A Novel Nonradioactive Method for Measuring Aromatase Activity Using a Human Ovarian Granulosa-Like Tumor Cell Line and an Estrone ELISA

Ken Ohno*, Naohiro Araki*, Toshihiko Yanase, Hajime Nawata{dagger} and Mitsuru Iida*,1

* EcoScreen R & D Section, Endocrine Disrupting Chemical Analysis Center, Otsuka Life Science Initiative, Otsuka Pharmaceutical Co., Ltd. 224-18 Ebisuno Hiraishi, Kawauchi-cho, Tokushima, 771-0195, Japan; and {dagger} Department of Medicine and Bioregulatory Science (Third Department of Internal Medicine), Graduate School of Medicine Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

Received July 30, 2004; accepted September 7, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Aromatase is a key enzyme in steroidogenesis and plays an important role in sexual differentiation, fertility, and carcinogenesis. Importantly, a variety of chemicals in the environment may influence its activity and thereby disrupt endocrine function. In the current studies, we developed a novel nonradioactive method for measuring aromatase activity that uses a specific ELISA for estrone along with KGN human ovary granulosa-like carcinoma cells. This cell line has relatively high aromatase activity, and because it lacks 17{alpha}-hydroxylase, it secretes little or no androstenedione, 17ß-estradiol, or estrone. Therefore, aromatase activity can be assayed simply by measuring the production of estrone in the culture medium after addition of the substrate, androstenedione. Furthermore, by making a slight change in the commercial ELISA kit and optimizing the experimental conditions, we developed a sensitive aromatase assay that could measure a wide range of estrone concentrations with very low interference by androgens. We used this assay to investigate the effects of 23 chemicals that have been previously reported to affect aromatase activity in vitro. We confirmed that 17 of 23 test chemicals had inhibitory or inducible effects, although the specific effects of some were different than previously reported. In conclusion, we have developed a simple, sensitive, and nonradioactive assay that can be used for large-scale screening of compounds that can disrupt endocrine function by influencing aromatase activity.

Key Words: aromatase; endocrine disrupter; screening assay; KGN cell line; benomyl.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
A variety of environmental contaminants and chemicals used in commercial products are suspected endocrine disrupters that may lead to abnormalities in sexual differentiation, reproductive capacity, growth, and development (McLachlan, 2001Go). Current research has mainly focused on the interactions of these chemicals with sex hormone receptors, such as the estrogen and androgen receptors (Kelce et al., 1997Go; Lambright et al., 2000Go). However, these chemicals may also disrupt biological function by other mechanisms, such as altering hormone biosynthetic pathways.

Aromatase is a key enzyme in the conversion of androgens to estrogens and has an important role in maintaining a homeostatic balance between them. Some flavonoid chemicals, for example, {alpha}-naphthoflavone, apigenin, and chrysin, are known to inhibit aromatase activity in vitro (Campbell and Kurzer, 1993Go; Jeong et al., 1999Go; Kellis and Vickery, 1984Go; Le Bail et al., 1998Go; Pelissero et al., 1996Go). In addition, the herbicide atrazine has recently been shown to induce aromatase activity, and various imidazole-like fungicides have been shown to be aromatase inhibitors (Sanderson et al., 2002Go). Also, the biocides triphenyltin (TPT) and tributyltin (TBT), which are used in antifouling paints and wood preservatives, are suspected to inhibit aromatase activity and cause imposex in gastropods (Heidrich et al., 2001Go; Saitoh et al., 2001Go).

Two kinds of in vitro assay have been developed to measure aromatase activity: a cell-free assay using human placental microsomes (Njar et al., 1995Go; Vinggaard et al., 2000Go) or human recombinant aromatase protein; and a cell-based assay using mammalian cell lines, such as the human JEG-3 (Drenth et al., 1998Go; Yue and Brodie, 1997Go) and JAr (Brueggemeier et al., 1997Go) cell lines. In either case, aromatase activity is determined by measuring the amount of 3H-water released upon enzymatic conversion of radiolabeled androstenedione. However, these assays require the use of radioactive materials and specialized equipment for radiometric measurement. An alternative fluorescent cell-free method has recently been developed using human recombinant aromatase protein (Stresser et al., 2000Go), but this method cannot detect aromatase induction because it utilizes a cell-free system.

