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Bhas 42 Cell Transformation Assay for Genotoxic and Non-Genotoxic Carcinogens

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Genotoxicity and DNA Repair

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Cell transformation assays (CTAs) are in vitro carcinogenicity tests measuring morphological transformation of cells either as transformed colonies (SHE cells) or foci (C3H/10T1/2 and BALB/c 3T3 including Bhas 42 cells) derived from a single cell. CTAs such as Bhas 42 CTA can detect both genotoxic and non-genotoxic carcinogens. When used as an initiation assay to test tumor-initiating activity, cells at low density are treated with a test chemical for 3 days, whereas a promotion assay to test for tumor-promoting activity, near-confluent cells are treated with a test chemical for a period of 10 days. The Bhas 42 CTA has advantages compared with BALB/c 3T3 and other CTAs due to its simplicity, higher sensitivity, less time needed for assay performance, and robustness (exemplified by its adaptation to a high-throughput method). The Bhas 42 CTA has been validated together with other CTAs and recommended for development of an OECD guideline. The assay has already been applied in testing various chemical and physical agents including particles and nanomaterials. Protocols for both 6 and 96-well plate formats of initiation and promotion assays are described in detail.

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References

  1. Berwald Y, Sachs L (1963) In vitro cell transformation with chemical carcinogens. Nature 200:1182–1184

    Article  CAS  PubMed  Google Scholar 

  2. Kakunaga T (1973) A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3. Int J Cancer 12:463–473

    CAS  PubMed  Google Scholar 

  3. Reznikoff CA, Bertram JS, Brankow DW et al (1973) Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division. Cancer Res 33:3239–3249

    CAS  PubMed  Google Scholar 

  4. Evans CH, DiPaolo JA (1981) In vitro mammalian cell transformation for identification of carcinogens, cocarcinogens, and anticarcinogens. In: Stich HF, San RHC (eds) Short-term tests for chemical carcinogens. Springer, New York, pp 306–322

    Chapter  Google Scholar 

  5. Sakai A, Iwase Y, Nakamura Y et al (2002) Use of a cell transformation assay with established cell lines, and a metabolic cooperation assay with V79 cells for the detection of tumor promoters: a review. Altern Lab Anim 30:33–59

    CAS  PubMed  Google Scholar 

  6. Schechtman LM (2012) Rodent cell transformation assays—a brief historical perspective. Mutat Res 744:3–7

    Article  CAS  PubMed  Google Scholar 

  7. Sasaki K, Mizusawa H, Ishidate M (1988) Isolation and characterization of ras-transfected BALB/3T3 clone showing morphological transformation by 12-O-tetradecanoyl-phorbol-13-acetate. Jpn J Cancer Res 79:921–930

    Article  PubMed  Google Scholar 

  8. Sasaki K, Mizusawa H, Ishidate M (1990) Establishment of a highly reproducible transformation assay of a ras-transfected BALB 3T3 clone by treatment with promoters. In: Kuroda Y, Shankel DM, Waters MD et al (eds) Antimutagenesis and anticarcinogenesis mechanisms II, vol 52, Basic life sciences. Plenum, New York, pp 411–416

    Chapter  Google Scholar 

  9. Asada S, Sasaki K, Tanaka N et al (2005) Detection of initiating as well as promoting activity of chemicals by a novel cell transformation assay using v-Ha-ras-transfected BALB/c 3T3 cells (Bhas 42 cells). Mutat Res 588:7–21

    Article  CAS  PubMed  Google Scholar 

  10. Sakai A, Sasaki K, Muramatsu D et al (2010) A Bhas 42 cell transformation assay on 98 chemicals: the characteristics and performance for the prediction of chemical carcinogenicity. Mutat Res 702:100–122

    Article  CAS  PubMed  Google Scholar 

  11. Sakai A (2008) BALB/c 3T3 cell transformation assays for the assessment of chemical carcinogenicity. AATEX 14(Special Issue):367–373

    Google Scholar 

  12. Ohmori K, Sasaki K, Asada S et al (2004) An assay method for the prediction of tumor promoting potential of chemicals by the use of Bhas 42 cells. Mutat Res 557:191–202

    Article  CAS  PubMed  Google Scholar 

  13. Muramatsu D, Sasaki K, Kuroda S et al (2009) Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB/c 3T3 cell transformation assay. Mutat Res 675:66–70

