**2. Body**

### **2.1. MicroRNAs in fish cells**

MicroRNAs (miRNAs) form a class of endogenously expressed small, non-coding RNAs, that play key roles in the regulation of gene expression of a broad spectrum of biological processes. Figure 1 summarizes crucial steps in microRNA processing. MiRNAs are tran‐ scribed by RNA polymerases II or III as primary transcripts (pri-miRNAs), which are fur‐

© 2013 Brzuzan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Brzuzan et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ther processed by the nuclear RNase III enzyme Drosha to stem-loop-structured miRNA precursor molecules (pre-miRNAs). The pre-miRNAs are subsequently transported to the cytoplasm where the RNase III enzyme Dicer cleaves off the double stranded (ds) portion of the hairpin and generates a short-lived dsRNA of about 19–23 nucleotides (nt) in size. The duplex is subsequently unwound and only one strand gives rise to the mature miR‐ NA, which is incorporated into miRNA-protein complexes (miRNPs) [1-2]. The mature miRNAs binds to partially complementary recognition sequences located in the 3'-un‐ translated regions (3'-UTRs) of mRNAs and target them for degradation or translational repression (reviewed in [3]).

In metazoans miRNA complementarity to their targets is far from perfect, so one miRNA can bind up to 200 targets, and each mRNA could have recognition sites for more than one miRNA. It is estimated that about 30% of the human protein-coding genes are nega‐ tively regulated by miRNA, which suggests that miRNAs are very important regulators of gene expression process [3]. Although specific functions and target mRNAs have been assigned to only a few dozen of miRNAs, much experimental evidence suggests that miR‐ NAs participate in the regulation of a vast spectrum of biological processes. miRNAs con‐ trol diverse cellular processes including animal development and growth, cell differentiation, signal transduction, cancer, neuronal disease, virus-induced immune de‐ fense, programmed cell death, insulin secretion, and metabolism (see [4] and references therein). Understanding of RNA interference (RNA*i*) has been made possible through a variety of experimental and bioinformatics approaches using different model organisms,

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To discover aberrantly expressed miRNAs in fish and to determine how altered miRNA function contributes to a disease, new RNA*i* technologies may be applied (Figure 2). In toxicological studies attention is focused on the relationship between exposure to a chem‐ ical and adverse effects it produces in cells, tissues or organisms. So, when a treatment study is carried out small RNA may be collected from a tissue to generate miRNA libra‐ ries, from either control or exposed fish. That is the first important step to establish the full repertoire of miRNAs that are differentially regulated in treated fish. Then the miR‐ NA libraries are subjected to massively parallel sequencing, a next generation sequencing technique, which is a combination of emulsion PCR and pyrosequencing [7]. In compari‐ son to microarray analyses, this approach is not limited to previously identified miRNAs and is expected to have superior sensitivity at high sequencing depth. Such approaches have expanded the catalogue of differentially expressed miRNA genes in various fish tis‐ sues [6]. The genome-wide screen for regulated miRNAs should yield candidate miRNAs for further profiling (Real Time qPCR) and functional analyses (e.g. Renilla luciferase re‐

As miRNAs regulate many different pathways and orchestrate integrated responses in cells and tissues, it is reasonable to think that they also play key roles in coordinating networks in the poisoned organs. Indeed, there are reports concluding that miRNAs may be key mole‐ cules involved in aberrant gene expression in liver cells exposed to hepatotoxic agents, other than MC-LR. For example, Fukushima and co-workers [8] have shown that two well known hepatotoxicants which induce hepatocellular injuries and necrosis, acetaminophen or carbon tetrachloride, were capable of modulating expression of two miRNAs (miR-298 and miR-370) in rats, and that those effects were accompanied by impaired liver metabolism. The observation that miRNAs levels in rat livers were changed by hepatotoxic compounds prompted our team to investigate the role of fish microRNAs in the context of liver-specific

including fish [5-6].

porter assay).

MC-LR toxicity.

**Figure 1.** miRNA processing and target recognition. The pri-miRNA is processed by the Drosha enzyme to a stem-loopstructured miRNA precursor molecule (pre-miRNA). The pre-miRNAs is transported to the cytoplasm where the Dicer enzyme cleaves off the double stranded (ds) portion of the hairpin and generates the mature miRNA, which is incor‐ porated into miRNA-protein complexes (miRNPs). The mature miRNA binds to partially complementary recognition se‐ quences on 3'-UTRs of mRNAs and targets them for decay or translational repression.

