Enhancement by Estradiol 3-Benzoate in Thioacetamide-Induced Liver Cirrhosis of Rats

Jin Seok Kang*, Hideki Wanibuchi*, Keiichirou Morimura*, Rawiwan Puatanachokchai*, Elsayed I. Salim*, Atsushi Hagihara*, Shuichi Seki{dagger} and Shoji Fukushima*,1

* Department of Pathology and {dagger} Department of Hepatology, Graduate School of Medicine, Osaka City University Medical School, Osaka 545-8585, Japan

1 To whom correspondence should be addressed at Department of Pathology, Osaka City University Medical School, 1–4–3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail: fukuchan{at}med.osaka-cu.ac.jp.

Received December 17, 2004; accepted February 7, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As part of an investigation on the role of estrogen in liver disease, we tested the effects of estradiol-3-benzoate (EB) in the thioacetamide (TAA)-induced rat liver cirrhosis model. Male F344 rats (n = 100) were divided into six groups. Animals of groups 1–4 received TAA (0.03% in drinking water) for 12 weeks, and groups 5 and 6 served as controls without TAA. For the exposure period, EB pellets were implanted subcutaneously to give doses of 0 (groups 1 and 5), 1 (group 2), 10 (group 3), and 100 µg (groups 4 and 6) simultaneously. All animals were sacrificed at week 12. Significant increase of liver cirrhosis, liver weight, collagen content, and lipid peroxidation in the livers was evident in groups 3 and 4 (p < 0.05) compared with group 1. Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was significantly elevated in group 4 (p < 0.01), along with expression of alpha-smooth muscle actin (alpha-SMA) and stellate cell activation-associated protein (STAP), as determined by RT-PCR analysis (p < 0.01). However, there were no differences in liver weight, collagen content, lipid peroxidation, 8-OHdG formation, and alpha-SMA and STAP mRNA expression between groups 5 and 6. We conclude that EB treatment enhances TAA-induced cirrhosis, associated with increase of oxidative stress and activation of hepatic stellate cells.

Key Words: estradiol-3-benzoate; thioacetamide; cirrhosis; alpha-smooth muscle actin; Stellate cell activation-associated protein.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidemiological and experimental studies have found sex differences in the incidence of chronic liver disease (Becker and Gluud, 1991Go; Bosch et al., 1999Go; De Maria et al., 2002Go), indicating hormonal involvement. Estrogens exert diverse biological effects in the body, and in the liver they play important roles in the regulation of proliferation (Francavilla et al., 1989Go). Most of the actions of estrogens are mediated by two estrogen receptors (ERs), ER alpha and ER beta, cloned in 1986 (Green et al., 1986Go) and 1996 (Kuiper et al., 1996Go), respectively.

It has been reported that estrogens exert inhibitory effects on some liver diseases. In dimethylnitrosamine (DMN) and pig serum models, treatment with estradiol inhibited the activation of hepatic stellate cells (HSCs) in vivo and in vitro (Shimizu et al., 1999Go) and reduced fibrosis (Yasuda et al., 1999Go). The hormone was also found to inhibit lipid peroxidation, and decrease expression of I{kappa}B-{alpha} and NF-{kappa}B in rat liver cell culture (Omoya et al., 2001Go). However, in some situations, estrogens have negative roles on the progress of liver disease. It was reported estradiol in combination with epidermal growth factor increased growth and induced c-fos mRNA expression at early times in cell culture, which ultimately culminated in enhanced DNA synthesis (Lee and Edwards, 2001Go). Estrogens also stimulate proliferation of intrahepatic biliary epithelium (Alvaro et al., 2000Go), and estrogens antagonist treatment during bile duct ligation (BDL) decreased fibrosis (Alvaro et al., 2002Go).

It is known that estrogens can act as potent endogenous antioxidants, reducing lipid peroxidation levels in liver and serum (Lacort et al., 1995Go), and induce antioxidant enzymes such as copper-zinc SOD and glutathione peroxidase (Lu et al., 2004Go).

