Research
cDNA fingerprint from the hepatopancreatic glands of pond snails (Lymnaea stagnalis) exposed to benzo[a]pyrene
Maciej Woźny, Przemysław Kowal, Sławomir Ciesielski, Maciej Florczyk, Rafał Wiśniewski, Ewelina Malicka, Paweł Brzuzan
Abstract Identification of differentially expressed genes that could be potentially used as biomarkers of PAH exposure of common invertebrate animal (like freshwater snail) would be a valuable resource for investigators interested in toxicology and biomonitoring of aquatic environments. Therefore, the aim of this research was to investigate effects of waterborne benzo[a]pyrene (B[a]P) exposure on mRNA expression in the pond snail’s (Lymnaea stagnalis) hepatopancreatic gland. Toward this end, mature individuals of pond snail (L. stagnalis) were treated with 50µM B[a]P solution in a short 36h static exposure test. Differential Display PCR (DD-PCR) was used to generate a unique cDNA fingerprint of genes that were differentially expressed in the tissues of exposed and unexposed snails. To assess the putative identity of the isolated cDNA amplicons (ESTs), BLAST queries were performed to find similarities in their nucleotide sequence. Real-Time qPCR analysis was used to verify the DD-PCR expression profile. Finally, an additional independent exposure study, including higher dose of B[a]P (100µM), was conducted to validate the expression of selected ESTs. BLAST revealed that only 3 out of 9 isolated ESTs had meaningful information on their putative nucleotide sequence identity. The highest similarity was scored for EST-A1, identified as the transcript of UAP-like protein, found to be up-regulated after B[a]P exposure. The original expression pattern that was observed in DD-PCR step was coherent with results of the qPCR verification for 3 out of 5 analyzed ESTs. However, changes in the ESTs expression were modest and the treatment with B[a]P resulted in significant down-regulation for only 1 unidentified fragment (EST-G42, almost 2-fold; p<0.05) when compared to untreated snails. Although no significant changes were observed for EST-A1 and EST-G42 in the validation study, their expression pattern was consistent with that obtained from DD-PCR. Surprisingly, EST-C5 remained in contrast to the DD-PCR part, but it showed significant down-regulation in group of snails exposed to 100µM B[a]P (3.5-fold; p<0.05). The obtained results show that diverse genes may be involved in the molecular response of the pond snail’s hepatopancreas to treatment with B[a]P. However, further research is needed to confirm the utility of the discovered EST as PAH biomarkers in biomonitoring practices with L. stagnalis as bioindicator species.
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