The Influence of Zearalenone on Selected Hemostatic Parameters in Sexually Immature Gilts.

Vascular toxicity induced by xenobiotics is associated with dysfunctions or damage to endothelial cells, changes in vascular permeability or dysregulation of the vascular redox state. The aim of this study was to determine whether per os administration of zearalenone (ZEN) influences selected hemostatic parameters in prepubertal gilts. This study was performed on female gilts divided into a control group which received placebo and an experimental group which received ZEN at a dose of 5.0 µg·kg-1 b.w. × day-1. On days 14, 28 and 42, blood samples were collected from the animals for analyses of hematological, coagulation and fibrinolysis parameters, nitric oxide, von Willebrand factor antigen content and catalase activity. The results demonstrated that the treatment of gilts with ZEN at a dose below no observable adverse effect level did not affect the primary hemostasis and the blood coagulation cascade. However, ZEN could have temporarily affected the selected indicators of endothelial cell function (increase of von Willebrand factor, decrease of nitric oxide levels) and the oxidative status plasma (decrease of catalase activity) of the exposed gilts. In summary, these results suggest that the adaptive response to ZEN-exposure can induce a transient imbalance in the vascular system by acting on vascular endothelial cells.

Effects of a Low Dose of T-2 Toxin on the Percentage of T and B Lymphocytes and Cytokine Secretion in the Porcine Ileal Wall.

Plant materials used in the production of pig feed are frequently contaminated with mycotoxins. T-2 toxin is a secondary metabolite of selected Fusarium species, and it can exert a harmful influence on living organisms. Most mycotoxins enter the body via the gastrointestinal tract, and they can modulate the gut-associated lymphoid tissue (GALT) function. However, little is known about the influence of low T-2 toxin doses on GALT. Therefore, the aim of this study was to evaluate the effect of T-2 toxin administered at 50% of the lowest-observed-adverse-effect level (LOAEL) on the percentage of CD2+ T cells, CD4+ T helper cells, CD8+ cytotoxic T cells, CD4+CD8+ double-positive T cells, TCRγδ+ cells, CD5+CD8- B1 cells, and CD21+ B2 cells, and the secretion of proinflammatory (IFN-γ, IL-1ß, IL-2, IL-12/23p40, IL-17A), anti-inflammatory, and regulatory (IL-4, IL-10, TGF-ß) cytokines in the porcine ileal wall. The results of the study revealed that T-2 toxin disrupts the development of tolerance to food antigens by enhancing the secretion of proinflammatory and regulatory cytokines and decreasing the production of anti-inflammatory TGF-ß. T-2 toxin triggered the cellular response, which was manifested by an increase in the percentage of CD8+ T cells and a decrease in the percentage of B2 and Tγδ lymphocytes.

Molecular characterization of the cyclin-dependent protein kinase 6 in whitefish (Coregonus lavaretus) and its potential interplay with miR-34a.

Cyclin-dependent protein kinase 6 (CDK6) plays a pivotal role in the regulation of the cell cycle and cell proliferation in mammals, and disruption of its expression by various microRNAs has been implicated in the pathogenesis of multiple human cancers. In mammals, miR-34a acts as a downstream effector of p53, and thus indirectly targets Cdk6, abrogating its effects. However, no studies have been done so far to examine the mechanistic involvement of miR-34a in the silencing of cdk6 in fish. In the present study, we found that the cDNA sequence of whitefish cdk6 has a 3'UTR region that contains a binding site for miR-34a. Using a luciferase reporter assay, we demonstrated that whitefish cdk6 is a direct target of miR-34a in vitro. In order to confirm this relationship in vivo, we measured the miR-34a and cdk6 mRNA expression patterns in the liver of whitefish after short-term (8, 24, and 48 h) and long-term (14 and 28 days) exposure to microcystin-LR (MC-LR), a known hepatotoxin and tumor promoter. In contrast to the in vitro findings, we noticed an up-regulation of miR-34a and cdk6 expression after long-term MC-LR treatment. While these results indicate that both, miR-34a and cdk6 are responsive to MC-LR treatment, they do not support the presence of a miR-34a:cdk6 mRNA regulatory pair in the MC-LR-challanged whitefish liver in vivo. On the other hand, our findings suggests that cell regulatory elements, partnering with either miR-34a or cdk6, are worthy of further screening to better understand the molecular mechanisms that underlie the physiological response of fish challenged with hepatotoxic environmental pollutants like microcystins.

Microcystin-LR-Triggered Neuronal Toxicity in Whitefish Does Not Involve MiR124-3p.

Microcystin-LR (MC-LR) is a potent hepatotoxin that has also been pointed out of causing neurotoxicity, but the exact mechanisms of action still remain ambiguous and need to be elucidated. Data from studies on mammals show that pathology of astrocyte cells points to perturbations of microRNA signaling. Glial fibrillary acidic protein (GFAP), a neuronal cell/astrocyte-specific protein, and a microRNA-124-3p (MiR124-3p) are among putative triggers and regulators of neuronal cell/astrocyte reactivity. In the present study on whitefish (Coregonus lavaretus), we found that gfap mRNA contains a putative target site for MIR124-3p, to potentially affect its expression changes. qPCR expression study of gfap:MiR124-3p pair in the midbrain of juvenile whitefish, during 28 days of exposure to a repeated subacute dose of MC-LR (100 µg kg-1 body mass), showed marginally significant up-regulation of gfap only on the 7th day of exposure period which suggests neuronal toxicity. During the whole exposure period, neither midbrain nor blood plasma levels of MiR124-3p were changed. Furthermore, double luciferase gene reporter assay confirmed the lack of MiR124-3p involvement in mediating control over gfap mRNA expression. These data show that, although MC-LR may trigger neuronal toxicity in whitefish, this does not involve MiR124-3p in response to the treatment.

