Selenium Supplementation Decreases Hepatic Fibrosis in Mice After Chronic Carbon Tetrachloride Administration
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- Ding, M., Potter, J.J., Liu, X. et al. Biol Trace Elem Res (2010) 133: 83. doi:10.1007/s12011-009-8414-x
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Oxidative stress stimulates fibrogenesis, and selenium (Se) has antioxidant properties. This study determined whether Se supplementation affects CCl4-induced liver injury and fibrosis. Mice were administered CCl4 over 4 weeks, while controls received olive oil. Se was provided as sodium selenite in the drinking water. Se increased liver Se-dependent glutathione peroxidase activity and decreased liver malondialdehyde after CCl4. Se decreased liver inflammation but not necrosis caused by CCl4. Se increased hepatocyte apoptosis after CCl4 and the pro-apoptotic BAX and Bcl Xs/l proteins. Stellate cell apoptosis occurred only after CCl4 in Se-supplemented mice. Se decreased stellate cell number and fibrosis after CCl4. Liver matrix metalloproteinase-9 increased after CCl4 with Se supplementation. In conclusion, Se supplementation decreased hepatic fibrosis after CCl4 in the setting of decreased inflammation but increased apoptosis. The principal mechanisms for the decreased fibrosis are a lower number of collagen-producing stellate cells and increased collagen degradation.
Oxidative stress stimulates fibrogenesis . Lipid peroxidation products enhance α1(I) collagen expression and collagen synthesis by stellate cells in culture [2, 3]. Reactive oxygen species (ROS) are mostly generated by increased NAD(P)H oxidase which produces superoxide anion (O2•−) from oxygen (O2). Superoxide anion in turn generates H2O2 and hydroxyl radical (OH−). Liver fibrosis is less after chronic CCl4 administration in mice deficient in the gp91phox subunit of the NADPH complex than in wild-type control mice [4, 5].
Selenium is a trace element which has antioxidant properties because it is an essential component of some oxidoreductase enzymes . Most notable is the selenium-dependent glutathione peroxidase enzyme (Se-GSH-PX) which catalyses the reduction of hydrogen peroxide (H2O2) to water (H2O) by conversion of reduced glutathione (GSH) to its oxidized form (GSSG). Selenium and Se-GSH-PX deficiency are associated with higher levels of ROS and lipid peroxidation , which can be reversed by Se supplementation [6, 7]. Serum and liver Se are decreased in alcoholic and nonalcoholic liver diseases, most likely due to decreased dietary intake [8, 9]. In one study, administration of vitamin E together with Se and zinc resulted in a lower 1-month mortality in patients with alcoholic hepatitis . In other studies, however, administration of Se together with vitamins A and E did not affect survival of patients with alcoholic hepatitis . Selenium-enriched lactobacillus decreased liver injury and lipid peroxidation induced by CCl4 administration in mice , while supplementation of the diet with vitamin E and Se decreased hepatic fibrosis produced in rats by acute and chronic CCl4 administration .
The purpose of this study was to determine whether Se supplementation affects liver injury and fibrosis produced by chronic CCl4 administration in mice.
Materials and Methods
Animals and Materials
Male C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). All animals received humane care in compliance with the guidelines from the Animal Care and Use Committee of The Johns Hopkins University. Sirius Red was obtained from Polysciences, Warrington, PA, USA. Carbon tetrachloride (CCl4), sodium selenite, and goat anti-mouse α-smooth muscle actin Cy3 conjugate antibody were purchased from Sigma Chemical, St. Louis, MO, USA. Anti-mouse thioesther s-methyltransferase (TEMT) antibody was a gift from Dr. Linda A. Toth from the Southern Illinois University School of Medicine, Springfield, IL, USA. Caspase 3 and caspase 8 fluorometric assay kits were obtained from BioVision (Mountain View, CA, USA).