In the current study, we developed a novel nonradioactive cell-based assay that can detect both inhibition and induction of aromatase. This assay was performed using KGN cells, which are a steroidogenic human ovarian granulosa-like tumor cell line that was established from a patient with invasive granulosa cell carcinoma (Nishi et al., 2001Go). This cell line possesses normal properties of granulosa-like cells, including relatively high aromatase activity that is stimulated by follicle stimulating hormone or cAMP. In addition, the KGN cells cannot synthesize androgen or estrogen by themselves due to the absence or low level of 17{alpha}-hydroxylase. Therefore, the aromatase activity can be evaluated simply by culturing the cells with androstenedione and measuring the estrone level in the culture medium with a specific enzyme-linked immunosorbent assay (ELISA). This novel assay should be useful for the high-throughput screening of chemicals for aromatase inhibition or induction.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Cell culture. KGN cells were grown in Dulbecco's modified Eagle medium/Ham's F-12 nutrient mix (DMEM/F-12) medium (Invitrogen, Carlsbad, CA) supplemented with 5% (v/v) fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS), 50 U/ml penicillin, and 50 µg/ml streptomycin in 175-cm2 cell culture flasks (Nalge Nunc International, Rochestar, NY) at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged every 2 to 3 days when they reached confluence by treating them for 4 min with 0.05% trypsin and then replating them at (1.5 to 2.5) x 106 cells/flask.

Chemical preparations. Test chemicals (Table 1) and androstenedione (Sigma, St. Louis, MO) were dissolved in DMSO at a concentration of 10 mM and stored at –20°C. The maximum concentration of each chemical used in the experiments was 10 µM, 1 µM, or 100 nM, depending on its solubility in the cell culture medium. Then the test chemicals and the substrate were diluted with serum-free DMEM/F-12 medium by 50 and 100 folds, respectively. And there were six dose points (by 10 folds diluted from maximum concentrations) tested in this study. The final concentration of DMSO in the cell growth medium was 0.15% (v/v), and we confirmed that 0.15% DMSO had no statistically significant effect on the estrone production in KGN cells by Student's t-test (p = 0.65, n = 8). All treatments were tested in triplicate, except for the controls, which were performed in sextuplicate.


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TABLE 1 Summary of Tested Chemicals and Effects on Aromatase Activity

 
Aromatase assay. A 180-µl volume of 5 x 104 KGN cells/ml was added to each well of a 96-well tissue culture plate. Cells were grown at 37°C in DMEM/F-12 medium containing 5% (v/v) charcoal/dextran-treated FBS (Hyclone, Logan, UT). After 2 days, 10 µl of test chemicals were added, and cells were incubated for 24 h. Solvent control wells were dosed with media plus 0.15% DMSO. Next, 10 µl of 0.1 µM androstenedione was added to each well, and cells were incubated for another 24 h. A 120-µl sample of the culture medium was removed from each well and transferred to a second 96-well tissue culture plate. The estrone concentration in each of these wells was measured using an estrone ELISA kit (Otsuka Pharmaceuticals Co., Ltd., Tokushima, Japan), and cell viability was determined using the cells remaining in the original 96-well plate. In some cases, plates were stored at –20°C prior to measurement of estrone levels.

Estrone ELISA. The estrone ELISA was carried out as described in the manufacturer's instructions, except that 3,3',5,5'-tetramethylbenzidine solution and Stop Buffer (Scytech, Logan, UT) was used instead of the o-phenylenediamine and the stop buffer provided in the kit. The absorbance in each well was measured at 450 nm using an ARVOsx 1420 multilabel counter (Wallac, Turku, Finland), and the estrone concentration in each well was calculated based on a standard curve and using SOFTmax Pro 4.0 (Molecular Devices, Sunnyvale, CA).

