Skip to main content

Advertisement

SpringerLink
Log in

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

  • Molecular Toxicology
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Andersson KB, Winer LH, Mørk HK, Molkentin JD, Jaisser F (2010) Tamoxifen administration routes and dosage for inducible Cre-mediated gene disruption in mouse hearts. Transgenic Res 19:715–725

    Article  CAS  Google Scholar 

  • Cardoso CM, Almeida LM, Custódio JB (2004) Protection of tamoxifen against oxidation of mitochondrial thiols and NAD(P)H underlying the permeability transition induced by prooxidants. Chem Biol Interact 148:149–161

    Article  CAS  Google Scholar 

  • Daurio NA, Tuttle SW, Worth AJ, Song EY, Davis JM, Snyder NW, Blair IA, Koumenis C et al (2016) AMPK activation and metabolic reprogramming by tamoxifen through estrogen receptor-independent mechanisms suggests new uses for this therapeutic modality in cancer treatment. Cancer Res 76(11):3295–3306. https://doi.org/10.1158/0008-5472.CAN-15-2197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ding BS, Nolan DJ, Butler JM, James D, Babazadeh AO, Rosenwaks Z et al (2010) Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 468(7321):310–315

    Article  CAS  Google Scholar 

  • Edwards MJ, Keller BJ, Kauffman FC, Thurman RG (1993) The involvement of Kupffer cells in carbon tetrachloride toxicity. Toxicol Appl Pharmacol 119(2):275–279

    Article  CAS  Google Scholar 

  • Francis PA, Pagani O, Fleming GF, Walley BA, Colleoni M, Láng I et al (2018) Tailoring adjuvant endocrine therapy for premenopausal breast cancer. N Engl J Med. https://doi.org/10.1056/NEJMoa1803164

    Article  PubMed  Google Scholar 

  • Gogiashvili M, Edlund K, Gianmoena K, Marchan R, Brik A, Andersson JT et al (2017) Metabolic profiling of ob/ob mouse fatty liver using HR-MAS 1H-NMR combined with gene expression analysis reveals alterations in betaine metabolism and the transsulfuration pathway. Anal Bioanal Chem 409:1591–1606

    Article  CAS  Google Scholar 

  • Greenhalgh SN, Conroy KP, Henderson NC (2015) Cre-activity in the liver: transgenic approaches to targeting hepatic nonparenchymal cells. Hepatology 61:2091–2099

    Article  Google Scholar 

  • Hammad S, Hoehme S, Friebel A, von Recklinghausen I, Othman A, Begher-Tibbe B et al (2014) Protocols for staining of bile canalicular and sinusoidal networks of human, mouse and pig livers, three-dimensional reconstruction and quantification of tissue microarchitecture by image processing and analysis. Arch Toxicol 88:1161–1183

    Article  CAS  Google Scholar 

  • Henderson NC, Arnold TD, Katamura Y, Giacomini MM, Rodriguez JD, McCarty JH et al (2013) Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nat Med 19:1617–1624

    Article  CAS  Google Scholar 

  • Huh WJ, Khurana SS, Geahlen JH, Kohli K, Waller RA, Mills JC (2012) Tamoxifen induces rapid, reversible atrophy, and metaplasia in mouse stomach. Gastroenterology 142(1):21–24.e7

    Article  Google Scholar 

  • Kam MK, Lee KY, Tam PK, Lui VC (2012) Generation of NSE-MerCreMer transgenic mice with tamoxifen inducible Cre activity in neurons. PLoS One 7:e35799

    Article  CAS  Google Scholar 

  • Kisseleva T, Cong M, Paik Y, Scholten D, Jiang C, Benner C et al (2012) Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc Natl Acad Sci USA 109:9448–9453

    Article  CAS  Google Scholar 

  • Konstandi M, Cheng J, Gonzalez FJ (2013) Sex steroid hormones regulate constitutive expression of Cyp2e1 in female mouse liver. Am J Physiol Endocrinol Metab 304:E1118–E1128

    Article  CAS  Google Scholar 

  • Koop DR, Morgan ET, Tarr GE, Coon MJ (1982) Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits. J Biol Chem 257:8472–8480

    CAS  PubMed  Google Scholar 

  • Lee SS, Buters JT, Pineau T, Fernandez-Salguero P, Gonzalez FJ (1996) Role of CYP2E1 in the hepatotoxicity of acetaminophen. J Biol Chem 17:271:12063–12067

    Article  Google Scholar 

  • Lemaigre FP (2015) Determining the fate of hepatic cells by lineage tracing: facts and pitfalls. Hepatology 61:2100–2103

    Article  Google Scholar 

  • Lu Y, Zhuge J, Wang X, Bai J, Cederbaum AI (2008) Cytochrome P450 2E1 contributes to ethanol-induced fatty liver in mice. Hepatology 47:1483–1494

