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A cross-kingdom assay model for evaluating estrogenic activity: application of transgenic Arabidopsis thaliana callus

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Abstract

Phytoestrogens have attracted attention in the last few decades as potential therapeutic candidates targeting several ailments including osteoporosis and menopausal syndrome. Many herbal preparations possess phytoestrogenic activity without solid evidence due the lack of an easy and feasible assay protocol in evaluating their activity. In 2005, the first report of a cross-kingdom bioassay using transgenic Arabidopsis assay system was published by our group to evaluate the estrogenic activity of potent natural products. In the current investigation, calli induced from the Arabidopsis pER8:GUS line were utilized for the first time to evaluate the estrogenic activity of 17β-estradiol. Calli, induced on solid 1DK1 medium, showed significant GUS activity in a dose-dependent manner, and the minimum detectable concentration of 17β-estradiol was 0.0001 µM. Furthermore, calli from liquid media ɩ1N1B and ɩ1/2 1N1B produced by suspension culture exhibited significant sensitivity towards 17β-estradiol in a dose-dependent manner. However, calli of ɩ1NK5, ɩ1/2 1NK5, ɩ1DK1, or ɩ1/2 1DK1 media could not show reliable activity toward GUS expression, which was demonstrated by the irregular fluorometric levels. Calli production avoided gene variation from the sexual generational shift and thus ensured uniformity and accuracy of the results. The optimal methods of transgenic calli induction, production, and estrogenic activity assay were established. This method can be used to investigate new sources of phytoestrogens and to detect environmental estrogenic compounds. This is the first developed protocol specialized in evaluating animal function using plant callus.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

4-MU:

4-Methylumbelliferone

4-MUG:

4-Methylumbelliferyl-β-D-glucuronide

6-BA:

6-Benzylaminopurine

E2 :

17β-Estradiol

ER:

Estrogen receptor

GUS:

β-Glucuronidase

Kn:

Kinetin

MS:

Murashige and Skoog medium

NAA:

α-Naphthalene acetic acid

X-Gluc:

5-Bromo-4-chloro-3-indolyl-β-D-glucuronic acid

References

  • Bae H, Sicher R, Natarajan S, Bailey B (2009) In situ expression of trehalose synthesizing genes, TPS1 and TPPB, in Arabidopsis thaliana using the GUS reporter gene. Plant Cell Tiss Organ Cult 98:311–319

    Article  CAS  Google Scholar 

  • Brand L, Hörler M, Nüesch E, Vassalli S, Barrell P, Yang W, Jefferson RA, Grossniklaus U, Curtis MD (2006) A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis. Plant Physiol 141:1194–1204

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chang FR, Hayashi KI, Chua NH, Kamio S, Huang ZY, Nozaki H, Wu YC (2005) The transgenic arabidopsis plant system, pER8-GFP, as a powerful tool in searching for natural product estrogen-agonists/antagonists. J Nat Prod 68:971–973

    Article  CAS  PubMed  Google Scholar 

  • Chlebowski RT, Manson JE, Anderson GL, Cauley JA, Aragaki AK, Stefanick ML, Lane DS, Johnson KC, Wactawski-Wende J, Chen C (2013) Estrogen plus progestin and breast cancer incidence and mortality in the Women’s Health Initiative Observational Study. J Natl Cancer Inst 105:526–535

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cos P, De Bruyne T, Apers S, Berghe DV, Pieters L, Vlietinck AJ (2003) Phytoestrogens: recent developments. Planta Med 69:589–599

    Article  CAS  PubMed  Google Scholar 

  • Dixon RA (2004) Phytoestrogens. Annu Rev Plant Biol 55:225–261

    Article  CAS  PubMed  Google Scholar 

  • Engelmann NJ, Reppert A, Yousef G, Rogers RB, Lila MA (2009) In vitro production of radiolabeled red clover (Trifolium pratense) isoflavones. Plant Cell Tiss Organ Cult 98:147–156

    Article  CAS  Google Scholar 

  • Hayashi K, Jones AM, Ogino K, Yamazoe A, Oono Y, Inoguchi M, Kondo H, Nozaki HJ (2003) Yokonolide B, a novel inhibitor of auxin action, blocks degradation of AUX/IAA factors. J Biol Chem 278:23797–23806