Some of the many actions of estradiol may not be caused by estradiol per se, but may result from the formation of active estrogen metabolite(s) (Zhu and Conney, 1998Go). Among the metabolites, catechol estrogens can be oxidized to semiquinones, which in the presence of molecular oxygen are oxidized to quinones with formation of superoxide and subsequently hydroxyl radicals (Cavalieri et al., 2000Go), and estradiol-derived metabolites, 2- and 4-hydroxyestradiol, can exert either pro- or antioxidant actions (Martinez et al., 2002Go). These data let us set the hypothesis that some metabolites of estrogens might increase oxidative stress in some diseases which are related to the formation of reactive oxygen species.

Thioacetamide (TAA) causes liver cirrhosis at medium-term treatment (Muller et al., 1988Go) and hepatic neoplasms at long term treatment in animals (Becker, 1983Go). Because this is associated with decreased catalase and glutathione peroxidase (Sanz et al., 1995Go), we here tested the hypothesis that estrogen treatment may enhance oxidative stress and act as an oxidant or pro-oxidant in TAA-treated animals.

For this purpose we investigated the action of estradiol-3-benzoate (EB) on parameters of liver cirrhosis, as well as expression of alpha-smooth muscle actin (alpha-SMA) as a marker of activated HSCs and stellate cell activation-associated protein (STAP), which was recently discovered to be a kind of haemoprotein activated in fibrosis (Asahina et al., 2002Go; Kawada et al., 2001Go). Modulation of ER alpha and ER beta expression by EB treatment was also assessed.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatment.
One hundred, 5-week-old, male F344 rats were obtained from Charles River Japan, Inc. (Atsugi, Japan) and housed in rooms maintained on a 12-h light/dark cycle at constant temperature and humidity. They were allowed free access to pellet chow diets (MF pellet diet, Oriental Yeast Co., Tokyo, Japan) during the experiment. All procedures were approved by the Institutional Animal Care and Use Committee.

At 6 weeks of age, the rats were divided into six groups. Animals of groups 1–4 (n = 20 for each group) received TAA (0.03% in drinking water) for 12 weeks, and animals of groups 5 and 6 (10 per group) received drinking water without TAA. During this exposure period, EB pellets were subcutaneously implanted to give doses of 0 (groups 1 and 5), 1 (group 2), 10 (group 3), and 100 µg (groups 4 and 6). The EB pellets were produced by the method described previously (Kang et al., 2004Go). Briefly, after mixing 0.132, 1.32, or 13.2 mg of EB with 2 g of cholesterol and 1.7 ml of olive oil, the preparation was introduced into medical grade silastic tubes (Silicon Medical Tube No. 2, 100–2N; Kaneka Medix Corporation, Japan). The total content of EB in each 1-cm tube was estimated as approximately 1, 10, or 100 µg. EB pellets were replaced with new ones every 4 weeks.

Body weights of all animals were measured every week. All animals were sacrificed at week 12 under ether anesthesia. At necropsy, liver weights were measured. Liver tissues were fixed in 10% phosphate-buffered formalin and routinely processed for embedding in paraffin, and staining of 4 µm sections with hematoxylin, and eosin staining for histopathological examination. The amount of total estradiol in serum was measured by 125I-radioimmunoassay kit (Coat-A-Count® Estradiol, Diagnostic Product Corporation, CA) as recommended protocol provided by producers. Detection limit of this kit is 10 pg/ml.

Fibrosis was categorized with the following criteria: +, portal fibrosis, characterized by mild fibrous expansion of portal tracts; ++, periportal fibrosis showing fine strands of connective tissue in zone 1 with only rare portal–portal septa; +++, septal fibrosis manifested by connective tissue bridges that link portal tracts and central veins, minimally distorted architecture, but no regenerative nodules. Liver cirrhosis was diagnosed on the basis of bridging fibrosis and the presence of regenerative hepatic nodules. Samples of liver from all animals were also snap-frozen in liquid nitrogen for biochemical and molecular analysis.