Mice 4–6 weeks of age and weighing 20–30 g were kept in a temperature-controlled room with an alternating 12-h dark and light cycle. Thirty-two mice were divided into four experimental groups of eight mice each as follows: (1) CCl4 injections; (2) CCl4 injections with the drinking water supplemented with Se; (3) olive oil injections; and (4) olive oil injections with the drinking water supplemented with Se. CCl4 was administered as biweekly intraperitoneal (i.p.) injections of 5 μl of a 20% solution of CCl4 in olive oil per gram body weight (1.0 ml/kg of CCl4). Se supplementation was provided as sodium selenite (4 mg of anhydrous sodium selenite/L) in the drinking water. This supplementation was initiated 2 days before starting the CCl4 or olive oil injections. The water volume ingested was recorded. The animals were on ad lib rodent diet (# 2018X) obtained from Harlan Teklad, Madison, WI, USA, which contains 200 μg of Se per kilogram of diet. The animals were killed 4 weeks after the start of these injections. At the time of killing, blood was obtained from the aorta for measurement of serum alanine aminotransferase (ALT), and the samples were stored at −20°C. The liver was removed, rinsed with phosphate-buffered saline (PBS), and divided into five portions: (a) fixed in 10% buffered formaldehyde formalin and embedded in paraffin; (b) snap frozen at −70°C for sectioning and immunohistochemistry; (c) homogenized in appropriate buffer(s) and aliquots frozen at −70°C for biochemical assays; (d) snap frozen at −70°C for Se assay; and (e) placed in RNA STAT-60 (from Tel-Test, Friendswood, TX, USA) solution, homogenized, and stored in −70°C for RNA isolation.
Liver Selenium and Selenium-Dependent Glutathione Peroxidase Activity
Liver Se was determined in liver homogenates by the Oscar E. Olson Biochemistry Laboratories of the South Dakota State University using the Association of Official Analytical Chemists official fluorometric method 996.16 . The activity of Se-dependent glutathione peroxidase was determined in the liver supernatant, obtained after centrifugation of the liver homogenate at 3,000×g for 10 min, with H2O2 as a substrate using the glutathione cellular activity assay kit obtained from Sigma-Aldrich, St Louis, MO, USA.
Liver Histology and Morphometric Collagen Determination
The liver sections imbedded in paraffin were cut (5 μm) and stained with hematoxylin–eosin (H&E), Masson's trichrome, or Sirius red. The extent of necrosis and inflammation was evaluated on blinded slides by M.S.T. from our Department of Pathology. Fibrosis was determined histologically by measuring the intensity of fibrosis in four to six (×100) digital images captured from slides of each mouse stained with Sirius red. The total fibrosis density score was determined by dividing the image intensity by the image area as described previously .
Quantitation of Stellate Cells
Immunofluorescent staining for α-smooth muscle actin (α-SMA) was done in deparaffinized liver sections. The slides were washed in PBS, followed by blocking using PBS–5% fetal bovine serum (FBS). The slides were incubated with Cy3 conjugated monoclonal α-SMA antibody (Sigma, 1:500 in PBS–5% FBS) for 1 h at room temperature and subsequently at 4°C overnight. After washing with PBS four times for 15 min, the slides were mounted and eight areas per slide captured by fluorescent microscopy (magnification ×100). Stellate cells were counted in the eight fields per slide for each mouse.
Serum Alanine Aminotransferase
Serum alanine aminotransferase was determined by the spectrophotometric method of Bergmeyer et al. .
Liver slices were homogenized with cold 1.15% KCl, and malondialdehyde was determined using thibarbituric acid by the method of Uchiyama and Mihara .