Measurement of cell viability. The cytotoxicity of the various test chemicals was assessed using AlamarBlue assay (Ahmed et al., 1994Go). An 8 µl volume of the AlamarBlue reagent (Serotec Ltd., Oxford, UK) was added to the wells, and cells were incubated for 3 to 4 h at 37°C according to the manufacture's protocol. The fluorescence was measured at 590 nm with excitation at 544 nm using the ARVOsx 1420 microplate reader. The cytotoxicity was determined by comparing the fluorescence in each well with the fluorescence of solvent control wells (0.15% DMSO).

Data and statistical analysis. Effects on aromatase activity and cell toxicity were expressed as a relative ratio of estrone concentrations at each dosing point divided by the estrone concentration in solvent controls. If there was more than 50% inhibition of aromatase activity, IC50 values were calculated by GraphPad PRISM ver 4.0 (GraphPad, San Diego, CA) using sigmoidal dose-response curve fitting (variable slope). Data were analyzed by one-way factorial ANOVA using SatView for Windows (SAS Institute Inc., Cary, NC).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Cross-Reactivity of the Estrone ELISA Kit
We first investigated the specificity of the commercially available estrone ELISA kit (Otsuka Pharmaceuticals) by examining its cross-reactivity with 17ß-estradiol, estriol, testosterone, progesterone, and androstenedione (Table 2). We found less than 0.005% cross-reactivity with the aromatase substrates androstenedione and testosterone. In contrast, there was 30% cross-reactivity with estradiol and 0.4% cross-reactivity with estriol. Finally, a representative estrone standard curve using the ELISA kit is shown in Figure 1. Collectively, these results confirmed that the ELISA kit can be used to determine the estrone level in the culture medium without interference by aromatase substrates.


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TABLE 2 Cross Activity of Estrone ELISA (Otsuka)

 


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FIG. 1. Example of a standard curve for the estrone ELISA. Data from eight estrone concentrations (0, 1.2, 4.8, 12.5, 78.1, 312.5, 1250, and 5000 pg/ml) are shown.

 
Optimization of the Assay Conditions
To optimize the sensitivity of the test system, we examined the relationship between the substrate concentration and estrone production. Two days after plating the cells, 10 µl of the serum-free medium was added in cell culture medium instead of samples. After 24 h, various concentrations of androstenedione were then added to the wells, and the estrone concentration in the culture medium was measured after 1, 2, 3, 6, 12, and 24 h using the estrone ELISA (Fig. 2). With the exception of 100 pM, there was a linear increase in estrone concentration with time at all androstenedione concentrations. In addition, the estrone level reached a 24-h maximum between 100 and 120 pg/ml at androstenedione concentrations above 10 nM. To be able to detect the effects of a competitive inhibitor, the concentration of substrate should be near the Km. Therefore, we chose 5 nM androstenedione as the substrate concentration for the analysis of test chemicals because it gave a reaction rate approximately half of the maximal velocity.



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FIG. 2. Effect of substrate concentration on estrone production. KGN cells were grown in 96-well plates for 48 h. Next, 10 µl of serum-free medium was added in cell culture medium instead of samples. After 24 h, various concentrations (10 µM, 1 µM, 100 nM, 10 nM, 5 nM, 1 nM, or 100 pM) of androstenedione were added in triplicate. After an additional 24 h, the estrone concentration in the medium of each well was assayed by estrone ELISA, and the average values were plotted.

 
Other test conditions were optimized, such as cell density, preincubation time, and incubation time with substrate, and effects of plate position were minimized (data not shown). The final conditions included seeding the KGN cells at 9 x 103 cells per well, 2 days growth following plating, 24-h incubation with substrate, and exclusion of wells on the edge of the plate.

