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Chronic CCl4 intoxication causes liver and bone damage similar to the human pathology of hepatic osteodystrophy: a mouse model to analyse the liver–bone axis

  • Organ Toxicity and Mechanisms
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Abstract

Patients with chronic liver diseases frequently exhibit decreased bone mineral densities (BMD), which is defined as hepatic osteodystrophy (HOD). HOD is a multifactorial disease whose regulatory mechanisms are barely understood. Thus, an early diagnosis and therapy is hardly possible. Therefore, the aim of our study consisted in characterizing a mouse model reflecting the human pathomechanism. Serum samples were collected from patients with chronic liver diseases and 12-week old C57Bl6/N mice after 6-week treatment with carbon tetrachloride (CCl4). Repetitive injections of CCl4 induced liver damage in mice, resembling liver fibrosis in patients, as assessed by serum analysis and histological staining. Although CCl4 did not affect primary osteoblast cultures, μCT analysis revealed significantly decreased BMD, bone volume, trabecular number and thickness in CCl4-treated mice. In both HOD patients and CCl4-treated mice, an altered vitamin D metabolism with decreased CYP27A1, CYP2R1, vitamin D-binding protein GC and increased 7-dehydrocholesterol reductase hepatic gene expression, results in decreased 25-OH vitamin D serum levels. Moreover, both groups exhibit excessively high active transforming growth factor-beta (TGF-β) serum levels, inhibiting osteoblast function in vitro. Summarizing, our mouse model presents possible mediators of HOD, e.g. altered vitamin D metabolism and increased active TGF-β. Liver damage and significant changes in bone structure and mineralization are already visible by μCT analysis after 6 weeks of CCl4 treatment. This fast response and easy transferability makes it an ideal model to investigate specific gene functions in HOD.

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Abbreviations

BMD:

Bone mineral density

CYP2R1:

Cytochrome P450 2R1

CYP27A1:

Cytochrome P450 27A1

DHCR7:

7-Dehydrocholesterol reductase

GC:

Vitamin D-binding protein

HOD:

Hepatic osteodystrophy

IGF-1:

Insulin-like growth factor

OPG:

Osteoprotegerin

RANKL:

Receptor activator of NF-κB ligand

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Acknowledgments

The work was partially supported by funding of the BG trauma clinic and by the Federal Ministry of Education and Research (BMBF—0315741—Virtual Liver).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Siegfried Weller Institute for Trauma Research, Eberhard-Karls-Universität Tübingen, Schnarrenbergstraße 95, 72076, Tübingen, Germany

    Andreas K. Nussler, Thomas Freude & Sabrina Ehnert

  2. Berlin Brandenburg Center for Regenerative Therapies (BCRT), Julius Wolff Institut, Charité, Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany

    Britt Wildemann

  3. Department of Traumatology, Klinikum rechts der Isar, Technische Universität München, Ismaninger Straße 22, 81675, Munich, Germany

    Christian Litzka, Petra Soldo & Karl F. Braun

  4. Department of Surgery, MRI, Klinikum rechts der Isar, Technische Universität München, Ismaninger Straße 22, 81675, Munich, Germany

    Helmut Friess

  5. Leibniz-Institut für Arbeitsforschung (ifado), TU Dortmund, Ardeystraße 67, 44139, Dortmund, Germany

    Seddik Hammad, Jan G. Hengstler & Patricio Godoy

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

    Seddik Hammad

  7. Faculty of Medicine, St. Joseph University, Beirut, Lebanon

    Viviane Trak-Smayra

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  1. Andreas K. Nussler
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  2. Britt Wildemann
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  3. Thomas Freude
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  4. Christian Litzka
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  10. Viviane Trak-Smayra
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  11. Patricio Godoy
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Corresponding author

Correspondence to Andreas K. Nussler.

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204_2013_1191_MOESM1_ESM.tif

Supplementary Figure: CCl4 treatment damages hepatocytes but not osteoblasts. (a) Primary human hepatocytes (N = 5, n ≥ 4) and primary human osteoblasts (N = 6, n = 4) were stimulated repeatedly (4 times) with 0, 1, 2 and 10 mM CCl4. After 96 h cell viability was determined by resazurin conversion. Reduced cell viability was observed only in hepatocytes but not in osteoblasts. (b) The CCl4 treated osteoblasts were further analysed for AP activity and matrix mineralization as functional characteristics. CCl4 treatment did not alter AP activity and matrix mineralization in osteoblasts. * p < 0.05, *** p < 0.001 as compared to untreated controls. (TIFF 1052 kb)

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Nussler, A.K., Wildemann, B., Freude, T. et al. Chronic CCl4 intoxication causes liver and bone damage similar to the human pathology of hepatic osteodystrophy: a mouse model to analyse the liver–bone axis. Arch Toxicol 88, 997–1006 (2014). https://doi.org/10.1007/s00204-013-1191-5

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