Archives of Toxicology

, Volume 78, Issue 6, pp 309–315 | Cite as

Role of the aryl hydrocarbon receptor and Cyp1b1 in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin

  • Kei Takemoto
  • Miki Nakajima
  • Yuto Fujiki
  • Miki Katoh
  • Frank J. Gonzalez
  • Tsuyoshi Yokoi
Molecular Toxicology


The role of aryl hydrocarbon receptor (AhR) and cytochrome P450 (Cyp) 1 family in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in vivo. Immature (21 days old) AhR, Cyp1a2, or Cyp1b1 knockout (−/−) mice were treated intraperitoneally with estradiol (E2, 20 ng/mouse per day, for 14 consecutive days) and/or TCDD (200 ng/mouse per day, on days 7, 9, 11, and 13). Uterine wet weight and uterine peroxidase activity (UPA) were measured as markers of estrogen responsiveness. UPA was a better marker of estrogen responsiveness than the uterine wet weight. In AhR wild-type (+/+) mice, UPA (208.1±81.6 units/g tissue) was increased by the administration of E2 (to 297.2±178.7 units/g). The administration of TCDD significantly (p<0.01) decreased the UPA (10.5±3.4 units/g) compared with that in the control mice. Co-administration of TCDD with E2 also significantly (p<0.05) decreased the UPA (18.8±19.9 units/g) compared with that in E2-treated mice. In AhR(−/−) mice, UPA (162.9±146.7 units/g) was significantly (p<0.01) increased by the administration of E2 (486.8±108.2 units/g). In contrast to the results in AhR(+/+) mice, UPA was not affected by the administration of TCDD (51.8±70.6 units/g) compared with control, and co-administration of TCDD with E2 (545.8±189.4 units/g) compared with that in E2-treated mice. In Cyp1a2/1b1(+/+) mice, UPA was significantly (p<0.05) increased by the administration of E2 (70.0±36.4 units/g). Co-administration of TCDD with E2 significantly (p<0.05) decreased the UPA (29.6±22.2 units/g) compared with that in E2-treated mice. In Cyp1a2(−/−) mice, co-administration of TCDD with E2 significantly (p<0.01) decreased the UPA (6.8±5.1 units/g) compared with that in E2-treated mice. In Cyp1b1(−/−) mice, UPA (5.5±8.1 units/g) was significantly (p<0.05) increased by the administration of E2 (56.6±34.1 units/g). In contrast to the results in Cyp1a2/1b1(+/+) mice or Cyp1a2(−/−) mice, UPA was not affected by the co-administration of TCDD and E2 (52.6±30.1 units/g) compared with that in E2-treated mice. This is the first demonstration that Cyp1b1 as well as AhR is involved in the antiestrogenic effects of TCDD.


Cyp1b1 Aryl hydrocaron receptor Estrogen receptor 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Antiestrogenic activity 



We thank Mr. Brent Bell for reviewing the manuscript. The experiments in the present study complied with the current laws in Japan.


  1. Abbott BD, Schmid JE, Pitt JA, Buckalew AR, Wood CR, Hel, GA, Diliberto JJ (1999) Adverse reproductive outcomes in the transgenic Ah receptor-deficient mouse. Toxicol Appl Pharmacol 155:62–70CrossRefPubMedGoogle Scholar
  2. Aoyama T, Korzekwa K, Nagata K, Gillette J, Gelboin HV, Gonzalez FJ (1990) Estradiol metabolism by cDNA expressed human cytochrome P450s. Endocrinology 126:3101–3106PubMedGoogle Scholar
  3. Astroff B, Eldridge B, Safe S (1991) Inhibition of the 17β-estradiol-induced and constitutive expression of the cellular protooncogene c-fos by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the female rat uterus. Toxicol Lett 56:305–315CrossRefPubMedGoogle Scholar
  4. Bradford A (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  5. Brodie A (1991) Aromatase and its inhibitors—an overview. J Steroid Biochem Mol Biol 40:255–261CrossRefPubMedGoogle Scholar
  6. Buchanan DL, Sato T, Peterson RE, Cooke PS (2000) Antiestrogenic effects of 2,3,7,8-tetrachlordibenzo-p-dioxin in mouse uterus: critical role of the aryl hydrocarbon receptor in stromal tissue. Toxicol Sci 57:302–311CrossRefPubMedGoogle Scholar
  7. Bunyagidj C, MaLachlan JA (1988) Catechol estrogen formation in mouse uterus. J Steroid Biochem 31:795–801CrossRefPubMedGoogle Scholar
  8. Buters JT, Sakai S, Richter T, Pineau, T, Alexander DL, Savas U, Doehmer J, Ward JM, Jefcoate CR, Gonzalez FJ (1999) Cytochrome P450 CYP1B1 determines susceptibility to 7,12-dimethylbenz[a]anthracene-induced lymphomas. Proc Natl Acad Sci USA 96:1977–1982CrossRefPubMedGoogle Scholar
  9. Dannan GA, Porubek DJ, Nelson SD, Waxman DJ, Guengerich FP (1986)17β-Estradiol 2- and 4-hydroxylation catalyzed by rat hepatic cytochrome P-450: roles of individual forms, inductive effects, developmental patterns, and alterations by gonadectomy and hormone replacement. Endocrinology 118:1952–1960PubMedGoogle Scholar
  10. Fernandez-Salguero P, Pineau T, Hilbert DM, McPhail T, Lee SS, Kimura S, Nebert DW, Rudikoff S, Ward JM, Gonzalez FJ (1995) Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268:722–726PubMedGoogle Scholar
  11. Fishman J (1976) The cathechol estrogens. Neuroendocrinology 22:363–374PubMedGoogle Scholar
  12. Gale SK, Sclafani A (1977) Comparison of ovarian and hypothalamic obesity syndromes in the female rat: effects of diet palatability on food intake and body weight. J Comp Physiol Psychol 91:381–392PubMedGoogle Scholar
  13. Gallo MA, Hesse EJ, Macdonald GJ, