Uterotropic Effects of Dietary Equol Administration in Ovariectomized Sprague-Dawley Rats

Written By: Sam Savage

Published Date: October 18, 2007 Last Edited: October 18, 2007

By Rachon, D Vortherms, T; Seidlova-Wuttke, D; Menche, A; Wuttke, W

Key words: UTERUS, MENOPAUSE, PHYTOESTROGENS, EQUOL, PROLIFERATING CELL NUCLEAR ANTIGEN, INSULIN-LIKE GROWTH FACTOR I, PROGESTERONE RECEPTOR, COMPLEMENT PROTEIN 3 ABSTRACT

Aim The aim of the present study was to evaluate the uterotropic effects of the administration of dietary equol, a metabolite of soy- derived daidzein or formononetin present in red clover, in an ovariectomized rat model of menopause.

Method Two doses of racemic equol were used (50 mg/kg of chow and 400 mg/kg of chow) and the results were compared with two doses of estradiol-3 benzoate (E2B) (4.3 mg/kg of chow and 17.3 mg/kg of chow). After 3 months, animals were sacrificed and the uteri were removed, weighed and paraffin-embedded for morphometrical and immunohistochemical evaluation. The expression of selected uterine estrogen-responsive genes was also measured using real-time reverse transcription-polymerase chain reaction.

Results Compared to controls, uterine weights in animals treated with high-dose equol were significantly higher, presented histologic features of mild estrogenic stimulation and had greater epithelial height and thickness of the uterine stroma and myometrium. Staining for the presence of the proliferating cell nuclear antigen (PCNA) also showed a greater prevalence of the PCNA-positive cells in the uterine stroma in animals treated with high-dose equol. Conversely, the percentage of PCNA-positive cells in the uterine epithelium was lower compared to the controls. Dietary high-dose equol treatment also increased significantly levels of uterine insulin-like growth factor 1, progesterone receptor and complement protein 3 mRNA. Although statistically significant, all these effects were, however, lower in magnitude compared to the effects of low- and high-dose E2B treatment. Low-dose equol did not have any effects on the above- studied parameters.

Conclusion Long-term high-dose dietary equol administration to ovariectomized rats exerts uterotropic effects at the cellular and molecular level which question the safety of uncontrolled and unlimited consumption of soy or red clover supplements by postmenopausal women with intact uteri.

INTRODUCTION

Soy (Glycine max) and red clover (Trifolium pratense) extracts are nowadays widely advertised as an effective treatment of climacteric complaints without the adverse side-effects attributed to estrogen use1’2. Yet the results of several recent clinical trials on the effectiveness of these preparations in ameliorating estrogen-deficiency symptoms in postmenopausal women have been inconsistent and question their safety3’4. Most of the uncertainties relate to the potential adverse effects of these preparations in estrogen-sensitive tissues. The uterus is one of the main estrogen targets and unopposed estrogen treatment in postmenopausal women induces endometrial hyperplasia and the risk of endometrial cancer5. Also, treatment with estrogens after menopause may increase the risk of uterine leiomyoma formation6. Results from the numerous studies investigating the effects of soy or red clover extracts on the human uterus have been largely inconclusive4’7-9. Recently, however, in a large randomized and placebo-controlled trial, a small but significant increase in the incidence of endometrial hyperplasia was observed in postmenopausal women receiving a daily dose of 150 mg purified isoflavones for a period of 5 years10.

Equol (7-hydroxy-3 – (4′-hydroxypheny 1 (-enroman) is the end- product of the biotransformation by the gastrointestinal tract microflora of the soyderived isoflavone daidzein or formononetin found in red clover. It is a chiral molecule existing as R and S enantiomeric forms11 and structurally similar to 17beta-estradiol (E2), having affinity for both estrogen receptors (ERa and ERjS)12- 14. This evidence has raised concern that uncontrolled and high- dose intake of soy and/or clover preparations after menopause may actually have adverse effects in estrogen-sensitive tissues. S- equol is formed in humans, but 30-50% of the adult population does not have the adequate gut flora for its production15.

The aim of the present study was to evaluate the uterotropic effects of dietary equol administration in an ovariectomized rat model of menopause. Two doses of equol were used and administered together with the rodent chow for a period of 3 months so as to mimic long-term use of soy- or red clover-containing supplements in postmenopausal women. The effects were evaluated on uterine weight, histology, staining for the presence of the proliferating cell nuclear antigen (PCNA) as well as the expression of three estrogen- responsive genes: insulin-like growth factor 1 (IGF-I), progesterone receptor (PR) and complement protein 3 (C3). All the obtained results were then compared with the effects of estradiol-3 benzoate (E2B).

MATERIALS AND METHODS

Animals

All experiments were performed in the year 2005 and approved by the Local Ethics Committee for Animal Care and Use at the Georg August University in Gottingen, according to the German animal welfare regulations, under permission given by the district authorities of Braunschweig, Germany (number 509.42502/01-36.03). Sixty virgin female Sprague-Dawley rats raised in the animal facility of the Gottingen University Clinic were used. Animals were kept in groups of six in Makrolon(R) cages (type 4) under a 12-h light, 12-h dark cycle, in a room temperature of 22-24[degrees]C and relative humidity 50-55%, with free access to water. In order to eliminate exposure to soyderived isoflavones found in regular rodent chow, they were fed with soy-free food (Ssniff Spezialdiaten GmbH, Soest, Germany). At the age of 3 months (mean body weight 244 g), the animals were bilaterally ovariectomized under ketamin (18.75 mg/ animal, Ketavet, Pharmacia & Upjohn, Erlangen, Germany) and xylazin (2.25 mg/animal, Rompun 2%, Bayer, Leverkusen, Germany) anesthesia. After ovariectomy, animals were randomized, placed in groups of six per cage and divided into four treatment groups (12 animals per group). The control group received soy-free food only. Low- and high- dose E2B groups received soy-free food with the addition of E2B (98% purity, Sigma-Aldrich Chemie GmbH, St. Louis, USA) at concentrations of 4.3 mg and 17.3 mg per kg of chow, respectively. The lowand high- dose equol groups received soy-free food with the addition of a racemic mixture of equol (98% purity, Changzhou Dahua Imp. & Exp. Group, China) at concentrations of 50 mg and 400 mg per kg of chow, respectively. The rodent chow was provided by Ssniff special diets GmbH (Soest, Germany) and was prepared by mixing the tested substances with the soy-free formulation Ssniff SM R/M (10 mm pellets) to homogeneity before the process of pelleting. Animals were fed ad libitum and, according to the records of food intake, the average consumption of the tested substances per animal throughout the whole experiment period was calculated. The average consumptions of E2B by the low- and high-dose groups were 0.07 mg and 0.20 mg per animal per day, respectively. The average consumptions of equol by the low- and high-dose groups were 0.92 mg and 6.54 mg per animal per day, respectively.

Serum and organ collection

After 3 months of treatment, animals were decapitated under CO2 anesthesia between 8:00 and 12:00. Blood was drained from the carcass into polypropylene tubes (Sarstedt, Nuembrecht, Germany) and kept at 4[degrees]C for 2-4 h. Serum was then obtained after centrifugation of the samples at 2500 rpm for 30 min at a temperature of 4[degrees]C. Samples were aliquoted in quadruplets into 1.5 ml polypropylene tubes (Eppendorf AG, Hamburg, Germany) and stored at – 20[degrees]C until further analyses. The abdominal cavity was opened with a longitudinal cut and the uteri were removed. Uterine horns were dissected free of adhering fat and mesentery. After weighing, one uterine horn was fixed in 10% buffered formalin for histological evaluation and immunohistochemistry studies. The contralateral horn was transferred into 2 ml polypropylene tubes (Sarstedt), frozen in liquid nitrogen, and stored at – 70[degrees]C for further analyses.