In metazoans miRNA complementarity to their targets is far from perfect, so one miRNA can bind up to 200 targets, and each mRNA could have recognition sites for more than one miRNA. It is estimated that about 30% of the human protein-coding genes are nega‐ tively regulated by miRNA, which suggests that miRNAs are very important regulators of gene expression process [3]. Although specific functions and target mRNAs have been assigned to only a few dozen of miRNAs, much experimental evidence suggests that miR‐ NAs participate in the regulation of a vast spectrum of biological processes. miRNAs con‐ trol diverse cellular processes including animal development and growth, cell differentiation, signal transduction, cancer, neuronal disease, virus-induced immune de‐ fense, programmed cell death, insulin secretion, and metabolism (see [4] and references therein). Understanding of RNA interference (RNA*i*) has been made possible through a variety of experimental and bioinformatics approaches using different model organisms, including fish [5-6].

ther processed by the nuclear RNase III enzyme Drosha to stem-loop-structured miRNA precursor molecules (pre-miRNAs). The pre-miRNAs are subsequently transported to the cytoplasm where the RNase III enzyme Dicer cleaves off the double stranded (ds) portion of the hairpin and generates a short-lived dsRNA of about 19–23 nucleotides (nt) in size. The duplex is subsequently unwound and only one strand gives rise to the mature miR‐ NA, which is incorporated into miRNA-protein complexes (miRNPs) [1-2]. The mature miRNAs binds to partially complementary recognition sequences located in the 3'-un‐ translated regions (3'-UTRs) of mRNAs and target them for degradation or translational

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

**Figure 1.** miRNA processing and target recognition. The pri-miRNA is processed by the Drosha enzyme to a stem-loopstructured miRNA precursor molecule (pre-miRNA). The pre-miRNAs is transported to the cytoplasm where the Dicer enzyme cleaves off the double stranded (ds) portion of the hairpin and generates the mature miRNA, which is incor‐ porated into miRNA-protein complexes (miRNPs). The mature miRNA binds to partially complementary recognition se‐

quences on 3'-UTRs of mRNAs and targets them for decay or translational repression.

repression (reviewed in [3]).

Applications

224

To discover aberrantly expressed miRNAs in fish and to determine how altered miRNA function contributes to a disease, new RNA*i* technologies may be applied (Figure 2). In toxicological studies attention is focused on the relationship between exposure to a chem‐ ical and adverse effects it produces in cells, tissues or organisms. So, when a treatment study is carried out small RNA may be collected from a tissue to generate miRNA libra‐ ries, from either control or exposed fish. That is the first important step to establish the full repertoire of miRNAs that are differentially regulated in treated fish. Then the miR‐ NA libraries are subjected to massively parallel sequencing, a next generation sequencing technique, which is a combination of emulsion PCR and pyrosequencing [7]. In compari‐ son to microarray analyses, this approach is not limited to previously identified miRNAs and is expected to have superior sensitivity at high sequencing depth. Such approaches have expanded the catalogue of differentially expressed miRNA genes in various fish tis‐ sues [6]. The genome-wide screen for regulated miRNAs should yield candidate miRNAs for further profiling (Real Time qPCR) and functional analyses (e.g. Renilla luciferase re‐ porter assay).

As miRNAs regulate many different pathways and orchestrate integrated responses in cells and tissues, it is reasonable to think that they also play key roles in coordinating networks in the poisoned organs. Indeed, there are reports concluding that miRNAs may be key mole‐ cules involved in aberrant gene expression in liver cells exposed to hepatotoxic agents, other than MC-LR. For example, Fukushima and co-workers [8] have shown that two well known hepatotoxicants which induce hepatocellular injuries and necrosis, acetaminophen or carbon tetrachloride, were capable of modulating expression of two miRNAs (miR-298 and miR-370) in rats, and that those effects were accompanied by impaired liver metabolism. The observation that miRNAs levels in rat livers were changed by hepatotoxic compounds prompted our team to investigate the role of fish microRNAs in the context of liver-specific MC-LR toxicity.

ponds and rivers used for recreational activities as well as in sources for drinking water preparation [9]. In surface waters, concentrations of total MCs (cell-bound and dissolved) measured with ELISA may reach high levels, of up to 1300 μg/l [9], and thus the toxins may pose a threat to aquatic organisms and humans [10]; the World Health Organization recom‐ mends 1 μg/l as the maximum acceptable level for microcystin-LR (MC-LR) in drinking wa‐ ter [11]. So far, more than 100 different structural analogues of MCs have been identified,

