Skip to main content

Advertisement

SpringerLink
Log in

Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells

  • Genotoxicity and Carcinogenicity
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

Abstract

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens (“true positives”), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect (“true negatives”) and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays (“false positives”). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ≥90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Andersen KE, Angers-Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Leite SB, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tähti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM (2011) Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch Toxicol 85:367–485

    Google Scholar 

  • Akyüz N, Wiesmüller L (2003) Proof of principle: Detection of genotoxicity by a fluorescence-based recombination test in mammalian cells. ALTEX 20:77–84

    PubMed  Google Scholar 

  • Akyüz N, Boehden GS, Süsse S, Rimek A, Preuss U, Scheidtmann KH, Wiesmüller L (2002) DNA substrate-dependence of p53-mediated regulation of double-strand-break-repair. Mol Cell Biol 22:6306–6317

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Allen C, Ashley AK, Hromas R, Nickoloff JA (2011) More forks on the road to replication stress recovery. J Mol Cell Biol 3:4–12

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Billington N, Cahill P, Hughes C, Tate M, Walmsley RM (2010) A pre-validation transferability study of the GreenScreen HC GADD45a-GFP assay with a metabolic activation system (S9). Mutat Res 700:44–50

    Article  CAS  Google Scholar 

  • Birrell L, Cahill P, Hughes C, Tate M, Walmsley RM (2010) GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests. Mutat Res 695:87–95

    Article  CAS  PubMed  Google Scholar 

  • Boehden GS, Restle A, Marschalek R, Stocking C, Wiesmüller L (2004) Recombination at chromosomal sequences involved in leukaemogenic rearrangements is differentially regulated by p53. Carcinogenesis 8:1–9

    Google Scholar 

  • Böhringer M, Wiesmüller L (2010) Fluorescence-based quantification of pathway-specific DNA double-strand break repair activities: a powerful method for the analysis of genome destabilizing mechanisms. Subcell Biochem 50:297–306

    Article  PubMed  CAS  Google Scholar 

  • Brennan RJ, Schiestl RH (2004) Detecting carcinogens with the yeast DEL assay. Methods Mol Biol 262:111–124

    CAS  PubMed  Google Scholar 

  • Concin N et al (2003) Comparison of p53 mutational status with mRNA and protein expression in a panel of 24 human breast carcinoma cell lines. Breast Cancer Res Treat 79:37–46

    Article  CAS  PubMed  Google Scholar 

  • Corvi R, Aardema MJ, Gribaldo L, Hayashi M, Hoffmann S, Schechtman L, Vanparys P (2012) ECVAM prevalidation study on in vitro cell transformation assays: general outline and conclusions of the study. Mutat Res 744:12–19

    Article  CAS  PubMed  Google Scholar 

  • Danesi CC, Bellagamba BC, Dihl RR, de Andrade HH, Cunha KS, Lehmann M (2010) Evaluation of the genotoxicity of cisplatin, paclitaxel and 5-fluorouracil combined treatment in the Drosophila wing-spot test. Food Chem Toxicol 48:3120–3124

    Article  CAS  PubMed  Google Scholar 

  • Digue L, Orsière T, De Méo M, Mattéi MG, Depetris D, Duffaud F, Favre R, Botta A (1999) Evaluation of the genotoxic activity of paclitaxel by the in vitro micronucleus test in combination with fluorescent in situ hybridization of a DNA centromeric probe and the alkaline single cell gel electrophoresis technique (comet assay) in human T-lymphocytes. Environ Mol Mutagen 34:269–278

    Article  CAS  PubMed  Google Scholar 

  • Elhajouji A, Likamowicz M, Cammerer Z, Kirsch-Volders M (2011) Potential thresholds for genotoxic effect by micronucleus scoring. Mutagenesis 26:199–204

    Article  CAS  PubMed  Google Scholar 

  • Essmann F, Engels IH, Totzke G, Schulze-Osthoff K, Jänicke RU (2004) Apoptosis resistance of MCF-7 breast carcinoma cells to ionizing radiation is independent of p53 and cell cycle control but caused by the lack of caspase-3 and a caffeine-inhibitable event. Cancer Res 64:7065–7072

    Article  CAS  PubMed  Google Scholar 

  • Filipič M (2012) Mechanisms of cadmium induced genomic instability. Mutat Res 733:69–77