    Article  CAS  PubMed  Google Scholar 

  14. Ohmori K, Umeda M, Tanaka N et al (2005) An inter-laboratory collaborative study by the Non-Genotoxic Carcinogen Study Group in Japan, on a cell transformation assay for tumour promoters using Bhas 42 cells. Altern Lab Anim 33:619–639

    CAS  PubMed  Google Scholar 

  15. Tanaka N, Sasaki K, Hayashi K et al (2009) An interlaboratory collaborative study on a cell transformation assay using Bhas 42 cells. AATEX 14:831–848

    Google Scholar 

  16. Sakai A, Sasaki K, Hayashi K et al (2011) An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity. Mutat Res 725:57–77

    Article  CAS  PubMed  Google Scholar 

  17. Hayashi M, Kojima H, Corvi R et al (2012) Bhas 42 cell transformation assay validation study report. EURL ECVAM Recommendations, Ispra, Italy

    Google Scholar 

  18. Ohmori K (2009) In vitro assays for the prediction of tumorigenic potential of non-genotoxic carcinogens. J Health Sci 55:20–30

    Article  CAS  Google Scholar 

  19. Vanparys P, Corvi R, Aardema MJ et al (2012) Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics. Mutat Res 744:111–116

    Article  CAS  PubMed  Google Scholar 

  20. Balls M, Clothier R (2010) A FRAME response to the Draft Report on Alternative (Non-animal) Methods for Cosmetics Testing: Current Status and Future Prospects—2010. Altern Lab Anim 38:345–353

    CAS  PubMed  Google Scholar 

  21. Corvi R, Aardema MJ, Gribaldo L et al (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 

  22. Maire MA, Pant K, Poth A et al (2012) Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0 for assessment of carcinogenic potential of chemicals. Mutat Res 744:64–75

    Article  CAS  PubMed  Google Scholar 

  23. Pant K, Bruce SW, Sly JE et al (2012) Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7 for assessment of carcinogenic potential of chemicals. Mutat Res 744:54–63

    Article  CAS  PubMed  Google Scholar 

  24. Tanaka N, Bohnenberger S, Kunkelmann T et al (2012) Prevalidation study of the BALB/c 3T3 cell transformation assay for assessment of carcinogenic potential of chemicals. Mutat Res 744:20–29

    Article  CAS  PubMed  Google Scholar 

  25. Benigni R, Bossa C, Tcheremenskaia O (2013) In vitro cell transformation assays for an integrated, alternative assessment of carcinogenicity: a data-based analysis. Mutagenesis 28:107–116

    Article  CAS  PubMed  Google Scholar 

  26. IARC/NCI/EPA Working Group (1985) Cellular and molecular mechanisms of cell transformation and standardization of transformation assays of established cell lines for the prediction of carcinogenic chemicals: overview and recommended protocols. Cancer Res 45:2395–2399

    Google Scholar 

  27. Kerckaert GA, Brauninger R, LeBoeuf RA et al (1996) Use of the Syrian hamster embryo cell transformation assay for carcinogenicity prediction of chemicals currently being tested by the National Toxicology Program in rodent bioassays. Environ Health Perspect 104:1075–1084

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Hasegawa G, Shimonaka M, Ishihara Y (2013) Differential genotoxicity of chemical properties and particle size of rare metal and metal oxide nanoparticles. J Appl Toxicol 32:72–80

    Article  Google Scholar 

  29. Mazzotti F, Sabbioni E, Ponti J et al (2002) In vitro setting of dose–effect relationships of 32 metal compounds in the Balb/3T3 cell line, as a basis for predicting their carcinogenic potential. Altern Lab Anim 30:209–217

    CAS  PubMed  Google Scholar 

  30. Ponti J, Munaro B, Fischbach M et al (2007) An optimised data analysis for the BALB/c 3T3 cell transformation assay and its application to metal compounds. Int J Immunopathol Pharmacol 20:673–684

    CAS  PubMed  Google Scholar 

  31. Ponti J, Sabbioni E, Munaro B et al (2009) Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3T3 mouse fibroblasts. Mutagenesis 24:439–445

