Bovine Peripheral Lymphocytes Research Articles

Bifenox induces programmed cell death in bovine mammary epithelial cells by impairing calcium homeostasis, triggering ER stress, and altering the signaling cascades of PI3K/AKT and MAPK

Bifenox (methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate), a nitrophenyl ether herbicide, was first introduced in the 1980s to control broadleaf weeds. As a result of its wide and frequent application in diverse agricultural settings and reports on residual traces, potential adverse effects of bifenox have been studied extensively in rat hepatocytes, bovine peripheral lymphocytes, and mice. Despite the reported risks of bifenox exposure in dairy cows, the toxicity of bifenox on bovine lactation system has not been extensively investigated. Therefore, we used bovine mammary epithelial (MAC-T) cells to study the toxic effects of bifenox on mammary glands. We found that bifenox inhibited MAC-T cells proliferation and disturbed the cell cycle, especially in the sub-G1 and G1 phases. Bifenox also disrupted the calcium homeostasis within the cell and impaired mitochondrial membrane potential. We also examined phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) signaling cascades. The findings indicated hyperactivation of phosphorylated protein kinase B (AKT), p70 ribosomal S6 kinase (p70S6K), S6, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and c-Jun, as well as endoplasmic reticulum (ER) stress caused by bifenox treatment. In conclusion, based on our in vitro study employing MAC-T cells, we report that bifenox can induce damage to the bovine mammary glands, potentially impacting milk production.

Assessment of Cytotoxic, Genotoxic, and Oxidative Stress of Dibutyl Phthalate on Cultured Bovine Peripheral Lymphocytes.

Recently, there have been numerous reports showing that phthalates have negative human health impacts and may cause several diseases such as asthma, breast cancer, obesity, type II diabetes, and male infertility. Animals are also exposed to phthalates through the environment and can cause adverse health effects on them. Several studies have been found on the cytogenetic effects of dibutyl phthalate (DBP) on different organisms but no documented evidence has been found on the cytotoxic and genotoxic effects of dibutyl phthalate (DBP) on bovine cultured lymphocytes. MTT assay was performed on different series of DBP concentrations (10 μM, 20 μM, 30 μM, 50 μM, 70 μM, 100 μM). A concentration-dependent decrease in cell viability was observed by the DBP. The LD50, LD50/2, and 2∗LD50 were found to be 50 μM, 30 μM, and 80 μM on bovine lymphocytes, respectively. Then, these concentrations of DBP were utilized to perform comet, micronucleus assays, and oxidative stress. A concentration-dependent increase in DNA damage, oxidative stress, and micronuclei formation was observed in lymphocytes by the DBP as compared to the control group. Highest genotoxic effects were observed at a concentration of 2∗LD50. Similarly, total oxidative stress was found higher, and antioxidative stress was lower in concentration-dependent manner by the DBP. The current study revealed a significant cytotoxic, genotoxic, and oxidative stress of DBP on cultured bovine lymphocytes.

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.

Doramectin induced cytotoxic and genotoxic effects on bovine peripheral lymphocytes and cumulus cells in vitro

The effect of doramectin (DOR) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PL) and cumulus cells (CC). The cytotoxicity and genotoxicity of DOR were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, single cell gel electrophoresis assay (SCGE) and cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Both cells were treated with three concentrations of DOR (20, 40, 60 ng mL–1) for 24 h. The results obtained from PL demonstrated that DOR was able to induce cytotoxic effect and DNA damage with all concentrations tested. Additionally, DOR increased micronuclei (MNi) frequency and nuclear buds (NBuds) with 20, 40, 60 ng mL–1, and nucleoplasmic bridges (NPBs) only with 40 ng mL–1. On the other hand, the three concentrations of DOR were not able to induce cytotoxic effect and DNA damage using SCGE in the bovine CC. Nevertheless, the two higher concentrations of DOR (20, 40 µg mL–1) significantly increased the frequency of micronucleus formation in bovine CC. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by DOR on bovine PL and CC.

Cytotoxic and genotoxic effects induced by enrofloxacin-based antibiotic formulation Floxagen® in two experimental models of bovine cells in vitro: peripheral lymphocytes and cumulus cells.

