On the contrary, a few changes were determined in the SOX9 exon 2 sequences: G instead of A at position 103; C instead of reference T at position 115; GCG instead of reference CGC at position 138-140; T instead of reference C at positions 161, 164 and 167.

Potential chromosome damage, cell-cycle kinetics/and apoptosis induced by epoxiconazole in bovine peripheral lymphocytes in vitro.

The epoxiconazole was tested in vitro for its potential on induction of chromosome damage and/or cell cycle kinetics in cultured bovine peripheral lymphocytes. Cytogenetic endpoints such as: Chromosome Aberrations (CA); Sister Chromatid Exchanges (SCE); Micronuclei (MN); Mitotic Index (MI); Proliferation Index (PI); and Cytokinesis Block Proliferation Index (CBPI) were investigated for 24 h and 48 h of incubation. The cultured lymphocytes were exposed to the epoxiconazole at concentrations of 2.5, 5, 10, 25, 50 and 100 µg mL-1. From our results is evident that treatment of bovine peripheral lymphocytes with the epoxiconazole was not related to DNA damage; no genotoxic effect and/or clastogenic/aneugenic effects were recorded. However, epoxiconazole has ability to significantly affect cell cycle kinetics/and induce apoptosis. A decrease of proliferation in the MI, CBPI and identically in the PI were observed; hence, cytostatic/cytotoxic effects of epoxiconazole have been recorded. The prolonged time of exposure at the highest concentration caused an inhibition of the replication. Electrophoretic analysis confirmed the epoxiconazole potential to induce ladder-like patterns of DNA fragments that are a hallmark of apoptosis.

Evaluation of cytotoxic and genotoxic activity of fungicide formulation Tango® Super in bovine lymphocytes.

Tango® Super is a two-compound fungicide formulation widely employed in grain protection. However, details of Tango® Super effects on cell cultures have not been fully investigated. In this study, bovine lymphocytes were exposed to a concentration range 0.5; 1.5; 3; 6; and 15 µg mL-1 for 4 h to assess the cytotoxicity and genotoxicity of the fungicide. Our experiments revealed that this fungicide treatment reduced cell viability, decreased cell proliferation and provoked apoptotic cell death. Cell cycle analysis showed predominant accumulation of cells in the G0/G1 phase of the cell cycle. The fungicide was able to induce mitochondrial superoxide production accompanied by elevated levels of carbonylated proteins and changes in the lipid membrane composition. The fungicide did not induce micronuclei production, but stimulated both DNA double-strand breaks and the formation of p53 binding protein, which is accumulated during the DNA repair process at the site of double-strand breaks. Based on the obtained data we suppose that the fungicide-induced DNA damage is the result of oxidative stress, which may contribute to higher occurrence of apoptotic cell death. Because ergosterol biosynthesis-inhibiting fungicides are widely used in agriculture to ensure higher crop yields and may cause health impairment of animals and humans, there is a need for further testing to elucidate their potential genotoxic effects using in vivo and/or in vitro systems.

Evaluation of potential genotoxic/cytotoxic effects induced by epoxiconazole and fenpropimorph-based fungicide in bovine lymphocytes in vitro.

Potential genotoxic/cytotoxic effects of the epoxiconazole/fenpropimorph-based fungicide were investigated using single cell gel electrophoresis and cytogenetic assays: chromosomal aberrations, sister chromatid exchanges, micronuclei and fluorescence in situ hybridization in cultured bovine lymphocytes. No statistically significant elevations of DNA damage and increases in cytogenetic endpoints were seen. However, evident cytotoxic effect presented as a decrease in mitotic and proliferation indices were recorded after exposure of bovine lymphocytes to the fungicide for 24 and 48 h at concentrations ranging from 3 to 15 µg mL(-1) (P < 0.05, P < 0.01, P < 0.001). Similarly, for 24 h an inhibition in the cytokinesis block proliferation index (CBPI) was obtained after exposure to the fungicide at concentrations ranging from 1.5 to 15 µg mL(-1) (P < 0.01, P < 0.001) in each donor.

The effect of thiacloprid formulation on DNA/chromosome damage and changes in GST activity in bovine peripheral lymphocytes.

The potential genotoxic effect of thiacloprid formulation on bovine peripheral lymphocytes was evaluated using the comet assay and the cytogenetic endpoints: chromosome aberrations (CAs), sister chromatid exchanges (SCEs) and micronuclei (MNi). Whole blood cultures were treated with the insecticide at concentrations of 30, 60, 120, 240 and 480 µg mL(-1) for 24, 48 h and/or 2 h of incubation. A statistically significant increase in the frequency of DNA damage, as well as in unstable chromosome aberrations (% breaks) were found after exposure to the insecticide at concentrations ranging from 120 to 480 µg mL(-1) (P < 0.05, P < 0.01, P < 0.001). For the detection of stable structural chromosome aberrations (e.g., translocations) and numerical aberrations by the FISH method, three whole chromosome painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) were used in our experiments. We observed numerical aberrations, but without any statistical significance. Regarding the sister chromatid exchanges, no significant elevation in the SCE frequencies was found after 24-h exposure to the insecticide. A dose-related response in the SCE induction was obtained in bovine cultures after the prolonged time of exposure (48 h) to thiacloprid formulation at concentrations ranging from 120 to 480 µg mL(-1) in each donor (P < 0.05, P < 0.01), which was associated with a reduction of the PI (P < 0.05, P < 0.01). The insecticide failed to produce MNi; however, a significant reduction of CBPI was observed. Using real-time PCR, a decrease in the expression of bovine glutathione S-transferase M3 (GSTM3) was detected at the lowest dose. The higher concentrations of thiacloprid formulation caused an increase in the mRNA expression.

