Abstract
Gene expression analysis has been established as a tool for the characterization of genotoxic mechanisms of chemical mutagens. It has been suggested that expression analysis is capable of distinguishing compounds that cause DNA damage from those that interfere with mitotic spindle function. Formaldehyde (FA) is known to be a DNA-reactive substance which mainly induces chromosomal damage in cultured mammalian cells. However, there has been concern that FA might also induce leukemia-specific aneuploidies, although recent cytogenetic studies excluded a relevant aneugenic potential of FA. We now investigated whether gene expression profiling can be used as a molecular tool to further characterize FA’s genotoxic mode of action and to differentiate between clastogenic and aneugenic activity. TK6 cells were exposed to FA for 4 and 24 h, and changes in gene expression were analyzed using a whole-genome human microarray. Results were compared to the expression profiles of two DNA-damaging clastogens (methyl methanesulfonate and ethyl methanesulfonate) and two aneugens (colcemid and vincristine). The genotoxic activity of FA, MMS and EMS under these conditions was confirmed by comet assay experiments. The gene expression profiles indicated that clastogens and aneugens induce discriminable gene expression patterns. Exposure of TK6 cells to FA led to a discrete gene expression pattern, and all toxicogenomics analyses revealed a closer relationship of FA with clastogens than with aneugens.
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Amundson SA, Do KT, Vinikoor L, Koch-Paiz CA, Bittner ML, Trent JM, Meltzer P, Fornace AJ Jr (2005) Stress-specific signatures: expression profiling of p53 wild-type and -null human cells. Oncogene 24:4572–4579
Casanova M, Morgan KT, Steinhagen WH, Everitt JI, Popp JA, Heck HD (1991) Covalent binding of inhaled formaldehyde to DNA in the respiratory tract of rhesus monkeys: pharmacokinetics, rat-to-monkey interspecies scaling, and extrapolation to man. Fundam Appl Toxicol 17:409–428
Dickinson DA, Warnes GR, Quievryn G, Messer J, Zhitkovich A, Rubitski E, Aubrecht J (2004) Differentiation of DNA reactive and non-reactive genotoxic mechanisms using gene expression profile analysis. Mutat Res 549:29–41
Doerjer G, Bedell A, Oesch F (1984) DNA adducts and their biological relevance. In: Obe G (ed) Mutations in Man. Springer, Berlin, pp 20–34
Elhajouji A, Lukamowicz M, Cammerer Z, Kirsch-Volders M (2011) Potential thresholds for genotoxic effects by micronucleus scoring. Mutagenesis 26:199–204
Ellinger-Ziegelbauer H, Fostel JM, Aruga C, Bauer D, Boitier E, Deng S, Dickinson D, Le Fevre AC, Fornace A Jr, Grenet O, Gu Y, Hoflack JC, Shiiyama M, Smith R, Snyder RD, Spire C, Tanaka G, Aubrecht J (2009) Characterization and interlaboratory comparison of a gene expression signature for differentiating genotoxic mechanisms. Toxicol Sci 110:341–352
Godderis L, Thomas R, Hubbard AE, Tabish AM, Hoet P, Zhang L, Smith MT, Veulemans H, McHale CM (2012) Effect of chemical mutagens and carcinogens on gene expression profiles in human TK6 cells. PLoS ONE 7:e39205
Greenwood SK, Hill R, Sun J, Armstrong MJ, Johnson TE, Gara JP, Galloway SM (2004) Population doubling: a simple and more accurate estimation of cell growth suppression in the in vitro assay for chromosomal aberrations that reduces irrelevant positive results. Environ Mol Mutagen 43:36–44
Hu T, Gibson DP, Carr GJ, Torontali SM, Tiesman JP, Chaney JG, Aardema MJ (2004) Identification of a gene expression profile that discriminates indirect-acting genotoxins from direct-acting genotoxins. Mutat Res 549:5–27
IARC (International Agency for Research on Cancer) (2012) Chemical agents and related occupations. Monogr Eval Carcinog Risks Hum 100F:401–435
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:249–264
Islaih M, Li B, Kadura IA, Reid-Hubbard JL, Deahl JT, Altizer JL, Watson DE, Newton RK (2004) Comparison of gene expression changes induced in mouse and human cells treated with direct-acting mutagens. Environ Mol Mutagen 44:401–419
Islaih M, Halstead BW, Kadura IA, Li B, Reid-Hubbard JL, Flick L, Altizer JL, Thom DJ, Monteith DK, Newton RK, Watson DE (2005) Relationships between genomic, cell cycle, and mutagenic responses of TK6 cells exposed to DNA damaging chemicals. Mutat Res 578:100–116
Kuehner S, Schlaier M, Schwarz K, Speit G (2012) Analysis of leukemia-specific aneuploidies in cultured myeloid progenitor cells in the absence and presence of formaldehyde exposure. Toxicol Sci 128:72–78
Le Fevre AC, Boitier E, Marchandeau JP, Sarasin A, Thybaud V (2007) Characterization of DNA reactive and non-DNA reactive anticancer drugs by gene expression profiling. Mutat Res 619:16–29
Magkoufopoulou C, Claessen SM, Jennen DG, Kleinjans JC, van Delft JH (2011) Comparison of phenotypic and transcriptomic effects of false-positive genotoxins, true genotoxins and non-genotoxins using HepG2 cells. Mutagenesis 26:593–604