Uterine histology and immunohistochemistry

Formalin-fixed uterine horns were paraffinembedded, cut transversally into 5^m-thick slices and stained with hematoxylin- eosin. For morphological analyses, known E2-induced features were evaluated. Shape and polarity of the lamina propria cells, epithelial mitotic figures and necrosis, determination of hypertrophy and hyperplasia of glands and endometrial epithelium, and pathologic features like squamous metaplasia and pyometra were recorded. Also, the height of the endothelial cells (magnification 250 x ) and the thickness of the uterine stroma and myometrium (magnification 50 x ) were measured in ten animals from each group using a computer-based morphometry system (Soft Imaging System, Munster, Germany). Ten measurements were obtained in randomly selected areas in the middle transversal tissue cuts and a mean value was calculated for each animal. For immunohistochemistry studies, a monoclonal antibody for PCNA (PC-10, sc 56, Santa Cruz Biotechnology) was used after the antigen retrieval technique with microwave radiation in citrate buffer. The last immune reaction was performed with peroxidase and DAB (EnVision(TM)-DAB, Dako Cytomation, K5007). Ten representative high-power fields (? 400) were examined and 100 cells in each field were counted. The PCNA labeling index was determined as the number of cells with unequivocal nuclear staining, divided by the total number of cells counted (per 1000), expressed as a percentage. All the above- mentioned histological evaluations were performed blinded to treatment group. Serum 17beta-estradiol and equol measurements

Serum E2 levels were measured on a Roche Elecsys 2010 immunoassay analyzer using Estradiol II reagent kit (Roche Diagnostics GmbH, Mannheim). The lowest detection limit for this assay is 18.4 pmol/l and the mean intra- and intervariation coefficients are 2.7% and 3.4%, respectively. Serum equol levels were measured by high pressure liquid chromatography (HPLC) and ultraviolet detection at 260 nm. The HPLC conditions were gradient elution with 0 min 70% A 30% B, 15 min 25% A 75% B (A, water containing 0.085% 0-phosphoric acid; B, acetonitril; 250 x 4 mm C^sub 18^ reverse-phase column, flow rate 1 ml/min).

RNA extraction and reverse transcription

Before RNA extraction, frozen uteri were disrupted and homogenized in liquid nitrogen in precooled Teflon vessels, in the presence of stainless steel beads, by rapid agitation for 20 s at 2500 rpm using a Mikro-Dismembrator (B. Braun Biotech International GmbH, Melsungen, Germany). During the whole procedure, the samples were kept in liquid nitrogen or on dry ice. The tissue powder was then transferred back into the polypropylene tubes and kept at – 70[degrees]C. For RNA isolation, 30 mg of the tissue homogenate was used and the extraction process was carried out with the RNeasy Total RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The integrity of the isolated total RNA was checked by using the Agilent 2100 bioanalyzer and RNA 6000 LabChip kit (Agilent Technologies, Waldbronn, Germany) and its concentration was measured with an ultraviolet spectrophotometer (Biophotometer, Eppendorf, Hamburg, Germany) at wave lengths of 260 nm and 280 nm. Afterwards, each sample was diluted with RNase-free water to achieve a final concentration of 20 ng/[mu]l. The reverse transcription reaction was carried out in a total volume of 20 [mu]l containing 1 x reaction buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl^sub 2^, 50 mM dithiothreitol) (Promega, Madison, USA), 100 ng random hexamer primers (Invitrogen, Carlsbad, CA, USA), 0.5 mM dNTPs (Invitrogen), 200 U M-MMLV reverse transcriptase (Promega, Madison, USA), 4 U RNasin and 200 ng of total RNA (Promega, Madison, USA). Samples were incubated for 10 min at 22[degrees]C to allow primer annealing; reverse transcription was at 42[degrees]C for 50 min. At the end of incubation, the samples were heated at 95[degrees]C for 10 min to inactivate the enzyme and denature RNA-cDNA hybrids. The whole reaction was run in a Trio-Thermoblock (Biometra, Gottingen, Germany).

Real-time polymerase chain reaction

Real-time polymerase chain reactions were based on the 5′- nuclease assay16 which was run on an ABI Prism 7700 sequence detection system (TaqMan, PE Applied Biosystems, Foster City, CA, USA). Primer and probe sequences for C3, IGF-1 and progesterone receptors (PR) as well as the concentrations used have been described previously17. They were all purchased from Eurogentec (Seraing, Belgium). Real-time PCR reactions were run on 96-well microtiter plates (MicroAmp Optical 96-Well reaction Plate, PE Applied Biosystems, Weiterstadt, Germany) in a 25 [mu]l volume containing 1 x TaqMan Universal PCR Master Mix (PE Applied Biosystems), adequate primers and hybridization probe concentrations, and 2 [mu]l cDNA. Subsequently, samples were amplified over 40 cycles. Each cycle consisted of a denaturation phase of 15 s at 95[degrees]C and a hybridization/elongation phase of 1 min at 60 [degrees]C.