    Article  PubMed  CAS  Google Scholar 

  • Fowler P, Smith R, Smith K, Young J, Jeffrey L, Kirkland D, Pfuhler S, Carmichael P (2012) Reduction of misleading (“false”) positive results in mammalian cell genotoxicity assays. II. Importance of accurate toxicity measurement. Mutat Res 747:104–117

    Article  CAS  PubMed  Google Scholar 

  • Gallagher CM, Chen JJ, Kovach JS (2010) Environmental cadmium and breast cancer risk. Aging 2:804–814

    CAS  PubMed Central  PubMed  Google Scholar 

  • Gebel TW, Leister M, Schumann W, Hirsch-Ernst K (2002) Low-level self-tolerance to arsenite in human HepG2 cells is associated with a depressed induction of micronuclei. Mutat Res 514:245–255

    Article  CAS  PubMed  Google Scholar 

  • Godoy P et al (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87:1315–1530

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hartwig A (2013) Metal interaction with redox regulation: an integrating concept in metal carcinogenesis. Free Radic Biol Med 55:63–72

    Article  CAS  PubMed  Google Scholar 

  • Hastwell PW, Chai LL, Roberts KJ, Webster TW, Harvey JS, Rees RW, Walmsley RM (2006) High-specificity and high-sensitivity genotoxicity assessment in a human cell line: validation of the GreenScreen HC GADD45a-GFP genotoxicity assay. Mutat Res 607:160–175

    Article  CAS  PubMed  Google Scholar 

  • Hastwell PW, Webster TW, Tate M, Billinton N, Lynch AM, Harvey JS, Rees RW, Walmsley RM (2009) Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP “GreenScreen HC” genotoxicity assay. Mutagenesis 24:455–463

    Article  CAS  PubMed  Google Scholar 

  • Hengstler JG, Utesch D, Steinberg P, Platt KL, Diener B, Ringel M, Swales N, Fischer T, Biefang K, Gerl M, Böttger T, Oesch F (2000a) Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction. Drug Metab Rev 32:81–118

    Article  CAS  PubMed  Google Scholar 

  • Hengstler JG, Ringel M, Biefang K, Hammel S, Milbert U, Gerl M, Klebach M, Diener B, Platt KL, Böttger T, Steinberg P, Oesch F (2000b) Cultures with cryopreserved hepatocytes: applicability for studies of enzyme induction. Chem Biol Interact 125:51–73

    Article  CAS  PubMed  Google Scholar 

  • Hewitt NJ, Lechón MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, Lohmann C, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothuis GM, Hengstler JG (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 39:159–234

    Article  CAS  PubMed  Google Scholar 

  • Jagger C, Tate M, Cahill PA, Hughes C, Knight AW, Billinton N, Walmsley RM (2009) Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay. Mutagenesis 24:35–50

    Article  CAS  PubMed  Google Scholar 

  • Keimling M, Wiesmüller L (2009) DNA double-strand break repair activities in mammary epithelial cells-influence of endogenous p53 variants. Carcinogenesis 30:1260–1268

    Article  CAS  PubMed  Google Scholar 

  • Kern JC, Kehrer JP (2002) Acrolein/induced cell death: a caspase-influenced decision between apoptosis and oncosis/necrosis. Chem Biol Interact 139:79–95

    Article  CAS  PubMed  Google Scholar 

  • Khoury L, Zalko D, Audebert M (2013) Validation of high-throughput assay screening using γH2AX In-cell western assay on HepG2 cells. Environ Mol Mutagen 54:737–746

    Article  CAS  PubMed  Google Scholar 

  • Kirkland D, Aardema M, Henderson L, Muller L (2005) Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative productivity. Mutat Res 584:1–256

    Article  CAS  PubMed  Google Scholar 

  • Kirkland D, Pfuhler S, Tweats D, Aardema M, Corvi R, Darroudi F, Elhajouji A, Glatt H, Hastwell P, Hayashi M, Kasper P, Kirchner S, Lynch A, Marzin D, Maurici D, Meunier JR, Müller L, Nohynek G, Parry J, Parry E, Thybaud V, Tice R, van Benthem J, Vanparys P, White P (2007) How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: report of an ECVAM workshop. Mutat Res 628:31–55

    Article  CAS  PubMed  Google Scholar 

  • Kirkland D, Kasper P, Müller L, Corvi R, Speit G (2008) Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop. Mutat Res 653:99–108

    Article  CAS  PubMed  Google Scholar 

  • Kirkland D, Reeve L, Gatehouse D, Vanparys P (2011) A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins. Mutat Res 721:27–73

