Toxicological Research

Korean Society of Toxicology & Korea Environmental Mutagen Society (한국독성학회)
권호 표지

Screening Assay for Identification of Endocrine Disruptors with Androgen Activities using LNCaP Cells

LNCaP 세포주를 이용한 내분비계장애물질중 안드로겐성 확인시험을 위한 검색법

  • 김진호 (식품의약품안전청 국립독성연구소) ;
  • 정혜주 (식품의약품안전청 국립독성연구소) ;
  • 김영옥 (식품의약품안전청 국립독성연구소) ;
  • 정승태 (식품의약품안전청 국립독성연구소) ;
  • 박재현 (식품의약품안전청 국립독성연구소) ;
  • 조대현 (식품의약품안전청 국립독성연구소) ;
  • 김동섭 (식품의약품안전청 국립독성연구소)
  • Published : 2002.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Substantial evidences have been accumulated about the hormone-like effects of exogenous substances such as pesticides and industrial chemicals during past years. The effects of these substances on the endocrine system are believed to be either enhancing or reducing of various endocrine action. It is necessary to identify putative causal agents by the batter system and to assess their ability to disrupt the endocrine system. A variety of in vitro and In vivo approaches have been used to determine the androgenic effects of environmental chemicals. To establish the method for assessment of the putative endocrine disruptors with androgenic activity, we carried out the cell proliferation assay by MTS method after treatment with the various concentration of testosterone in LNCaP cells (human prostatic cancer cell line) and also observed the expression of androgen-related genes by quantitative RT-PCR. In the cell proliferation assay, the results showed that the grouth of LNCaP cells increased within level of at least 10pM testosterone. We measured by quantitative RT-PCR method on the effects of testosterone on mRNA expression of androgen receptor (AR), prostate-specific antigen (PSA), bone morphogenetic protein (BMP) and BMP receptor (BMPR) In LNCaP cells. The results demonstrated that mRNA expression of PSA and BMPR-IB was observed differently within level of at least 0.01 pM testosterone compared with non-treated control. These observations suggest that the detection of PSA and BMPR-IB mRNA by the quantitative RT-PCR in LNCaP cells is very sensitive method to identify the endocrine disruptors to have the androgenic effects.