Determination of Messenger RNA by Real-Time Quantitative Polymerase Chain Reaction
The 7900 HT (Applied Biosystems, Foster City, CA, USA) and the SDS 2.2.1 software were used to perform real-time quantitative polymerase chain reaction (RT-qPCR) at The Johns Hopkins DNA Analysis Facility. Total cellular RNA from a portion of liver was placed in RNA STAT 60 reagent, and following their protocol, RNA was purified and isolated. The concentration of the isolated RNA was determined from the optical density at 260 nm and its purity from the 260/280 nm OD ratio. The isolated RNA was stored at −80°C. RT-qPCR for α1(I) collagen mRNA was performed using sequence-specific probes from TaqMan gene expression assays of Applied Biosystems. The probes for mouse α1(I) collagen and β-actin (as endogenous control) were obtained from Applied Biosystems. Superscript III first-strand synthesis from Invitrogen (Carlsbad, CA, USA) was used to synthesize first-strand cDNA from the purified RNA. Gene-transcript levels of the above probes were compared to β-actin, the housekeeping endogenous control. Variation in the amount of the transcripts was corrected by the level of expression of the β-actin gene in each individual sample.
Western Blot Analysis
Liver sections were homogenized in 50 mM Tris–HCl buffer pH 7.6 containing 150 mM NaCl, 10 mM CaCl2, 0.25% Triton-X, 0.1 μM phenylmethanesulfonyl fluoride, 10 μM leupetin, 10 μM pepstatin, 0.1 mM iodoacetamide, and 25 μg aprotonin and then centrifuged at 3,000×g for 10 min at 4°C. The cytosol protein in the supernatant was initially stored at −80°C. The proteins were separated on mini-SDS gels at 100 V for 1 h and electrotransfered to nitrocellulose transblot membranes (BioRad, Hercules, CA). The membranes were washed in PBS, pH 7.6, containing 0.1% Tween 20 (PBS-T), blocked with 5% (W/V) dry nonfat milk in PBS-T for 1 h, rinsed with PBS-T, and then incubated with either rabbit anti-mouse antibodies to Fas, Bax, BclXs/l, Bcl-2, cytochrome C, MMP-2, MMP-9, TIMP-1, TIMP-2, or β-actin, obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA. The membranes were also incubated with rabbit anti-mouse antibody to TEMT, kindly provided by Dr. Linda A Toth. After repeated washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution; Amersham Biosciences, Piscataway, NJ, USA) at room temperature for 1 h. The membranes were then washed again and visualized by enhanced chemiluminescence reaction (ECL Plus; Amersham Biosciences). Densitometry was determined using Image J v 1.30 obtained from NIH.
Terminal Deoxynucleotidyl Transferase-Mediated Nick-End Labeling Assay
Terminal deoxynucleotidyl transferase-mediated in situ nick-end labeling (TUNEL) assay was performed on paraffin-embedded liver slices with the cell death detection kit from Roche Applied Science (Nutley, NJ, USA). Fluorescence microscope using the fluorescein isothiocyanate filter revealed the apoptotic bodies which were counted.
Apoptosis in Stellate Cells
Apoptosis in stellate cells was determined in 5 μM sections of cryopreserved liver tissue with the cell death detection kit from Roche Applied Science. Following TUNEL staining, the sections were blocked in 5% FBS for 60 min and incubated with Cy3-conjugated monoclonal α-smooth muscle actin antibody (Sigma, 1:500 in PBS–5% fetal bovine serum) for 1 h at room temperature. 4′,6-Diamidino-2-phenylindole (DAPI) for nuclear staining was added during the last 10 min of the incubation. The sections were washed in PBS for 1 h with frequent changes. The immunostained slices were evaluated and photographed with laser confocal microscopy.
The activities of caspase 3 and caspase 8 were determined in liver homogenates by measuring proteolytic cleavage of the specific fluorogenic substrates, DEVD-7-amino-4-trifluoromethyl coumarin (AFC) and IETD-AFC, respectively (BioVision). The results are expressed as relative units per milligram of protein. The concentration of protein was determined by the method of Lowry et al. .