    Article  CAS  PubMed  Google Scholar 

  32. Ohmori K, Sato Y, Nakajima D et al (2013) Characteristics of the transformation frequency at the tumor promotion stage of airborne particulate and gaseous matter at ten sites in Japan. Environ Sci Process Impacts 15:1031–1040

    Article  CAS  PubMed  Google Scholar 

  33. Weisensee D, Poth A, Roemer E et al (2013) Cigarette smoke-induced morphological transformation of Bhas 42 cells in vitro. Altern Lab Anim 41:181–189

    CAS  PubMed  Google Scholar 

  34. Uboldi C, Giudetti G, Broggi F et al (2012) Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts. Mutat Res 745:11–20

    Article  CAS  PubMed  Google Scholar 

  35. Ponti J, Broggi F, Mariani V et al (2013) Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential. Nanotoxicology 7:221–233

    Article  CAS  PubMed  Google Scholar 

  36. Kakunaga T, Crow JD (1980) Cell variants showing differential susceptibility to ultraviolet light-induced transformation. Science 209:505–507

    Article  CAS  PubMed  Google Scholar 

  37. Pontes H, Carvalho M, de Pinho PG et al (2008) Ethanol, the forgotten artifact in cell culture. Arch Toxicol 82:197–198

    Article  CAS  PubMed  Google Scholar 

  38. Arai S, Sakai A, Hayashi K et al (2013) A high-throughput cell transformation assay applicable to automation for detecting potential chemical carcinogens using Bhas 42 cells. AATEX 18:1–19

    Google Scholar 

  39. Heidelberger C, Freeman AE, Pienta RJ et al (1983) Cell transformation by chemical agents—a review and analysis of the literature. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat Res 114:283–385

    Article  CAS  PubMed  Google Scholar 

  40. Arai S, Tanaka N, Sasaki K et al (2010) A study on the dose setting of test chemicals for the promotion assay in Bhas 42 cell transformation assay. AATEX 15:6–13

    Google Scholar 

  41. Sasaki K, Sakai A, Tanaka N (2013) High-throughput quantification of morphologically transformed foci in Bhas 42 cells (v-Ha-ras transfected Balb/c 3T3) using spectrophotometry. In: Steinberg P (ed) High-throughput screening methods in toxicity testing. Wiley, New York, pp 317–339

    Chapter  Google Scholar 

  42. Suganuma M, Kurusu M, Suzuki K et al (2005) New tumor necrosis factor-α-inducing protein released from Helicobacter pylori for gastric cancer progression. J Cancer Res Clin Oncol 131:305–313

    CAS  PubMed  Google Scholar 

  43. Suganuma M, Okabe S, Marino MW et al (1999) Essential role of tumor necrosis factor α (TNF-α) in tumor promotion as revealed by TNF-α-deficient mice. Cancer Res 59:4516–4518

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the EC FP7 QualityNano [INFRA-2010-1.131], contract no: 214547-2, EC FP7 NANoREG, [NMP.2012.1.3-3] contract no: 310584, and by the NEDO grant (New Energy and Industrial Technology Development Organization).

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Authors and Affiliations

  1. Laboratory of Cell Carcinogenesis, Division of Alternative Toxicology Test Hatano Research Institute, Food and Drug Safety Center, Hadano, Japan

    Kiyoshi Sasaki & Noriho Tanaka

  2. Health Effects Laboratory, Department of Environmental Chemistry, NILU (Norwegian Institute for Air Research), Kjeller, Norway

    Anna Huk, Naouale El Yamani & Maria Dusinska

Authors
  1. Kiyoshi Sasaki
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  2. Anna Huk
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  3. Naouale El Yamani
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  4. Noriho Tanaka
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  5. Maria Dusinska
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Corresponding author

Correspondence to Maria Dusinska .

Editor information

Editors and Affiliations

  1. Universidad de Oviedo, Oviedo, Spain

    L. María Sierra

  2. Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal

    Isabel Gaivão

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Sasaki, K., Huk, A., Yamani, N.E., Tanaka, N., Dusinska, M. (2014). Bhas 42 Cell Transformation Assay for Genotoxic and Non-Genotoxic Carcinogens. In: Sierra, L., Gaivão, I. (eds) Genotoxicity and DNA Repair. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1068-7_20

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  • DOI: https://doi.org/10.1007/978-1-4939-1068-7_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1067-0

  • Online ISBN: 978-1-4939-1068-7

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