Long-term effects of low-dose mouse liver irradiation involve ultrastructural and biochemical changes in hepatocytes that depend on lipid metabolism
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The aim of the study was to investigate long-term effects of radiation on the (ultra)structure and function of the liver in mice. The experiments were conducted on wild-type C57BL/6J and apolipoprotein E knock-out (ApoE−/−) male mice which received a single dose (2 or 8 Gy) of X-rays to the heart with simultaneous exposure of liver to low doses (no more than 30 and 120 mGy, respectively). Livers were collected for analysis 60 weeks after irradiation and used for morphological, ultrastructural, and biochemical studies. The results show increased damage to mitochondrial ultrastructure and lipid deposition in hepatocytes of irradiated animals as compared to non-irradiated controls. Stronger radiation-related effects were noted in ApoE−/− mice than wild-type animals. In contrast, radiation-related changes in the activity of lysosomal hydrolases, including acid phosphatase, β-glucuronidase, N-acetyl-β-d-hexosaminidase, β-galactosidase, and α-glucosidase, were observed in wild type but not in ApoE-deficient mice, which together with ultrastructural picture suggests a higher activity of autophagy in ApoE-proficient animals. Irradiation caused a reduction of plasma markers of liver damage in wild-type mice, while an increased level of hepatic lipase was observed in plasma of ApoE-deficient mice, which collectively indicates a higher resistance of hepatocytes from ApoE-proficient animals to radiation-mediated damage. In conclusion, liver dysfunctions were observed as late effects of irradiation with an apparent association with malfunction of lipid metabolism.
KeywordsIonizing radiation Heart Liver Ultrastructure Lysosomes
The liver is regarded as a resistant organ which maintains its homeostatic function throughout the lifespan and may be spared even when a body is affected by atherosclerosis, diabetes and hypertension, probably thanks to its dual blood supply, abundant reserves and regenerative capacity (Abdel-Moneim et al. 2015; Stell and Wall 2003). In congestive heart failure, delivery of blood to the body is impaired, with a potential impact on the function of other organs, such as the liver. An increase in venous blood pressure caused by heart failure can lead to atrophy of hepatocytes and can cause perisinusoidal edema that can impair diffusion of oxygen and nutrients to hepatocytes. This, in turn, results in hepatic congestion, mild jaundice and abnormalities in liver enzymes (Alvarez and Mukherjee 2011).
Lipid metabolism is an essential function of the liver. Free fatty acids entering hepatocytes are transformed into triglycerides and are stored along with cholesterol in the form of lipid vesicles. Excessive storage of lipids may jeopardize functions of the liver, leading to many metabolic diseases including insulin resistance and predisposition to diabetes type 2, as well as cardiovascular diseases (Singh et al. 2009; Cursio et al. 2015; Kuipers et al. 1997). Such high lipid levels are observed in mice lacking the gene encoding for apolipoprotein E (ApoE), which belongs to glycoproteins and is a component of many plasma lipoproteins (including chylomicrons, VLDL, LDL, IDL, and HDL) playing a vital role in regulation of plasma lipids metabolism (Ayala-Lopez et al. 2010; Balcerzyk and Zak 2004; Hauser et al. 2011; Kuipers et al. 1997; Vasquez et al. 2012). Moreover, ApoE is involved in inflammation, platelet function, apoptosis, and response to oxidative stress. Therefore, ApoE is associated with numerous disorders, including atherosclerosis, ischemic heart and brain disease and some types of cancer (Posse de Chaves and Narayanaswami 2008; Ćwiklińska et al. 2015). Both deficiency and excess expression of ApoE may have detrimental effects and can lead to hypertriglyceridemia or cardiovascular diseases (Vasquez et al. 2012). Insufficient amounts of ApoE decrease the quality of the process of removal of triglycerides and their remnants. Its excess, however, promotes the excessive production of VLDL in the liver and impairs lipolysis (Ahn et al. 2012; Jawien et al. 2007). Unlike wild-type mice, ApoE−/− animals suffer from distorted intercellular homeostasis (Bandorowicz-Pikuła et al. 2011), elevated plasma cholesterol levels and develop arterial lesions with age. Therefore, they are widely used to study age-related atherosclerosis (Bonomini et al. 2013; Kumarathasan et al. 2013; Pendse et al. 2009; Zhang et al. 2010).
