Environmental Science and Pollution Research

, Volume 25, Issue 5, pp 4012–4022 | Cite as

p53 induction and cell viability modulation by genotoxic individual chemicals and mixtures

  • Carolina Di PaoloEmail author
  • Yvonne Müller
  • Beat Thalmann
  • Henner Hollert
  • Thomas-Benjamin Seiler
Effect-related evaluation of anthropogenic trace substances, -concepts for genotoxicity, neurotoxicity and, endocrine effects


The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.


p53 tumor suppression protein p53 pathway DNA damage Genotoxicity Cytotoxicity Reporter gene cell line bioassay 



Thanks to the RWTH colleague Simone Hotz for the support during the establishment of the cell line and assay in our laboratory. Thanks to BioDetection Systems BV (BDS, Amsterdam, The Netherlands) for supplying the cell line and respective culture and method protocols. Thanks to Promega GmbH, Germany, and to Tecan Group Ltd., Switzerland, for their contribution to this study as a partner of the Students Lab “Fascinating Environment” at Aachen Biology and Biotechnology (ABBt). This study was supported by the EDA-EMERGE ITN project within the EU Seventh Framework Program (FP7-PEOPLE-2011-ITN) under the grant agreement number 290100.


  1. Arlt VM, Cole KJ, Phillips DH (2004) Activation of 3-nitrobenzanthrone and its metabolites to DNA-damaging species in human B lymphoblastoid MCL-5 cells. Mutagenesis 19:149–156CrossRefGoogle Scholar
  2. Asiri YA (2010) Probucol attenuates cyclophosphamide-induced oxidative apoptosis, p53 and Bax signal expression in rat cardiac tissues. Oxidative Med Cell Longev 3:308–316CrossRefGoogle Scholar
  3. Beckerman R, Prives C (2010) Transcriptional regulation by p53. Cold Spring Harb Perspect Biol 2(8):a000935Google Scholar
  4. Bensaad K, Rouillard D, Soussi T (2001) Regulation of the cell cycle by p53 after DNA damage in an amphibian cell line. Oncogene 20:3766–3775CrossRefGoogle Scholar
  5. Berridge MV, Herst PM, Tan AS (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 11:127–152Google Scholar
  6. Bhaskaran A, May D, Rand-Weaver M, Tyler CR (1999) Fish p53 as a possible biomarker for genotoxins in the aquatic environment. Environ Mol Mutagen 33:177–184CrossRefGoogle Scholar
  7. Briat A, Vassaux G (2008) A new transgenic mouse line to image chemically induced p53 activation in vivo. Cancer Sci 99:683–688CrossRefGoogle Scholar
  8. Brinkmann M, Blenkle H, Salowsky H, Bluhm K, Schiwy S, Tiehm A et al (2014) Genotoxicity of heterocyclic PAHs in the micronucleus assay with the fish liver cell line RTL-W1. PLoS One 9:e85692CrossRefGoogle Scholar
  9. Brüsehafer K, Manshian BB, Doherty AT, Zaïr ZM, Johnson GE, Doak SH et al (2016) The clastogenicity of 4NQO is cell-type dependent and linked to cytotoxicity, length of exposure and p53 proficiency. Mutagenesis 31:171–180CrossRefGoogle Scholar
  10. Česen M, Kosjek T, Busetti F, Kompare B, Heath E (2016) Human metabolites and transformation products of cyclophosphamide and ifosfamide: analysis, occurrence and formation during abiotic treatments. Environ Sci Pollut Res 23:11209–11223CrossRefGoogle Scholar
  11. Chen C-S, Ho D-R, Chen F-Y, Chen C-R, Ke Y-D, Su J-GJ (2014) AKT mediates actinomycin D-induced p53 expression. Oncotarget 5:693–703Google Scholar
  12. Choong ML, Yang H, Lee MA, Lane DP (2009) Specific activation of the p53 pathway by low dose actinomycin D: a new route to p53 based cyclotherapy. Cell Cycle 8:2810–2818CrossRefGoogle Scholar
  13. Clewell RA, Sun B, Adeleye Y, Carmichael P, Efremenko A, McMullen PD et al (2014) Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage. Toxicol Sci 142:56–73CrossRefGoogle Scholar
  14. Duerksen-Hughes PJ, Yang J, Ozcan O (1999) p53 induction as a genotoxic test for twenty-five chemicals undergoing in vivo carcinogenicity testing. Environ Health Perspect 107:805–812CrossRefGoogle Scholar