Effects of Test Chemicals on Aromatase and Cell Viability
Using the optimized assay, we tested the effect of 23 chemicals that had previously been reported to affect aromatase activity. Most of these were selected from a draft of a detailed review paper on aromatase by the U. S. Environmental Protection Agency (2002)Go. Included in the 23 chemicals were flavonoids, a pesticide, pharmaceutical compounds, and organotin compounds. We first tested the eight flavonoid chemicals for effects on aromatase activity and cell viability (Fig. 3). All flavonoids were found to inhibit aromatase activity at relatively high concentrations, and flavone and flavanone were very weak inhibitors. These chemicals did not have cell toxicity on KGN cells, excepting for apigenin, which had some cell toxicity at maximum concentration. Next, we investigated the effects of 10 pesticides, pharmaceutical compounds, and organotins (Fig. 4). With the exception of o,p-DDT (o,p-dichloro-1, 1-diphenyl-2,2,2-trichloroethane), all of the chemicals inhibited the aromatase activity. The organotins also decreased aromatase activities, but they were very cytotoxic. These inhibitory effects on aromatase activity by organotins seemed to be affected by these cell toxicities. Finally, we tested five chemicals that are suspected to induce aromatase activity (Fig. 5). We found that benomyl and forskolin induced aromatase activity, but the other three, atrazine, diclobutrazole, and vinclozolin, had no inducible effects. A summary of these results and the related chemical information is shown in Table 1. The table also shows the mean IC50 values (n = 3) calculated for compounds that inhibited aromatase activity as well as any previously reported IC50 values. Overall, the IC50 values increased the following order: 4-hydroxyandrostenedione (4-OHA) < imazalil << {alpha}-naphthoflavone < propiconazole < 7-methoxyflavone < chrysin < fenarimol < aminoglutethimide < naringenin < apigenin < triadimefon << 7-hydroxyflavone < p,p'-DDD (p,p'-dichlorodiphenyldichloroethane).



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FIG. 3. Effects of flavonoid compounds on aromatase activity and cell viability. The bar graph represents the cell viability as measured by AlamarBlue reagent. The line graph represents the aromatase activity as evaluated by measurement of estrone concentration with ELISA. Values were calculated as the ratio of the measured activity versus control, and the plots show one set of triplicate measurements. The error bars show the SD from the triplicate determinations. IC50s shown are the means of three investigations ± SD. Asterisks denote statistical significance compared to respective control: **p < 0.001, ***p < 0.0001.

 


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FIG. 4. Effect of various classes of suspected aromatase inhibitors on aromatase activity and cell viability. Experiments were carried out and data are presented as in Fig. 3.

 


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FIG. 5. Effect of suspected aromatase inducers on aromatase activity and cell viability. Experiments were carried out and data are presented as in Fig. 3.

