**3. Conclusions**

The experiment allows one to determine which miRNAs change expression as a group or as a cluster. Genes that function together may define regulatory networks and regulate a com‐ mon set of regulated genes. Using clustering software, we divided the significantly regulat‐ ed miRNAs into different groups. In Figure 4 there were four different types of expression profiles among the miRNAs and genes. Some groups showed transient changes in the ex‐ pression profile (clusters B and C) while others stably increase (cluster A) or decrease (clus‐ ter D) during the treatment with MC-LR. Bearing in mind a variety of likely silencing targets for, and the onset of, the aberrant miRNAs expression (Table 2; [33]) it may be concluded that they are involved in diverse molecular pathways, such as liver cell metabolism, cell cy‐ cle regulation and apoptosis, and may contribute to the early phase of MC-LR induced hep‐ atotoxicity. Whereas, this argues that at least some of miRNAs listed in Table 2 are good candidates to pursue in future studies, a key to further elucidation of the miRNA role in the toxicity mechanism is the generation of more complete lists of their numbers and expression

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**MicroRNA\* Fold change Reported silencing targets Reference** let-7c 6.8 Rat sarcoma viral oncogene, RAS [34]

miR-9b 4.4 Caudal related homebox protein, CDX2 [35] miR-34a 4.0 B-cell lymphoma 2, BCL2 [46]

miR-16a 3.6 B-cell lymphoma 2, BCL2 [36] miR-122 2.7 Cationic amino acid transporter, CAT-1 [48]

**Table 2.** Reported mammalian silencing targets for differentially expressed miRNAs in MC-LR treated whitefish

ously confirmed to be a negative regulator of p53 in both zebrafish and humans [51].

On the other hand, the lack of p53 stabilization observed in our study infers the presence of alternate checkpoint mechanisms for deregulated growth signals and/or DNA damage in whitefish cells and may suggest post-transcriptional regulation of *p53*. Indeed, recent work by Liu and coworkers [49] suggest that two checkpoint kinases, ATM and ATR, which act upstream of p53, are promising candidates for the role. Further studies should also reveal if the lack of p53 induction in fish liver following exposure to many compounds known to cause DNA damage and DNA replication defects [49-50], is controlled by the miRNA net‐ work, a role it is known to fulfill in other organisms. For example, miR-125b has been previ‐

Myelocytomatosis viral related oncogene, neuroblastoma derived

Myelocytomatosis viral related oncogene, c-MYC [45]

[47]

changes in healthy and challenged fish.

Applications

232

(avian), MYCN

(100μg/kg body weight) after 24 h of the challenge [33].

\*Only miRNAs which were significantly up-regulated (p<0.05) are included in the column.

We are only beginning to understand the complexities of miRNA-mediated gene regulatory networks in fish cells. It should be expected that environmental contaminants that have the potential to induce oxidative stress and hypoxia in animal cells, like MCs, will also be agents deregulating miRNA expression. In our initial studies [4, 33] we observed rapid changes in liver microRNA levels of whitefish following MC-LR exposure. Bearing in mind a variety of likely silencing targets for and the onset of the aberrant miRNAs expression observed in the study, one may conclude that they are involved in various molecular pathways and may contribute to the early phase of MC-hepatotoxicity. This argues that studied miRNAs are good candidates to pursue in future studies, however, a key to further elucidation of the miRNA role in the toxicity mechanism will be the generation of more complete lists of their numbers and expression changes in healthy and challenged fish, using next generation se‐ quencing methods (Figure 2). As miRNA field continues to evolve, the new markers should help elucidating a variety of issues intrinsic to MC toxicity. As more profiling studies are performed after MC-LR treatment, and on different model organisms, it might be possible to obtain a miRNA snapshot map, the "core of the MC-LR toxicity connectivity grid". Finally, the revealed miRNA pathways underlying hepatotoxic effects of MC-LR may provide thera‐ peutic targets for a variety of liver diseases.