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MCs have strong affinity to serine/threonine specific protein phosphatases (PP1 and PP2A), thereby acting as inhibitors of the enzymes [13]. The acute toxicity of MC can be explained by the PP inhibition, which leads to an excessive phosphorylation of cell proteins, to altera‐ tions in the cytoskeleton, and a loss of cell shape [14]. Another biochemical feature of MC toxicity is the production of reactive oxygen species (ROS). MC-related ROS generation has been reported using both *in vitro* approaches with different cell lines of fish and mammals [15-16], as well as in a number of *in vivo* studies in rodent liver, heart and reproductive sys‐ tem [17-19]. This process is related to mitochondrial metabolism and it may lead to cell death and to genotoxicity [20]. Oxidative stress caused by MC exposure is believed to be in‐ volved in a series of heart, liver and kidney pathologies [19, 21], neurodegenerative effects

In recent years, new insights on the key molecules involved in the signal-transduction and toxicity have been reported [24], which highlighted the complexity of the interaction of these toxins with animal cells (Figure 3). Key proteins involved in MC up-take, biotransformation and excretion have been identified, demonstrating the ability of aquatic animals to metabo‐ lize and excrete the toxin. After having caused damage to intestinal (or gill) cells these toxins penetrate liver cell membranes through a bile acid carrier. In liver cells MCs inhibit serine/ threonine-specific protein phosphatases, PP1 and PP2A, through the binding to them, thus perturbing signaling pathway controlled by the enzymes. The consequences are induction of mitochondrial permeability and loss of mitochondrial membrane potential leading to dys‐ function of the mitochondria, induction of reactive oxygen species (ROS), DNA damage (through lowered expression of DNA-PK), and cell apoptosis (through increase Ca2+ levels, CaMKII). MC activity leads to the differential expression/activity of transcriptional factors (e.g. c-myc, p53) and protein kinases (NeK2) involved in the pathways of cellular differen‐

In the field of toxinology, a science of naturally occurring toxins, the relationship between toxicity and microRNA expression is poorly understood. However, based on current knowl‐ edge about genes involved in the animal cell response on the exposure to environmental stressors, putative targets for miRNA regulation emerge. Genes of transcription factors, *p53* and mapk (mitogen activated protein kinases) regulated proto-oncogenes e.g. *c-myc*, that are involved in MC-LR toxicity (Figure 3), are good candidates for tight and robust regulation by microRNAs. The nuclear phosphoprotein p53 is induced in response to cellular stress. It plays a role as a transcriptional trans-activator in DNA repair, apoptosis and tumor suppres‐

among which MC-LR is one of the most common and abundant [12].

tiation, proliferation, tumor promotion activity, and metastasis [25].

**2.3. Likely silencing targets in MC-exposed fish cells**

[22] and embryotoxicity [23].

**Figure 2.** Studying fish miRNAs. MicroRNA discovery has been recently revolutionized by next-generation sequencing. Following ligation of specific linkers to small RNAs (which comprise miRNAs), cDNAs can be produced, which are ideal‐ ly suited to sequencing using short-read platforms. Databases now offer online catalogues of known microRNAs, which may further be examined for their pathways and functions through a variety of approaches, such as target functional analysis of candidate miRNAs using luciferase reporter assays and miRNA profiling with Real Time PCR.

#### **2.2. Microcystins as potent cyanobacterial toxins**

Microcystins (MCs) are potent hepatotoxins produced by cyanobacteria of the genera *Plank‐ tothrix*, *Microcystis*, *Aphanizomenon*, *Nostoc*, or *Anabaena*, which have received worldwide concern in recent decades. Mass growths of cyanobacteria, leading to production of blooms, scums and mats, can occur in nutrient-enriched waterbodies (particularly with phosphorus and nitrogen), enhanced by higher temperature and pH values. MCs can be found in lakes, ponds and rivers used for recreational activities as well as in sources for drinking water preparation [9]. In surface waters, concentrations of total MCs (cell-bound and dissolved) measured with ELISA may reach high levels, of up to 1300 μg/l [9], and thus the toxins may pose a threat to aquatic organisms and humans [10]; the World Health Organization recom‐ mends 1 μg/l as the maximum acceptable level for microcystin-LR (MC-LR) in drinking wa‐ ter [11]. So far, more than 100 different structural analogues of MCs have been identified, among which MC-LR is one of the most common and abundant [12].

