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3 Polish Journal of Veterinary Sciences

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

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Brzuzan P. , Jurczyk L. , Luczynski M. K. , Gora M.

tom 09

nr 2

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 • 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°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.

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

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Brzuzan P. , Wozny M. , Wolinska-Niziol L. , Piasecka A. , Florczyk M. , Jakimiuk E. , Gora M. , Luczynski M. K. , Gajecki M.

nr 1

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 |ig/kg/d), DON (12 |ig/kg/d), ZEN+DON (40 + 12 |ig/kg/d), and placebo (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 facilitate understanding of the miRNA-regulated biological effects of mycotoxins.

Modification of zearalenone structure in model and natural conditions

63%

Gora M. , Luczynski M. K. , Smoczynski L. , Obremski K. , Polak M. , Swist M. , Zielonka L. , Gajecki M.

nr 3

Zearalenone is a mycotoxin widely occurring in cereals and animal feed, and it is associated with hyperestrogenism and other reprodutive disorders in animals. A new method of detoxication of feed- stuffs involves alkaline hydrolysis of toxic macrolactone (1) (as well as model compounds (2a, 2b)). The method caused modification of zearalenone structure under mild conditions and the toxin underwent irreversible hydrolysis with high efficiency.