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.

Comparative genomic hybridization in detection of DNA changes in canine lymphomas.

In this study, chromosomal imbalances in tumor tissues (lymphomas) and nucleotide changes in tumor suppressor TP53 were studied in a Bernese Mountain dog bitch and a cross breed bitch. Using comparative genomic hybridization, numerous chromosomal rearrangements were detected, which indicated the heterogeneity in tumor growth: in the cross breed bitch, a deletion on the chromosome 9, and duplications on chromosomes 5, 8 and 17 have been found. In the Bernese Mountain Dog bitch, losses on chromosomes 1, 5, 8, 12, 18, 22, 27, 29 and gains on chromosomes 1, 2, 9, 11, 15, 16, 18, 20, 23, 24, 25, 28, 29, 30, 34, 36, 37 and 38 were identified. With the sequencing of the TP53 gene, one silent mutation, transition A/G at position 138 in exon 5 was detected, without changing the amino acid.

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.

Induction of SCEs and DNA fragmentation in bovine peripheral lymphocytes by in vitro exposure to tolylfluanid-based fungicide.

The potential for genotoxic and cytotoxic effects of tolylfluanid-based fungicide (50% active agent) was evaluated using sister chromatid exchange (SCE) and proliferation indices (PI) in cultured bovine peripheral lymphocytes. For the detection of possible genetic damage, DNA fragmentation assay was also applied. Bovine lymphocytes cultured for 72 h were treated with the fungicide at the final concentrations of 1.75, 3.5, 8.75, and 17.5 µg/mL for the last 24 and 48 h of culture without S9 metabolic activation, and during the last 2 h of culture with S9 metabolic activation. In the SCE assays no evidence for genotoxic activity of the fungicide was found in treatments of 24 h without and 2 h with S9. After the 24 h exposure to tolylfluanid, a weak decrease in the PI was observed. With the prolonged exposure time (48 h), dose dependence in the increase of SCE frequencies was observed. Moreover, after 48 h exposure slight fragmentation of DNA at the concentrations of 3.5 and 8.75 µg/mL was demonstrated. SCE quantification is the most widely used approach for the assessment of genotoxic/cytogenetic effects of chemical compounds. Positive results in the assay at 48 h exposure indicated a potential of the fungicide to increase frequency of chromosomal damage (replication injuries) that is the confirmation of early effect of exposure.

Induction of SCEs and DNA fragmentation in bovine peripheral lymphocytes by in vitro exposure to tolylfluanid-based fungicide

The potential for genotoxic and cytotoxic effects of tolylfluanid-based fungicide (50 percent active agent) was evaluated using sister chromatid exchange (SCE) and proliferation indices (PI) in cultured bovine peripheral lymphocytes. For the detection of possible genetic damage, DNA fragmentation assay was also applied. Bovine lymphocytes cultured for 72 h were treated with the fungicide at the final concentrations of 1.75, 3.5, 8.75, and 17.5 µg/mL for the last 24 and 48 h of culture without S9 metabolic activation, and during the last 2 h of culture with S9 metabolic activation. In the SCE assays no evidence for genotoxic activity of the fungicide was found in treatments of 24 h without and 2 h with S9. After the 24 h exposure to tolylfluanid, a weak decrease in the PI was observed. With the prolonged exposure time (48 h), dose dependence in the increase of SCE frequencies was observed. Moreover, after 48 h exposure slight fragmentation of DNA at the concentrations of 3.5 and 8.75 µg/mL was demonstrated. SCE quantification is the most widely used approach for the assessment of genotoxic/cytogenetic effects of chemical compounds. Positive results in the assay at 48 h exposure indicated a potential of the fungicide to increase frequency of chromosomal damage (replication injuries) that is the confirmation of early effect of exposure.

Effect of fungicide Euparen Multi (Tolylfluanid) on the induction of chromosomal aberations in cultivated bovine lymphocytes.

The effect of the fungicide Euparen Multi (containing 50% tolylfluanid) was investigated on the induction of chromosomal aberrations (CA) in cultured bovine peripheral lymphocytes. Cultures from two healthy donors were treated with tolylfluanid-based fungicide at concentrations ranging from 1.7 to 17.5 µg/ml for the last 24 and 48 hours of cultivation. Conventional cytogenetic method (CA assay) with Giemsa staining as well as fluorescence in situ hybridization (FISH) with whole bovine chromosomes 1 and 5 painting probes were used in the experiment. In the CA assay, no clastogenic effect of the fungicide was found after Euparen Multi treatment for 24 hours. On the contrary, significant elevation in polyploidy induction was observed with dose-dependence in one of the donors. Using prolonged time of exposure to the fungicide (the last 48 h of the cultivation), a slight clastogenic effect was detected at the doses of 8.75 and 17.5 µg/ml (P < 0.05, P < 0.01, respectively) in donor 1 and at the dose of 8.75 µg/ml (P < 0.05) in donor 2. The highest doses tested caused reduction of the mitotic indices (MI) (P < 0.05, P < 0.01) in both donors as well as both treatment times. The evaluation of stable structural aberrations in lymphocytes by two-colour FISH (48 h exposure) using bovine chromosome painting probes revealed the presence of nonreciprocal translocations at two examined concentrations (3.5 µg/ml and 8.75 µg/ml).

Effect of N-metylcarbamate pesticide bendiocarb on cattle lymphocytes after in vitro exposure.

Bendiocarb is a carbamate broad-spectrum insecticide used to control disease vectors such as mosquitoes and flies, as well as household and agricultural pests. Nowadays, only few papers reporting cytogenetic or possible genotoxic effect of this insecticide on mammalian cells are available. In the present study 24-hour exposure to bendiocarbamate at concentrations ranging from 20 to 160 µg/ml was used for investigation of unstable chromosomal aberrations (CA), sister chromatid exchanges (SCE) and stable chromosomal aberration induction in cultured bovine peripheral lymphocytes. The slight but no significant increase of chromatide breaks frequency was observed after the exposure of lymphocytes to 80 µg/ml of bendiocarb. At the highest concentration added to the cell cultures (160 µg/ml) mitotic index decrease was shown in both donors (p < 0.05; p < 0.01). Both statistically significant elevation of SCEs (p < 0.05) and a reduction of proliferative indices (PI) (p < 0.01) were shown at a dose of 80 µg/ml. By means of two fluorescent-labelled whole chromosome-painting probes, stable aberrations such as bovine chromosome 1 and 5 translocation as well as numerical aberrations (polyploidies, heteroploidies) were visualised under fluorescent microscope in some examined metaphases.

FISH detection of chromosome 1 aberration in human interphase and metaphase lymphocytes after exposure to benzene.

Benzene is a relatively common environmental and occupational contaminant with carcinogenic and clastogenic properties. Therefore, further understanding of the adverse effect of benzene is still a matter of interest. In the present study, induction of aberrations in the pericentromeric region of chromosome 1 (1q12) was examined by fluorescence in situ hybridisation (FISH) in both interphase and metaphase human lymphocytes after in vitro exposure to benzene at two concentrations (50 and 100 mumol/l). A weak but not significant increase of interphase cells micronuclei frequency was recorded at 100 micromol/l concentration in both donors examined (chi(2) test, p > 0.05). No fluorescent signal indicating the presence of chromosome 1 was observed in adjacent micronuclei. In metaphase cells, hypoploidy (monosomy) and polyploidy (tetraploidy) were the types of numerical aberrations most often exhibiting classical satellite probe signal. Chromosome breakage in the investigated pericentromeric region was assumed in lymphocyte metaphase cultures of donor 2 exposed to a dose of 100 micromol/l.

Chromosomal changes induced in mouse bone marrow cells following six weeks inhalation of cyclohexanol.

The effects of low doses of cyclohexanol exposure were studied in mouse bone marrow cells including chromosome aberrations (CA), micronucleus (MN) and sister chromatid exchanges (SCE) as biomarkers. Capillaries with a tested agent that was evaporated continuously were placed in an experimental chamber for six weeks. No clastogenic and/or aneugenic effect of CA and MN induction was observed. A significant elevation of induced damage was achieved in the SCE study (p < 0.001) that has confirmed the early exposure of cyclohexanol to mice.

Cytogenetic effect of technical glyphosate on cultivated bovine peripheral lymphocytes.

A technical herbicide containing isopropyl amine salt of glyphosate was tested for induction of chromosome aberrations (CA) and sister chromatid exchanges (SCE) in cultured bovine peripheral lymphocytes. Cultures were exposed to a glyphosate formulation at concentrations ranging from 28 to 1120 micromol/l without and with metabolic activation. No clastogenic effect of the herbicide was found. Its genotoxic effect was confirmed in the SCE assay after 24 h of incubation. A statistically significant elevation in SCE induction was observed in each of the donors after application of the product at doses ranging from 56 to 1120 micromol/l. The highest concentrations (560 and 1120 micromol/l) also caused reduction of mitotic and proliferation indices. In the 2 h-assay with metabolic activation a statistically significant frequency of SCE was observed only in cultures treated with the agent at a concentration of 140 micromol/l.

Chromosomal aberrations in humans induced by benzene.

Adverse effects associated with occupational exposure to benzene have often been reported in humans. It has been shown, that benzene causes chromosomal aberrations, sister chromatid exchanges and micronuclei in lymphocytes of exposed workers. In addition to evidence by conventional cytogenetic methods, the genotoxic effect of benzene has also been proved by a more specific approach based on fluorescence in situ hybridization with DNA probes. In the present paper, the nature of benzene-induced chromosomal aberrations and supposed consequence on human health is reviewed. The new possibilities in chromosomal alterations identification by molecular cytogenetic methods are also presented.

The induction of micronuclei in bovine lymphocytes by exposure to benzene and S-9 mix.

Benzene is a widespread human carcinogen, inducing leukaemia and hematotoxicity. It has been shown to be a multi-organ carcinogen in animals. The effect of benzene was studied using induction of micronuclei (MN) in whole blood lymphocytes cultures after treatment with different concentrations of benzene (5, 10, 50, 100, 500 and 1000 microM) with and without metabolic activation (S-9 mix). A significant elevation in the induction of micronuclei was found after application of benzene at doses of 50 and 100 microM in both donors. Treatment of bovine lymphocytes did not result in the induction of micronuclei in a dose-dependent manner. The addition of an external metabolic factor (10 % S-9 mix for 2 h) in blood cultures treated with benzene indicated an increase of the genotoxic activity of benzene (at concentrations ranging from 10-100 microM).