MCs have strong affinity to serine/threonine specific protein phosphatases (PP1 and PP2A), thereby acting as inhibitors of the enzymes [13]. The acute toxicity of MC can be explained by the PP inhibition, which leads to an excessive phosphorylation of cell proteins, to altera‐ tions in the cytoskeleton, and a loss of cell shape [14]. Another biochemical feature of MC toxicity is the production of reactive oxygen species (ROS). MC-related ROS generation has been reported using both *in vitro* approaches with different cell lines of fish and mammals [15-16], as well as in a number of *in vivo* studies in rodent liver, heart and reproductive sys‐ tem [17-19]. This process is related to mitochondrial metabolism and it may lead to cell death and to genotoxicity [20]. Oxidative stress caused by MC exposure is believed to be in‐ volved in a series of heart, liver and kidney pathologies [19, 21], neurodegenerative effects [22] and embryotoxicity [23].

In recent years, new insights on the key molecules involved in the signal-transduction and toxicity have been reported [24], which highlighted the complexity of the interaction of these toxins with animal cells (Figure 3). Key proteins involved in MC up-take, biotransformation and excretion have been identified, demonstrating the ability of aquatic animals to metabo‐ lize and excrete the toxin. After having caused damage to intestinal (or gill) cells these toxins penetrate liver cell membranes through a bile acid carrier. In liver cells MCs inhibit serine/ threonine-specific protein phosphatases, PP1 and PP2A, through the binding to them, thus perturbing signaling pathway controlled by the enzymes. The consequences are induction of mitochondrial permeability and loss of mitochondrial membrane potential leading to dys‐ function of the mitochondria, induction of reactive oxygen species (ROS), DNA damage (through lowered expression of DNA-PK), and cell apoptosis (through increase Ca2+ levels, CaMKII). MC activity leads to the differential expression/activity of transcriptional factors (e.g. c-myc, p53) and protein kinases (NeK2) involved in the pathways of cellular differen‐ tiation, proliferation, tumor promotion activity, and metastasis [25].

### **2.3. Likely silencing targets in MC-exposed fish cells**

**Figure 2.** Studying fish miRNAs. MicroRNA discovery has been recently revolutionized by next-generation sequencing. Following ligation of specific linkers to small RNAs (which comprise miRNAs), cDNAs can be produced, which are ideal‐ ly suited to sequencing using short-read platforms. Databases now offer online catalogues of known microRNAs, which may further be examined for their pathways and functions through a variety of approaches, such as target functional analysis of candidate miRNAs using luciferase reporter assays and miRNA profiling with Real Time PCR.

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Applications

226

Microcystins (MCs) are potent hepatotoxins produced by cyanobacteria of the genera *Plank‐ tothrix*, *Microcystis*, *Aphanizomenon*, *Nostoc*, or *Anabaena*, which have received worldwide concern in recent decades. Mass growths of cyanobacteria, leading to production of blooms, scums and mats, can occur in nutrient-enriched waterbodies (particularly with phosphorus and nitrogen), enhanced by higher temperature and pH values. MCs can be found in lakes,

**2.2. Microcystins as potent cyanobacterial toxins**

In the field of toxinology, a science of naturally occurring toxins, the relationship between toxicity and microRNA expression is poorly understood. However, based on current knowl‐ edge about genes involved in the animal cell response on the exposure to environmental stressors, putative targets for miRNA regulation emerge. Genes of transcription factors, *p53* and mapk (mitogen activated protein kinases) regulated proto-oncogenes e.g. *c-myc*, that are involved in MC-LR toxicity (Figure 3), are good candidates for tight and robust regulation by microRNAs. The nuclear phosphoprotein p53 is induced in response to cellular stress. It plays a role as a transcriptional trans-activator in DNA repair, apoptosis and tumor suppres‐

