Effect of a toxic Microcystis aeruginosa lysate on the mRNA expression of proto-oncogenes and tumor suppressor genes in zebrafish
Introduction
Over the past few decades, the frequency of occurrence and the global distribution of cyanobacteria blooms in water bodies has become a worldwide problem (Yan et al., 2012). This concern is mainly due to the capacity of several species of cyanobacteria to produce a variety of cyanotoxins (Cui et al., 2011). Microcystins (MCs) are commonly found in the aquatic environment and are produced by species of several genera of cyanobacteria, such Microcystis sp. (Amado and Monserrat, 2010). MC-LR is a very common hepatotoxin and its toxicity in diverse biological systems has been investigated (Abdel-Rahman et al., 1993, Qiao et al., 2013).
Studies have demonstrated the appearance of hepatic lesions in fish exposed to MC-LR in the laboratory (Fischer and Dietrich, 2000, Mezhoud et al., 2008). One of the most studied toxic mechanisms for MC-LR is inhibition of the phosphatases PP1 and PP2A, which can lead to protein hyperphosphorylation and cytoskeletal alterations (Campos and Vasconcelos, 2010, Mezhoud et al., 2008). On the other hand, MC-LR could also induces oxidative stress in mammal cells by alteration in intracellular reduced glutathione (GSH), reactive oxygen species (ROS) production and lipid peroxidation (Bouaïcha and Maatouk, 2004).
Tumor promoter activity caused by MCs has been attributed to PP2A inhibition, involving regulation of mitogen activated protein kinases (MAPKs) (Gehringer, 2004, Wang et al., 2013). MAPKs, once activated, regulate the expression of proto-oncogenes. For example, the proto-oncogenes fosab and junba are well-known targets of the MAPK pathway (Delaney et al., 2008, Zegura et al., 2011) and initiate transcription of genes involved in growth, differentiation and cellular proliferation (Gehringer, 2004, Li et al., 2009, Wang et al., 2013, Zegura et al., 2008). The transcriptional induction of proto-oncogenes has been linked to the promotion of tumor activity in different organs in rats, such as kidney, testis, brain and liver (De Felipe and Hunt, 1994, Li et al., 2009, Wang et al., 2013).
Some studies have provided evidence that MC-LR induces the expression of c-Jun, c-Fos and another proto-oncogene, called myca, in primary cultures of hepatocytes from rats and zebrafish (Li et al., 2009, Sueoka et al., 1997, Wei et al., 2008). The protein c-Jun is a positive regulator of proliferation and induces other regulators of cell cycle progression (Szremska et al., 2003, Wei et al., 2008) and c-Fos has oncogenic activity with frequent overexpression in tumor cells (Verde et al., 2007). According to Fan et al. (2014), the altered expression of myc proto-oncogene contributes to tumor development. This gene is activated in 20% of all human cancers and has been found to be active in tumors of other species (Dang, 2012, Dang et al., 1999). On the other hand, the organisms possess a variety of defense mechanisms against cellular stressors and tumor suppressor genes are one mechanism responsible for preventing severe damage to the cell. These genes often encode proteins that function as negative regulators of cell proliferation (Smart et al., 2008) and are necessary for maintaining cell integrity and cellular content. The well-known tumor suppressor gene, p53, is the gene most often mutated in cancer (Storer and Zon, 2010) and is conserved in structure and function, being very similar in mammals and zebrafish.
The p53 gene is a central factor in cellular stress responses, governing the adaptive and protective responses following several types of damage, such as DNA damage, hypoxia, nucleotide imbalance and oxidative stress (Levine, 1997). Several reports have argued that oxidative stress is a toxicological consequence of the exposure to MCs in different organisms (Amado and Monserrat, 2010; Yan et al., 2012) and plays a critical role in apoptosis (Fu et al., 2005) and genotoxic potential (Bouaïcha et al., 2005). After oxidative stress status caused by MC-LR, p53 can be activated and induces the expression of genes related to cell cycle arrest, apoptosis and DNA repair (Fu et al., 2005, Zegura et al., 2008). Similarly to what have been observed for animal cells, MC-LR induced mRNA expression of p53 in fish too, suggesting its involvement in the promotion of cell cycle arrest and apoptosis (Brzuzan et al., 2012, Brzuzan et al., 2009).