In most measurements, the mean and the standard error of the mean were calculated. The data was analyzed with the Student's t test or by two-way analysis of variance when comparing means of more than two groups.
Liver Selenium and Selenium-Dependent Glutathione Peroxidase
Liver malondialdehyde was not increased significantly by CCl4 (Fig. 1). Se supplementation decreased liver malondialdehyde in the control (p < 0.05) and CCl4-treated (p < 0.01) mice.
Liver Injury and Fibrosis
Serum alanine aminotransferase was elevated to a similar degree after CCl4 administration in Se-supplemented and control mice (Fig. 2).
This study shows that dietary selenium (Se) supplementation reduces hepatic fibrosis produced in mice by chronic CCl4 administration. Hepatic fibrosis after CCl4 administration was previously shown to be decreased in rats by dietary supplementation with vitamin E and Se . The lesser accumulation of fibrosis in the Se-supplemented mice after CCl4 administration in our study was associated with a lower number of stellate cells. A combination of decreased formation and increased degradation of collagen was considered as causes of the lesser accumulation of collagen in the liver. The similar increases in α1(I) collagen mRNA in the Se-supplemented as compared to the control mice after chronic CCl4 administration indicates that Se does not affect Type I collagen transcription but does not rule out effects of Se on collagen translation. Furthermore, there is evidence in this study that increased collagen degradation contributes to the lesser accumulation of hepatic fibrosis in the Se-supplemented mice. Increased collagen formation from CCl4 administration [19, 20] and from other insults  is associated with an increase in collagen degradation. In this study, MMP-9 was increased after CCl4 in the Se-supplemented mice, while MMP-2 remained unchanged. TIMP-1 also remained unchanged after CCl4 treatment, while TIMP-2 increased after CCl4 in the Se-supplemented mice. TIMPs inhibit secretion and activation of metalloproteinases, hence inhibiting collagen degradation (22]. TIMP-2 is not only a specific inhibitor of MMP-2, but it is also required for the activation of MMP-2 ; however, in our study, the increase in TIMP-2 was not associated with a change in MMP-2. Of note is that methylselenol increased pro-MMP-2 and TIMP-2 but decreased active MMP-2 in HT1080 tumor cells in culture .
The lesser accumulation of collagen after CCl4 administration in the Se-supplemented mice was associated with less hepatocellular inflammation but similar grades of necrosis and similar elevations of serum ALT than seen in the nonsupplemented mice. Se is known to have anti-inflammatory properties . Se supplementation was shown to decrease inflammation in lung tissues induced by bacterial endotoxin lipopolysaccharide (LPS) in Se-deficient mice . As regards this study, LPS is known to contribute to CCl4 hepatotoxicity .
Se supplementation increased hepatocyte apoptosis in the mice treated with CCl4. In a previous study, Se supplementation increased stellate cell apoptosis after acute CCl4 administration . Large concentrations of sodium selenite (10 μM) induce oxidative stress and apoptosis in cultured HepG2 cells . This effect of high levels of Se is the result of depletion of GSH by its reaction with the oxidized form of selenium (Se042−) .
Apoptosis of stellate cells was only found in the CCl4-treated mice that had received Se supplementation. Iredale et al.  showed that apoptosis of stellate cells contributes to resolution of fibrosis after discontinuation of chronic CCl4 administration in rats. The exact mechanism for the increased apoptosis in the Se-supplemented mice remains uncertain. Pro-apoptotic BAX and Bcl Xs/l proteins were only increased after CCl4 treatment in the Se-supplemented mice, while the anti-apoptotic protein Bcl-2 was not changed significantly. Cytosolic cytochrome c was not changed significantly by 4 weeks of CCl4 treatment or Se supplementation. Most likely, the increase in cytosolic cytochrome c is an earlier event of CCl4-induced hepatic injury. The Fas (CD95/APO-1) receptor which mediates apoptosis caused by a variety of insults , principally via the extrinsic death receptor pathway, was increased after CCl4 treatment in the Se-supplemented mice. However, the activities of caspase 3 and caspase 8 were increased to a lesser extent after CCl4 in the Se-supplemented mice.