    Article  CAS  Google Scholar 

  • Mahli A, Thasler WE, Patsenker E, Müller S, Stickel F, Müller M et al (2015) Identification of cytochrome CYP2E1 as critical mediator of synergistic effects of alcohol and cellular lipid accumulation in hepatocytes in vitro. Oncotarget 6:41464–41468

    Article  Google Scholar 

  • Manavski Y, Abel T, Hu J, Kleinlützum D, Buchholz CJ, Belz C et al (2017) Endothelial transcription factor KLF2 negatively regulates liver regeneration via induction of activin A. Proc Natl Acad Sci USA 114(15):3993–3998

    Article  CAS  Google Scholar 

  • Manubolu M, Goodla L, Ravilla S, Thanasekaran J, Dutta P, Malmlöf K, Obulum VR (2014) Protective effect of Actiniopteris radiata (Sw.) Link. against CCl4-induced oxidative stress in albino rats. J Ethnopharmacol 153:744–752

    Article  CAS  Google Scholar 

  • Mederacke I, Hsu CC, Troeger JS, Huebener P, Mu X, Dapito DH et al (2013) Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun 4:2823

    Article  Google Scholar 

  • Michelotti GA, Xie G, Swiderska M, Choi SS, Karaca G, Krüger L et al (2013) Smoothened is a master regulator of adult liver repair. J Clin Invest 123:2380–2394

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nakamura E, Nguyen MT, Mackem S (2006) Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton. Dev Dyn 235:2603–2612

    Article  CAS  Google Scholar 

  • Ng TB, Gao W, Li L, Niu SM, Zhao L, Liu J et al (2005) Rose (Rosa rugosa)-flower extract increases the activities of antioxidant enzymes and their gene expression and reduces lipid peroxidation. Biochem Cell Biol 83:78–85

    Article  CAS  Google Scholar 

  • Ohrnberger S, Thavamani A, Braeuning A, Lipka DB, Kirilov M, Geffers R et al (2015) Dysregulated serum response factor triggers formation of hepatocellular carcinoma. Hepatology 61:979–989

    Article  CAS  Google Scholar 

  • Osborne CK (1998) Tamoxifen in the treatment of breast cancer. N Engl J Med 339(22):1609–1618

    Article  CAS  Google Scholar 

  • Pimeisl IM, Tanriver Y, Daza RA, Vauti F, Hevner RF, Arnold HH et al (2013) Generation and characterization of a tamoxifen-inducible Eomes(CreER) mouse line. Genesis 51:725–733

    Article  CAS  Google Scholar 

  • Recknagel RO, Glende EA Jr, Dolak JA, Waller RL (1989) Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther 43:139–154 (review)

    Article  CAS  Google Scholar 

  • Riggs BL, Hartmann LC (2003) Selective estrogen-receptor modulators—mechanisms of action and application to clinical practice. N Engl J Med 348(7):618–629

    Article  CAS  Google Scholar 

  • Ryu SH, Chung YH, Lee JK, Kim JA, Shin JW, Jang MK et al (2009) Anti-fibrogenic effects of tamoxifen in a rat model of periportal hepatic fibrosis. Liver Int 29:308–314

    Article  CAS  Google Scholar 

  • Scholten D, Osterreicher CH, Scholten A, Iwaisako K, Gu G, Brenner DA et al (2010) Genetic labeling does not detect epithelial-to-mesenchymal transition of cholangiocytes in liver fibrosis in mice. Gastroenterology 139:987–998

    Article  CAS  Google Scholar 

  • Schwartz B, Marks M, Wittler L, Werber M, Währisch S, Nordheim A et al (2014) SRF is essential for mesodermal cell migration during elongation of the embryonic body axis. Mech Dev 133:23–35

    Article  CAS  Google Scholar 

  • Shan L, Ding Y, Fu Y, Zhou L, Dong X, Chen S et al (2016) mTOR overactivation in mesenchymal cells aggravates CCl4-induced liver fibrosis. Sci Rep 6:36037

    Article  CAS  Google Scholar 

  • Shido K, Chavez D, Cao Z, Ko JL, Rafii S, Ding BS (2017) Platelets prime hematopoietic-vascular niche to drive angiocrine-mediated liver regeneration. Signal Transduct Targ Ther 2:e16044

    Article  Google Scholar 

  • Sin YY, Ballantyne LL, Mukherjee K, St Amand T, Kyriakopoulou L, Schulze A et al (2013) Inducible arginase 1 deficiency in mice leads to hyperargininemia and altered amino acid metabolism. PLoS One 8:e80001