sion pathways. Interestingly, the protein is a substrate of PP2A [26] and therefore its activity is likely to be regulated, in part, by MC-LR. Furthermore, p53 is a regulator of the expression of the anti- and pro-apoptotic genes including members of the BCL-2 family such as *BCL-2* and *BAX*, as well as *CDKN1A*, encoding p21Cip1, which is a cyclin dependent kinase inhibitor (CDKI), an important effector that acts by inhibiting CDK activity in p53-mediated cell cycle arrest in response to various agents. Indeed, we have shown previously that intraperitoneal injection of whitefish, *Coregonus lavaretus*, with MC-LR at subacute dose of 100 μg/kg body weight induced mRNA expression of tumor suppressor p53 and cyclin dependent kinase in‐ hibitor 1 (cdkn1a) in the liver of exposed fish [27]. Interestingly, it was proven in human cell lines that p53 is a transcription factor for some miRNAs, such as miR-34a [7]. miR-34a medi‐ ates some of the well-known effects of p53, i.e. cell cycle arrest or apoptosis, and reduced miR-34a levels can serve as a biomarker for any dysfunction along the p53 axis [28]. Yet, its role in controlling miRNA network in fish awaits investigation (Figure 3).

venously with MC-LR, with higher levels registered in liver [30]. Expression of these genes suggest that a possible mechanism for the tumor-promoting activity of the toxin could be controlled by MAPKs. Importantly, c-MYC controls expression of let-7 miRNA members by binding to their promoters. The levels of let-7 have been reported to decrease in models of MYC-mediated tumorigenesis, and to increase when MYC is inhibited by chemicals [31]. It is also found that MYC can repress p21Cip1 transcription (Figure 1), thereby overriding a p21-

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In 2008, we began a study of MC-LR induced transcriptional changes in European whitefish, *Coregonus lavaretus* L., a sentinel organism frequently used for pollution monitoring in aquatic systems [27]. To obtain necessary information for the study, full-length cDNA of p53 or cdkn1a of whitefish were determined, using molecular cloning and rapid amplification of cDNA ends (RACE). The *short term* treatment study showed that MC-LR at a dose of 100 μg/kg body weight induced hepatocyte cell DNA fragmentation and up-regulated mRNA expression of p53 and cdkn1a genes in whitefish liver. Interestingly, the elevated transcript levels of both genes were observed only from 48 through the 72 h of exposure, and were ac‐ companied by pathological signs of severe injury of the liver and loss of normal organ func‐

Whereas, the above study confirms that MC-LR exposure underlies various acute and chronic effects in fish, it is still little known about aberrant gene expression profiles and mo‐ lecular pathways involved in the liver of MC-LR challenged organisms. Therefore, to im‐ prove our knowledge about adverse effects of MC-LR on hepatocyte cell responses in fish, we performed an initial microRNA study to examine the abundance of 9 selected miRNAs (omy-miR-21, omy-miR-21t, omy-miR-122, omy-miR-125a, omy-miR-125b, omy-miR-125t, omy-miR-199-5a, omy-miR-295, omy-let-7a), in liver samples of whitefish exposed for 24 or 48h to MC-LR at a dose of 100μg/kg body weight [4]. Interestingly, the study showed that MC-LR treatment affected expression levels of two miRNAs, omy-miR-125a (up-regulation)

Following the early demonstration that MC-LR modulates expression of let-7a and miR-125a, in our most recent work [33] we aimed at profiling expression of other 6 miRNAs and 8 mRNAs (Table 1) in the liver of challenged whitefish during the first 48 h after single intraperitoneal injection. From studies on mammals we chose miRNAs which play regulato‐ ry roles in pathways of signal transduction (let-7c, [34]; miR-9b, [35]), apoptosis and cell cy‐ cle (miR-16a, [36]; miR-21a, [37]; miR-34a, [7]) and fatty-acid metabolism (miR-122, which is a liver specific miRNA, [38]). The selection of mRNA targets (Table 1) was based on their reported aberrant tissue expression on exposure to environmental stressors, and included mRNAs involved in apoptosis and cell cycle (bax, [20]; cas6, cdkn1a, p53, [27]), signal trans‐ duction (p-ras, [39]), cellular iron homeostasis (frih, [40]), gene silencing by miRNAs (dcr, [41]), and nucleosome assembly (h2a, [4]). Together with the RNA expression, we analyzed levels of tumor suppressor protein p53 to assess its potential contribution in molecular

tions (elevated levels of blood AspAT AlaAT, and hepatosomatic index; [27]).

mediated cell cycle checkpoint [32].

and omy-let-7a (down-regulation) [4].

mechanisms of liver toxicity induced by MCs in fish.

**2.4. miRNA expression in whitefish exposed to MC-LR**

**Figure 3.** Suggested pathways of MC up-take, toxicity, biotransformation and excretion in vertebrates. Based on the current knowledge, microRNAs (e.g. let-7 or miR-34a) may play roles in MC-LR dependent cell proliferation, cell-cycle arrest or apoptosis.