The in vitro effect of enrofloxacin (EFZ) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PLs) and cumulus cells (CCs). The cytotoxicity and genotoxicity of this veterinary antibiotic were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, single-cell gel electrophoresis (SCGE) assay, and cytokinesis-block micronucleus cytome (CBMN cyt) assay. Cells were treated during 24h, and three concentrations were tested (50μg/mL, 100μg/mL, 150μg/mL). When EFZ was tested in PLs, the results demonstrated that the antibiotic was able to induce cell death and DNA damage with all concentrations. In addition, 50μg/mL and 100μg/mL EFZ increased frequencies of micronuclei (MNi). On the other hand, the highest EFZ concentration occasioned cellular cytotoxicity in CCs as evidenced by mitochondrial activity alterations. Nevertheless, EFZ was not able to induce DNA damage and MNi in CCs. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by EFZ in bovine PLs and CCs.

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.

Micronucleus assay in genotoxicity assessment

Three different pesticide formulations were tested for micronuclei production and cell cycle arrest. Micronucleus test represents a suitable method for assessing chromosome damage because both chromosome breakage (clastogenicity) and chromosome loss (aneugenicity) can be measured simultaneously. In the experiments for 24 and/or 48 h exposure, no significant increase (p < 0.05) in MN frequency was found in comparison with negative control. On the contrary, the values of CBPI (cytochalasin blocked proliferative index) were significantly reduced in a dose dependent manner (p < 0.05, p < 0.01, p < 0.001) for both exposure time. Our results indicated an expressive cytotoxic effect of various pesticides in cultivated bovine peripheral lymphocytes.

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.

Evaluation of DNA damage and cytotoxicity induced by triazole fungicide in cultured bovine lymphocytes

The aim of the present study was to investigate the effect of triazole fungicide Prosaro® (tebuconazole/prothioconazole) on the induction of micronuclei (MNs) and DNA damage in bovine peripheral lymphocytes. Whole blood cultures or isolated lymphocytes were exposed to 1.5, 3, 7.5, 15 and 30 μg ml−1 concentrations of triazole formulation for 24 and 48 h, or 2 h for MN and Comet assays. For 24 and 48 h of incubation with the fungicide no significant elevations in the MN frequencies in bovine lymphocytes were found. However, a clear reduction of cytochalasin-blocked proliferation index (CBPI) was observed in the donors (p < 0.05, p < 0.01, and p < 0.001). After the prolonged time of exposure the highest concentrations resulted in nearly total inhibition of cell proliferation (30 μg ml−1). After the use of Comet assay, only a moderate increase in DNA damage without statistical significance was recorded, except the highest concentration (30 μg ml−1; p < 0.05).

Bovine macrophages sense Escherichia coli Shiga toxin 1.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections in cattle are asymptomatic; however, Stx impairs the initiation of an adaptive immune response by targeting bovine peripheral and intraepithelial lymphocytes. As presumptive bovine mucosal macrophages (Mø) are also sensitive to Stx, STEC may even exert immune modulatory effects by acting on steps preceding lymphocyte activation at the Mø level. We therefore studied the expression of the Stx receptor (CD77), cellular phenotype and functions after incubation of primary bovine monocyte-derived Mø with purified Stx1. A significant portion of bovine Mø expressed CD77 on their surface, with the recombinant B-subunit of Stx1 binding to >50% of the cells. Stx1 down-regulated significantly surface expression of CD14, CD172a and co-stimulatory molecules CD80 and CD86 within 4 h of incubation, while MHC-II expression remained unaffected. Furthermore, incubation of Mø with Stx1 increased significantly numbers of transcripts for IL-4, IL-6, IL-10, IFN-γ, TNF-α, IL-8 and GRO-α but not for IL-12, TGF-β, MCP-1 and RANTES. In the course of bovine STEC infections, Stx1 appears to induce in Mø a mixed response pattern reminiscent of regulatory Mø, which may amplify the direct suppressive effect of the toxin on lymphocytes.

Micronucleus assay in bovine lymphocytes after exposure to bisphenol A in vitro

Bisphenol A (BPA) [2,2-bis-(4-hydroxyphenyl) propane] is an important industrial agent, made by combining acetone and phenol, that is used extensively as a monomer in the production of polycarbonate plastics and as a precursor of epoxy resins. Micronucleus assays have served as an index of cytogenetic damage in in vivo and in vitro studies. We studied the genotoxic and cytotoxic effects of BPA on bovine peripheral lymphocytes in vitro. Lymphocyte cultures from two donors were exposed to four different concentrations of BPA (1 × 10(-4), 1 × 10(-5), 1 × 10(-6), and 1 × 10(-7) mol.L(-1)) for 48 h. The highest concentration of BPA (1 × 10(-4) mol.L(-1)) resulted in a significant increase in the number of micronuclei in comparison with the negative control (67.50 ± 2.121/1,000 binucleated cells versus 36.0 ± 5.657/1,000 binucleated cells in the DMSO control, P = 0.018). BPA did not affect the nuclear division index at any treatment concentrations. The present results thus demonstrate a significant genotoxic effect by BPA on bovine peripheral lymphocytes in vitro, only at the highest 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 48h 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 60μgmL−1. Statistical significant increases were seen in the CA assays at concentrations ranging from 6 to 30μgmL−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 15μgmL−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 60μgmL−1 in each donor for 24h. The highest concentrations also caused a statistical significant decrease in the PI. The treatment for 48h failed to exhibit any genotoxic activity of the fungicide.

Improving the analysis of quantitative PCR data in veterinary research

! 2011 Elsevier Ltd. All rights reserved. In recent years we have seen the emergence of molecular veterinary medicine, prompted by the availability of sequence information on pathogens, as well as on mammalian species relevant for economic (e.g. chicken, pig, cattle) and social (e.g. horse, dog, cat) purposes. Molecular biological tools are being integrated rapidly in the diverse specialities of veterinary research, allowing diseases to be described in molecular terms, both at the genetic level (DNA) and at the functional genomics level, including mRNA, non-coding RNA (ncRNA) and microRNA. Species-specific single nucleotide polymorphism (SNP) platforms are available to resolve the genetic background of inherited diseases. Expression profiling or functional genomics aims to identify differential gene signatures or ncRNA profiles associated with various experimental and/or clinical conditions. This is commonly carried out using species-specific micro-array platforms that measure relative expression levels of large numbers of gene products or miRNAs. Results need to be verified using independent techniques; the most popular validation technique is real-time, fluorescence based, reverse transcription quantitative PCR (RT-qPCR). The highly sensitive RT-qPCR technique is a complex, multi-step procedure that can be prone to numerous errors, potentially leading to misinterpretation of data. Hence, it is important that the technical information provided in a manuscript is complete, that protocols are validated and that results and conclusions are based on appropriate methods of analysis. Numerous publications, mainly in non-veterinary journals, have argued for the need for appropriate validation of the reference genes used to report relative expression levels of specific gene products with accuracy. It is abundantly clear that calculations of gene expression levels relative to a single, unvalidated, reference gene, without any stability expression evaluation, can be highly misleading (Dheda et al., 2005). A quick survey of papers published in The Veterinary Journal in 2009 and 2010 revealed only two papers in which several reference genes were used to calculate relative gene expression. Veronica Spalenza and colleagues at the University of Turin evaluated several reference genes in bovine peripheral lymphocytes (Spalenza et al., 2011). In a paper by Eric Zini and colleagues at the Vetsuisse Faculty, University of Zurich, three reference genes were used for relative gene expression calculations (Zini et al., 2010). Very few veterinary publications provide information on RNA quality and integrity and there is little information on the reverse transcription step or on the efficiency of the subsequent PCR. This makes it difficult to evaluate the relevance of any data reported in these journals or to attempt to reproduce the data in a different laboratory. Standardisation of reporting procedures and, indeed, reporting of a minimum amount of relevant technical information of molecular strategies is of paramount importance if gene expression studies are to be more reliable. The so-called ‘Minimum Information for publication of Quantitative real-time PCR Experiments’ (MIQE) guidelines aim to improve the standard of publications utilising qPCR by providing a checklist of the critical information required to enhance readers’ appreciation of the data, facilitate the repeatability of these experiments and enhance the comparison between different studies (Bustin et al., 2009). These guidelines are gradually being put into practice, with more than 300 citations appearing in the peer-reviewed literature in the last year; their general adoption by veterinary researchers would serve to strengthen the research field of veterinary medicine. A recently published modified standardisation approach, MIQEprecis (Bustin et al., 2010) offers simplified guidelines and a Microsoft Excel-based checklist aimed at improving transparency and 1090-0233/$ see front matter ! 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.06.044 ⇑ Corresponding author. Tel.: +31 3

Gene expression and inducibility of the aryl hydrocarbon receptor-dependent pathway in cultured bovine blood lymphocytes