Statistical analyses

All data are presented as arithmetic mean +- standard deviation. Relative changes of mRNA levels were analyzed in the real-time PCR experiments. The mean value of the absolute data measured in the control group was set at 100% and all other values determined in the respective assays were expressed in relation to this average value. One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons was performed to compare the differences between the studied groups, p values

RESULTS

Effects on serum 17beta-estradiol and equol concentrations

As expected, animals fed with low- and high-dose E2B- supplemented chow had significantly higher mean serum E2 levels compared to controls (133 +- 17 pmol/l and 312 +- 61 pmol/l vs. 85 +- 7 pmol/l, p 0.05, respectively). Mean serum equol concentrations in animals fed with equol-containing chow at low and high dose were 33 +- 49 nmol/ l and 1095 +- 564 nmol/l, respectively. Equol was undetectable in serum of the controls and E2B-treated animals.

Effects on uterine weight

As shown in Table 1, uterine weights in low-dose equol-treated animals did not differ from those in the controls. In contrast, animals fed with highdose equol had significantly higher uterine weights in comparison with control animals (p

Effects on uterine tissue morphology

Histological analysis of the uteri from animals treated with low- dose equol did not show any features of estrogenic stimulation and they were similar to those of the control group. The endometrium was composed of low cuboidal epithelium lining the uterine lumen and uterine stroma was composed of unresponsive stromal cells. In contrast, in six out of 12 animals from the highdose equol-treated group, the endometrial epithelium was composed of tall columnar cells which presented increased mitotic activity and some differentiation in the stroma. Animals treated with dietary E2B at low and high doses showed typical features of estrogenic stimulation. The endometrium was composed of tall, pseudostratified columnar cells with high mitotic activity. In the high-dose E2B- treated group, signs of squamous metaplasia were also present. Uterine stroma was composed of well-differentiated, responsive stromal cells. In one animal treated with the low E2B dose and three animals treated with the high E2B dose, eosinophilic and neutrophilic infiltration of the endometrium was seen. Morphometric analyses showed that, compared to controls, animals treated with high-dose dietary equol had greater endometrial epithelium height (10.3 +- 1.3 pm vs. 9.0 +- 0.7 pm, p

Table 1 Effects of 3-month dietary equol and estradiol-3 benzoate (E2B) treatment on body and uterine weight in ovariectomized Sprague- Dawley rats. Data are presented as mean +- standard deviation

Also, compared to the control group, the immunohistochemical staining of the uteri for the presence of PCNA expression showed a greater prevalence of PCNA-positive cells in the uterine stroma of animals treated with high-dose equol (3.4 +- 0.3% vs. 2.4 +- 0.4%, p

Effects on uterine IGF-1, PR and C3 gene expression

Uterine expression of IGF-1, progesterone receptor and C3 is up- regulated through ERa-mediated pathways and can serve as a sensitive marker of ligand-dependent estrogen receptor activation within the uterus19. Therefore, in the last step of our experiment, we evaluated the effects of dietary equol administration on the uterine expression of the above-mentioned genes using the quantitative real- time PCR method. The obtained results were then compared with the effects of E2B. Compared to controls, uterine IGF-1 mRNA levels were significantly higher in high-dose equol-treated animals (194 +- 75% vs. 100 +- 27%, p

