Benzo[a]pyrene-induced transcriptomic responses in primary hepatocytes and in vivo liver: Toxicokinetics is essential for in vivo–in vitro comparisons
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The traditional 2-year cancer bioassay needs replacement by more cost-effective and predictive tests. The use of toxicogenomics in an in vitro system may provide a more high-throughput method to investigate early alterations induced by carcinogens. Recently, the differential gene expression response in wild-type and cancer-prone Xpa −/− p53 +/− primary mouse hepatocytes after exposure to benzo[a]pyrene (B[a]P) revealed downregulation of cancer-related pathways in Xpa −/− p53 +/− hepatocytes only. Here, we investigated pathway regulation upon in vivo B[a]P exposure of wild-type and Xpa −/− p53 +/− mice. In vivo transcriptomics analysis revealed a limited gene expression response in mouse livers, but with a significant induction of DNA replication and apoptotic/anti-apoptotic cellular responses in Xpa −/− p53 +/− livers only. In order to be able to make a meaningful in vivo–in vitro comparison we estimated internal in vivo B[a]P concentrations using DNA adduct levels and physiologically based kinetic modeling. Based on these results, the in vitro concentration that corresponded best with the internal in vivo dose was chosen. Comparison of in vivo and in vitro data demonstrated similarities in transcriptomics response: xenobiotic metabolism, lipid metabolism and oxidative stress. However, we were unable to detect cancer-related pathways in either wild-type or Xpa −/− p53 +/− exposed livers, which were previously found to be induced by B[a]P in Xpa −/− p53 +/− primary hepatocytes. In conclusion, we showed parallels in gene expression responses between livers and primary hepatocytes upon exposure to equivalent concentrations of B[a]P. Furthermore, we recommend considering toxicokinetics when modeling a complex in vivo endpoint with in vitro models.
KeywordsToxicogenomics Carcinogenesis Benzo[a]pyrene Xpa−/−p53+/− Physiologically based kinetic modeling
Analysis of variance
False discovery rate
Gene Map Annotator and Pathway Profiler
Kyoto Encyclopedia of Genes and Genomes
Physiologically based kinetic
Principal component analysis
Xeroderma pigmentosum A
Xpa −/− p53 +/−
We thank R. Vlug, J. Bos, H. Strootman and T. van de Kuil for their support with the hepatocytes isolations. We also thank Prof. Dr. J. Hengstler for his advice on hepatocyte isolation and culturing. This work was supported by the Technology Foundation STW [grant MFA6809].
Conflict of interest
The authors declare that they have no conflict of interest.
- 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 (5):367–485Google Scholar
- Fielden MR, Nie A, McMillian M, Elangbam CS, Trela BA, Yang Y, Dunn RT, Dragan Y, Fransson-Stehen R, Bogdanffy M, Adams SP, Foster WR, Chen SJ, Rossi P, Kasper P, Jacobson-Kram D, Tatsuoka KS, Wier PJ, Gollub J, Halbert DN, Roter A, Young JK, Sina JF, Marlowe J, Martus HJ, Aubrecht J, Olaharski AJ, Roome N, Nioi P, Pardo I, Snyder R, Perry R, Lord P, Mattes W, Car BD (2008) Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat. Toxicol Sci 103(1):28–34PubMedCrossRefGoogle Scholar
- Godschalk RW, Maas LM, Van Zandwijk N, van ‘t Veer LJ, Breedijk A, Borm PJ, Verhaert J, Kleinjans JC, van Schooten FJ (1998) Differences in aromatic-DNA adduct levels between alveolar macrophages and subpopulations of white blood cells from smokers. Carcinogenesis 19(5):819–825PubMedCrossRefGoogle Scholar
- Klaassen R, Olling M, Lusthof KJ (1996) The influence of the use of animal cages with wire floor or macrolon cages with middle size saw on the kinetics of benzo(a)pyrene in the rat. RIVM Report 658603 006Google Scholar
- Uehara T, Minowa Y, Morikawa Y, Kondo C, Maruyama T, Kato I, Nakatsu N, Igarashi Y, Ono A, Hayashi H, Mitsumori K, Yamada H, Ohno Y, Urushidani T (2011) Prediction model of potential hepatocarcinogenicity of rat hepatocarcinogens using a large-scale toxicogenomics database. Toxicol Appl Pharmacol 255(3):297–306PubMedCrossRefGoogle Scholar
- van Kesteren PC, Zwart PE, Pennings JL, Gottschalk WH, Kleinjans JC, van Delft JH, van Steeg H, Luijten M (2011) Deregulation of cancer-related pathways in primary hepatocytes derived from DNA repair-deficient Xpa −/− p53 +/− mice upon exposure to benzo[a]pyrene. Toxicol Sci 123(1):123–132PubMedCrossRefGoogle Scholar
- van Steeg H, de Vries A, van Oostrom CT, van Benthem J, Beems RB, van Kreijl CF (2001) DNA repair-deficient Xpa and Xpa/p53 +/− knock-out mice: nature of the models. ToxicolPathol 29(Suppl):109–116Google Scholar
- Zeilmaker MJ, van Eijkeren JCH, Olling M (1999) A PBPK-model for B(a)P in the rat relating dose and liver DNA-adduct level. RIVM Report 658603 008Google Scholar