Elevated risk of cardiac disease is a delayed side effect of radiotherapy for breast cancer (Darby et al. 2010). Exposure of the heart to low and medium doses of ionizing irradiation causes a low but statistically significant increase in the risk of cardiovascular diseases and death (Marmagkiolis et al. 2016; Stewart et al. 2006). Heart rhythm disorders, ischemia, myocarditis, and pericarditis and sometimes even acute heart failure occurring after radiotherapy have also detrimental effects on the function of other organs including liver (Alvarez and Mukherjee 2011). Hence, increased attention is being paid to long-term effects of local heart radiation exposure in organs that were not exposed, yet could be affected by the radiation-induced malfunction of the cardiovascular system.
The present study focused on changes in livers of wild type and ApoE−/− C57BL/6J mice, whose hearts were selectively exposed to a high doses (2 and 8 Gy) of a low linear energy transfer (LET) radiation. Due to scatter radiation, the livers received negligibly low doses of no more than 30 and 120 mGy, respectively. Morphology and ultrastructure of hepatocytes, activities of lysosomal enzymes in liver, and levels of plasma markers of a liver damage were analyzed 60 weeks after exposure.
Materials and methods
Animals and irradiation procedure
Male C57BL/6J mice, either wild-type (wt) or homozygous ApoE deficient (ApoE−/−), were purchased from Charles River Laboratories (Research Models and Services, Germany GmbH). The age of the animals at the time of irradiation was 8 ± 1 weeks. The animals were kept at room temperature (21°) in a naturally controlled ratio of light and dark 12:12 and were given laboratory chow ad libitum. Mice were immobilized (without anesthesia) in specially designed jigs and locally irradiated to the heart using a YXLON MG325 device (Yxlon International X-ray GmbH, Germany) operated at 200 kV, with a tube current of 20 mA and a beam filter of 0.6 cm Cu, resulting in a dose rate of 0.8 Gy/min. Single local heart doses of 2 or 8 Gy were applied; a control group was sham-irradiated (0 Gy). Ten mice were irradiated per treatment group. The whole heart (plus about 20% of lung volume) was irradiated with a prescribed dose, whereas the rest of the animal, including the liver, was shielded with lead plates as previously reported (Monceau et al. 2013). The exact position of heart before irradiation was assessed by radiography. A dose distribution in tissues adjacent to the heart was estimated based on a mouse phantom measurements. According to this estimation, livers used for further experiments received below 1.5% of a prescribed dose (i.e., below 30 or 120 mGy); the dose distribution in an animal is illustrated in the Supplementary Material Figure S1. Animals were irradiated in Dresden, Germany, and shipped to Gliwice 1–2 months after irradiation. The mice were kept on a regular diet. At 60 weeks after irradiation, the animals were sacrificed by cervical dislocation and liver tissues were immediately extracted for further analyses. The protocol was approved by the Committee on the Ethics of Animal Experiments of Landesdirektion Dresden (file no. 24-1968.1-11/2009-10, Germany) and Directive 2010/63/EU of the European Parliament.
Analysis of histomorphology
Tissue samples from the distal part of the left liver lobe were embedded in Epoxy resin (Agar Scientific Ltd, Stansted, England), and semi-thin sections (500 nm) were prepared using a Reichert-Jung ultramicrotome (Reichert, Vienna, Austria), stained with toluidine blue and examined using a light microscope ZEISS Axio Scope.A1 with a black and white digital camera. Areas of lipid deposits were determined with the Photoshop CS6 software (Adobe), using the sample color area tool for black (R = 0; G = 0; B = 0) and then recording the number of pixels in a histogram window. Pixels were counted on three random pictures in each group. In order to compare the groups, p values were calculated using the two-tailed Student’s t test for independent and normally distributed samples (Statistica 10, StatSoft Inc.).
Analysis of ultrastructure
Immediately after resection of the distal part of the left liver lobe, the lower part of the left lateral lobe was cut into proper size pieces (2 mm3) and fixed by immersion in buffered 3% glutaraldehyde in cacodylate buffer (pH 7.2) for at least 2 h at 4 °C. The tissue specimens were then post-fixed in 1% osmium tetroxide in cacodylate buffer (pH 7.2) for 1 h at 4 °C. Dehydration of the fixed tissues was performed using an ascending series of ethanol and then transferred into Epoxy resin via propylene oxide (Marzella and Glaumann 1980a). Finally, the liver samples were embedded in a mixture of DDSA/NMA/Embed-812 (Agar Scientific Ltd). Ultra-thin sections (40–60 nm) were cut on a Reichert-Jung ultramicrotome and double stained with uranyl acetate and lead citrate. Evaluation of ultrastructure was performed using a transmission electron microscope Tesla BS-500 with Frame Transfer-1K-CCD-Camera (TRS, Germany). The percentage of abnormal mitochondria was estimated in each TEM picture by counting 1000 randomly chosen mitochondria. The significance of differences between experimental groups was estimated by the U-Mann–Whitney test (Statistica 10).