The exposure to dioxin-like (DL) compounds, an important class of persistent environmental pollutants, results in the altered expression of target genes. This occurs through the binding to the aryl hydrocarbon receptor (AhR), the subsequent dimerization with the AhR nuclear translocator (ARNT), and the binding of the complex to DNA responsive elements. A number of genes are up-regulated, including, among others, the AhR repressor (AHRR) and several biotransformation enzymes, such as the members of CYP1 family and NAD(P)H-quinone oxidoreductase (NOQ1). The expression and the inducibility of the above genes were investigated in mitogen-stimulated cultured blood lymphocytes from cattle, which represent a notable source of DL-compound human exposure through dairy products and meat. As assessed by real-time PCR, all the examined genes except CYP1A2 and NQO1 were detected under basal conditions. Cell exposure to the DL-compounds PCB126 or PCB77 in the 10−6–10−9M concentration range resulted in a 2–4-fold induction of CYPIA1 and CYP1B1, which was antagonized by α-naphthoflavone or PCB153. This study demonstrates for the first time the presence and inducibility of the AhR pathway in easily accessible cells like bovine peripheral lymphocytes and prompts further investigations to verify whether similar changes could occur under in vivo conditions.

Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes

Gene expression studies in blood cells, particularly lymphocytes, are useful for monitoring potential exposure to toxicants or environmental pollutants in humans and livestock species. Quantitative PCR is the method of choice for obtaining accurate quantification of mRNA transcripts although variations in the amount of starting material, enzymatic efficiency, and the presence of inhibitors can lead to evaluation errors. As a result, normalization of data is of crucial importance. The most common approach is the use of endogenous reference genes as an internal control, whose expression should ideally not vary among individuals and under different experimental conditions. The accurate selection of reference genes is therefore an important step in interpreting quantitative PCR studies. Since no systematic investigation in bovine lymphocytes has been performed, the aim of the present study was to assess the expression stability of seven candidate reference genes in circulating lymphocytes collected from 15 dairy cows. Following the characterization by flow cytometric analysis of the cell populations obtained from blood through a density gradient procedure, three popular softwares were used to evaluate the gene expression data. The results showed that two genes are sufficient for normalization of quantitative PCR studies in cattle lymphocytes and that YWAHZ, S24 and PPIA are the most stable genes.

Evaluation of cytogenetic effects on bovine peripheral lymphocytes after the treatment with tebuconazole

Tebuconazole-based fungicide (group of triazole fungicides, with 25% of active agent) was evaluated for their ability to induce sister chromatid exchange (SCE) and proliferation indices (PI) in cultured bovine peripheral lymphocytes. The cultures were treated with the fungicides at the concentrations ranged from 3 to 60 µg.ml -1 for the last 24 and 48 h of incubation. Positive results in the SCE induction were obtained after the exposure to tebuconazole for the last 24h of incubation. The highest concentration of the fungicide also reflected in reduction of proliferation activity, but only insufficient number of cells could be analysed. Our results correspond to our previous finding from the CA assay (chromosomal aberrations) in bovine peripheral lymphocytes treated with the fungicide. Prolonged time of exposure provided inadequate evidence for the genotoxic activity of the fungicide.

Identification of chromosomes and aberrations after exposure to pesticide in bovine lymphocytes

Tebuconazole belongs to the group of triazole fungicides. As a systemic foliar fungicide it was widely used in agriculture for crops protection, such as barley, wheat, peanuts and orchard fruits. Associations between exposure to pesticides and health outcomes in humans, including different kinds of cancer were reported by several authors. Effects in immune, hematological, nervous, endocrine and reproductive systems have also been described. Data about genotoxic effects of pesticides are rarely reported. We are interested in the detection of structural and numerical aberrations in bovine peripheral lymphocytes using fluorescence in situ hybridization (FISH) in vitro. Three whole chromosome painting probes (WCPs), BTA1 for the bovine chromosome 1 (red colour), BTA 5 for chromosome 5 (green colour) and BTA 7 for chromosome 7 (red colour), as well, were used in experiments. Aneuploidy and unstable structural aberrations were detected more precisely than those seen in conventional chromosome aberrations assays, but only on the marked chromosomes could be analysed.

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).

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.

Effects of two selected fungicides on the frequency of SCE in bovine peripheral lymphocytes

Effects of two selected fungicides on the frequency of SCE in bovine peripheral lymphocytes