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. 2022 May 21;4(7):100508.
doi: 10.1016/j.jhepr.2022.100508. eCollection 2022 Jul.

A new mouse model of radiation-induced liver disease reveals mitochondrial dysfunction as an underlying fibrotic stimulus

Nicolas Melin  1   2 , Tural Yarahmadov  1   2 , Daniel Sanchez-Taltavull  1   2 , Fabienne E Birrer  1   2 , Tess M Brodie  1   2 , Benoît Petit  3 , Andrea Felser  4 , Jean-Marc Nuoffer  4 , Matteo Montani  5 , Marie-Catherine Vozenin  3 , Evelyn Herrmann  6 , Daniel Candinas  1 , Daniel M Aebersold  6 , Deborah Stroka  1   2
Affiliations

A new mouse model of radiation-induced liver disease reveals mitochondrial dysfunction as an underlying fibrotic stimulus

Nicolas Melin et al. JHEP Rep. .

Abstract

Background & aims: High-dose irradiation is an essential tool to help control the growth of hepatic tumors, but it can cause radiation-induced liver disease (RILD). This life-threatening complication manifests itself months following radiation therapy and is characterized by fibrosis of the pericentral sinusoids. In this study, we aimed to establish a mouse model of RILD to investigate the underlying mechanism of radiation-induced liver fibrosis.

Methods: Using a small animal image-guided radiation therapy platform, an irradiation scheme delivering 50 Gy as a single dose to a focal point in mouse livers was designed. Tissues were analyzed 1 and 6 days, and 6 and 20 weeks post-irradiation. Irradiated livers were assessed by histology, immunohistochemistry, imaging mass cytometry and RNA sequencing. Mitochondrial function was assessed using high-resolution respirometry.

Results: At 6 and 20 weeks post-irradiation, pericentral fibrosis was visible in highly irradiated areas together with immune cell infiltration and extravasation of red blood cells. RNA sequencing analysis showed gene signatures associated with acute DNA damage, p53 activation, senescence and its associated secretory phenotype and fibrosis. Moreover, gene profiles of mitochondrial damage and an increase in mitochondrial DNA heteroplasmy were detected. Respirometry measurements of hepatocytes in vitro confirmed irradiation-induced mitochondrial dysfunction. Finally, the highly irradiated fibrotic areas showed markers of reactive oxygen species such as decreased glutathione and increased lipid peroxides and a senescence-like phenotype.

Conclusions: Based on our mouse model of RILD, we propose that irradiation-induced mitochondrial DNA instability contributes to the development of fibrosis via the generation of excessive reactive oxygen species, p53 pathway activation and a senescence-like phenotype.

Lay summary: Irradiation is an efficient cancer therapy, however, its applicability to the liver is limited by life-threatening radiation-induced hepatic fibrosis. We have developed a new mouse model of radiation-induced liver fibrosis, that recapitulates the human disease. Our model highlights the role of mitochondrial DNA instability in the development of irradiation-induced liver fibrosis. This new model and subsequent findings will help increase our understanding of the hepatic reaction to irradiation and to find strategies that protect the liver, enabling the expanded use of radiotherapy to treat hepatic tumors.

Keywords: 4HNE, 4-hydroxynonenal; CV, central vein; ECM, extracellular matrix; ETC, electron transfer chain; GSH, reduced glutathione (glutathione); GSSG, oxidized glutathione (glutathione disulfide); HSCs, hepatic stellate cells; IGRT, image-guided radiation therapy; IHC, immunohistochemistry; IMC, imaging mass cytometry; MDA, malondialdehyde; RILD, radiation-induced liver disease; RNAseq, RNA sequencing; ROS; ROS, reactive oxygen species; RT, radiation therapy; SASP, senescence-associated secretory phenotype; SNP, single nucleotide polymorphism; SOS, sinusoidal obstruction syndrome; fibrosis; image guided radiation therapy (IGRT); mitochondrial dysfunction; mitochondrial-DNA; mouse model; mtDNA, mitochondrial DNA; mtROS, mitochondrial reactive oxygen species; p53; radiation-induced liver disease (RILD); rcf, relative centrifuge force; senescence; sinusoidal obstruction syndrome.