Among the various p53 target genes, gadd45α is one that operates in cell cycle control and DNA repair processes. The gadd45α gene removes a variety of DNA lesions or interrupts the cell cycle, preventing the replication of damaged DNA (Smart et al., 2008, Svircev et al., 2010, Zegura et al., 2008). If DNA damage is too severe, p53 can induce apoptosis through the regulation of genes that stimulate apoptotic pathways (Smart et al., 2008), such as baxa (Wang et al., 2013). Exposure to MC-LR is reported to cause a persistent increase of its transcriptional and protein levels in hepatocytes and testicular cells and it is responsible for cell death with cytochrome c release and expression of caspases (Fu et al., 2005, Wang et al., 2013).
Previous evidences indicated for biphasic effects of the MC-LR in fish liver, characterized by a severe injury at the beginning of the exposure and long term reconstruction of the liver parenchyma with inflammatory responses (Wozny et al., 2016). Thus, it would be interesting to see if biphasic effects were also observed for tumor-related genes in fish exposed to a toxic a M. aeruginosa. The alteration of the expression of proto-oncogenes and tumor suppressor genes has been studied with the aim to improve the understanding of toxicity of MC-LR and its carcinogenic potential (Li et al., 2009, Zegura et al., 2011). The present study evaluated the transcriptional response of the proto-oncogenes, fosab, junba and myca, and tumor suppressor genes, baxa, gadd45α and p53, in zebrafish (Danio rerio) after exposure to Microcystis aeruginosa lysate containing 3.5 and 54.6 µg L−1 [D-Leu1] MC-LR for 6, 24, 96 and 384 h.
Section snippets
Microcystis aeruginosa lysate
The cyanobacteria lysate used was obtained from a culture of M. aeruginosa originally isolated from the Patos Lagoon Estuary, Rio Grande, RS, Brazil. M. aeruginosa cells of RST 9501 strain producing cyanotoxin were cultivated at the Laboratory of Cyanobacteria and Phycotoxins, Oceanographic Institute of Federal University of Rio Grande (IO-FURG). Characterization of the MCs produced was previously reported by Matthiensen et al. (2000). The most abundant MC variant in that strain was a [D-Leu1]
Results
Mortality of fish was observed only with the longer exposure (384 h), where three fish died, one in the control group and two in the group exposed to the higher concentration of lysate.
All genes that were evaluated in liver of fish, including the proto-oncogenes fosab, junba and myca and the tumor suppressor genes baxa, gadd45α and p53, were repressed after exposure to M. aeruginosa lysate (p < 0.05 in respect to control). This transcriptional repression was biphasic, since some genes were
Discussion
In the present study, both tumor suppressor genes and proto-oncogenes were regulated at the transcriptional level. This response was demonstrated by a time-dependent downregulation of those genes. The results suggest that tumor suppressor genes are altered with shorter periods of exposure, while proto-oncogenes are altered with longer exposure times. Transcriptional repression can occur in a local or global manner by involving numerous cellular processes and can be limited to the time when
Conclusion
This study shows that exposure to M. aeruginosa lysate containing [D-Leu1] MC-LR caused suppression of gene expression in time-biphasic manner, affecting all genes analyzed in the liver of zebrafish, but also led to overexpression of p53 with exposure for 384 h to a moderate concentration. Most of the studies investigating the impact of cyanotoxins have focused on purified cyanotoxins (e.g. MC-LR) and have used in vitro tests or intraperitoneal injection. We used a lysate of M. aeruginosa and
Acknowledgements
Viviane Barneche Fonseca received support and financial assistance from a post-graduate scholarship funded by CAPES. This study was supported by funds from the Brazilian agency CNPq, approved by Juliano Zanette (480708/2013-4). J. Zanette and J. Sarkis Yunes are productivity research fellows from CNPq-Brazil.
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