Estimation of collagen content.
Two sections of liver were cut approximately 15-µm thick, deparaffinized and hydrated. They were then place in distilled water at 4°C and incubated in Fast green FCF (Sigma), and subsequently in Fast green FCF and Sirius red F3B (Aldrich). After adding 0.05 N NaOH in 50% aqueous methanol, eluted color was read in a spectrophotometer (Ultraspec 3000, UV/Visible Spectrophotometer; Pharmacia Biotech, Tokyo, Japan) at 530 nm and 605 nm. The collagen content was calculated according to the previously described method (Gascon-Barre et al., 1989Go). Data are the mean ± SD from ten samples per group and two independent experiments.

Quantification of lipid peroxidation.
Frozen livers were homogenized in sodium phosphate buffer, with addition of trichloroacetic acid (TCA, nacalai tesq, Japan). After centrifuge, the supernatant were mixed with 2-thiobarbituric acid (TBA, nacalai tesq, Japan), and the samples were boiled at 100°C for 10 min. They were mixed with butanol and were centrifuged, and the absorbance was measured using a spectrophotofluorometer (Hitachi F4500, Japan), and 2-thiobarbituric acid-reacting substances (TBARS), expressed as malondialdehyde equivalent (MDA eq) values, were calculated from a standard curve, with reference to protein content measured by the Lowry method. Data are the mean ± SD from five samples per group and three independent experiments.

Quantification of 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation.
DNA samples isolated from pieces of frozen liver weighing 500 mg were digested into deoxynucleosides by combined treatment with nuclease P1 and alkaline phosphatase. Levels of 8-OHdG were determined by high-performance liquid chromatography as described earlier (Nakae et al., 1997Go) and expressed as the number of 8-OHdG residues/105 total deoxyguanosines (dG). Data are the mean ± SD from ten samples per group and two independent experiments.

RNA preparation.
Total RNA was isolated from frozen liver using ISOGEN (Nippon Gene Co. Ltd, Tokyo, Japan), isopropanol precipitated, dissolved in DEPC-treated distilled water, and stored at –80°C until use. RNA concentrations were determined with a spectrophotometer (Ultraspec 3000, UV/Visible Spectrophotometer; Pharmacia Biotech, Tokyo, Japan). For cDNA synthesis, 3 µg of total RNA was heated to 70°C for 10 min and then placed immediately on ice for 10 min. To each sample, 4 µl of 5x first strand buffer, 2 µl of 0.1 M DTT, 4 µl of 2 mM each dNTP mix, 1 µl of oligo(dT) primer, and 1 µl of Superscript II reverse transcriptase (Invitrogen, CA) were added. Reverse transcription was then carried out at 42°C for 50 min, followed by heating to 70°C for 15 min, and cDNA samples were stored at –20°C until assayed.

RT-PCR for Alpha-SMA mRNA, STAP mRNA, ER Alpha mRNA, ER Beta mRNA, and Beta-Actin mRNA.
cDNAs were amplified using specific oligonucleotide primers for rat alpha-SMA, STAP, ER alpha, ER beta, and beta-actin as follows: rat alpha-SMA sense, 5'-ACT GGG ACG ACA TGG AAA AG-3' and antisense, 5'-CAT CTC CAG AGT CCA GCA CA-3'; rat STAP sense, 5'-ATG GAG AAA GTG CCG GGC GAC-3', antisense, 5'-TGG CCC TGA AGA GGG CAG TGT-3'; rat ER alpha, sense, 5'-CTG CCA AGG AGA CTC GCT ACT G-3', antisense, 5'-TCT TCC TCC GGT TCT TAT CGA TGG-3'; rat ER beta, sense 5'-AAC AAG GGC ATG GAA CAT CTG CT-3', antisense, 5'-TCC GCC TCA GGC CTG GCC ATC A-3'; and rat beta-actin, sense: 5'-ACC ACA GCT GAG AGG GAA ATC G-3', antisense, 5'-AGA GGT CTT TAC GGA TGT CAA CG-3'.