 
Reproducibility of this Aromatase Assay
The interassay reproducibility of the system was evaluated by calculation of coefficients of variation (CV), where CV = 100% x SD/mean. The overall average CV for aromatase activity in the chemical tests was 10.94% (n = 161), and for cell viability, the overall average CV was 6.01% (n = 161). Intra-assay reproducibility in the aromatase assay was determined by comparing the IC50s calculated for each of the triplicate dose-response curves for 4-OHA and imazalil, which were chosen because their inhibitory effects covered a wide concentration range. The average calculated IC50 value for 4-OHA was 1.15 ± 0.28 x 10–9 M (CV = 24.7%), and for imazalil, 4.44 ± 1.28 x 10–9 M (CV = 28.9%).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
In this study, we developed a novel nonradioactive cell-based assay that can measure both inhibition and induction of aromatase by chemical compounds. This was made possible by the use of KGN cells in combination with an estrone ELISA. The ELISA kit used in this study had a low cross-reactivity toward substrate compounds, and only 17ß-estradiol, which is not an aromatase substrate, showed significant (30%) cross-reactivity. If there is a relatively high 17ß-hydroxysteroid dehydrogenase (17ß-HSD) activity in KGN cells, most of the estrone would be converted to 17ß-estradiol, which could interfere with quantitative measurement of aromatase activity. Nishi et al. (2001)Go reported that estrone (E1) and 17ß-estradiol (E2) were produced in a ratio of approximately 1:1.7 when KGN cells were incubated with 10 µM androstenedione, a concentration 2000-fold higher than we used in this study. However, using an ELISA for 17ß-estradiol, we previously found that incubation of KGN cells with E1 for 24 h resulted in only 10% or less conversion to E2 (data not shown). Therefore, the activity of 17ß-HSD in KGN cells may not be enough to interfere with in the measurement of estrone. However, both of these experiments were used the immunoassay technique; a more careful consideration was required for cross-reactivity to various steroids. Further studies using HPLC or LC/MS/MS may be necessary to confirm this problem, but in general, the assay appears to reflect actual aromatase activity.

Our testing of potential endocrine disrupters showed that the eight flavonoids, which are weak inhibitors of aromatase in vitro (Ibrahim and Abul-Hajj, 1990Go; Kao et al., 1998Go; Kellis et al., 1984Go; Le Bail et al., 2001Go; Saarinen et al., 2001Go), also inhibited aromatase activity in our cell-based assay. In addition, flavone and flavanone, which are weaker aromatase inhibitors than other flavonoids, could be detected using our assay, indicating that the method has high sensitivity. However, IC50 values were not calculated for these two compounds because the highest concentrations tested produced less than 50% inhibition.

Among agricultural chemicals, pesticides, and fungicides suspected to be inhibitors of aromatase, fenarimol, triadimefon, imazalil, propiconazole, and p,p'-DDD had inhibitory effects in our assay, whereas o,p'-DDT had little or no effect at the concentration range we tested (100 pM–10 µM). On the contrary, o,p'-DDT was reported to inhibit aromatase activity at 10 µM in H295R cells (Sanderson et al., 2002Go). Although the reason for this discrepancy is unclear, it may be due to the difference in cell type or origin. We also found that the pharmaceutical compounds 4-OHA (also called formestane) and imazalil strongly inhibited the aromatase activity. In fact, the IC50 of imazalil was lower in our assay than in previous reports (Sanderson et al., 2002Go). Sanderson et al. (2002)Go investigated imazalil at concentration from 0.1 µM to 1 mM, and their IC50 value of imazalil was 0.1 µM in H295R cell. Our estimated IC50 value of imazalil was about 20-folds lower than that of H295R results (Table 1).

Organotin compounds were very toxic to the KGN cells. In fact, both TBT (tributyltin chloride) and TPT (triphenyltin chloride) had previously been reported to have strong cell toxicity (Saitoh et al., 2001Go). They showed TBT and TPT had inhibitory effect on aromatase activity in KGN cells with no cell toxicity at concentration lower than 20 ng/ml. In this study, decrease of the cell viability was also observed in TBT and TPT (Fig. 4). In TBT, the reduction of aromatase activity was also observed, however the cell viability was described in parallel. Therefore we considered reduction of aromatase activity in TBT mostly depended on its cell toxicity. TPT also had cell toxicity at concentration of 1000 ng/ml; however aromatase activity at 200 ng/ml was clearly decreased without cytotoxic effect. TPT could inhibit aromatase activity, but that range was very limited. We have used 24 h for preincubation of chemicals with KGN cells and an additional 24-h incubation for aromatase reaction with 96-well plate; meanwhile Saitoh's have 48 h and 6 h incubation with petri dish, respectively. Cell culture methods and exposure time with KGN cells and/or enzyme reaction time may have contributed to the difference about organotin compound between two studies. Although Bettin et al. (1996)Go concluded that the induction of imposex in gastropods by organotin compounds may be due to inhibition of aromatase (Bettin et al., 1996Go), our results as well as those of Sanderson et al. (2000) and Morcillo and Porte (1999)Go suggest that the induction of imposex in gastropods by TBT or TPT occurs via mechanisms other than inhibition of aromatase activity.