A comparative FISH mapping of LCA5L gene in cattle, sheep, and goats.

In this study, chromosomal position and nucleotide sequencing of the LCA5L exons were analyzed in cattle, sheep, and goats. Using fluorescence in situ hybridization, the LCA5L gene was localized at the distal region of BTA1q44 in cattle, OAR1q43 in sheep, and CHI1q44 in goats. Sequencing of selected LCA5L exons revealed a high identity of the gene that was in accordance with the previously described high homology of autosomes in Bovidae. Three silent single nucleotide polymorphisms (SNPs) were found: a mismatch at position 1016 (A/G) in bovine exon 4 (rs109149293) and two newly identified mutations at position 1903 (C/T) and 137094787 (C/T) in sheep and goats, respectively.

Effect of triazole pesticide formulation on bovine culture cells.

To date, most data about the possible genotoxic effect of triazole pesticides are focused on laboratory animals resulting in limited information on further non-target organisms such as cattle. The objective of the present study was to investigate the effect of triazole (tebuconazole/prothioconazole) fungicide formulation on the induction of chromosomal aberrations (CAs), sister chromatid exchanges (SCEs) and DNA fragmentation in bovine cultured lymphocytes. Our results showed that the fungicide formulation did not induce significant number of CAs in bovine cells after 24 h treatment. Nevertheless, the dose-dependent reduction of mitotic division was observed, with the strongest effect at 30.0 µg mL(-1) in both donors (P < 0.01 and P < 0.001, respectively). Prolonged 48 h exposure caused the increased level of breaks in treated cultures (3.0-15.0 µg mL(-1); P < 0.05) and significant decrease in mitotic index (MI). The tested fungicide failed to produce any statistical changes in the SCE frequency neither after 24 h nor 48 h treatment. However, the significant decline of the proliferation index (PI) was observed after 24 h indicating the fungicide influence on cell cycle kinetics. Prolonged 48 h exposure caused cytotoxicity reflecting in lower PI value relative to control mainly at the highest fungicide concentrations (30.0 µg mL(-1), P < 0.001). Using painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) only low levels of aneuploidies were detected. Significant increase of polyploidy cells (P < 0.05) was induced by a 3.0 µg mL(-1) dose of the fungicide after 48 h. DNA fragmentation assay didn't reveal the presence of DNA nucleosome ladder in cell cultures at any time (24 h and 48 h) and fungicide concentration.

Assessment of cytogenetic damage in bovine peripheral lymphocytes exposed to in vitro tebuconazole-based fungicide.

The tebuconazole-based fungicide was tested in vitro for its potential genotoxic and cytotoxic effects on cultured bovine peripheral lymphocytes. Following 24h and 48 h of incubation, several cytogenetic endpoints were investigated such as: Chromosome Aberrations (CAs); Sister Chromatid Exchanges (SCEs); Micronuclei (MN); Mitotic Index (MI); Proliferation Index (PI); and Cytokinesis Block Proliferation Index (CBPI). The cultured lymphocytes were exposed to the fungicide formulation at concentrations of 3, 6, 15, 30 and 60 µg mL(-1). Statistical significant increases were seen in the CA assays at concentrations ranging from 6 to 30 µg mL(-1) for 24h. The higher doses caused a decrease or total inhibition of chromosome damages in comparison to the last active dose, or the control values. The Fluorescence in situ Hybridisation (FISH) technique was also used for the study of stable/unstable structural chromosomal aberrations and numerical aberrations of aneuploidy/polyploidy at the concentrations of 6 and 15 µg mL(-1). Under conditions of our study, no reciprocal translocations were detected. The more frequent types of aberrations were trisomies and monosomies; both have been identified in association with either bovine chromosome 5 or 7. No statistical significant value was seen in the induced MN; but, the clear, evident reduction of the CBPI was observed. Significant elevations of SCE were observed after the applications of the fungicide formulation at doses from 15 to 60 µg mL(-1) in each donor for 24h. The highest concentrations also caused a statistical significant decrease in the PI. The treatment for 48 h failed to exhibit any genotoxic activity of the fungicide.

Disorder of sexual development in a Yorkshire terrier (78, XY; SRY-positive).

A 9-month-old Yorkshire terrier was admitted to the clinic because of abnormal sexual behaviour and clitoral hypertrophy. External examination confirmed standard development of caudal genital organs: vagina, vulva and cervix uteri. Serum profile of gonadotropin hormones 17 ß-estradiol (<10.0 pg.ml(-1)) and testosterone (9.1 ng.ml(-1)) revealed the presence of testicular tissue. A midline laparotomy was performed to detect the cranial parts of the genital system. Gonads resembling testicles, structures indicating epididymis and rudimentary deferent ducts were resected, along with adherent part of the uterus. Cytogenetic analysis showed a male chromosomal complement 78, XY in all metaphases of the studied Yorkshire terrier dog. The chromosomal constitution was confirmed by fluorescence in situ hybridisation (FISH) with whole-chromosome painting probes specific for chromosomes X and Y, as well as by polymerase chain reaction (PCR) amplification of the 271-bp Y-linked fragment of SRY (the sex-determining region on the Y chromosome) gene. Sequencing of the dog's SRY gene coding region did not reveal any mutation. To search for potential mutation in the SOX9 gene (Sry-box containing gene 9), which is considered to be one of the key genes involved in the sex determination process, the PCR fragments of exons 1, 2 and 3 originating from the canine patient were sequenced in order to compare with both male and female healthy control dogs. In the analysed regions of the SOX9 gene, no mutation was found.