In the other pathway (Figure 3), mitogen-activated protein kinases (MAPKs) regulate the ex‐ pression of proto-oncogenes which on the other hand regulate the transcription of genes in‐ volved in the growth and differentiation [29]. Expression of MAPKs is mediated by PP2A and are likely to be regulated by MC. The expression of three proto-oncogenes c-fos, c-jun and c-myc were reported to increase in liver, kidney and testis of Wistar rats injected intra‐ venously with MC-LR, with higher levels registered in liver [30]. Expression of these genes suggest that a possible mechanism for the tumor-promoting activity of the toxin could be controlled by MAPKs. Importantly, c-MYC controls expression of let-7 miRNA members by binding to their promoters. The levels of let-7 have been reported to decrease in models of MYC-mediated tumorigenesis, and to increase when MYC is inhibited by chemicals [31]. It is also found that MYC can repress p21Cip1 transcription (Figure 1), thereby overriding a p21 mediated cell cycle checkpoint [32].

### **2.4. miRNA expression in whitefish exposed to MC-LR**

sion pathways. Interestingly, the protein is a substrate of PP2A [26] and therefore its activity is likely to be regulated, in part, by MC-LR. Furthermore, p53 is a regulator of the expression of the anti- and pro-apoptotic genes including members of the BCL-2 family such as *BCL-2* and *BAX*, as well as *CDKN1A*, encoding p21Cip1, which is a cyclin dependent kinase inhibitor (CDKI), an important effector that acts by inhibiting CDK activity in p53-mediated cell cycle arrest in response to various agents. Indeed, we have shown previously that intraperitoneal injection of whitefish, *Coregonus lavaretus*, with MC-LR at subacute dose of 100 μg/kg body weight induced mRNA expression of tumor suppressor p53 and cyclin dependent kinase in‐ hibitor 1 (cdkn1a) in the liver of exposed fish [27]. Interestingly, it was proven in human cell lines that p53 is a transcription factor for some miRNAs, such as miR-34a [7]. miR-34a medi‐ ates some of the well-known effects of p53, i.e. cell cycle arrest or apoptosis, and reduced miR-34a levels can serve as a biomarker for any dysfunction along the p53 axis [28]. Yet, its

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

**Figure 3.** Suggested pathways of MC up-take, toxicity, biotransformation and excretion in vertebrates. Based on the current knowledge, microRNAs (e.g. let-7 or miR-34a) may play roles in MC-LR dependent cell proliferation, cell-cycle

In the other pathway (Figure 3), mitogen-activated protein kinases (MAPKs) regulate the ex‐ pression of proto-oncogenes which on the other hand regulate the transcription of genes in‐ volved in the growth and differentiation [29]. Expression of MAPKs is mediated by PP2A and are likely to be regulated by MC. The expression of three proto-oncogenes c-fos, c-jun and c-myc were reported to increase in liver, kidney and testis of Wistar rats injected intra‐

arrest or apoptosis.

Applications

228

role in controlling miRNA network in fish awaits investigation (Figure 3).

In 2008, we began a study of MC-LR induced transcriptional changes in European whitefish, *Coregonus lavaretus* L., a sentinel organism frequently used for pollution monitoring in aquatic systems [27]. To obtain necessary information for the study, full-length cDNA of p53 or cdkn1a of whitefish were determined, using molecular cloning and rapid amplification of cDNA ends (RACE). The *short term* treatment study showed that MC-LR at a dose of 100 μg/kg body weight induced hepatocyte cell DNA fragmentation and up-regulated mRNA expression of p53 and cdkn1a genes in whitefish liver. Interestingly, the elevated transcript levels of both genes were observed only from 48 through the 72 h of exposure, and were ac‐ companied by pathological signs of severe injury of the liver and loss of normal organ func‐ tions (elevated levels of blood AspAT AlaAT, and hepatosomatic index; [27]).

Whereas, the above study confirms that MC-LR exposure underlies various acute and chronic effects in fish, it is still little known about aberrant gene expression profiles and mo‐ lecular pathways involved in the liver of MC-LR challenged organisms. Therefore, to im‐ prove our knowledge about adverse effects of MC-LR on hepatocyte cell responses in fish, we performed an initial microRNA study to examine the abundance of 9 selected miRNAs (omy-miR-21, omy-miR-21t, omy-miR-122, omy-miR-125a, omy-miR-125b, omy-miR-125t, omy-miR-199-5a, omy-miR-295, omy-let-7a), in liver samples of whitefish exposed for 24 or 48h to MC-LR at a dose of 100μg/kg body weight [4]. Interestingly, the study showed that MC-LR treatment affected expression levels of two miRNAs, omy-miR-125a (up-regulation) and omy-let-7a (down-regulation) [4].