Keywords

References

  1. J. Appl. Toxicol. v.20 The peripubertal male rat assay as an alternative to the Hershberger castrated male rat assay for the detection of antiandrogens, oestrogens and metabolic modulators Ashby, J.;Lefevre, P.A. https://doi.org/10.1002/(SICI)1099-1263(200001/02)20:1<35::AID-JAT633>3.0.CO;2-8
  2. Regul. Toxicol. Pharmacol. v.26 The weanling male rat as an assay for endocrine disruption: preliminary observations Ashby J.;Lefevre P.A. https://doi.org/10.1006/rtph.1997.1177
  3. Can. J. Physiol. Pharmacol. v.74 Assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures using in vitro recombinant receptor/reporter gene assays Balaguer, P.;Joyeux, A.;Denison, M.;Vincent, R.;Gillesby, B.;Zacharewski, T. https://doi.org/10.1139/cjpp-74-2-216
  4. B.M.J. v.309 no.6946 Decline in sperm counts: an artefact of changed reference range of "normal?" Bromwich P.;Cohen J.;Stewart I.;Walker A. https://doi.org/10.1136/bmj.309.6946.19
  5. Urol. Res. v.16 no.5 Effects of Cimetidine, Progesterone, Cannitracin and Tolazoline on the weight and DNA content of the testosterone-induced hyperplastic prostate of the rat Chen, W.;Zhou, X.M.;Chen, D.Y.;Kang, J.S.
  6. Environ. Health Perspect v.101 Developmental effects of endocrine-disrupting chemicals in wildlife and humans Colborn, T.;vom Saal, F.S.;Soto, A.M. https://doi.org/10.2307/3431890
  7. Endocrinology v.137 no.12 Activin Inhibition of prostate cancer cell growth : selective actions on androgen-responsive LNCap cells Dalkin A.C.;Gilrain J.T.;Bradshaw D.;Myers C.E. https://doi.org/10.1210/en.137.12.5230
  8. The Prostate v.29 Androgen independence of primary epithelial culture of the prostate is associated with a down-regulation of androgen receptor gene expression Grant, E.S.;Batchelor, K.W.;Habib, F.K. https://doi.org/10.1002/(SICI)1097-0045(199612)29:6<339::AID-PROS1>3.0.CO;2-3
  9. Toxicol. Lett. v.102-103 Tiered screening and testing strategy for xenoestrogens and antiandrogens Gray, L.E. Jr. https://doi.org/10.1016/S0378-4274(98)00287-2
  10. Annu. Rev. Pharmacol. Toxicol. v.22 Comparative toxicology of chlorodecone (epone) in humans and experimental animals Guzelian P.S. https://doi.org/10.1146/annurev.pa.22.040182.000513
  11. Clin. Chim. Acta. v.132 A versatile method for the determination of serum cortisol globulin binding capacities Hammond, G.L.;Lahteenmaki, P.L.A. https://doi.org/10.1016/0009-8981(83)90237-1
  12. J. Biol. Chem. v.268 no.36 A complex response element in intron 1 of the androgen-regulated 20-kDa protein gene displays cell type-dependent androgen receptor specificity Ho K.C.;Marschke K.B.;Tan J.;Power S.G.;Wilson E.M.;French F.S.
  13. Cancer Res. v.57 Growth regulation of human prostate cancer cells by bone morphogenetic protein-21 Ide H.;Yoshida T.;Matsumoto N.;Aoki K.;Osada Y.;Sugimura T.;Terada M.
  14. Reprod. Fertil. Dev. v.10 no.1 Antiandrogens as environmental endocrine disruptors Kelce, W.R.;Gray, L.E.;Wilson, E.M. https://doi.org/10.1071/R98051
  15. vivo confirmation of an androgen receptor-mediated mechanism;Toxicol. Appl. Pharmacol. v.142 no.1 Vinclozolin and p,p'DDE alter androgen-dependent gene expression Kelce W.R.;Lambright C.R.;Gray L.E Jr.;Roberts K.P.
  16. LNCaP. Endocrinology v.137 no.3 Transforming growth factor-betal is a mediator of androgen-regulated growth arrest in an androgen-responsive prostatic cancer cell line Kim, I.Y.;Kim, J.H.;Zelner, D.J.;Ahn, H.J.;Sensibar, J.A.;Lee, C. https://doi.org/10.1210/en.137.3.991
  17. Toxicol. Sci. v.42 no.1 Subchronic toxicity of Di(2-ethylhexyl)phthalate in common marmosets: lack of hepatic peroxisome proliferation, testicular atrophy, or pancreatic acinar cell hyperplasia Kurata, Y.;Kidachi, F.;Yokoyama, M.;Toyota, N.;Tsuchitani, M.;Katoh, M.
  18. LNCap;nude mice;The Prostate v.226 Growth of an androgen-sensitive human prostate cancer cell line Lim D.J.;Liu X.;Sutkowski D.M.;Braun E.J.;Lee C.;Koslowski J.M.
  19. Toxicol. Sci. v.43 no.1 Male reproductive tract malformations in rats following gestational and lactational exposure to Di(n-butyl)phthalate: an antiandrogenic mechanism? Mylchreest, E.;Cattley, R.C.;Foster, P.M. https://doi.org/10.1093/toxsci/43.1.47
  20. Fertil. Steril. v.63 no.4 Have sperm counts been reduced 50 percent in 50 years? A statistical model revisited Olsen G.W.;Bodner K.M.;Ramlow J.M.;Ross C.E.;Lipshultz L.I.
  21. Toxicol. Sci. v.54 Evaluation of a Tier I screening battery for detecting endocrine-Active compounds (EACs) using the positive controls testosterone, coumestrol, progesterone, and RU 486 Connor, J.C.;Davis, L.G.;Frame S.r.;Cook J.C.;Ross, C.E.;Lipshultz, L.I. https://doi.org/10.1093/toxsci/54.2.338
  22. Regul. Toxicol. Pharmacol. v.26 Human exposure to endocrine-active chemicals:hazard assessment problems Safe, S.;Connor, K.;Ramamoorthy, K.;Gaido, K.;Maness, S. https://doi.org/10.1006/rtph.1997.1118
  23. J. Steroid Biochem. Molec. Biol. v.65 An updated review of environmental estrogen and androgen mimics and antagonists Sonnenschein, C.;Soto, A.M. https://doi.org/10.1016/S0960-0760(98)00027-2
  24. Cancer. Res. v.49 Negative controls of cell proliferation : human prostate cancer cells and androgens Sonnenxchein C.;Olea N.;Pasanen M.E.;Soto A.M.
  25. T. A. M. A. v.140 Occupational oligospermia Sunger, O.L. https://doi.org/10.1001/jama.1949.02900500057039
  26. J. Biol. Chem. v.267 no.11 Response elements of the androgen-regulated C3 gene Tan, J.;Marschke, K.B.;Ho, K.C.;Perry, S.T.;Wilson, E.M.;French, F.S.
  27. J. Biol. Chem. v.25;270 no.34 Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide Wong, C.;Kelce, W.R.;Sar, M.;Wilson E.M.
  28. Environ. Health. Perspect. v.106 no.sup.2 Identification and assessment of endocrine disruptors ; limitations of in vivo and in vitro assays Zacharewski, T.
  29. Mol. Endocrinol. v.9 no.2 Specificity of ligand-dependent androgen receptor stabilization: receptor domain interactions influence ligand dissociation and receptor stability Zhou Z.X.;Lane M.V.;Kemppainen J.A.;French F.S.;Wilson E.M. https://doi.org/10.1210/me.9.2.208