miR-122-5p as a plasma biomarker of liver injury in fish exposed to microcystin-LR.

Recent studies have shown the presence of large amounts of microRNAs (miRNAs; miRs) from damaged cells in the peripheral blood. In this study, we investigated the levels of miRNAs circulating in the blood plasma of whitefish (Coregonus lavaretus) after exposure to microcystin-LR. We used real-time PCR to examine the relative expression of plasma levels of 4 miRNAs (miR-122-5p and let-7c-5p, the liver-enriched microRNAs, miR-148a-3p which promotes the hapatospecific phenotype in mammals, and miR-92a-3p, a cell proliferation and angiogenesis promoter, potentially hepatocarcinogenic) during the first 48 h after exposure to MC-LR. We observed a rapid increase of miR-122-5p levels 8 h after exposure (P < 0.05), which continued to the end of the experiment. Our results demonstrated that the plasma miR-122-5p was indicative of MC-LR-induced liver injury, exhibiting areas under the curve close to 1 in ROC analysis (AUC = 0.976, P < 0.001). Although plasma levels of miR-148a-3p and miR-92a-3p were significantly elevated by the end of the experiment, their discriminative power was lower than reported for the miR-122-5p. Based on these results and reports on miRNA-based diagnosis of liver injuries in mammals, plasma miR-122-5p could be considered as a robust, new generation diagnostic biomarker in fish, helpful for the non-invasive diagnosis of liver damage.

MicroRNA expression profiles in liver and colon of sexually immature gilts after exposure to Fusarium mycotoxins.

To improve our knowledge of the role of microRNAs (miRs) in responses of the porcine digestive system to two Fusarium mycotoxins, zearalenone (ZEN) and deoxynivalenol (DON), we examined the expression of 7 miRs (miR-9, miR-15a, miR-21, miR-34a, miR-122, miR-125b, and miR-192), previously found to be deregulated in diseased liver and colon cells. In this study, immature gilts were exposed to NOEL doses of ZEN (40 µg/kg/d), DON (12 µg/kg/d), ZEN + DON (40 + 12 µg/kg/d), andplacebo (negative control group) for 7, 14, 21, 28, 35, and 42 days. Before the treatment, expression levels of the selected miRs were measured in the liver, the duodenum, the jejunum, and the ascending and the descending colon of the gilts. Hierarchical clustering of the tissues by their miR expression profiles was consistent with what would be expected based on the anatomical locations and the physiological functions of the organs, suggesting that functions of the miRs are related to the specificities of the tissues in which they are expressed. A subset of 2 pairs of miRs (miR-21+miR-192 and miR-15a+miR-34a), which were assigned to two distinct clusters based on their tissue abundance, was then evaluated in the liver and the ascending and the descending colon during the treatment. The most meaningful results were obtained from the ascending colon, where a significant effect of the treatment was observed, suggesting that during the exposure to mycotoxins, the pathways involved in cell proliferation and survival were disordered. Changes in miR expression in the liver and the descending colon of the treated gilts were smaller, and were associated more with treatment duration than the exposure to ZEN, DON, or ZEN + DON. Further research should focus on identification of genes whose expression is regulated by these aberrantly expressed miRs. This should facili- tate understanding of the miRNA-regulated biological effects of mycotoxins.

c-myc in whitefish (Coregonus lavaretus): structure, expression, and insights into possible posttranscriptional regulatory mechanism.