Mathijs K, Brauers KJ, Jennen DG, Boorsma A, van Herwijnen MH, Gottschalk RW, Kleinjans JC, van Delft JH (2009) Discrimination for genotoxic and nongenotoxic carcinogens by gene expression profiling in primary mouse hepatocytes improves with exposure time. Toxicol Sci 112:374–384
Mathijs K, Brauers KJ, Jennen DG, Lizarraga D, Kleinjans JC, van Delft JH (2010) Gene expression profiling in primary mouse hepatocytes discriminates true from false-positive genotoxic compounds. Mutagenesis 25:561–568
Merk O, Speit G (1998) Significance of formaldehyde-induced DNA-protein crosslinks for mutagenesis. Environ Mol Mutagen 32:260–268
Moll UM, Petrenko O (2003) The MDM2-p53 interaction. Mol Cancer Res 1:1001–1008
Neuss S, Holzmann K, Speit G (2010) Gene expression changes in primary human nasal epithelial cells exposed to formaldehyde in vitro. Toxicol Lett 198(2):289–295
OECD (2010) Guideline for the testing of chemicals No. 487: in vitro mammalian cell micronucleus test (Mnvit). OECD, Paris
Pennie W, Pettit SD, Lord PG (2004) Toxicogenomics in risk assessment: an overview of an HESI collaborative research program. Environ Health Perspect 112:417–419
Platel A, Gervais V, Sajot N, Nesslany F, Marzin D, Claude N (2010) Study of gene expression profiles in TK6 human cells exposed to DNA-oxidizing agents. Mutat Res 689:21–49
Schmid O, Speit G (2007) Genotoxic effects induced by formaldehyde in human blood and implications for the interpretation of biomonitoring studies. Mutagenesis 22:69–74
Simon R, Lam A, Li MC, Ngan M, Menenzes S, Zhao Y (2007) Analysis of gene expression data using BRB-ArrayTools. Cancer Inform 3:11–17
Snel B, Lehmann G, Bork P, Huynen A (2000) STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene. Nucleic Acids Res 28:3442–3444
Speit G, Merk O (2002) Evaluation of mutagenic effects of formaldehyde in vitro: detection of crosslinks and mutations in mouse lymphoma cells. Mutagenesis 17:183–187
Speit G, Rothfuss A (2012) The comet assay: a sensitive genotoxicity test for the detection of DNA damage and repair. Methods Mol Biol 920:79–90
Speit G, Schmid O, Neuss S, Schütz P (2008) Genotoxic effects of formaldehyde in the human lung cell line A549 and in primary human nasal epithelial cells. Environ Mol Mutagen 49:300–307
Speit G, Gelbke HP, Pallapies D, Morfeld P (2010) Occupational exposure to formaldehyde, hematotoxicity and leukemia-specific chromosome changes in cultured myeloid progenitor cells. Cancer Epidemiol Biomarkers Prev 19:1882–1884
Speit G, Kuehner S, Linsenmeyer R, Schutz P (2011) Does formaldehyde induce aneuploidy? Mutagenesis 26:805–811
Speit G, Linsenmeyer R, Schutz P, Kuehner S (2012) Insensitivity of the in vitro cytokinesis-block micronucleus assay with human lymphocytes for the detection of DNA damage present at the start of the cell culture. Mutagenesis 27:743–747
Thybaud V, Le Fevre AC, Boitier E (2007) Application of toxicogenomics to genetic toxicology risk assessment. Environ Mol Mutagen 48:369–379
Tibshirani R, Hastie T, Narasimhan B, Chu G (2002) Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc Natl Acad Sci USA 99:6567–6572
Van Delft JH, van Agen E, van Breda SG, Herwijnen MH, Staal YC, Kleinjans JC (2004) Discrimination of genotoxic from non-genotoxic carcinogens by gene expression profiling. Carcinogenesis 25:1265–1276
Wright GW, Simon RM (2003) A random variance model for detection of differential gene expression in small microarray experiments. Bioinformatics 19:2448–2455
Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, Sunshine M, Narasimhan S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, Barrett JC, Weinstein JN (2003) GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol 4:R28
Zhang L, Honma M, Hayashi M, Suzuki T, Matsuoka A, Sofuni T (1995) A comparative study of TK6 human lymphoblastoid and L5178Y mouse lymphoma cell lines in the in vitro micronucleus test. Mutat Res 347:105–115
Zhang L, Tang X, Rothman N, Vermeulen R, Ji Z, Shen M, Qiu C, Guo W, Liu S, Reiss B, Freeman LB, Ge Y, Hubbard AE, Hua M, Blair A, Galvan N, Ruan X, Alter BP, Xin KX, Li S, Moore LE, Kim S, Xie Y, Hayes RB, Azuma M, Hauptmann M, Xiong J, Stewart P, Li L, Rappaport SM, Huang H, Fraumeni JF Jr, Smith MT, Lan Q (2010) Occupational exposure to formaldehyde, hematotoxicity, and leukemia-specific chromosome changes in cultured myeloid progenitor cells. Cancer Epidemiol Biomarkers Prev 19:80–88
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The financial support by the European Chemical Industry Council (CEFIC) is gratefully acknowledged.
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The authors declare that the views in this paper are those of the authors they have no conflict of interest in presenting their opinions.
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Kuehner, S., Holzmann, K. & Speit, G. Characterization of formaldehyde’s genotoxic mode of action by gene expression analysis in TK6 cells. Arch Toxicol 87, 1999–2012 (2013). https://doi.org/10.1007/s00204-013-1060-2
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DOI: https://doi.org/10.1007/s00204-013-1060-2