Elsevier

Ecotoxicology and Environmental Safety

Volume 161, 15 October 2018, Pages 729-734
Ecotoxicology and Environmental Safety

Effect of a toxic Microcystis aeruginosa lysate on the mRNA expression of proto-oncogenes and tumor suppressor genes in zebrafish

https://doi.org/10.1016/j.ecoenv.2018.06.007Get rights and content

Highlights

  • Tumor-related genes were evaluated in zebrafish exposed to a toxic M. aeruginosa.

  • p53, baxa and gadd45α were downregulated in early times of exposure.

  • In longer times of exposure, fosab, junba, myca were repressed.

  • p53 was induced by exposure to M. aeruginosa for 384 h.

Abstract

Cyanobacterial blooms of Microcystis aeruginosa represent a significant risk to the environment and have become a worldwide concern. M. aeruginosa can produce the hepatotoxins microcystins (MCs) with potential for tumor promotion. The present study evaluated the time-dependent effects in the transcription of tumor-related genes in the zebrafish, Danio rerio, exposed to dilutions of a M. aeruginosa lysate containing 3.5 and 54.6 µg L−1 MCs. We used a cultured M. aeruginosa strain, RST 9501, which contains mainly the variant [D-Leu1] MC-LR and originated from the Patos Lagoon Estuary (RS, Brazil). The exposure caused short-term repression of tumor suppressor genes and long-term repression of proto-oncogenes. These responses were more evident for p53 that was repressed with exposure for 6, 24 and 96 h, and fosab and myca that were consistently repressed with exposure for 384 h, when fish were exposed to both M. aeruginosa lysate dilutions, compared to controls (p < 0.05). The suppressor genes, baxa and gadd45α, and the proto-oncogene, junba, were suppressed mainly at 96 h, where both dilutions of the lysate caused repression compared to controls (p < 0.05). The p53 gene was the only gene to be induced; this occurred in fish exposed to lysate containing 3.5 µg L−1 for 384 h. This is the first study to show that M. aeruginosa containing an environmentally relevant concentration of [D-Leu1] MC-LR could cause time-dependent repression of proto-oncogenes and tumor suppressor genes in fish. The results suggest that short-term repression of tumor suppressor genes could participate in the mechanism of tumor promotion caused by M. aeruginosa in fish.

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.

References (55)

  • W.J. Fischer et al.

    Pathological and biochemical characterization of microcystin-induced hepatopancreas and kidney damage in carp (Cyprinus carpio)

    Toxicol. Appl. Pharmacol.

    (2000)
  • M.M. Gehringer

    Microcystin-LR and okadaic acid-induced cellular effects: a dualistic response

    FEBS Lett.

    (2004)
  • K. Hercog et al.

    Genotoxic potential of the binary mixture of cyanotoxins microcystin- LR and cylindrospermopsin

    Chemosphere

    (2017)
  • H. Li et al.

    In vivo study on the effects of microcystin extracts on the expression profiles of proto-oncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins

    Toxicon

    (2009)
  • X. Li et al.

    Alterations in transcription and protein expressions of HCC-related genes in HepG2 cells caused by microcystin-LR

    Toxicol. In Vitro

    (2017)
  • A. Matthiensen et al.

    [D-Leu1]Microcystin-LR, from the cyanobacterium Microcystis RST9501 and from a Microcystis bloom in the Patos Lagoon estuary, Brazil

    Eur. J. Phytochem.

    (2000)
  • K. Mezhoud et al.

    Proteomic and phosphoproteomic analysis of cellular responses in medaka fish (Oryzias latipes) following oral gavage with microcystin-LR

    Toxicon

    (2008)
  • S. Pavagadhi et al.

    Biochemical response of diverse organs in adult Danio rerio (zebrafish) exposed to sub-lethal concentrations of microcystin-LR and microcystin-RR: a balneation study

    Aquat. Toxicol.

    (2012)
  • Q. Qiao et al.

    Female zebrafish (Danio rerio) are more vulnerable than males to microcystin-LR exposure, without exhibiting estrogenic effects

    Aquat. Toxicol.

    (2013)
  • R. Samarakoon et al.

    Linking cell structure to gene regulation: signaling events and expression controls on the model genes PAI-1 and CTGF

    Cell. Signal.

    (2010)
  • S.R. Saraf et al.

    Effects of Microcystis on development of early life stage Japanese medaka (Oryzias latipes): comparative toxicity of natural blooms cultured Microcystis and microcystin-LR

    Aquat. Toxicol.

    (2018)
  • A.P. Szremska et al.

    Jun B inhibits proliferation and transformation in B-lymphoid cells

    Blood

    (2003)
  • L. Vergani et al.