The PCR program cycles were set as follows: for alpha-SMA and beta-actin, initial denaturing at 95°C for 9 min, followed by 28 cycles or 24 cycles (95°C for 1 min, 58°C for 1 min, and 72°C for 1 min), respectively, and a final extension at 72°C for 5 min; for STAP, initial denaturing at 95°C for 9 min, following by 34 cycles (95°C for 1 min, 68°C for 1 min, and 72°C for 1 min), and a final extension at 72°C for 5 min; for ER alpha, initial denaturing at 95°C for 9 min, followed by 30 cycles (95°C for 1 min, 60°C for 1 min, and 72°C for 1 min), and a final extension at 72°C for 5 min. For ER beta, the PCR program cycle was set to denature at 95°C for 30 sec, to anneal at 65°C for 15 sec, and to extend at 72°C for 15 sec for a total of 20 cycles, the annealing temperature being reduced 0.5 °C in every cycle, and then set to denature at 95°C for 30 sec, to anneal at 51°C for 15 sec, and to extend at 72°C for 15 sec for a total of 30 cycles. For confirmation of ER beta PCR product, DNA was recovered using EasytrapTM Ver. 2 (Takara, Japan) from gel slices with the manufacturer's protocol, and purified in a Microspin S-300 HR column (Amersham Bioscience, NJ) for examination of the sequence by ABI PRISM 3100-Avant (Applied Biosystem, USA).

Beta-actin mRNA was employed as an internal standard, and alpha-SMA, STAP, ER alpha, and ER beta mRNA expression were determined by quantitative PCR and normalized against beta-actin mRNA levels. The PCR products were electrophoresed on a 2% Nusieve agarose gel and stained with ethidium bromide. The gels were then scanned and analyzed using a FLA-5100 and Multi Gauge Ver 2.0 software (Fujifilm, Japan). All PCR products were amplified in a linear cycle. Data are the mean ± SD from five samples per group and three independent experiments.

Statistical analysis.
Statistical analyses were performed with the Tukey-Kramer method using the JMP program (SAS Institute, Cary, NC). For all comparisons, probability values less than 5% (p < 0.05) were considered to be statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body and Relative Liver Weight
Marked growth retardation was noted during TAA and EB treatment, with significant decrease in final body weights and increase of relative liver and spleen weight in groups 3 and 4 at week 12 compared with group 1 (p < 0.01). The body weight in group 6 was also significantly lower than in group 5 (p < 0.01) (Table 1). During the experiment, unscheduled deaths occurred in groups 1 and 2 (n = 1, respectively), and group 4 (n = 3). With the three doses of EB, dose-dependent increase of serum estradiol was achieved (Table 1). There was elevation of estradiol level in TAA-treated animals compared with those of normal animals.


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TABLE 1 Final Body Weight, Relative Liver and Spleen Weights, and E2 Level in Serum

 
Histopathological Examination of Liver
Fibrosis and/or cirrhosis were observed in all animals treated with TAA. There was significant increase of cirrhosis incidence in groups 3 and 4 by chi-square analysis at the value of p < 0.01 and p < 0.05, respectively (Table 2). In groups 3 and 4, intensity of fibrosis showed a similar pattern.


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TABLE 2 Pathological Finding of Livers

 
Cirrhosis cases demonstrated marked hepatic nodules, separated by fibrous septa (Fig. 1A) with a dense collagenous matrix, and many myofibroblast-like cells and fibroblasts. There were also dysplastic bile-ductule-like structures and metaplastic intestinal-like glands, surrounded by spindle shape cells. EB treatment with TAA represented severe advanced cirrhosis, showing more marked hepatic nodules and thicker fibrous septa (Fig. 1B).



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FIG. 1. Effects of estradiol-3-benzoate (EB) on liver cirrhosis. (A) Liver section from a rat treated with 0.03% thioacetamide (TAA) (group 1) in drinking water for 12 weeks, showing bridging fibrosis (arrow) and nodular regeneration. There was a dense collagenous matrix containing many myofibroblast-like cells and fibroblasts. (B) Liver section from a rat treated with TAA + EB 100 µg (group 4) for 12 weeks, exhibiting advanced cirrhosis. There were more marked hepatic nodules and thicker fibrous septa (arrow) compared with those of (A). H&E, magnification, x40.