  15. Ferre-Aracil J, Valcárcel Y, Negreira N, de Alda ML, Barceló D, Cardona SC et al (2016) Ozonation of hospital raw wastewaters for cytostatic compounds removal. Kinetic modelling and economic assessment of the process. Sci Total Environ 556:70–79CrossRefGoogle Scholar
  16. Garner E, Raj K (2008) Protective mechanisms of p53-p21-pRb proteins against DNA damage-induced cell death. Cell Cycle 7:277–282CrossRefGoogle Scholar
  17. Groten JP, Feron VJ, Suhnel J (2001) Toxicology of simple and complex mixtures. Trends Pharmacol Sci 22:316–322CrossRefGoogle Scholar
  18. Han H, Pan Q, Zhang B, Li J, Deng X, Lian Z et al (2007) 4-NQO induces apoptosis via p53-dependent mitochondrial signaling pathway. Toxicology 230:151–163CrossRefGoogle Scholar
  19. Harris SL, Levine AJ (2005) The p53 pathway: positive and negative feedback loops. Oncogene 24:2899–2908CrossRefGoogle Scholar
  20. IARC. IARC Working Group on the Evaluation of Carcinogenic Risk to Humans. Diesel and gasoline engine exhausts and some nitroarenes. Lyon (FR): International Agency for Research on Cancer (2014) (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, No. 105.) 3-NITROBENZANTHRONE. Available from: 2014
  21. Imazawa T, Nishikawa A, Toyoda K, Furukawa F, Mitsui M, Hirose M (2003) Sequential alteration of apoptosis, p53 expression, and cell proliferation in the rat pancreas treated with 4-hydroxyaminoquinoline 1-oxide. Toxicol Pathol 31:625–631CrossRefGoogle Scholar
  22. Ivashkevich A, Redon CE, Nakamura AJ, Martin RF, Martin OA (2012) Use of the γ-H2AX assay to monitor DNA damage and repair in translational cancer research. Cancer Lett 327:123–133CrossRefGoogle Scholar
  23. Jin L, Gaus C, Escher BI (2015) Adaptive stress response pathways induced by environmental mixtures of bioaccumulative chemicals in dugongs. Environ Sci Technol 49:6963–6973CrossRefGoogle Scholar
  24. Kester HA, Sonneveld E, van der Saag PT, van der Burg B (2003) Prolonged progestin treatment induces the promoter of CDK inhibitor p21Cip1, Waf1 through activation of p53 in human breast and endometrial tumor cells. Exp Cell Res 284:262–271CrossRefGoogle Scholar
  25. Knight AW, Little S, Houck K, Dix D, Judson R, Richard A et al (2009) Evaluation of high-throughput genotoxicity assays used in profiling the US EPA ToxCast chemicals. Regul Toxicol Pharmacol 55:188–199CrossRefGoogle Scholar
  26. Kumari R, Kohli S, Das S (2014) p53 regulation upon genotoxic stress: intricacies and complexities. Mol Cell Oncol 1:e969653CrossRefGoogle Scholar
  27. Kuo LJ, Yang LX (2008) Gamma-H2AX—a novel biomarker for DNA double-strand breaks. In Vivo 22:305–309Google Scholar
  28. Landvik NE, Arlt VM, Nagy E, Solhaug A, Tekpli X, Schmeiser HH et al (2010) 3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells. Mutat Res Fundam Mol Mech Mutagen 684:11–23CrossRefGoogle Scholar
  29. Lavin MF, Gueven N (2006) The complexity of p53 stabilization and activation. Cell Death Differ 13:941–950CrossRefGoogle Scholar
  30. Lutzker SG, Mathew R, Taller DR (2001) A p53 dose-response relationship for sensitivity to DNA damage in isogenic teratocarcinoma cells. Oncogene 20:2982–2986CrossRefGoogle Scholar
  31. Mischo HE, Hemmerich P, Grosse F, Zhang S (2005) Actinomycin D induces histone γ-H2AX foci and complex formation of γ-H2AX with Ku70 and nuclear DNA helicase II. J Biol Chem 280:9586–9594CrossRefGoogle Scholar
  32. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  33. Nagy E, Adachi S, Takamura-Enya T, Zeisig M, Möller L (2007) DNA adduct formation and oxidative stress from the carcinogenic urban air pollutant 3-nitrobenzanthrone and its isomer 2-nitrobenzanthrone, in vitro and in vivo. Mutagenesis 22:135–145CrossRefGoogle Scholar
  34. Øya E, Øvrevik J, Arlt VM, Nagy E, Phillips DH, Holme JA (2011) DNA damage and DNA damage response in human bronchial epithelial BEAS-2B cells following exposure to 2-nitrobenzanthrone and 3-nitrobenzanthrone: role in apoptosis. Mutagenesis 26(6):697–708Google Scholar