    Modifications of chromatin structure and gene expression following induced alterations of cellular shape

    Int. J. Biochem. Cell Biol.

    (2004)
  • X. Wang et al.

    Microcystin (-LR) induced testicular cell apoptosis via up-regulating apoptosis-related genes in vivo

    Food Chem. Toxicol.

    (2013)
  • L. Wei et al.

    Gene expression profiles in liver of zebrafish treated with microcystin-LR

    Environ. Toxicol. Pharmacol.

    (2008)
  • M. Wozny et al.

    Intraperitoneal exposure of whitefish to microcystin-LR induces rapid liver injury followed by regeneration and resilience to subsequent exposures

    Toxicol. Appl. Pharmacol.

    (2016)
  • W. Yan et al.

    Water-borne exposure to microcystin-LR alters thyroid hormone levels and gene transcription in the hypothalamic–pituitary–thyroid axis in zebrafish larvae

    Chemosphere

    (2012)
  • Cited by (8)

    • The revised European Directive 2020/2184 on the quality of water intended for human consumption. A step forward in risk assessment, consumer safety and informative communication

      2022, Environmental Research
      Citation Excerpt :

      The main target of microcystin toxicity is the liver, as microcystins cross cell membranes mainly through the bile acid transporter (World Health Organization, 2017). Microcystin-LR, the most investigated variant of this group, is a highly effective tumour promoter (classified as 2B on the International Agency for Research on Cancer, IARC, carcinogenic ladder) (IARC, 2021) whose action, mediated by inhibition of the protein phosphatases PP1/PP2A, induces oxidative DNA damage (Zegura et al., 2003), activation of the proto-oncogenes c-jun, c-fos, c-myc (Li et al., 2009) and the nuclear factor Nrf2 (erythroid-related factor 2) (Gan et al., 2014), repression of the tumour suppressor genes baxa, gadd45α and the proto-oncogene junba (Fonseca et al., 2018). Studies on polluted lakes have highlighted the risk of human exposure to microcystins through both direct (drinking water, recreational activities) and indirect (fish consumption) routes (Zhang et al., 2009; Chen et al., 2009; Bruno et al., 2020).

    • Imidacloprid and thiamethoxam affect synaptic transmission in zebrafish

      2021, Ecotoxicology and Environmental Safety
      Citation Excerpt :

      Firstly, fosab is a member of the IEG family, widely used as the neuronal excitability and synaptic activity marker in rodents and zebrafish (Faria et al., 2018). Moreover, fosab often accounted for addiction and stress in the locomotive analysis (Duart-Castells et al., 2019; Fan et al., 2021; Fonseca et al., 2018). The FOSAB protein was analyzed in animal models to verify the effect of stress on the central nervous system and the appearance of aggressive behavior (Felippe et al., 2021).

    • Proteomic analysis of zebrafish brain damage induced by Microcystis aeruginosa bloom

      2021, Science of the Total Environment
      Citation Excerpt :

      Above of all, we promise that all experiments were performed in compliance with the national guideline, “Laboratory animal - Guideline for ethical review of animal welfare (GB/T 35892-2018)”. Microcystin-LR (MC-LR), which is one of the most toxic cyanotoxins, has been widely discussed (Fonseca et al., 2018; Watson et al., 2017). In our study, after the exposure period, the intra- and extracellular concentration of MC-LR were determined in the culture medium by high performance liquid chromatography as 571.95 ± 15.71 μg L−1 and 55.57 ± 5.34 μg L−1, respectively.

    • Acute exposure to N-Ethylpentylone induces developmental toxicity and dopaminergic receptor-regulated aberrances in zebrafish larvae

      2021, Toxicology and Applied Pharmacology
      Citation Excerpt :

      Th2, a homolog of th, identified as another marker of DA neurons (Ren et al., 2013), was upregulated in two NEP-treated groups (Fig. 10c–d, P < 0.0001 for 1.5 μM and 15 μM NEP). Fosb and fosab, often accounted for addiction and stress in the locomotive analysis (Fonseca et al., 2018; Duart-Castells et al., 2019), were also analyzed. Fosab was upregulated in NEP-treated groups (Fig. 10c–d, P < 0.001), while the level of fosb expression was increased only by 15 μM NEP (Fig. 10d, P < 0.001).

    • Proteomic analysis of the hepatotoxicity of Microcystis aeruginosa in adult zebrafish (Danio rerio) and its potential mechanisms

      2019, Environmental Pollution
      Citation Excerpt :

      All experiments were conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. As one of the most toxic cyanotoxins, the toxicity of microcystin-LR (MC-LR) is commonly investigated (Fonseca et al., 2018; Watson et al., 2017). At the end of our exposure, the concentrations of MC-LR in the culture medium were measured.

    View all citing articles on Scopus
    View full text