 
Collagen Content, Lipid Peroxidation, and 8-Ohdg Formation in the Liver
Data for hepatic collagen content, lipid peroxidation, and 8-OHdG formation are summarized in Table 3. Collagen contents in groups 3 and 4 were significantly increased compared with group 1 (p < 0.01, p < 0.05, respectively). Biochemical analysis of lipid peroxidation showed significant increase in groups 3 and 4 compared with group 1 (p < 0.01), and there were significant difference between group 1 and group 5 (p < 0.01). HPLC analysis of 8-OHdG formation showed significant increase in group 4 compared with group 1 (p < 0.01). However, collagen content, lipid peroxidation, and 8-OHdG formation in group 6 did not differ from the group 5 values.


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TABLE 3 Collagen Content, Lipid Peroxidation, and 8-OHdG Formation in the Liver

 
Alpha-SMA, STAP, ER Alpha, and ER Beta mRNA Expression
RT-PCR analysis showed a significant increase of hepatic alpha-SMA and STAP mRNA expression with TAA+EB 100 µg (group 4) as compared to TAA alone (group 1). However, EB 100 µg (group 6) did not cause any change from the control values (group 5) (Fig. 2). Regarding ER alpha mRNA expression, there was a significant increase with EB 100 µg (group 6) as compared to TAA alone (group 1) and the control (group 5) (p < 0.01). However, there were no differences in ER beta expression among the groups (Fig. 3).



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FIG. 2. RT-PCR analysis of alpha-SMA and STAP mRNA expression. (A) Two representative samples per group are shown. (B) RT-PCR analysis of alpha-SMA mRNA expression in the liver. Alpha-SMA mRNA was quantified and normalized with beta-actin mRNA expression as described in the Materials and Methods. Note the significant increase in alpha-SMA mRNA expression with TAA+EB 100 µg (group 4) compared to TAA (group 1) (p < 0.01), but no difference between Control (group 5) and EB 100 µg (group 6). (C) Stellate cell activation-associated protein (STAP) mRNA expression in the liver, normalized to beta-actin mRNA. Note significant increase of STAP mRNA expression with TAA+EB 100 µg (group 4) as compared to TAA (group 1) (p < 0.01), but no difference between Control (group 5) and EB 100 µg (group 6). **Significantly different from TAA (group 1) (p < 0.01); NS: Not significant.

 


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FIG. 3. RT-PCR analysis of ER alpha and ER beta mRNA expression. (A) Two representative samples per group are shown. (B) ER alpha mRNA expression in the liver was quantified and normalized to beta-actin mRNA as described in the Materials and Methods. Note significant increase with EB 100 µg (group 6) compared to TAA (group 1) and Control (group 5) (p < 0.01). (C) ER beta mRNA expression in the liver was quantified and normalized to beta-actin mRNA. Note the lack of variation among the groups; **Significantly different from TAA (group 1) (p < 0.01). #Significantly different from Control (group 5) (p < 0.01).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, increases of liver weight, cirrhosis, and collagen content in TAA-treated animals were dose-dependently enhanced by EB treatment, associated with oxidative stress as evidenced by lipid peroxidation and 8-OHdG formation, and activation of HSCs showing increased expression of alpha-SMA and STAP.

The results are thus not in line with the reported antioxidant action of estrogen (Lacort et al., 1995Go) and the induction of antioxidant enzymes (Lu et al., 2004Go). In DMN and pig serum models, treatment with estradiol resulted in suppression of hepatic fibrosis (Shimizu et al., 1999Go) and reduced hepatic collagen deposition (Yasuda et al., 1999Go), and inhibition of lipid peroxidation in rat liver cell culture has been documented (Omoya et al., 2001Go). However, estrogen treatment enhanced fibrosis or cirrhosis in BDL model (Alvaro et al., 2000Go, 2002Go), as in the TAA model in this study. This difference in action appears to reflect variation in metabolic activation and interaction with the hepatotoxin.