  35. Rao B, van Leeuwen IMM, Higgins M, Campbell J, Thompson AM, Lane DP et al (2010) Evaluation of an actinomycin D/VX-680 aurora kinase inhibitor combination in p53-based cyclotherapy. Oncotarget 1:639–650Google Scholar
  36. Rutkowski R, Hofmann K, Gartner A (2010) Phylogeny and function of the invertebrate p53 superfamily. Cold Spring Harb Perspect Biol 2:a001131CrossRefGoogle Scholar
  37. Salazar AM, Ostrosky-Wegman P, Menendez D, Miranda E, Garcia-Carranca A, Rojas E (1997) Induction of p53 protein expression by sodium arsenite. Mutat Res Fundam Mol Mech Mutagen 381:259–265CrossRefGoogle Scholar
  38. Salazar AM, Sordo M, Ostrosky-Wegman P (2009) Relationship between micronuclei formation and p53 induction. Mutat Res Genet Toxicol Environ Mutagen 672:124–128CrossRefGoogle Scholar
  39. SCHER, SCCS, SCENIHR (2012) Opinion on the toxicity and assessment of chemical mixtures. European Commission, BrusselsGoogle Scholar
  40. Sohn TA, Bansal R, Su GH, Murphy KM, Kern SE (2002) High-throughput measurement of the Tp53 response to anticancer drugs and random compounds using a stably integrated Tp53-responsive luciferase reporter. Carcinogenesis 23:949–958CrossRefGoogle Scholar
  41. Steger-Hartmann T, Kümmerer K, Hartmann A (1997) Biological degradation of cyclophosphamide and its occurrence in sewage water. Ecotoxicol Environ Saf 36:174–179CrossRefGoogle Scholar
  42. Storer NY, Zon LI (2010) Zebrafish models of p53 functions. Cold Spring Harb Perspect Biol 2:a001123CrossRefGoogle Scholar
  43. Strauss G, Westhoff MA, Fischer-Posovszky P, Fulda S, Schanbacher M, Eckhoff SM et al (2007) 4-Hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG. Cell Death Differ 15:332–343CrossRefGoogle Scholar
  44. van der Linden SC, von Bergh ARM, van Vught-Lussenburg BMA, Jonker LRA, Teunis M, Krul CAM et al (2014) Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress. Mutat Res Genet Toxicol Environ Mutagen 760:23–32CrossRefGoogle Scholar
  45. van Leeuwen IM, Higgins M, Campbell J, Brown CJ, McCarthy AR, Pirrie L et al (2011) Mechanism-specific signatures for small-molecule p53 activators. Cell Cycle 10:1590–1598CrossRefGoogle Scholar
  46. Wernersson A-S, Carere M, Maggi C, Tusil P, Soldan P, James A et al (2015) The European technical report on aquatic effect-based monitoring tools under the water framework directive. Environ Sci Eur 27:1–11CrossRefGoogle Scholar
  47. Xiao H, Kuckelkorn J, Nüßer LK, Floehr T, Hennig MP, Roß-Nickoll M et al (2016) The metabolite 3,4,3′,4′-tetrachloroazobenzene (TCAB) exerts a higher ecotoxicity than the parent compounds 3,4-dichloroaniline (3,4-DCA) and propanil. Sci Total Environ 551–552:304–316CrossRefGoogle Scholar
  48. Yang J, Duerksen-Hughes P (1998) A new approach to identifying genotoxic carcinogens: p53 induction as an indicator of genotoxic damage. Carcinogenesis 19:1117–1125CrossRefGoogle Scholar
  49. Yeh RYL, Farré MJ, Stalter D, Tang JYM, Molendijk J, Escher BI (2014) Bioanalytical and chemical evaluation of disinfection by-products in swimming pool water. Water Res 59:172–184CrossRefGoogle Scholar
  50. Zajkowicz A, Gdowicz-Klosok A, Krzesniak M, Scieglinska D, Rusin M (2015) Actinomycin D and nutlin-3a synergistically promote phosphorylation of p53 on serine 46 in cancer cell lines of different origin. Cell Signal 27:1677–1687CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of Ecosystem Analysis, Institute for Environmental Research, Aachen Biology and Biotechnology (ABBt)RWTH Aachen UniversityAachenGermany
  2. 2.College of Resources and Environmental ScienceChongqing UniversityChongqingChina
  3. 3.College of Environmental Science and Engineering and State Key Laboratory of Pollution Control and Resource ReuseTongji UniversityShanghaiChina
  4. 4.State Key Laboratory of Pollution Control and Resource Reuse, School of the EnvironmentNanjing UniversityNanjingChina

Personalised recommendations