    Article  CAS  PubMed  Google Scholar 

  • Kirpnick Z, Homiski M, Rubitski E, Repnevskaya M, Howlett N, Aubrecht J, Schiestl RH (2005) Yeast DEL assay detects clastogens. Mutat Res 582:116–134

    Article  CAS  PubMed  Google Scholar 

  • Komissarova EV, Saha SK, Rossman TG (2005) Dead or dying: the importance of time in cytotoxicity assays using arsenite as an example. Toxicol Appl Pharmacol 202:99–107

    Article  CAS  PubMed  Google Scholar 

  • Lacroix M, Robert-Alain Toillon RA, Leclercq G (2006) p53 and breast cancer, an update. Endocr Relat Cancer 13:293–325

    Article  CAS  PubMed  Google Scholar 

  • Law JC, Ritke MK, Yalowich JC, Leder GH, Ferrell RE (1993) Mutational inactivation of the p53 gene in the human erythroid leukemic K562 cell line. Leuk Res 17:1045–1050

    Article  CAS  PubMed  Google Scholar 

  • Levy JA, Virolainen M, Defendi V (1968) Human lymphoblastoid lines from lymph node and spleen. Cancer 22:517–524

    Article  CAS  PubMed  Google Scholar 

  • Lozzio CB, Lozzio BB (1975) Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45:321–344

    CAS  PubMed  Google Scholar 

  • Lynch A, Sasaki J, Elespuru R (2011) New and emerging technologies for genetic toxicity testing. Environ Mol Mutagen 52:205–223

    Article  CAS  PubMed  Google Scholar 

  • Mahdi T, Alcalay D, Cognard C, Tanzer J, Kitzis A (1998) Rescue of K562 cells from MDM2-modulated p53-dependent apoptosis by growth factor-induced differentiation. Biol Chem 90:15–27

    Google Scholar 

  • McGahon A, Bissonnette R, Schmitt M, Cotter KM, Green DR, Cotter TG (1994) BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death. Blood 83:1179–1187

    CAS  PubMed  Google Scholar 

  • Meyer C et al (2013) The MLL recombinome of acute leukemias in 2013. Leukemia 27:2165–2176

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mirault ME, Boucher P, Tremblay A (2006) Nucleotide-resolution mapping of topoisomerase-mediated and apoptotic DNA strand scissions at or near an MLL translocation hotspot. Am J Hum Genet 79:779–791

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mondal S, Bhattacharya K, Mallick A, Sangwan R, Mandal C (2012) Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during withanolide D-induced apoptosis. PLoS ONE 7:e34277

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Muntean AG, Hess JL (2012) The pathogenesis of mixed-lineage leukemia. Annu Rev Pat 7:283–301

    Article  CAS  Google Scholar 

  • Nagai S, Heun P, Gasser SM (2010) Roles for nuclear organization in the maintenance of genome stability. Epigenomics 2:289–305

    Article  CAS  PubMed  Google Scholar 

  • O′Donovan M (2012) A critique of methods to measure cytotoxicity in mammalian cell genotoxicity assays. Mutagenesis 27:615–621

    Article  PubMed  CAS  Google Scholar 

  • Olaharski A, Albertini S, Kirchner S, Platz S, Uppal H, Lin H, Kolaja K (2009) Evaluation of the GreenScreen GADD45α-GFP indicator assay with non-proprietary and proprietary compounds. Mutat Res 627:10–16

    Article  CAS  Google Scholar 

  • Olaharski AJ, Albertini S, Mueller L, Zeller A, Struwe M, Gocke E, Kolaja K (2011) GADD45α induction in the GreenScreen HC indicator assay does not occur independently of cytotoxicity. Environ Mol Mutagen 52:28–34

    Article  CAS  PubMed  Google Scholar 

  • Pfuhler S, Fellows M, van Benthem J, Corvi R, Curren R, Dearfield K, Fowler P, Frötschl R, Elhajouji A, Hégarat Le L, Kasamatsu T, Kojima H, Ouédraogo G, Scott A, Speit G (2011) In vitro genotoxicity test approaches with better predictivity: summary of an IWGT workshop. Mutat Res 723:101–107

    Article  CAS  PubMed  Google Scholar 

  • Restle A, Färber M, Baumann C, Böhringer M, Scheidtmann KH, Müller-Tidow C, Wiesmüller L (2008) Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants. Nucleic Acids Res 36:5362–5375

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rothkamm K, Krüger I, Thompson LH, Löbrich M (2003) Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol Cell Biol 23:5706–5715