It should be remembered that catechol estrogens can exert pro-oxidant activities (Martinez et al., 2002Go), and the fact that TAA treatment causes decrease in catalase and glutathione peroxidase, with glutathione depletion (Sanz et al., 1995Go), might have played a role. In this study, estrogen actually enhanced collagen content, lipid peroxidation, and alpha-SMA and STAP expression in TAA-treated animals. Reduction of intracellular glutathione and increase of lipid peroxidation in biliary epithelial damages were evident in the BDL model (Tsuneyama et al., 2002Go), in which antiestrogen treatment inhibited cholangiocyte proliferation and biliary fibrosis (Alvaro et al., 2000Go).

Hepatic fibrosis and cirrhosis are considered to result from direct injury to hepatocytes or bile ducts, with excess deposition of extracellular matrix (ECM) components that are produced by HSCs (Friedman, 2000Go). Upon activation, HSCs may undergo proliferation and differentiation, including a phenotypic shift to myofibroblastic-like cells that are positive for alpha-SMA (Friedman, 2000Go, 2003Go). Activation of HSCs is associated with oxidative stress and change of composition and organization of ECM (Eng and Friedman, 2000Go). Since EB treatment here enhanced lipid peroxidation in liver, this may have been the main cause of increase of alpha-SMA expression. It has been reported that STAP, recently renamed as cytoglobulin/STAP (Cygb/STAP) (Nakatani et al., 2004Go), is activated under fibrotic and cirrhotic conditions (Asahina et al., 2002Go; Kawada et al., 2001Go). Cygb/STAP is a cytoplasmic protein that is induced during the activation of HSCs which are fibulin-2 positive (Tateaki et al., 2004Go).

At the molecular level, estrogens can lead to the formation of DNA adducts (Shimomura et al., 1992Go) and have been shown to induce 8-hydroxylation of guanine bases in kidney and liver DNA in male Syrian hamsters (Han and Liehr, 1994Go) and 8-OHdG formation in a breast cancer cell line (Mobley and Brueggemeier, 2004Go). It has been reported that some synthetic estrogens have promoting properties (Yager and Liehr, 1996Go), and estrogen has been considered a genotoxic carcinogen (Cavalieri et al., 2000Go). However, in the present study EB treatment did not enhance 8-OHdG production in normal animals, so that this action could be dependent on a disease-specific background.

Liver lesions from groups 3 and 4 showed a similar pattern, and significant decrease of final body weight was found in groups 3 and 4 as compared to group 1. Treatment of high-dose EB is too toxic in TAA-treated animals, which may influence induction and severity of fibrosis and cirrhosis. However, there was also a decrease of final body weight in group 6 compared to group 5. Thus, it appears that body weight is not a major factor for enhancing cirrhosis by EB treatment.

It has been reported that estrogen treatment enhances ER levels in isolated hepatocytes (Vickers and Lucier, 1991Go), and endothelial and Kupffer cells (Vickers and Lucier, 1996Go). The ER level was significantly correlated with CuZn-SOD level and inversely proportional to MDA production (Shimizu et al., 2001Go). In our study, EB treatment induced ER alpha expression significantly in normal animals, but in TAA-treated animals there was a tendency for increase. Because surface receptor molecules that allow cells to respond to hormones and cytokines can be inactivated during lipid peroxidation (Halliwell and Gutteridge, 1999Go), there may be a retardation of estrogen signal transduction under certain conditions.


    ACKNOWLEDGMENTS
 
We would like to thank Miss Kaori Touma, Masayo Imanaka, and Shoko Araki for their technical assistance, and Mari Dokoh, Yuko Onishi, and Yoko Shimada for their help during preparation of this manuscript. This research was supported by a grant from the Ministry of Health, Labour and Welfare of Japan.


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