    Article  CAS  Google Scholar 

  • Tumurbaatar B, Sun Y, Chan T, Sun J (2007) Cre-estrogen receptor-mediated hepatitis C virus structural protein expression in mice. J Virol Methods 146:5–13

    Article  CAS  Google Scholar 

  • Wend K, Wend P, Krum SA (2012) Tissue-specific effects of loss of estrogen during menopause and aging. Front Endocrinol (Lausanne) 3:19. https://doi.org/10.3389/fendo.2012.00019

    Article  Google Scholar 

  • Werfel S, Jungmann A, Lehmann L, Ksienzyk J, Bekeredjian R, Kaya Z et al (2014) Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV-mediated expression of Cre recombinase. Cardiovasc Res 104:15–23

    Article  CAS  Google Scholar 

  • Wong FW, Chan WY, Lee SS (1998) Resistance to carbon tetrachloride-induced hepatotoxicity in mice whichmice, which lack CYP2E1 expression. Toxicol Appl Pharmacol 153:109–118

    Article  CAS  Google Scholar 

  • Wu D, Cederbaum AI (2008) Development and properties of HepG2 cells that constitutively express CYP2E1. Methods Mol Biol 447:137–150

    Article  CAS  Google Scholar 

  • Yang L, Jung Y, Omenetti A, Witek RP, Choi S, Vandongen HM et al (2008) Fate-mapping evidence that hepatic stellate cells are epithelial progenitorsin adult mouse livers. Stem Cells 26:2104–2113

    Article  CAS  Google Scholar 

  • Yoshikawa Y, Miyashita T, Higuchi S, Tsuneyama K, Endo S, Tsukui T et al (2012) Mechanisms of the hepatoprotective effects of tamoxifen against drug-induced and chemical-induced acute liver injuries. Toxicol Appl Pharmacol 264:42–50

    Article  CAS  Google Scholar 

  • Zellmer S, Schmidt-Heck W, Godoy P, Weng H, Meyer C, Lehmann T et al (2010) Transcription factors ETF, E2F, and SP-1 are involved in cytokine-independent proliferation of murine hepatocytes. Hepatology 52:2127–2136

    Article  CAS  Google Scholar 

Download references

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.

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.

Author information

Author notes

  1. Seddik Hammad and Amnah Othman are equally contributing authors.

Authors and Affiliations

  1. Molecular Hepatology Section, Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, 68167, Mannheim, Germany

    Seddik Hammad, Christoph Meyer, Bedair Dewidar, Julia Werle, Zeribe Chike Nwosu, Christof Dormann, Yan Gao, Kerry Gould, Mei Han, Xiaodong Yuan & Steven Dooley

  2. Department of Forensic Medicine and Veterinary Toxicology, Faculty of Veterinary Medicine, South Valley University, 83523, Qena, Egypt

    Seddik Hammad

  3. Leibniz Institut für analytische Wissenschaften, ISAS e.V., 44139 Dortmund, Germany

    Amnah Othman, Ahmad Telfah, Joerg Lambert, Mikheil Gogiashvili & Roland Hergenröder

  4. Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tanta University, 31527, Tanta, Egypt

    Bedair Dewidar

  5. Institute of Biochemistry (Emil-Fischer Zentrum), Friedrich-Alexander University Erlangen-Nürnberg, 91054, Erlangen, Germany

    Abdo Mahli & Claus Hellerbrand

  6. Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology and University of Tuebingen, Stuttgart, Germany

    Maria Thomas

  7. Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, 68167, Mannheim, Germany

    Matthias Philip Ebert

  8. Department of Veterinary Medicine, Institute of Veterinary Physiology, Free University of Berlin, 14163, Berlin, Germany

    Salah Amasheh

  9. Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund (IfADo), 44139, Dortmund, Germany

    Jan G. Hengstler

Authors
  1. Seddik Hammad
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amnah Othman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christoph Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ahmad Telfah
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joerg Lambert
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bedair Dewidar
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Julia Werle
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zeribe Chike Nwosu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Abdo Mahli
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Christof Dormann
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yan Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Kerry Gould
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Mei Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Xiaodong Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Mikheil Gogiashvili
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Roland Hergenröder
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Claus Hellerbrand
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Maria Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Matthias Philip Ebert
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Salah Amasheh
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Jan G. Hengstler
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Steven Dooley
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to Seddik Hammad or Steven Dooley.

Ethics declarations

Conflict of interest

All authors declare no conflict of interests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

204_2018_2254_MOESM1_ESM.jpg

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

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

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

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

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)

Supplementary material 6 (DOCX 14 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hammad, S., Othman, A., Meyer, C. et al. Confounding influence of tamoxifen in mouse models of Cre recombinase-induced gene activity or modulation. Arch Toxicol 92, 2549–2561 (2018). https://doi.org/10.1007/s00204-018-2254-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-018-2254-4

Keywords

Search

Navigation