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Genotoxic effects of the cyanobacterial hepatotoxin cylindrospermopsin in the HepG2 cell line

  • Genotoxicity and Carcinogenicity
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Abstract

The cyanobacterial alkaloid cylindrospermopsin (CYN) is being increasingly identified in drinking water supplies worldwide. It is a potent protein synthesis inhibitor and causes human intoxications and animal mortality. The few genotoxicity studies available indicate that CYN is genotoxic, generally implying that it is pro-genotoxic. We evaluated CYN genotoxicity in the human hepatoma cell line, HepG2, analyzing the induction of DNA strand breaks, with the alkaline comet assay, and micronuclei (MNi), nuclear bud (NBUD), and nucleoplasmic bridge (NPB) formation, with the cytokinesis block micronucleus (CBMN) assay. In addition, changes in the expression of genes involved in the response to DNA damage (P53, CDKN1A, GADD45α, and MDM2) and genes presumably involved in CYN metabolism (genes from the Cytochrome P450 family: CYP1A1 and CYP1A2) were determined, using quantitative real-time PCR. Non-cytotoxic concentrations of CYN induced increased DNA damage after 12 and 24 h of exposure and increased the frequency of MNi, NBUDs, and NPBs after 24 h exposure. Moreover, CYN up-regulated the expression of the CYP1A1 and CYP1A2 genes. Although no changes in the expression of the P53 tumor-suppressor gene were found, CYN up-regulated the expression of the P53 downstream-regulated genes CDKN1A, GADD45α, and MDM2. Our results provide new evidence that CYN is genotoxic and strongly suggest that it needs to be considered in the human health risk assessment.

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Abbreviations

BaP:

Benzo[a]pyrene

BNC:

Binucleated cells

CYN:

Cylindrospermopsin

CYP450:

Cytochrome p-450

CBMN:

Cytokinesis block micronucleus assay

MNed:

Micronucleated

MNi:

Micronuclei

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NDI:

Nuclear division index

NBUD:

Nuclear bud

NPB:

Nucleoplasmic bridge

References

  • Bain P, Shaw G, Patel B (2007) Induction of p53-Regulated Gene Expression in Human Cell Lines Exposed to the Cyanobacterial Toxin Cylindrospermopsin. Journal of Toxicology & Environmental Health: Part A 70(19):1687–1693

    Article  CAS  Google Scholar 

  • Bazin E, Mourot A, Humpage AR, Fessard V (2010) Genotoxicity of a freshwater cyanotoxin, cylindrospermopsin, in two human cell lines: Caco-2 and HepaRG. Environ Mol Mutagen 51(3):251–259

    PubMed  CAS  Google Scholar 

  • Bressac B, Galvin KM, Liang TJ, Isselbacher KJ, Wands JR, Ozturk M (1990) Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proceedings of the National Academy of Sciences 87(5):1973–1977

    Article  CAS  Google Scholar 

  • Byth S (1980) Palm Island mystery disease. Med J Aust 2(1):40–42

    PubMed  CAS  Google Scholar 

  • Carmichael WW, Azevedo SM, An JS et al (2001) Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins. Environ Health Perspect 109(7):663–668

    Article  PubMed  CAS  Google Scholar 

  • Ellinger-Ziegelbauer H, Aubrecht J, Kleinjans JC, Ahr H-J (2009) Application of toxicogenomics to study mechanisms of genotoxicity and carcinogenicity. Toxicol Lett 186(1):36–44

    Article  PubMed  CAS  Google Scholar 

  • EPA (2001) Creating a Cyanotoxin Target List for the Unregulated Contaminant Monitoring Rule. US Environmental Protection Agency, Technical Service Center, Cincinnati, OH. http://www.epa.gov/safewater/ucmr/ucmr1/pdfs/meeting_ucmr1_may2001.pdf accessed September 28, 2010

  • Falconer IR (2008) Health effects associated with controlled exposures to cyanobacterial toxins Cyanobacterial Harmful Algal Blooms: State of the Science and Research Needs. pp 607–612

  • Falconer IR, Humpage AR (2001) Preliminary evidence for in vivo tumour initiation by oral administration of extracts of the blue-green alga Cylindrospermopsis raciborskii containing the toxin cylindrospermopsin. Environ Toxicol 16(2):192–195

    Article  PubMed  CAS  Google Scholar 

  • Falconer IR, Hardy SJ, Humpage AR, Froscio SM, Tozer GJ, Hawkins PR (1999) Hepatic and renal toxicity of the blue-green alga (cyanobacterium) Cylindrospermopsis raciborskii in male Swiss albino mice. Environ Toxicol 14(1):143–150

    Article  CAS  Google Scholar 

  • Fenech M (2000) The in vitro micronucleus technique. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 455(1–2):81–95