DISCUSSION

The aim of the present study was to investigate the uterotropic effects of equol in an ovariectomized rat model of menopause. Our results clearly demonstrate that long-term equol consumption at a relatively high dose has apparent uterotropic effects in ovariectomized Sprague-Dawley rats. Apart from the increase in uterine weight, signs of estrogenic stimulation were noted at the cellular and molecular level. Compared to controls, uteri from animals fed with high-dose equol exhibited characteristic features of estrogenic stimulation, such as an increase in endometrial epithelium height and thickness of the uterine stroma and myometrium. Although statistically significant, these changes, however, were lower in magnitude compared to the effects of E2B given at low and high doses. PCNA, a 36 kDa nuclear protein, is expressed in proliferating cells during the S phase of the cell cycle and immunostaining for its presence has been shown to be of practical value in assessing cell proliferation20 as well as a useful tool in cancer prognosis research21’22. The PCNA immunostaining method has been also shown to correlate with other measures of cell proliferation, such as thymidine labeling index and S-phase fraction determined by flow cytometry23,24. Therefore, in our study we also evaluated the number of PCNA-positive cells in the endometrial epithelial cells and the uterine stroma. Both stromal and epithelial cells of the endometrium proliferate rapidly during estrogen exposure, and estrogen withdrawal results in their apoptosis25’26. Therefore, our result, that high-dose equol or E2B treatment increased the number of PCNA-positive cells in the stroma, was not surprising. In contrast, the finding that long-term dietary treatment with high-dose equol and low- or high-dose E2B decreased the percentage of PCNA-positive cells in the endometrial epithelium was rather unexpected. In intact, normally cycling rats, the expression of PCNA in the luminal epithelium increases from diestrus to proestrus, when estrogen levels are high. However, during estrus, when estrogen levels decrease, apoptosis occurs markedly and the expression of PCNA disappears27. Also, it has been shown that short- term (2 weeks) subcutaneous estrogen treatment increases PCNA expression in the luminal and glandular epithelium of ovariectomized rats27. In our experiment, although high-dose equol and low- or high- dose E2B treatment increased the height of the luminal epithelium, the expression of the PCNA was decreased compared to the control animals. An explanation for this contradictory finding may be that longterm estrogen exposure leads to the down-regulation of the PCNA expression in the endometrial epithelial cells. In a study of Diel and colleagues 28, it has been demonstrated that uterine PCNA mRNA levels increased after 7 h following oral daidzein administration but were already decreased after 24 h to levels of ovariectomized controls. This assumption, however, warrants further studies.

Figure 2 The prevalence of PCNA-positive cells in uterine stroma and epithelium in ovariectomized rats treated for 3 months with dietary equol and estradiol-3 benzoate (E2B). (a) Animals treated with high-dose dietary equol had a higher percentage of PCNA- positive cells in the uterine stroma compared to the controls. This effect was also apparent in animals treated with low- and high-dose E2B. (b) Conversely, high-dose equol as well as low- and high-dose E2B treatment decreased the percentage of PCNA-positive cells in the uterine epithelium. *p

Uterotropic effects of estrogens are due to the activation of the uterine IGF-1 expression which, in turn, stimulates the proliferation of uterine tissues (myometrium and endometrium)29’30. In addition, exposure of the uterus to estrogens dramatically increases uterine expression of PR and C319,31, effects which are primarily mediated by ERalpha32 and are regarded as a more estrogensensitive parameter than the increase in uterine weight19,33. Therefore, in our experiment, we also measured uterine mRNA levels of IGF-1, PR and C3. High-dose equol treatment significantly increased uterine expression of all the three abovementioned genes. Although statistically significant, all these effects were, however, 2-fold, 3-fold and over 60-fold lower for IGF- 1, PR and C3 gene expression, respectively, compared with the effects of E2B. To our knowledge, this is the first report on the effects of dietary equol on uterine IGF-1, PR and C3 gene expression in ovariectomized rats. Nevertheless, the soy-derived equol precursor daidzein has already been shown to notably up-regulate uterine C3 expression in the rat uterus19 and in a rat endometrial adenocarcinoma cell line34. Endometrial PR gene expression has also been shown to be up-regulated in ovariectomized cynomolgus monkeys fed soy isoflavones (genistein and daidzein) for 28-33 days35. Yet, the role of ERalpha-mediated increase of C3 gene expression in the uterus during exposure to estrogens or estrogen agonists is unknown. C3 is the key protein taking part in the complement activation cascade, which is an important component of the innate immunity against bacterial infections. Therefore, we may only speculate that its increased expression during estrogen exposure, as in proestrus for example or during pregnancy, may protect the uterus and the urogenital tract from bacterial infections, thereby improving the chances of conception and pregnancy. Also, ERalpha-mediated up- regulation of uterine PR expression during estrogen exposure allows the endometrium to become more responsive to progesterone action, thereby facilitating the implantation of a fertilized egg and successful pregnancy36. IGF-1, in turn, plays a role in mediating the mitogenic effects of estrogens in the uterus30, leading to its increased mass and growth. In vitro studies, however, have shown that IGF-1 may also stimulate the proliferation of endometrial cancer cells37 as well as promote leiomyoma cell growth38 , which explains the high correlation between long-term estrogen exposure and the prevalence of these gynecological disorders in older women5’39. Therefore, uterine IGF-1 expression may affect several different aspects of uterine physiology as well as pathology.