Se is detoxified by successive methylation reactions catalyzed first by the enzyme thiol s-methyltransferase that converts hydrogen selenide to methylselenol and dimethylselenide, with subsequent conversion of dimethylselenide to trimethylselenomium catalyzed by thioether s-methyltransferase. Dimethylselenide is eliminated by the lung, while trimethylselenomium, which is water-soluble, is excreted in the urine . Both selenium (selenite) and methylseleninic acid, an oxidation product of methylselenol, cause apoptosis in DU-145 cancer cells. While methylseleninic acid causes apoptosis via caspase 8 and the mitochondrial pathway with cytochome c release, selenite causes apoptosis by a different pathway without those changes . In this study, the TEMT was detected in the liver by Western blot and shown to be decreased by chronic CCl4 administration regardless of Se supplementation. The lower TEMT concentration may result in increased retention of the methylated forms of selenium.
This study shows that Se supplementation decreased liver malondialdehyde in the control and CCl4-treated mice. The hepatic damage caused by CCl4 is due its metabolism to trichloromethyl (CCl3⋅) and trichloromethylperoxyl (CCl3OO⋅) reactive free radicals . GSH defends against reactive free radicals by glutathione peroxidase enzymes which inactivate ROS via conversion of GSH to its oxidized form GSSG  or by donating an electron to the free radical R to form RH . CCl4 administration decreases liver GSH as we showed previously  and also decreases the Se-dependent glutathione peroxidase enzyme, as shown in this study, allowing for the accumulation of free radical metabolites resulting in lipid peroxidation and liver damage. Oxidative stress is also well known to enhance collagen synthesis but can also enhance collagen degradation. Reactive oxygen species enhance stellate cell activation  and lipid peroxidation products, such as malondialdehyde, and stimulate α1(I) collagen expression and collagen synthesis by stellate cells in culture [2, 3]. ROS is generated by increased NAD(P)H oxidase which produces superoxide anion (O2•−) from oxygen (O2), and the amount of hepatic fibrosis after chronic CCl4 administration is less in NAD(P)H-deficient mice than in wild-type mice [4, 5]. Oxidative stress, however, also increases active MMPs, which degrade collagen and other extracellular matrix proteins [35, 36]. The most likely cause for the effect of Se supplementation in decreasing hepatic fibrosis caused by CCl4 is a decrease in oxidant stress resulting in reduced collagen formation and increased collagen degradation. The postulated reduced collagen formation, which is associated with a lower number of stellate cells, may be due to a decrease in collagen translation and triple helix formation since it is not associated with a lower α1(I) collagen mRNA.
In conclusion, this study shows that Se supplementation has a protective effect on hepatic fibrosis induced by CCl4 in mice. The decreased hepatic fibrosis after chronic CCl4 administration in mice supplemented with Se occurred in the setting of decreased inflammation but increased apoptosis. The principal mechanism for the decrease in fibrosis is a decrease in the number of collagen-producing stellate cells, which is accompanied by increased collagen degradation. Apoptosis of stellate cells is a mechanism for the lesser number of stellate cells after CCl4 administration in Se-supplemented mice. Se deficiency in conjunction with enhanced oxidative stress, which is common in liver disease, most likely enhance liver injury and fibrosis caused by multiple agents such as alcohol and hepatitis C. Se supplementation in such cases could ameliorate liver injury and slow the progression of fibrosis.
This work was supported by grant AA000626 from the US Public Health Service. M. D. was a postdoctoral fellow on National Research Service Award 2 T32 AA07467 from the National Institute of Alcohol Abuse and Alcoholism (NIH). The authors acknowledge assistance from The Hopkins Digestive Diseases Basic Research Development Center (NIH grant 2464388) in the performance of this study.