c-myc has a crucial function in growth control, differentiation, and apoptosis of vertebrate cells. Despite the important role of c-myc in mediating the biological effects, studies of c-myc gene expression and factors that control it in organisms other than mammals, such as fish, have been rare. In the current study, we asked whether c-myc mRNA of whitefish, a feasible organism for pollution monitoring in aquatic systems and a model in toxicological research, contains activity sites for regulatory motifs in its 5'- and 3'-UTRs, similar to those found in mammals. We were particularly interested in whether miRNA-34, a known negative regulator of c-myc's in mammals, is able to regulate c-myc in fish. To answer these questions, we determined the mRNA sequence of whitefish c-myc and inferred the structure of the protein that it codes for. We found that the active sites of mRNA and structures of the inferred c-myc protein are similar to those found in mammals and other fish. Remarkably, levels of c-myc mRNA expression were very high in ovaries compared to other tissues of whitefish, thus corroborating previous data in fish. Using bioinformatic searches on c-myc 3'-UTR, we confirmed the presence of two miRNA-34a (miR-34a) response elements. Luciferase reporter assay showed that activity of reporters containing either the miR response elements or entire c-myc 3'-UTR was significantly reduced (p < 0.001) by ectopic expression of miR-34a. Therefore, we further investigated possible involvement of miR-34a in c-myc gene silencing by profiling the expression of both genes in livers of whitefish treated for 8, 24, 48 h with MC-LR, a potent c-myc inducer in mammals. Although the difference was only significant at p = 0.08, the expression of c-myc mRNA in challenged whitefish after 24 h of the treatment was notably higher than that in livers of control fish. Concurrently, we noticed slight but significant up-regulation of miR-34a after 24 and 48 h of the challenge (p < 0.05); however, we found no significant correlation of the c-myc mRNA levels and miR-34a expression. Together, these results suggest that miR-34a might regulate c-myc gene expression in whitefish liver; however, their involvement in MC-LR hepatotoxicity should be clarified in future studies.

Evaluation of selected serum biochemical and haematological parameters in gilts exposed per os to 100 ppb of zearalenone.

Zearalenone (ZEN) widely contaminates animal feed of plant origin. The recommended safe concentrations of ZEN in feeds for various animal species are set mainly based on the mycotoxin's hormonal properties (NOEL). Our growing knowledge about biologically active concentrations of ZEN, molecular mechanisms and cells/tissues targeted by ZEN indicates that the harmful effects exerted by this mycotoxin on animals may be far greater than previously believed. This experiment was performed on pre-pubertal gilts divided into a control group (n=9) and an experimental group (ZEN, n=9). The control group received placebo, whereas the experimental group was administered ZEN at a dose of 0.1 mg/kg feed (equivalent to 5 µg/kg BW/day) for 42 days. On days 14, 28 and 42 blood samples were collected from the animals to determine the concentrations of selected zearalenols, serum biochemical and haematological parameters. Conjugated ZEN was found in the blood serum of the experimental gilts. Changes in the analysed biochemical parameters included a transient increase in albumin and cholesterol levels. A statistically significant increase in the concentrations of neutrophilic and acidophilic granulocytes was observed in the white blood cell system. The results indicate that long-term per os exposure of pre-pubertal gilts to low doses of ZEN (below NOEL) has a modulatory effect on liver function and white blood cells.

The effect of T-2 toxin on percentages of CD4+, CD8+, CD4+ CD8+ and CD21+ lymphocytes, and mRNA expression levels of selected cytokines in porcine ileal Peyer's patches.

The immune system is one of the main toxicity targets of the T-2 toxin. In view of scant research data demonstrating the effect of T-2 on cellular and humoral responses in gut-associated lymphoid tissue (GALT), this study set out to investigate the effects of chronic exposure to low doses of the T-2 toxin (200 microg T-2 toxin kg(-1) feed) on percentages of CD4+ and CD8+ T lymphocytes, CD4+/CD8+ double-positive T lymphocytes, CD21+ B cells, and IL-2, IFN-gamma, IL-4 and IL-10 mRNA expression levels in porcine ileal Peyer's patches. The investigated material comprised ileum sections sampled from piglets (aged 8-10 weeks, body weight of 15-18 kg) on days 14, 28 and 42 of the experiment. After 42 days of exposure to T-2, a significant drop in the quantity of the IL-10 product was observed (R = 0.94; S.E. 0.49-0.79; p < 0.001). A gradual decrease in the amount of IL-4 and IFN-gamma cytokine transcripts was found throughout the experiment, but the reported trend was not significant. On experimental days 14 and 42, a significant increase in the percentage of CD8+ T lymphocytes was observed in comparison with the control (p = 0.04 and p = 0.05, respectively), whereas on day 28, a significant decrease in the percentage of the above subpopulation was noted (p = 0.00). The percentage of CD21+ B cells in the experimental group decreased steadily in comparison with the control, and the observed drop was significant on days 28 and 42 (p = 0.06 and p = 0.00, respectively). On days 14 and 28, the percentages of CD4+ and CD8+ T lymphocytes were lower in the experimental animals than in the control group, and the drop reported on day 28 was statistically significant (p = 0.03).