    Article  CAS  PubMed  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed Central  CAS  PubMed  Google Scholar 

  • Knasmüller S, Gottmann E, Steinkellner H, Fomin A, Pickl C, Paschke A, Göd R, Kundi M (1998) Detection of genotoxic effects of heavy metal contaminated soils with plant bioassays. Mutat Res, Genet Toxicol Environ Mutagen 420:37–48

    Article  Google Scholar 

  • Kovalchuk O, Telyuk P, Kovalchuk L, Kovalchuk I, Titov V (2003) Novel plant bioassays for monitoring the genotoxicity of drinking water from the inhabited areas of the Ukraine affected by the Chernobyl accident. Bull Environ Contam Toxicol 70:847–853

    Article  CAS  PubMed  Google Scholar 

  • Lai WC, Wang HC, Chen GY, Yang JC, Korinek M, Hsieh CJ, Nozaki H, Hayashi KI, Wu CC, Wu YC, Chang FR (2011) Using the pER8: GUS reporter system to screen for phytoestrogens from Caesalpinia sappan. J Nat Prod 74:1698–1706

    Article  CAS  PubMed  Google Scholar 

  • Legler J, Zeinstra LM, Schuitemaker F, Lanser PH, Bogerd J, Brouwer A, Vethaak AD, Voogt PD, Murk AT, Burg BV (2002) Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity. Environ Sci Technol 36:4410–4415

    Article  CAS  PubMed  Google Scholar 

  • Loyola-Vargas VM, Vázquez-Flota F (2006) Plant cell culture protocols. Springer, New York

    Google Scholar 

  • Monis PC, Altmann T (1994) Tissue culture and transformation. In: Meyerowitz EM, Somerville CR (eds) Arabidopsis. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 173–222

    Google Scholar 

  • Mueller SO (2002) Overview of in vitro tools to assess the estrogenic and antiestrogenic activity of phytoestrogens. J Chromatogr B 777:155–165

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Ochatt SJ, Sangwan RS (2008) In vitro shortening of generation time in Arabidopsis thaliana. Plant Cell Tiss Organ Cult 93:133–137

    Article  Google Scholar 

  • Orzechowska M, Stępień K, Kamińska T, Siwińska D (2013) Chromosome variations in regenerants of Arabidopsis thaliana derived from 2- and 6-week-old callus detected using flow cytometry and FISH analyses. Plant Cell Tiss Organ Cult 112:263–273

    Article  CAS  Google Scholar 

  • Padidam M (2003) Chemically regulated gene expression in plants. Curr Opin Plant Biol 6:169–177

    Article  CAS  PubMed  Google Scholar 

  • Pennacchio M, Jefferson LV, Havens K (2005) Arabidopsis thaliana: a new test species for phytotoxic bioassays. J Chem Ecol 31:1877–1885

    Article  CAS  PubMed  Google Scholar 

  • Prossnitz ER, Barton M (2011) The G-protein-coupled estrogen receptor GPER in health and disease. Nat Rev Endocrinol 7:715–726

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ribnicky DM, Ilic N, Cohen JD, Cooke TJ (1996) The effects of exogenous auxins on endogenous indole-3-acetic acid metabolism (the implications for carrot somatic embryogenesis). Plant Physiol 112:549–558

    PubMed Central  CAS  PubMed  Google Scholar 

  • Rogers JA, Metz L, Yong VW (2013) Review: endocrine disrupting chemicals and immune responses: a focus on bisphenol-A and its potential mechanisms. Mol Immunol 53:421–430

    Article  CAS  PubMed  Google Scholar 

  • Schug TT, Janesick A, Blumberg B, Heindel JJ (2011) Endocrine disrupting chemicals and disease susceptibility. J Steroid Biochem Mol Biol 127:204–215

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Summart J, Panichajakul S, Prathepha P, Thanonkeo P (2008) Callus induction and influence of culture condition and culture medium on growth of Thai aromatic rice, Khao Dawk Mali 105, cell culture. World Appl Sci J 5:246–251

    Google Scholar 

  • Van-Patten CL, Olivotto IA, Chambers GK, Gelmon KA, Hislop TG, Templeton E, Wattie A, Prior JC (2002) Effect of soy phytoestrogens on hot flashes in postmenopausal women with breast cancer: a randomized, controlled clinical trial. J Clin Oncol 20:1449–1455