    Article  CAS  Google Scholar 

  • Fenech M (2006) Cytokinesis-block micronucleus assay evolves into a “cytome” assay of chromosomal instability, mitotic dysfunction and cell death. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 600(1–2):58–66

    Article  CAS  Google Scholar 

  • Fessard V, Bernard C (2003) Cell alterations but no DNA strand breaks induced in vitro by cylindrospermopsin in CHO K1 cells. Environ Toxicol 18(5):353–359

    Article  PubMed  CAS  Google Scholar 

  • Froscio SM, Humpage AR, Burcham PC, Falconer IR (2001) Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin. Environ Toxicol 16(5):408–412

    Article  PubMed  CAS  Google Scholar 

  • Froscio SM, Humpage AR, Burcham PC, Falconer IR (2003) Cylindrospermopsin-induced protein synthesis inhibition and its dissociation from acute toxicity in mouse hepatocytes. Environ Toxicol 18(4):243–251

    Article  PubMed  CAS  Google Scholar 

  • Griffiths DJ, Saker ML (2003) The Palm Island mystery disease 20 years on: A review of research on the cyanotoxin cylindrospermopsin. Environ Toxicol 18(2):78–93

    Article  PubMed  CAS  Google Scholar 

  • Harkin DP, Bean JM, Miklos D et al (1999) Induction of GADD45 and JNK/SAPK-Dependent Apoptosis following Inducible Expression of BRCA1. Cell 97(5):575–586

    Article  PubMed  CAS  Google Scholar 

  • Hawkins PR, Runnegar MT, Jackson AR, Falconer IR (1985) Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju isolated from a domestic water supply reservoir. Appl Environ Microbiol 50(5):1292–1295

    PubMed  CAS  Google Scholar 

  • Hollander MC, Kovalsky O, Salvador JM et al (2001) Dimethylbenzanthracene Carcinogenesis in Gadd45a-null Mice Is Associated with Decreased DNA Repair and Increased Mutation Frequency. Cancer Res 61(6):2487–2491

    PubMed  CAS  Google Scholar 

  • Humpage AR, Fenech M, Thomas P, Falconer IR (2000) Micronucleus induction and chromosome loss in transformed human white cells indicate clastogenic and aneugenic action of the cyanobacterial toxin, cylindrospermopsin. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 472(1–2):155–161

    Article  CAS  Google Scholar 

  • Humpage A, Fontaine F, Froscio S, Burcham P, Falconer I (2005) Cylindrospermopsin genotoxicity and cytotoxicity: role of cytochrome P-450 and oxidative stress. Journal of Toxicology & Environmental Health: Part A 68(9):739–753

    Article  CAS  Google Scholar 

  • Jin S, Antinore MJ, Lung F-DT et al (2000) The GADD45 Inhibition of Cdc2 Kinase Correlates with GADD45-mediated Growth Suppression. J Biol Chem 275(22):16602–16608

    Article  PubMed  CAS  Google Scholar 

  • Knasmuller S, Mersch-Sundermann V, Kevekordes S et al (2004) Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants; current state of knowledge. Toxicology 198(1–3):315–328

    Article  PubMed  CAS  Google Scholar 

  • Lankoff A, Wojcik A, Lisowska H, Bialczyk J, Dziga D, Carmichael WW (2007) No induction of structural chromosomal aberrations in cylindrospermopsin-treated CHO-K1 cells without and with metabolic activation. Toxicon 50(8):1105–1115

    Article  PubMed  CAS  Google Scholar 

  • Maire MA, Bazin E, Fessard V, Rast C, Humpage AR, Vasseur P (2010) Morphological cell transformation of Syrian hamster embryo (SHE) cells by the cyanotoxin, cylindrospermopsin. Toxicon 55(7):1317–1322

    Google Scholar 

  • Michael D, Oren M (2002) The p53 and Mdm2 families in cancer. Curr Opin Genet Dev 12(1):53–59

    Article  PubMed  CAS  Google Scholar 

  • Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63

    Article  PubMed  CAS  Google Scholar 

  • Norris RLG, Seawright AA, Shaw GR et al (2002) Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse. Toxicon 40(4):471–476

    Article  PubMed  CAS  Google Scholar 

  • Ohtani I, Moore RE, Runnegar MTC (1992) Cylindrospermopsin: a potent hepatotoxin from the blue-green alga Cylindrospermopsis raciborskii. J Am Chem Soc 114(20):7941–7942

    Article  CAS  Google Scholar 

  • Pelkonen O, Turpeinen M, Hakkola J, Honkakoski P, Hukkanen J, Raunio H (2008) Inhibition and induction of human cytochrome P450 enzymes: current status. Arch Toxicol 82(10):667–715

    Article  PubMed  CAS  Google Scholar 

  • Runnegar MT, Kong S-M, Zhong Y-Z, Lu SC (1995) Inhibition of reduced glutathione synthesis by cyanobacterial alkaloid cylindrospermopsin in cultured rat hepatocytes. Biochem Pharmacol 49(2):219–225