In our investigation of aromatase inducers, we found that forskolin, a known inducer of cAMP, strongly enhanced aromatase activity. This is not surprising, because cAMP promotes aromatase gene expression by binding to a cAMP response element upstream of the aromatase gene. At 10 mM, benomyl, one of fungicides, induces aromatase activity twofold, which is consistent with previously reported results in KGN cells (Morinaga et al., 2004Go). They further reported that benomyl and its metabolite carbendazim induce aromatase activity through stimulation of CYP19 (aromatase) expression. The mechanism was unclear, although it was confirmed to not be through elevation of intracellular cAMP. This suggests that long-term exposure of wildlife and humans to chemicals like benomyl might lead to estrogen-mediated pathologies, such as tumor promotion, inappropriate sexual differentiation, and inappropriate feminization. Therefore, it is urgent to further investigate the physiological effects of this and similar compounds.

In contrast to forskolin and benomyl, there was no enhancement of aromatase by atrazine, o,p'-DDT, diclobutrazole, or vinclozolin, even though they have been reported to increase aromatase activity in H295R cells (Sanderson et al., 2002Go). Particularly, atrazine has been reported to cause over twofold induction on aromatase activity at 30 µM in H295R cells (Heneweer et al., 2004Go; Sanderson et al., 2002Go). However we could not observe such inducible effects at 5 µM or 50 µM in KGN cells. Because the H295R cell line is derived from a human adrenocortical carcinoma cell, the observed discrepancy may be due to the different origins of the cells. This could also be due to alternative transcriptional mechanisms; for example, the promoter sites in the aromatase (CYP19) gene are different in various tissues and are regulated by cellular conditions or signaling pathways (Bulun et al., 2004Go; Simpson, 2004Go). In fact, one study reported that atrazine induces aromatase activity in H295R but not rat R2C (Heneweer et al., 2004Go). Furthermore, another report using KGN cells and a radioisotope-based aromatase assay showed no enhancement of activity by 10 µM of atrazine (Morinaga et al., 2004Go). Also, although atrazine has been reported to enhance cAMP levels in H295R cells (Sanderson et al., 2002Go), it had no effect on aromatase in KGN cells despite the fact that forskolin promoted aromatase in our assay. Although the reason for this is unclear, again, it could be due to alternative promoter site usage. Considering these results, if the objective is to screen endocrine disruptors, it would be better to use cells from the reproductive tissue origin.

In summary, in the current studies we introduced a novel method for measuring aromatase activity. Whether KGN cells are the most suitable for aromatase screening remains to be determined, but we expect that this assay will be very useful for the screening of large numbers of samples because the assay is simple, nonradioactive, and adaptable to high-throughput screening. Therefore, it should be possible to perform large-scale screening for identification of chemicals that can affect aromatase activity and thereby lead to abnormal steroidogenesis.


    SUPPLEMENTARY DATA
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
Supplementary Data is available online.


    ACKNOWLEDGMENTS
 
We thank Dr. K. Kitamura (Yanaihara Institute), Dr. H. Morinaga (Kyushu University), and Dr. T. Kato. This work was supported by the Japanese Ministry of Economy, Trade, and Industry as part of the Millennium Project. We also thank Ms. E. Nishikawa and K. Yamada for their skillful technical assistance.

The KGN cells are available from the Riken Cell Bank as the stock number RCB No.1154 (http://www.brc.riken.go.jp/).


    NOTES
 

1 To whom correspondence should be addressed. Fax: 81-88-665-3613. E-mail: iidam{at}hq.otsuka.co.jp.


    REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 REFERENCES
 
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