Following the early demonstration that MC-LR modulates expression of let-7a and miR-125a, in our most recent work [33] we aimed at profiling expression of other 6 miRNAs and 8 mRNAs (Table 1) in the liver of challenged whitefish during the first 48 h after single intraperitoneal injection. From studies on mammals we chose miRNAs which play regulato‐ ry roles in pathways of signal transduction (let-7c, [34]; miR-9b, [35]), apoptosis and cell cy‐ cle (miR-16a, [36]; miR-21a, [37]; miR-34a, [7]) and fatty-acid metabolism (miR-122, which is a liver specific miRNA, [38]). The selection of mRNA targets (Table 1) was based on their reported aberrant tissue expression on exposure to environmental stressors, and included mRNAs involved in apoptosis and cell cycle (bax, [20]; cas6, cdkn1a, p53, [27]), signal trans‐ duction (p-ras, [39]), cellular iron homeostasis (frih, [40]), gene silencing by miRNAs (dcr, [41]), and nucleosome assembly (h2a, [4]). Together with the RNA expression, we analyzed levels of tumor suppressor protein p53 to assess its potential contribution in molecular mechanisms of liver toxicity induced by MCs in fish.


evolution. Indeed, the order of individual miRNA abundances in human liver (miR-122 > let-7c

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Our treatment study [33] identified miRNAs whose expression levels rose (from 2.7-fold for miR-122 to 6.8-fold for let-7c) in MC-LR treated fish, compared to the respective levels in control fish (Figure 4). The increase, which was most apparent at 24 h of the experiment, was correlated with a reduction in the expression of mRNAs: ferritin H (frih) and HNK Ras –like protein (p-ras) and an overexpression of bcl2-associated X protein (bax), cyclin dependent kinase inhibitor 1a (cdkn1a), dicer (dcr), histone 2A (h2a) and p53. Expression of the remain‐ ing caspase 6 (cas6) mRNA did not change over 48 h of the treatment. Moreover, exposure

**Figure 4.** Heat map and hierarchical clustering of differentially expressed genes and miRNAs in MC-LR treated white‐ fish. Each row represents one gene/miRNA and each column represents a mean of 5 replicates/duration of exposure. Colors represent expression levels of each individual gene/miRNA: red, up-regulation; green, down-regulation. Four distinct clusters (A through D) based on the observed expression profiles could be identified by the analysis. The analy‐ sis and visualization were performed using GenEx 5 software (MultiD Analyses AB; Sweden), based on raw expression

≈ miR-21 ≈ miR16 > miR-34a > miR-9; [43]) held in whitefish as well [33].

to MC-LR did not alter whitefish p53 protein levels [33].

data from our recent study [33].

\* based on literature review; see text for details.

\*\* putative *miR-34* gene is present in *Salmo salar* genome; Contig\_142190, whole genome shotgun se‐ quence, GenBank ACC. No. AGKD01142167.1, nucleotides from 5978 through 6053.

\*\*\* in terms of Gene Ontology Annotation (http://www.ebi.ac.uk/QuickGO).

**Table 1.** miRNA and mRNA targets selected under study.

Quantifying miRNAs in different tissues is an important initial step in investigating their biolog‐ ical functions. To this end, we determined the expression levels of 6 selected miRNAs in adult whitefish liver using Real-Time qPCR. Prominent expression of miR-122 in the liver of white‐ fish was observed which is consistent with other data from fish [4,6] and mammals [42]. Varia‐ ble expression levels of other miRNAs studied in the liver of whitefish corroborated results of previous work on normal human tissues [43], and they are also in agreement with available da‐ ta on the fish miRNome isolated from rainbow trout [6] and zebrafish [44] miRNA libraries. While the actual expression values of miRNAs can vary by orders of magnitude between whitefish and humans [43], their relative abundance in a particular tissue should tend to be more conserved in evolution. Indeed, the order of individual miRNA abundances in human liver (miR-122 > let-7c ≈ miR-21 ≈ miR16 > miR-34a > miR-9; [43]) held in whitefish as well [33].

miRNA Putative biological process\*

dre-miR-34\*\* cell cycle, signal transduction

mRNA [gene abbreviation] Biological process\*\*\*

bcl2-associated X protein

cyclin-dependent kinase inhibitor 1a

(bax)

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caspase 6 (cas6)

(cdkn1a)

ferritin heavy chain

tumor protein 53

HNK Ras –like protein

\* based on literature review; see text for details.