Analyses of lysosomal enzyme activity
Immediately after resection, the liver tissue was homogenized in a medium consisting of 0.25 M sucrose (1 g tissue per 7 mL sucrose) in a Potter–Elvehjem glass homogenizer with a Teflon piston operated at 200 rpm according to the modified method of Marzella and Glaumann (Marzella and Glaumann 1980b). The homogenates were fractionated by differential centrifugation. The first centrifugation was performed at 1000g for 10 min to remove cell debris, nuclei, and heavy mitochondria. The resulting supernatant was centrifuged at 20,000g for 20 min; the resulting pellet, i.e., the “lysosomal fraction” contained a mixture of lysosomes (the dominant component), light mitochondria, peroxisomes, and endoplasmic reticulum. The pellet was suspended in 5 mL of 0.1% TRITON X-100 to release latent lysosomal enzymes. The pellet was then frozen and stored at − 20 °C until analysis. For this, samples were thawed, transferred to 1.5 mL Eppendorf tubes, and centrifuged at 12,000g for 2 min to remove debris and insoluble material. The activity of acid phosphatase (AcP, EC 220.127.116.11) was determined according to the method of Hollander (1970), while the activity of β-glucuronidase (BGRD, EC 18.104.22.168), N-acetyl-β-d-hexosaminidase (HEX, EC 22.214.171.124), β-galactosidase (BGAL, EC 126.96.36.199) and α-glucosidase (AGLD, EC 188.8.131.52) was determined according to the method of Barrett (1972). The protein level was determined by a modification of Lowry’s method (Kirschke and Wiederanders 1984). The activity of enzymes was expressed in µM of products per mg of total protein per hour. Absorbance was measured with the use of a Spekol 1500 UV/VIS spectrophotometer (Analityk Jena AG). Statistical significance of differences between groups was estimated by the student’s t-test for unpaired data using the Statistica software (Ver.10. StatSoft Company, 2011).
Approximately 200–400 µL of peripheral blood was collected immediately after death by cardiac puncture. Blood from each animal was mixed with 3.2% of buffered sodium citrate in 1.8 mL microtube (Becton Dickinson) and centrifuged at 4.750×g for 10 min at room temperature. Next, plasma specimens were promptly collected and frozen at − 70 °C. All further analyses were performed using the semi-automatic biochemical analyzer COBAS INTEGRA® 400 plus system (Roche Diagnostics Ltd., Rotkreuz, Switzerland). For the liver function, the following parameters were measured: alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), lipase, high-density lipoprotein-cholesterol (HDL), low-density lipoprotein-cholesterol (LDL), total cholesterol, and triglycerides (TG). The code of all chemicals was inserted in equipment in accordance with the standards given by kits fabricant (Roche Diagnostics Ltd., Rotkreuz, Switzerland). Statistical significance of differences was estimated by the Student’s t test (Ver.10. StatSoft Company, 2011).
Figure 1a presents a fragment of liver tissue of a control wt mouse (0 Gy), characterized by normal hepatocytes, which maintain a polygonal morphology with distinct cellular boundaries. Hepatocytes have a round vesicular nucleus and are almost lipid free. Increased numbers of lipid droplets could be observed in tissues of animals exposed to radiation (Fig. 1b, c), yet differences were not statistically significant (Fig. 2). Nevertheless, the general morphological profile of hepatocytes and their nuclei corresponded to those observed in non-irradiated animals. Morphology of whole hepatocytes and cell nuclei were normal in ApoE-deficient mice, either controls or exposed to radiation (Fig. 1d–f). However, numerous fine lipid droplets were observed in tissue of control ApoE−/− mice; overall area of such lipid deposits was 4-times higher than in control wt mice. The number of lipid droplets further increased significantly in a dose-dependent mode in livers of irradiated ApoE−/− mice (Fig. 1e, f; Fig. 2).
