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Archives of Toxicology

, Volume 92, Issue 8, pp 2549–2561 | Cite as

Confounding influence of tamoxifen in mouse models of Cre recombinase-induced gene activity or modulation

  • Seddik Hammad
  • Amnah Othman
  • Christoph Meyer
  • Ahmad Telfah
  • Joerg Lambert
  • Bedair Dewidar
  • Julia Werle
  • Zeribe Chike Nwosu
  • Abdo Mahli
  • Christof Dormann
  • Yan Gao
  • Kerry Gould
  • Mei Han
  • Xiaodong Yuan
  • Mikheil Gogiashvili
  • Roland Hergenröder
  • Claus Hellerbrand
  • Maria Thomas
  • Matthias Philip Ebert
  • Salah Amasheh
  • Jan G. Hengstler
  • Steven Dooley
Molecular Toxicology
  • 373 Downloads

Abstract

Tamoxifen (TAM) is commonly used for cell type specific Cre recombinase-induced gene inactivation and in cell fate tracing studies. Inducing a gene knockout by TAM and using non-TAM exposed mice as controls lead to a situation where differences are interpreted as consequences of the gene knockout but in reality result from TAM-induced changes in hepatic metabolism. The degree to which TAM may compromise the interpretation of animal experiments with inducible gene expression still has to be elucidated. Here, we report that TAM strongly attenuates CCl4-induced hepatotoxicity in male C57Bl/6N mice, even after a 10 days TAM exposure-free period. TAM decreased (p < 0.0001) the necrosis index and the level of aspartate- and alanine transaminases in CCl4-treated compared to vehicle-exposed mice. TAM pretreatment also led to the downregulation of CYP2E1 (p = 0.0045) in mouse liver tissue, and lowered its activity in CYP2E1 expressing HepG2 cell line. Furthermore, TAM increased the level of the antioxidant ascorbate, catalase, SOD2, and methionine, as well as phase II metabolizing enzymes GSTM1 and UGT1A1 in CCl4-treated livers. Finally, we found that TAM increased the presence of resident macrophages and recruitment of immune cells in necrotic areas of the livers as indicated by F4/80 and CD45 staining. In conclusion, we reveal that TAM increases liver resistance to CCl4-induced toxicity. This finding is of high relevance for studies using the tamoxifen-inducible expression system particularly if this system is used in combination with hepatotoxic compounds such as CCl4.

Keywords

Tamoxifen Hepatotoxicity CCl4 Inducible Cre system 

Notes

Acknowledgements

The authors thank Elmar Kriek (IfADo-Dortmund) for his excellent technical assistance with the animal experiments, Nina Draeger (UMM Technician school) for her technical assistance in IHC staining and microscopy and Prof. Dr. Arthur Cederbaum (USA) for providing HepG2 cells overexpressing CYP2E1.

Author contributions

SH, AO, CM, AT, BD, JW, AM, CD, KG, YG, HM, XY, MG and MT conceived the study, data acquisition and performed data analyses and SH, AO, JGH and SD wrote the manuscript. JL, ZCN, RH, CH, MPE and SA performed critical revision of the manuscript and provided supervisory support. All authors read the final version of the manuscript.

Funding

This study was supported by the BMBF (German Federal Ministry of Education and Research) Project LiSyM (SD, SH and JGH), e:Bio-Modull-II: MS_DILI (SH and SD) and Sino-German Cooperation project, GZ1263, supported by Sino-German Scientific Center (SD). ISAS (AO, AT, JL, MG and RH) acknowledges financial support by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin, and the Bundesministerium für Bildung und Forschung.

Compliance with ethical standards

Conflict of interest

All authors declare no conflict of interests.