    Article  CAS  PubMed  Google Scholar 

  • Washburn S, Burke GL, Morgan T, Anthony M (1999) Effect of soy protein supplementation on serum lipoproteins, blood pressure, and menopausal symptoms in perimenopausal women. Menopause 6:7–13

    Article  CAS  PubMed  Google Scholar 

  • Zacharewski T (1997) In vitro bioassays for assessing estrogenic substances. Environ Sci Technol 31:613–623

    Article  CAS  Google Scholar 

  • Zheng M, Konzak C (1999) Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.). Plant Cell Rep 19:69–73

    Article  CAS  Google Scholar 

  • Zuo J, Niu QW, Chua NH (2000) An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J 24:265–273

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Prof. Nam-Hai Chua (Laboratory of Plant Molecular Biology, The Rockefeller University, 1230, York Avenue, New York, NY 10065, USA) for the pER8:GFP seeds sharing. We also thank the Institute of the Plant Biology and the Zurich-Basel Plant Science Centre, University of Zurich, Switzerland, for providing seeds of pER8:GUS. We would like to acknowledge the grants from the Ministry of Science and Technology of Taiwan (MOST 101-2325-B-039-004; 102-2628-B-037-003-MY3; MOST 102-2911-I-002-303; MOST 103-2911-I-002-303; MOST 104-2911-I-002-302; MOST 104-2911-I-037-501). This work was also supported by the Excellence for Cancer Research Center Grant, the Ministry of Health and Welfare, Executive Yuan, Taipei, Taiwan (MOHW104-TDU-B-212-124-003), National Health Research Institutes of Taiwan (NHRI-EX104-10241BI), and the grant for Health and Welfare Surcharge of Tobacco Products and Kaohsiung Medical University “Aim for the Top Universities Grant”, KMU-TP103, and in part from the grant from Chinese Medicine Research Center, China Medical University, Taiwan (the Ministry of Education, the Aim for the Top University Plan).

Author contribution

Y.-C. Tsai and S.-Y. Chiang did the major experimental work. Y.-C. Tsai, W.-C Lai, and F.-R. Chang conceived the project and designed the experiments. K.-I. Hayashi introduced the technology of plant tissue culture to our group and provided the needed materials. Y.-C. Tsai, M. El-Shazly, Y.-C. Wu, and F.-R. Chang drafted and revised the manuscript. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, 100 Shih Chuan 1st Rd., Kaohsiung, 807, Taiwan

    Yu-Chi Tsai, Wan-Chun Lai, Mohamed El-Shazly, Shang-Yu Chiang, Yang-Chang Wu & Fang-Rong Chang

  2. Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street, Abassia, Cairo, 11566, Egypt

    Mohamed El-Shazly

  3. Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama, 700-0005, Japan

    Ken-Ichiro Hayashi

  4. Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung, 404, Taiwan

    Yang-Chang Wu

  5. School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, 404, Taiwan

    Yang-Chang Wu

  6. Center for Molecular Medicine, China Medical University Hospital, Taichung, 404, Taiwan

    Yang-Chang Wu

  7. Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, 807, Taiwan

    Fang-Rong Chang

  8. Research Center for Natural Product and New Drug, Kaohsiung Medical University, Kaohsiung, 807, Taiwan

    Fang-Rong Chang

  9. Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, 807, Taiwan

    Fang-Rong Chang

Authors
  1. Yu-Chi Tsai
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  2. Wan-Chun Lai
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  3. Mohamed El-Shazly
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  4. Shang-Yu Chiang
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  5. Ken-Ichiro Hayashi
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  6. Yang-Chang Wu
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  7. Fang-Rong Chang
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Corresponding author

Correspondence to Fang-Rong Chang.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Tsai, YC., Lai, WC., El-Shazly, M. et al. A cross-kingdom assay model for evaluating estrogenic activity: application of transgenic Arabidopsis thaliana callus. Plant Cell Tiss Organ Cult 123, 427–433 (2015). https://doi.org/10.1007/s11240-015-0833-0

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  • DOI: https://doi.org/10.1007/s11240-015-0833-0

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