In the second step, the ultrastructure of hepatocytes was analyzed. Figure 3a shows a representative micrograph of a hepatocyte from a control ApoE-proficient animal, where normal structures of the nucleus, endoplasmic reticulum, lysosomes, and mitochondria could be observed. Ultrastructural changes could be noted in hepatocytes from animals irradiated with the lower dose of 2 Gy: many mitochondria were enlarged and more lysosomes, small lipid deposits and vacuoles were observed, together with swollen Golgi apparatus and small vacuoles in the proximity to the nucleus (Fig. 3b). More severe changes could be detected in animals exposed to the higher dose of 8 Gy, which included an increased number of enlarged mitochondria (yet with a preserved density of matrix and cristae), primary lysosomes and single vacuoles; however, no changes in the nucleus and nucleolus were observed (Fig. 3c). Significant differences between ApoE-proficient and ApoE-deficient mice were detected, which confirmed the observations made by light microscopy. A significant increase in the number of lipid deposits was observed in control ApoE−/− mice (Fig. 3d). Further changes were detected in the ultrastructure of hepatocytes from exposed ApoE−/− animals (Fig. 3e, f). These included a significant increase in the number of damaged mitochondria (Fig. 4), with discontinued membranes, as well as swelling and discontinued cristae. Moreover, multiple lysosomes and vacuoles, as well as markedly increased lipid droplets, were detected in such hepatocytes.
The biochemical analysis of hepatic lysosomal hydrolases revealed radiation-related changes in their activity. The activity of all analyzed enzymes (AcP, BGRD, HEX, BGAL, and AGLD) was significantly higher in the livers of ApoE-proficient animals exposed to radiation as compared to non-irradiated controls (Fig. 5). On the other hand, differences between irradiated and non-irradiated hearts were barely detectable in ApoE-deficient animals.
To assess for potential radiation-mediated damage of livers, several relevant plasma biomarkers were analyzed (Fig. 6). When control wt and ApoE−/− mice were compared, markedly higher plasma level of LDL and total cholesterol and lower levels ALT, AST, and GGT were observed in ApoE-deficient animals. Irradiation resulted in reduced plasma levels of ALT, AST, GGT, and lipase in ApoE-proficient, while plasma levels of GGT and lipase increased in ApoE-deficient animals. Moreover, levels of HDL, LDL, and total cholesterol increased after irradiation in plasma of ApoE-deficient mice, while remained low in plasma of ApoE-proficient animals.
The relationship between cardiac and hepatic dysfunction has been extensively investigated, as reviewed by Shah and Sass (2015). Cardiac disorders affect the liver and liver disorders affect the heart, and both effects may be accelerated by additional factors (Alvarez and Mukherjee 2011; Persson et al. 2005). Liver damage known as “cardiac hepatopathy” is a frequent feature of variable hemodynamic disturbances, and may be related to acute or chronic heart failure (Shah and Sass 2015). Exposure of the heart to ionizing radiation induces a deterioration of its functions, with possible further consequences (Darby et al. 2010; Marmagkiolis et al. 2016; Stewart et al. 2006). In the present study, we observed several structural and functional features of hepatocytes, that could be related to a systemic effect of damage induced by high doses of radiation delivered to the heart (Alvarez and Mukherjee 2011; Patties et al. 2015). Moreover, our data suggest an important role of lipid metabolism in heart-liver interactions, as documented by significant differences between ApoE-proficient and ApoE-deficient animals.
Tissue damage induced by ionizing radiation activates inflammation processes, not only local but also systemic, which result in increased plaque cell turnover and oxidative stress (Van Eeden and Sin 2013). These effects may favor the generation of lipid droplets via activation of protein complexes and generation of reactive oxygen species (ROS), which oxidize lipids already present in a liver and promote a generation of other ROS (Darby et al. 2010; Tanaka et al. 2005). Lipid and protein oxidation result in damage to membrane structures, which in turn cause further changes, such as in membrane potential, its permeability, and release of digestion enzymes to the intercellular matrix. Mitochondria are cellular structures which most sensitive to ROS, which could cause unspecific pores in the inner mitochondrial membrane leading to an imbalance between the intermembrane space and the mitochondrial matrix (Brunk and Terman 2002). The improper functioning of the respiratory chain results in increased production of ROS, and as a consequence, further malfunction of mitochondria and the whole cell (Duchen and Szabadkai 2010; Ivanova and Yankova 2013; Lagouge and Larsson 2013). Most disease processes originate from or result in disturbances in cellular energy balance; therefore, mitochondria swelling belongs to the most common early ultrastructural feature of affected cells (Cui et al. 2012; Duchen and Szabadkai 2010). Here, we observed large numbers of damaged mitochondria in hepatocytes from irradiated ApoE-deficient mice. On the other hand, hepatocytes from ApoE-proficient animals did not exhibit any significant damage to mitochondria, which may indicate their higher resistance to processes induced by irradiation.