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Schaap MM, Zwart EP, Wackers PF, Huijskens I, van de Water B, Breit TM, van Steeg H, Jonker MJ, Luijten M (2012) Dissecting modes of action of non-genotoxic carcinogens in primary mouse hepatocytes. Arch Toxicol 86:1717–1727

    Article  CAS  PubMed  Google Scholar 

  • Schiestl RH (1989) Nonmutagenic carcinogens induce intrachromosomal recombination in yeast. Nature 337:285–288

    Article  CAS  PubMed  Google Scholar 

  • Scott D, Galloway SM, Marshall RR, Ishidate M Jr, Brusick D, Ashby J, Myhr BC (1991) International commission for protection against environmental mutagens and carcinogens. Genotoxicity under extreme culture conditions. A report from ICPEMC Task Group 9. Mutat Res 257:147–205

    Article  CAS  PubMed  Google Scholar 

  • Sim SP, Liu LF (2001) Nucleolytic cleavage of the mixed lineage leukemia breakpoint cluster region during apoptosis. J Biol Chem 276:31590–31595

    Article  CAS  PubMed  Google Scholar 

  • Thybaud V, Aardema M, Casciano D, Dellarco V (2007) Relevance and follow-up of positive results in in vitro genetic toxicity assays: an ILSI-HESI initiative. Mutat Res 633:67–79

    Article  CAS  PubMed  Google Scholar 

  • Tsamou M, Jennen DG, Claessen SM, Magkoufopoulou C, Kleinjans JC, van Delft JH (2012) Performance of in vitro γH2AX assay in HepG2 cells to predict in vivo genotoxicity. Mutagenesis 27:645–652

    Article  CAS  PubMed  Google Scholar 

  • Un F (2007) G1 arrest induction represents a critical determinant for cisplatin cytotoxicity in G1 checkpoint-retaining human cancers. Anticancer Drugs 18:411–417

    Article  CAS  PubMed  Google Scholar 

  • Valenci-Quintana R, Gómez-Arroyo S, Waliszewski SM (2012) Evaluation of the genotoxic potential of dimethyl sulfoxide (DMSO) in meristematic cells of the root of Vicia faba. Toxicol Environ Health Sci 4:154–160

    Article  Google Scholar 

  • Wilkening S, Stahl F, Bader A (2003) Comparison of primary human hepatocytes and hepatoma cell line Hepg2 with regard to their biotransformation properties. Drug Metab Dispos 31:1035–1042

    Article  CAS  PubMed  Google Scholar 

  • Yvon AM, Wadsworth P, Jordan MA (1999) Taxol suppresses dynamics of individual microtubules in living human tumor cells. Mol Biol Cell 10:947–959

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhang F, Gu W, Hurles ME, Lupski JR (2009) Copy number variation in human health, disease, and evolution. Annu Rev Genomics Hum Genet 10:451–481

    Article  CAS  PubMed  Google Scholar 

  • Zhang QX, Yang GY, Li JT, Li WX, Zhang B, Zhu W (2011) Melamine induces sperm DNA damage and abnormality, but not genetic toxicity. Regul Toxicol Pharmacol 60:144–150

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Thomas Friedl, Ulm University, for help with the statistical evaluation of the data, and Günter Speit, Ulm University, for extremely helpful discussions and expert advice regarding genotoxicity testing. We thank Ms. Georgia Günther for competent support with isolation and cultivation of primary hepatocytes. This work was supported by the German state of Baden-Württemberg, Ministry of Nutrition and Agriculture, grant #315E, by the German Research Foundation (DFG, WI 3099/7-1 and -2) and by the FP7 EU projects DETECTIVE and NOTOX.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, University of Ulm, Prittwitzstrasse 43, 89075, Ulm, Germany

    Ivanildce C. Ireno, Cindy Baumann & Lisa Wiesmüller

  2. Leibniz Research Centre (IfADo), Ardeystraße 67, 44139, Dortmund, Germany

    Regina Stöber & Jan G. Hengstler

Authors
  1. Ivanildce C. Ireno
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cindy Baumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Regina Stöber
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jan G. Hengstler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lisa Wiesmüller
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lisa Wiesmüller.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 18 kb)

Supplementary material 2 (PDF 195 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ireno, I.C., Baumann, C., Stöber, R. et al. Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells. Arch Toxicol 88, 1141–1159 (2014). https://doi.org/10.1007/s00204-014-1229-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-014-1229-3

Keywords

Search

Navigation