    Article  PubMed  CAS  Google Scholar 

  • Runnegar MT, Xie C, Snider BB, Wallace GA, Weinreb SM, Kuhlenkamp J (2002) In Vitro Hepatotoxicity of the Cyanobacterial Alkaloid Cylindrospermopsin and Related Synthetic Analogues. Toxicol Sci 67(1):81–87

    Article  PubMed  CAS  Google Scholar 

  • Saker ML, Eaglesham GK (1999) The accumulation of cylindrospermopsin from the cyanobacterium Cylindrospermopsis raciborskii in tissues of the Redclaw crayfish Cherax quadricarinatus. Toxicon 37(7):1065–1077

    Article  PubMed  CAS  Google Scholar 

  • Serrano-García L, Montero-Montoya R (2001) Micronuclei and chromatid buds are the result of related genotoxic events. Environ Mol Mutagen 38(1):38–45

    Article  PubMed  Google Scholar 

  • Shaw GR, Seawright AA, Moore MR, Lam PKS (2000) Cylindrospermopsin, A Cyanobacterial Alkaloid: Evaluation of Its Toxicologic Activity. Ther Drug Monit 22(1):89–92

    Article  PubMed  CAS  Google Scholar 

  • Shen X, Lam PKS, Shaw GR, Wickramasinghe W (2002) Genotoxicity investigation of a cyanobacterial toxin, cylindrospermopsin. Toxicon 40(10):1499–1501

    Article  PubMed  CAS  Google Scholar 

  • Shimizu N, Itoh N, Utiyama H, Wahl GM (1998) Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase. J Cell Biol 140(6):1307–1320

    Article  PubMed  CAS  Google Scholar 

  • Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175(1):184–191

    Article  PubMed  CAS  Google Scholar 

  • Smith ML, Chen IT, Zhan Q et al (1994) Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 266(5189):1376–1380

    Article  PubMed  CAS  Google Scholar 

  • Smith ML, Kontny HU, Zhan Q, Sreenath A, O’Connor PM, Fornace AJ Jr (1996) Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin. Oncogene 13(10):2255–2263

    PubMed  CAS  Google Scholar 

  • Takekawa M, Saito H (1998) A Family of Stress-Inducible GADD45-like Proteins Mediate Activation of the Stress-Responsive MTK1/MEKK4 MAPKKK. Cell 95(4):521–530

    Article  PubMed  CAS  Google Scholar 

  • Thomas P, Umegaki K, Fenech M (2003) Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay. Mutagenesis 18(2):187–194

    Article  PubMed  CAS  Google Scholar 

  • Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408(6810):307–310

    Article  PubMed  CAS  Google Scholar 

  • Wang XW, Zhan Q, Coursen JD et al (1999) GADD45 induction of a G2/M cell cycle checkpoint. Proc Natl Acad Sci USA 96(7):3706–3711

    Article  PubMed  CAS  Google Scholar 

  • Westerink WMA, Schoonen WGEJ (2007) Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol In Vitro 21(8):1581–1591

    Article  PubMed  CAS  Google Scholar 

  • Yankiwski V, Marciniak RA, Guarente L, Neff NF (2000) Nuclear structure in normal and Bloom syndrome cells. Proc Natl Acad Sci USA 97(10):5214–5219

    Article  PubMed  CAS  Google Scholar 

  • Zegura B, Lah TT, Filipic M (2004) The role of reactive oxygen species in microcystin-LR-induced DNA damage. Toxicology 200(1):59–68

    Article  PubMed  CAS  Google Scholar 

  • Zegura B, Zajc I, Lah TT, Filipic M (2008) Patterns of microcystin-LR induced alteration of the expression of genes involved in response to DNA damage and apoptosis. Toxicon 51(4):615–623

    Article  PubMed  CAS  Google Scholar 

  • Zegura B, Straser A, Filipic M (2011) Genotoxicity and potential carcinogenicity of cyanobacterial toxins - a review. Mutation Research/Reviews in Mutation Research 727(1–2):16–41

    Article  CAS  Google Scholar 

  • Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in perspective. Nature 408(6811):433–439

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This study was supported by Slovenian Research Agency: Program P1-0245 and young researcher grant to AŠ.

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The authors report no conflict of interest.

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Authors and Affiliations

  1. Department for Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia

    Alja Štraser, Metka Filipič & Bojana Žegura

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  1. Alja Štraser
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  2. Metka Filipič
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  3. Bojana Žegura
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Correspondence to Bojana Žegura.

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Štraser, A., Filipič, M. & Žegura, B. Genotoxic effects of the cyanobacterial hepatotoxin cylindrospermopsin in the HepG2 cell line. Arch Toxicol 85, 1617–1626 (2011). https://doi.org/10.1007/s00204-011-0716-z

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