Influence of zearalenone on selected biochemical parameters in juvenile rainbow trout (Oncorhynchus mykiss).

Zearalenone (ZEA) is a mycoestrogen frequently found in food and animal feed materials all over the world. Despite its hydrophobic character, ZEA is also found in surface and ground waters which suggests an environmental risk for aquatic animals. Knowledge concerning mycotoxin-related mechanisms of toxicity is still incomplete, e.g. little is known about the influence of ZEA exposure on fish. The aim of this study was to investigate the effect of ZEA on selected biochemical parameters in juvenile rainbow trout after 24, 72, and 168 h of intraperitoneal exposure (10 mg/kg of body weight). The analysis showed a slight tendency towards prolonged blood clotting time and significant iron deficiency in the liver and ovary of exposed animals. However, no differences in aminotransferase (AlaAT, AspAT) activity or glucose levels in fish plasma was observed. The results of this study suggest that although trout exposed to ZEA did not exhibit any distinct symptoms of liver damage, the mycotoxin tested was able interfere with blood coagulation and iron-storage processes.

Expression profiling in vivo demonstrates rapid changes in liver microRNA levels of whitefish (Coregonus lavaretus) following microcystin-LR exposure.

At present, little is known about the role of miRNAs in liver response of fish to the cyanobacterial hepatotoxin microcystin-LR (MC-LR) treatment, despite the fact that the exposure is thought to underlie multiple acute and chronic effects. To address this question, we used the Real-Time PCR method to examine the differential expression of 6 miRNAs putatively playing roles in signal transduction (let-7c, miR-9b), apoptosis and cell cycle (miR-16a, miR-21a, miR-34a) and fatty acid metabolism (miR-122) in whitefish (Coregonus lavaretus) liver, during the first 48h after intraperitoneal injection of MC-LR (100 µg/kg body weight). In addition, we analyzed expression levels of 8 mRNAs and p53 protein, known to be involved in the cell response on the exposure to environmental stressors. Following the challenge we observed a rapid and transient increase in the mean (n=5) levels of individual miRNA expression (from 2.7-fold for miR-122 to 6.8-fold for let-7c), compared to the respective levels in control fish, which mostly peaked at 24h of the experiment. This increase was correlated with a reduction in the expression of mRNAs of genes coding for ferritin H (frih) and HNK Ras -like protein (p-ras) and an overexpression of mRNAs of genes coding for bcl2-associated X protein (bax), cyclin dependent kinase inhibitor 1a (cdkn1a), dicer (dcr), histone 2A (h2a) and p53. Expression of the remaining caspase 6 (cas6) mRNA did not change over 48 h of the treatment. Moreover, exposure to MC-LR did not alter whitefish p53 protein levels. Bearing in mind a variety of likely silencing targets for, and the onset of, the aberrant miRNA expression it may be concluded that they are involved in molecular pathways, such as liver cell metabolism, cell cycle regulation and apoptosis, and may contribute to the early phase of MC-LR induced hepatotoxicity.

The expression of genes encoding opioid precursors and the influence of opioid receptor agonists on steroidogenesis in porcine adrenocortical cells in vitro.