Supplementary material

204_2018_2254_MOESM1_ESM.jpg (6 mb)
Formalin-fixed livers were processed and stained with HE, CYP2E1, GS, F4/80 and CD45 to visualize and quantify hepatocellular necrosis, metabolizing enzymes, resident macrophages and recruited immune cells, respectively. Slides were scanned and the signal was randomly quantified in 15 images per liver. Yellow dashed lines indicate the necrotic areas. Yellow arrows refer to the positive signals. Scale bars are 200µm for HE and CYP2E1 and 100µm for GS, F4/80 and CD45 (JPG 6105 KB)
204_2018_2254_MOESM2_ESM.jpg (2.9 mb)
Primary mouse hepatocytes were isolated. a) Protein level of CYP2E1 was analyzed by co-immunofluorescence staining immediately after attachment (3h) and 24 hours later. b and c) Protein and mRNA levels of CYP2E1 were analyzed upon TAM pretreatment. d and e) Protein and mRNA levels of catalase were assessed upon TAM pretreatment. Bars represent the means±SD of 3 mice (JPG 3000 KB)
204_2018_2254_MOESM3_ESM.jpg (1.5 mb)
CYP2E1 activity in HepG2-E47 was measured. TAM inhibits (not significantly) the CYP2E1 activity compared to alcohol and DMSO-treated cells. C34 cell line was used as a negative control. Bars represent the means±SD of 3 biological replica. Student’s t test (paired t test) was used to check the statistical differences upon TAM incubation (JPG 1530 KB)
204_2018_2254_MOESM4_ESM.jpg (3.4 mb)
Heatmap shows deregulated phase I and phase II metabolic targets as well as transporter (a) and inflammatory assigned (b) genes as quantified by the Fluidigm platform in TAM/Oil and Veh/Oil-exposed mice. Red and green colors refer to upregulated and downregulated genes, respectively. The p value indicates the statistical difference between TAM/Oil and Veh/Oil groups (JPG 3455 KB)
204_2018_2254_MOESM5_ESM.jpg (2.8 mb)
Representative NMR spectra of different metabolites across the groups. b and c) NMR-based metabolomics heatmaps of liver tissue revealed different patterns upon TAM administration. Red and green colors refer to upregulated and downregulated genes, respectively. The p value indicates the statistical difference between the presented groups (JPG 2849 KB)
204_2018_2254_MOESM6_ESM.docx (14 kb)
Supplementary material 6 (DOCX 14 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Seddik Hammad
    • 1
    • 2
  • Amnah Othman
    • 3
  • Christoph Meyer
    • 1
  • Ahmad Telfah
    • 3
  • Joerg Lambert
    • 3
  • Bedair Dewidar
    • 1
    • 4
  • Julia Werle
    • 1
  • Zeribe Chike Nwosu
    • 1
  • Abdo Mahli
    • 5
  • Christof Dormann
    • 1
  • Yan Gao
    • 1
  • Kerry Gould
    • 1
  • Mei Han
    • 1
  • Xiaodong Yuan
    • 1
  • Mikheil Gogiashvili
    • 3
  • Roland Hergenröder
    • 3
  • Claus Hellerbrand
    • 5
  • Maria Thomas
    • 6
  • Matthias Philip Ebert
    • 7
  • Salah Amasheh
    • 8
  • Jan G. Hengstler
    • 9
  • Steven Dooley
    • 1
  1. 1.Molecular Hepatology Section, Department of Medicine II, Medical Faculty MannheimHeidelberg UniversityMannheimGermany
  2. 2.Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary MedicineSouth Valley UniversityQenaEgypt
  3. 3.Leibniz Institut für analytische WissenschaftenISAS e.V.44139 DortmundGermany
  4. 4.Department of Pharmacology and Toxicology, Faculty of PharmacyTanta UniversityTantaEgypt
  5. 5.Institute of Biochemistry (Emil-Fischer Zentrum)Friedrich-Alexander University Erlangen-NürnbergErlangenGermany
  6. 6.Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of TuebingenStuttgartGermany
  7. 7.Department of Medicine II, Medical Faculty MannheimHeidelberg UniversityMannheimGermany
  8. 8.Department of Veterinary Medicine, Institute of Veterinary PhysiologyFree University of BerlinBerlinGermany
  9. 9.Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund (IfADo)DortmundGermany

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