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Conflict of interest statement

The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Image-guided irradiation plan. Mouse liver is imaged by CT using a liver/spleen targeting contrast agent (A), allowing a 3D reconstruction of the liver in the abdominal cavity (B). The irradiation plane of a 50 Gy single-dose irradiation using a 180° arc irradiation (C). The dose-volume histogram depicting the dose delivered on the x-axis and the volume of the organ irradiated on the y-axis (D). A schematic view of the delivered doses allows to visually locate the dose delivered to the different liver lobe and the liver pieces used for the paired transcriptomic analysis; H for HIGH-irradiated, and L for LOW-irradiated (E).
Fig. 2
Fig. 2
50 Gy local liver irradiation leads to SOS and immune cell infiltration. Mice were fed with a LAO diet starting 2 weeks before the irradiation and during the experiment. The livers were collected at day-1 (n = 4), day-6 (n = 4), week-6 (n = 4), and week-20 (n = 3) (A). Schematic representation of the doses delivered to the left and median lobe. Histological evaluation focusing on the annotated hot spot (B). Histological evaluation of the livers using H&E staining (C), Masson’s trichrome staining (D), and Prussian blue (E). Imaging mass cytometry visualization of Ly6G, CD3e, CD11b, and DNA (F), and E-cadherin, CD31, α-SMA, and DNA (G) around the CV. CV, central vein; LAO, low antioxidant; SOS, sinusoidal obstruction syndrome.
Fig. 3
Fig. 3
Paired analysis of irradiated and non-irradiated liver transcriptome. Time-dependent PCA analysis displaying the control tissue in black, HIGH-irradiated tissue in red, and LOW-irradiated in blue. The paired HIGH- and LOW-irradiated tissue (coming from the same mouse) were represented with the same shape (A). Bar graph illustrating the numbers of upregulated and downregulated genes issued from comparing the paired HIGH- vs. LOW-irradiated sample (B). Venn diagram displaying the upregulated genes (C) and downregulated genes (D) at the different time post-irradiation. Pathway enrichment analysis depicting only the most relevant summary terms for each time point; day-1 p <5x10-15; day-6 p <4x10-4; week-6 p <1x10-10; week-20 p <5x10-15 (Hypergeometric test) (E-H). PCA, principal component analysis.
Fig. 4
Fig. 4
Irradiated mouse hepatocyte displays modified reparation post-irradiation. Percentage of mtDNA heteroplasmy to the reference mtDNA sequence (AJ512208.1) in the paired LOW- and HIGH-irradiated liver (n = 4) 24 h post-irradiation (paired t test) (A). Number (B) and confluency (C) of mouse hepatocyte 6 days after control (n = 4) or irradiation treatment (n = 4). Basal and maximal respiration of hepatocyte cultured following control or irradiation treatment, levels of significance displayed on the graph (paired t test) (D), and their basal respiration and complex I, I I+II, and IV activity normalized to the maximal respiration, level of significance displayed on the fig. (paired t test) (E). mtDNA, mitochondrial DNA.
Fig. 5
Fig. 5
RedOx balance following hepatic irradiation. GSH and GSSG in isolated hepatocytes at day-6 post-irradiation (t test) (A). GSH (B), and GSSG (C) in HIGH and its LOW samples at day-1, day-6, week-6, and week-20 following irradiation, levels of significance displayed on the graph (paired t test). 4HNE (D) and MDA (E) staining of the HIGH-irradiated liver and its LOW-irradiated counterpart at day-1, day-6, week-6, and week-20 following irradiation. 4HNE, 4-hydroxynonenal; GSSG, oxidized glutathione (glutathione disulfide); GSH, reduced glutathione (glutathione); MDA, malondialdehyde.
Fig. 6
Fig. 6
p53 and senescence promote profibrotic pathways following hepatic radiation. Regulation of genes within the p53 pathway (A), and cellular senescence (B) at the different time points. p16INK4a (Cdkn2a) staining 20-weeks post-irradiation at 0.5x and 35x (C). Heat map of regulated SASP (D). Regulation of genes within the extracellular matrix organization (E). ECM, extracellular matrix; SASP, senescence-associated secretory phenotype.
Fig. 7
Fig. 7
From hepatic irradiation to sinusoidal obstruction syndrome. Schematic representation of the proposed mechanism leading to radiation-induced liver disease. mtDNA, mitochondrial DNA; ROS, reactive oxygen species.
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