Endogenous opioid peptides are involved in the regulation of the HPA-axis function and stress response mechanism. However, there is a scarcity of data on opioid involvement in the regulation of the adrenocortical endocrine function. This study was performed to: 1) establish the expression of proenkephalin, POMC and prodynorphin genes in the porcine adrenal cortex and test in vitro the influence of ACTH, angiotensin II, CRH and epinephrine on this expression, and 2) determine the effects of opioid receptor agonists on basal and ACTH- or angiotensin II-affected secretion of cortisol, aldosterone and progesterone by the cultured adrenocortical cells. Our experiment has demonstrated the presence of mRNAs for opioid precursors in cells isolated from the adrenal cortex and the significant effects of ACTH and angiotensin II, but not CRH or epinephrine, on adrenocortical transcription of the analyzed genes. Angiotensin II reduced the expression of the POMC gene but stimulated that of prodynorphin. In turn, ACTH decreased the transcription of prodynorphin. The study has also demonstrated the effects of selective opioid receptor agonists - DPLPE (delta), FK33-824 (mu) and U50,488 (kappa) - on adrenal steroidogenesis in pigs. Basal secretion of cortisol was enhanced after the activation of mu or kappa receptors, whereas ACTH-stimulated cortisol output was increased only by the mu receptor agonist. Angiotensin II-treated cells significantly decreased aldosterone secretion in the presence of the kappa receptor agonist. The present results suggest that opioid peptides are synthesized in the porcine adrenal cortex, indicating their involvement in the regulation of adrenal steroidogenesis through autocrine and/or paracrine interactions.

Factors determining sensitivity of prepubertal gilts to hormonal influence of zearalenone.

Among large husbandry animals, swine are the most predisposed to zearalenone (ZEA) intoxication, mainly because cereal is an important component of their diet. Studies performed so far (in vivo, in vitro) suggest that ZEA and its metabolites, which may appear due to ZEA biotransformation (especially alpha-zearalenole; alpha-ZOL), can modify signaling cascades of endogenous sex steroids, through either receptor or non-receptor mechanisms. Of all age groups of swine, immature gilts are particularly predisposed to zearalenone intoxication, as manifested by the occurrence of genital tract tissue dysfunction on exposure to ZEA. The intensity of the adverse effects observed at either systemic or local level in gilts, when compared to sexually mature swine females, suggest that specific age-dependent physiological conditions may exist, which determine the high sensitivity of gilts to exogenous estrogen-like compounds, including ZEA.

Real-time PCR analysis of p53 mRNA levels in tissues of whitefish (Coregonus lavaretus) exposed to benzo[a]pyrene.

We developed a real-time PCR assay for measuring relative quantities (RQ) of p53 tumor suppressor mRNA in the whitefish (Coregonus lavaretus, Salmonidae, Teleostei). Real-time PCR primers for the p53 gene were designed from a region that was found to be conserved among salmonid p53 genes. To test for the usefulness of the assay we performed a treatment study, using benzo[a]pyrene (B[a]P) a putative p53-inducer. Two groups of hatchery raised whitefish, with an average body mass of 15 g and total length of 12 cm were either given an intraperitoneal injection (10 mg x kg(-1)) of B[a]P in corn oil (2 mg B[a]P ml(-1) corn oil) or corn oil alone (Control). After treatment (48 h, 7 degrees C), two random fish from each group were anesthetized and the liver, head kidney and brain were collected for mRNA isolation and analysis. In the control fish, relative quantification analysis based on the p53 mRNA levels in liver (RQ=1.00) showed higher basal levels of p53 mRNA in the head kidney (RQ= 1.69), and lower in the brain (RQ=0.41). In all three tissues sampled, p53 mRNA was affected by treatment with B[a]P. Liver tissue showed the greatest induction (RQ=1.53) from base levels (RQ=1.00), followed by brain (RQ=1.36), and head kidney (RQ=1.23). These results confirm that p53 mRNA is generally present at lower levels in differentiated tissues (liver and brain) than in those tissues with cell lines (head kidney), and demonstrate that p53 is moderately inducible by B[a]P in the whitefish. The approach presented here has the advantage of providing rapid and accurate measures of p53 induction in various tissues of fish responding to PAH contaminant exposure.

Androgenesis in rainbow trout using cryopreserved spermatozoa: the effect of processing and biological factors.