Under physiological homeostasis, lysosomal autophagy occurs at a relatively low level, contributing to the efficient exchange of proteins in a cell and preventing accumulation of damaged proteins (Ahmed 2005; Ogier-Denis and Codogno 2003; Madrigal-Matute and Cuervo 2016). In general, enhanced lysosomal autophagy is frequently associated with cellular damage as a process facilitating resistance and recovery from damage (Cursio et al. 2015; Zhang 2013; Yen and Klionsky 2008). Here, we observed an increased activity of lysosomal hydrolases in livers of ApoE-proficient animals 60 weeks after irradiation. This biochemical feature was accompanied by characteristic ultrastructural features observed in the cytoplasm of such hepatocytes, including numerous fine vacuoles and an increased number of primary lysosomes containing small vacuoles, which collectively indicated ongoing microautophagy (Cursio et al. 2015; Lamparska-Przybysz and Motyl 2005; Shintani and Klionsky 2004; Telbisz et al. 2002). In marked contrast, enhanced autophagy was not detected in hepatocytes of ApoE-deficient animals, which was reflected by barely affected lysosomal hydrolases in animals exposed to radiation.
Exposure to low-doses of radiation was shown cause deregulation of hepatic enzymes involved in lipid metabolism, which was evidenced as a long-term effect of radiation in different mice experimental models (Bakshi et al. 2015; Yi et al. 2018). Radiation-mediated downregulation of enzymes involved in lipid metabolism documented in these proteomics studies could contribute to an accumulation of lipid droplets in the hepatocyte. ApoE−/− mice constantly collect lipids in their cells owing to a general error in lipid homeostasis. This could be illustrated by an increased level of cholesterol in the blood of such animals described in the literature (Mitchel et al. 2011) and in our data. Interestingly, however, no major differences in plasma levels of triglycerides were observed in our experimental model between ApoE-proficient or deficient animals, neither irradiated nor non-exposed. Nevertheless, excessive accumulation of lipids in hepatocytes of ApoE-deficient mice could block the lysosomal autophagy that normally regulates cellular level of lipids (Levine and Kroemer 2008; Singh et al. 2009). Hence, accumulation of lipids related to malfunction of lipid metabolism in ApoE−/− mice causes further accumulation of lipids in hepatocytes of irradiated animals. Due to the presence of large amounts of lipid droplets, hepatocytes of ApoE-deficient mice are more vulnerable to oxidative processes, which could be additionally intensified due to other consequences of heart irradiation. Lipid oxidation promotes decreased stability of cell membranes and further generation of ROS (Cui et al. 2012; Kmiecik et al. 2013; Lagouge and Larsson 2013), which causes distortion of lysosomal autophagy required to maintain cell homeostasis (Brunk and Terman 2002; Kurz et al. 2007; Leach 2009; Levine 2005; Madrigal-Matute and Cuervo 2016; Shintani and Klionsky 2004). Among possible consequences of such changes is damage to mitochondria, which was clearly observed in our experimental model.
To assess for potential functional damage of hepatocytes mediated by radiation, the levels of several liver-related enzymes were analyzed in plasma of mice 60 weeks after irradiation. Increased activity of ALT, AST, GGT, and lipase in blood is characteristic for liver disease or damage (El-Missiry et al. 2007; Gharib et al. 2012). It is noteworthy that the levels of these liver damage markers were reduced in plasma of ApoE-proficient mice, that seemingly reflect higher resistance of hepatocytes to radiation-induced damage (the effect putatively related to cytoprotective autophagy activated in this animals). On the other hand, an increased level of hepatic lipase was observed in plasma of irradiated ApoE−/− mice, which could indicate radiation-mediated accumulation of lipids and damage of hepatocytes.
In conclusion, liver dysfunctions were observed at the (ultra)structural and biochemical level as for late effects of irradiation with an apparent association with malfunction of lipid metabolism. We postulate the existence of a functional interplay between different aspects of radiation-induced damage of hepatocytes in the sensitive ApoE-deficient animals: accumulation of damaged mitochondria, accumulation of lipids and reduced autophagy. Moreover, the results of our study demonstrate a link between possible heart damage induced by radiation and functions of the liver.
The research received funding from the European Atomic Energy Community’s Seventh Framework Programme (FP7/2007–2011) under the grant agreement no. 211403 (CARDIORISK) and from the National Science Center, Poland under the grant no. N-402685640. We thank Dr Marta Gawin for critical reading of the manuscript. We also acknowledge the contribution of Hermann Fuchs, PhD, regarding estimation of the dose to the liver.
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