The Effect of Deoxynivalenol on Selected Populations of Immunocompetent Cells in Porcine Blood-A Preliminary Study.

Deoxynivalenol (DON) is one of the most prevalent mycotoxins in Europe. Pigs are an animal species that is most susceptible to this mycotoxin. Deoxynivalenol causes significant losses in pig production by lowering feed intake, decreasing daily weight gains, disrupting immune responses, and increasing susceptibility to diseases. The aim of this experiment was to determine the influence of feed contaminated with DON at concentrations insignificantly higher than recommended by the European Commission (900 µg/kg). The experimental feed contained 1008 µg DON/kg. The experiment was performed on eight weaners from the same litter. The animals were randomly divided into two groups: an experimental group (M, n = 4) fed contaminated feed and a control group (C, n = 4) administered feed free of mycotoxins. The experiment lasted for six weeks, and peripheral blood samples were collected from the animals for analyses of selected morphological parameters and changes in the percentages of CD4⁺8-, CD4-8⁺, and CD4⁺8⁺ lymphocytes and antigen-presenting cells (APC) with CD14⁺172⁺ (monocytes), CD172ahigh4-14- (conventional dendritic cells, cDC), and CD172adim4⁺14- (plasmacytoid dendritic cells, pDC) phenotypes. The morphological parameters of porcine blood samples were determined by flow cytometry with non-fluorescent particle-size calibration standards, and no differences were observed between groups M and C. An immunophenotyping analysis of lymphocytes and dendritic cells (DC) revealed an increase in the percentage of CD4⁺8-, CD172ahigh4-14-, and CD172adim4⁺14- cells, and a decrease in the number of CD4-8⁺ cells in group M. The results of this experiment suggest that prolonged exposure to low doses of DON can change the proportions of immunocompetent cells (a shift towards humoral immunity), without affecting their overall counts.

miR-34a and bcl-2 expression in whitefish (Coregonus lavaretus) after microcystin-LR exposure.

Studies on mammals have demonstrated that the expression of miR-34a is associated with process of apoptosis in many cell types, by lowering expression of the anti-apoptotic protein Bcl-2. Despite the role of miR-34a, there is no data about the miR-34a:Bcl-2 interaction in lower vertebrates, especially in fish. In the current study, we determined the nucleotide sequence of miR-34a precursor, predicted its secondary structure, and shed light on the potential role of p53 in activation of miR-34a in whitefish, a salmonid fish species. In parallel, we determined a cDNA sequence of whitefish bcl-2, and gained insight into the primary structure and evolutionary relationship of the whitefish Bcl-2 protein that it codes for. In particular, we were interested whether whitefish bcl-2 3'UTR contains an active target site for miR-34a. Using a computational approach followed by luciferase reporter assay, we confirmed the direct interaction of miR-34a with the whitefish bcl-2 3'UTR. Therefore, we further investigated whether bcl-2 silencing via miR-34a occurs in liver samples of whitefish exposed for 48h to microcystin-LR (MC-LR), a known hepatotoxin and tumor promoter. We noticed a statistically unsignificant up-regulation of miR-34a expression, which was accompanied by a marginally significant increase of bcl-2 mRNA level and the significant increase of bax (pro-apoptotic) mRNA level. However, we found no significant correlation between bcl-2 and miR-34a expression in vivo, which suggests that their involvement in hepatocyte cell responses to MC-LR in whitefish is still questionable.

The effect of experimental long-term exposure to low-dose zearalenone on uterine histology in sexually immature gilts.

The objective of this study was to determine whether long-term (48-day) oral administration of low-dose zearalenone (ZEA) resulted in changes in uterine histology in sexually immature gilts. The study involved 12 clinically healthy 2-month-old gilts with a determined immune status. The animals were randomly divided into two experimental groups (E1, n=4; E2, n=4) and a control group (C, n=4). ZEA (20 µg/kg bw for group E1 and 40 µg/kg bw for group E2) was administered in gelatin capsules per os before the morning feeding for 48 days; group C was given placebo rather than ZEA. The animals were then sacrificed and the uteri were subjected to histological examination. Low doses of ZEA (50% and 100% of no observable adverse effect levels values) induced experimental hyperestrogenism and stimulated the proliferation of nearly all uterine wall tissues, as shown by significant increases in the index of proliferation values. The accompanying uterine hyperaemia caused uterine reddening and swelling. Atypical endometrial hyperplasia (hyperplasia simplex atypica) could be interpreted as the endometrium's physiological response to an excessive level of endogenous and/or exogenous estrogenic stimuli. The results of this study and the effects of ZEA in the uterus suggest that there is a possibility of detrimental health effects when the level of endogenous estrogens is low and the body is supplied with an additional dose of exogenous estrogens. Such effects probably results from synergic interaction that produce hyperestrogenism and lead to excessive estrogenic stimulation.