In addition to producing homozygous lines for biomedical and genomic research and monosex stocks for commercial purposes, androgenesis is the biotechnology considered most promising and reliable for recovering complete nuclear genome information from cryopreserved fish cells. That is because procedures of cryopreserving spermatozoa, contrary to procedures for oocytes or entire eggs, are being well developed. Application of androgenesis in genome banking programs addresses the needs of both the aquaculture industry (safeguard for valuable strains and lines) and natural resource conservation (in vitro protection of endangered species or populations). The present study was focused on successful production of an androgenetic rainbow trout stock using cryopreserved spermatozoa. Our report constitutes the first time a full factorial analysis of processing and biological factors affecting androgenesis efficacy has been presented. Also for the first time, the survival of androgenetic individuals during 2 years of life was recorded. Remarkably high survival rates were observed in one of the two experiments-up to 42.5 +/- 2.8% of hatched larvae, 22.5 +/- 0.1% of swim-up larvae and 10.5% of androgenetic alevins 0.4 g. Mortality rates in androgenetic groups were high especially during the first 6 months. In all, 114 androgenetic individuals (0.9%) survived 2 years. Cryopreservation of spermatozoa generally did not affect androgenesis efficiency significantly, however, this effect was significantly dependent on the method of ploidization shock and on the duration of treatment. Significant interactions were revealed between the irradiation dose and the magnitude of pressure applied, and between the treatment of sperm and duration of pressure shock. Individual variability of spermatozoa donors significantly affected androgenesis efficiency regardless of their genetic (outbred or inbred) origin. Genetic source of the oocytes, contrary to spermatozoa, proved to be an important factor. Following findings of other researchers that androgenesis using cryopreserved spermatozoa is possible, we demonstrated that viable stock could be successfully established from cryopreserved nuclear genome information. Complex statistical analysis of previously developed procedures resulted in information-rich data regarding factorial interactions helpful for developing protocols in genome-restoration programs.

Tandemly repeated sequences in mtDNA control region of whitefish, Coregonus lavaretus.

Length variation of the mitochondrial DNA control region was observed with PCR amplification of a sample of 138 whitefish (Coregonus lavaretus). Nucleotide sequences of representative PCR products showed that the variation was due to the presence of an approximately 100-bp motif tandemly repeated two, three, or five times in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA. This is the first report on the tandem array composed of long repeat units in mitochondrial DNA of salmonids.

Mitochondrial DNA sequence from an extinct population of European vendace, Coregonus albula L.

Nucleotide sequence of the mitochondrial DNA control region was determined for the formalin-preserved specimen of the individual European vendace, Coregonus albula, belonging to an extinct population. The specimen has been preserved and stored in formalin for 39 years. A comparison with two previously determined sequences obtained for specimens from other populations of vendace revealed both nucleotide substitutions and site heteroplasmy. The ability to sequence DNA from formalin-preserved museum specimens of rare or extinct taxa provides another sampling strategy for phylogenetic studies on fish.

Secretion of hatching enzyme and its proteolytic activity in coregoninae (Coregonus albula L andC. lavaretus L) embryos.

After the electrial stimulation Coregoninae embryos secreted the hatching enzyme (chorionase) within 0.1-0.5 h, and the dissolution of their chorions lasted 1.2-2.0 h, depending on embryo's developmental stage (DS 13 or DS 14) and water temperature (5.2 or 9.6-9.8°C).Crude chorionase (hatching liquid) ofCoregonus albula andC. lavaretus was collected in large quantities by means of the electric stimulation of eggs. In both species the temperature optimum of proteolytic activity of the crude chorionasc was 30°C; the activity was lost at temperatures < 3-2°C and > 35-40°C. The maximal proteolytic activity was observed at pH 8.5; a rapid decrease in enzyme activity was evident at pH < 7.0, and the activity was zero at pH 6.The temperature-activity curve of chorionase may reflect the adaptation of Coregoninae to hatching immediately after the ice cover recedes from lakes, whereas the rapid decrease of enzyme activity at pH 7 -pH 6 can affect adversely the process of hatching in acidified lakes.