**Table 1.** miRNA and mRNA targets selected under study.

dicer (dcr)

(frih)

(p53)

(p-ras)

histone 2A (h2a)

omy-miR-122 fatty-acid metabolism, maintenance of adult liver phenotype

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

apoptosis

apoptosis

cell cycle

gene silencing by miRNA

nucleosome assembly

signal transduction

\*\* putative *miR-34* gene is present in *Salmo salar* genome; Contig\_142190, whole genome shotgun se‐

Quantifying miRNAs in different tissues is an important initial step in investigating their biolog‐ ical functions. To this end, we determined the expression levels of 6 selected miRNAs in adult whitefish liver using Real-Time qPCR. Prominent expression of miR-122 in the liver of white‐ fish was observed which is consistent with other data from fish [4,6] and mammals [42]. Varia‐ ble expression levels of other miRNAs studied in the liver of whitefish corroborated results of previous work on normal human tissues [43], and they are also in agreement with available da‐ ta on the fish miRNome isolated from rainbow trout [6] and zebrafish [44] miRNA libraries. While the actual expression values of miRNAs can vary by orders of magnitude between whitefish and humans [43], their relative abundance in a particular tissue should tend to be more conserved in

quence, GenBank ACC. No. AGKD01142167.1, nucleotides from 5978 through 6053.

\*\*\* in terms of Gene Ontology Annotation (http://www.ebi.ac.uk/QuickGO).

cellular iron ion homeostasis

apoptosis, cell cycle, signal transduction

omy-let-7c signal transduction omy-miR-9b signal transduction omy-miR-16a apoptosis, cell cycle omy-miR-21a apoptosis, cell cycle

Our treatment study [33] identified miRNAs whose expression levels rose (from 2.7-fold for miR-122 to 6.8-fold for let-7c) in MC-LR treated fish, compared to the respective levels in control fish (Figure 4). The increase, which was most apparent at 24 h of the experiment, was correlated with a reduction in the expression of mRNAs: ferritin H (frih) and HNK Ras –like protein (p-ras) and an overexpression of bcl2-associated X protein (bax), cyclin dependent kinase inhibitor 1a (cdkn1a), dicer (dcr), histone 2A (h2a) and p53. Expression of the remain‐ ing 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 [33].

**Figure 4.** Heat map and hierarchical clustering of differentially expressed genes and miRNAs in MC-LR treated white‐ fish. Each row represents one gene/miRNA and each column represents a mean of 5 replicates/duration of exposure. Colors represent expression levels of each individual gene/miRNA: red, up-regulation; green, down-regulation. Four distinct clusters (A through D) based on the observed expression profiles could be identified by the analysis. The analy‐ sis and visualization were performed using GenEx 5 software (MultiD Analyses AB; Sweden), based on raw expression data from our recent study [33].

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 changes in healthy and challenged fish.

**3. Conclusions**

peutic targets for a variety of liver diseases.

**Acknowledgments**

**Author details**

**References**

project UWM No. 0809-0801.

Paweł Brzuzan1\*, Maciej Woźny<sup>1</sup>

2004;431(7006): 343-349.

sity of Warmia and Mazury in Olsztyn, Olsztyn, Poland

versity of Warmia and Mazury in Olsztyn, Olsztyn, Poland

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‐

Discovering the Role of MicroRNAs in Microcystin-Induced Toxicity in Fish

http://dx.doi.org/10.5772/52204

233

This work was supported by the Polish Ministry of Science and Higher Education (MNiSW),

1 Department of Environmental Biotechnology, Faculty of Environmental Sciences, Univer‐

2 Department of Chemistry, Faculty of Environmental Management and Agriculture, Uni‐

[1] Meister G, Tuschl T. Mechanisms of gene silencing by double-stranded RNA. Nature

and Michał K. Łuczyński<sup>2</sup>

, Lidia Wolińska<sup>1</sup>


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

**Table 2.** Reported mammalian silencing targets for differentially expressed miRNAs in MC-LR treated whitefish (100μg/kg body weight) after 24 h of the challenge [33].

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