Figure 3 Effects of dietary equol and estradiol-3 benzoate (E2) on uterine IGF-1, progesterone receptor (PR) and C3 gene expression, (a) Animals treated with high-dose dietary equol had significantly higher uterine IGF-1 mRNA levels compared to controls. Also, treatment with high-dose dietary equol significantly increased uterine PR mRNA (b) as well as C3 levels (c). These effects were, however, smaller in magnitude compared to the effects of low- and high-dose E2B treatment. *p

An additional finding in our experiment was that animals treated with low- and high-dose E2B had significantly lower body weight compared to controls. This was, however, not surprising, as data from our previous experiments and the results of others have clearly shown that estrogen treatment attenuates ovariectomy-induced body weight in rats18’40. This effect, however, did not have any confounding effect on the other parameters examined in this study.

To our knowledge, this is the first report on the uterotropic effects of dietary equol administration to ovariectomized Sprague- Dawley rats. Nevertheless, in an elegant study of Selvaraj and colleagues, daily subcutaneous injections of equol to ovariectomized C57BL/6 mice for 12 days also increased uterine weight and epithelial proliferation measured by the Ki-67 labeling index. Oral equol administration induced similar trends but, in the case of uterine weight gain, they did not reach statistical significance41. Parenteral equol administration has also been shown to be uterotropic in sheep42 and immature rats43. In contrast, Wood and colleagues did not observe any significant uterotropic effects of dietary equol administration to ovariectomized cynomolgus monkeys35. The authors note, however, that, in contrast to rodents, humans and monkeys produce significant levels of adrenal androgens which can be converted into estrone and further to 17beta-estradiol (‘estrogen background’), making their estrogen target tissues less sensitive to the estrogenic actions of equol35.

In view of its relevance to human physiology, the doses of equol used in our experiment also deserve discussion. While traditional soy-based Asian diets provide around 16 mg daidzein per day44, the commercially available and ‘over the counter’ soy preparations used to alleviate ‘hot flushes’ by postmenopausal women usually contain doses ranging from 50 mg to 70 mg per tablet45-47. Anecdotal evidence also points out that some women consume doses as high as 150 mg of pure daidzein per day, which would be equivalent to an average of 2.5 mg per kg body weight. Although one cannot predict how much of that amount can be transformed into pure equol by the gut flora in equol producers, pharmacologists claim that doses of any tested substance applied to rats must be 10-15-fold higher than in humans in order to exert equipotent effects48’49. Also, in an attempt to balance the fact that a racemic mixture of equol and not its S form, which is considered to be the more active isomer50, was used, the doses should be doubled. According to the calculations of mean food intake, daily average consumption of equol in the ‘high- dose’ group was 21.7 mgJkg body weight. Hence, the doses used in the present study may well be of importance for postmenopausal women’s health. In conclusion, long-term high-dose dietary equol administration to ovariectomized rats exerts uterotropic effects at the cellular and molecular level; this result questions the safety of uncontrolled and unlimited consumption of soy or red clover supplements by postmenopausal women with intact uteri.

Conflict of interest Nil.

Source of funding This work was funded by the European Commission Grants: EURISKED (contract no. EVKl -CT2002-00 128) and CASCADE (contract no. FOOD-CT-2004-506319).

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D. Rachon*,[dagger] T. Vortherms*, D. Seidlova-Wuttke*, A. Menche* and W. Wuttke*

*Department of Clinical and Experimental Endocrinology, University of Gottingen, Gottingen,

Germany; [dagger] Department of Immunology, Medical University of Gdansk, Gdansk, Poland

Correspondence: Dr D. Rachon, Department of Immunology, Medical University of Gdansk, ul. Debinki 1 , 80-2 1 0 Gdansk Poland

Received 29-12-06

Revised 1 2-03-07

Accepted 19-03-07

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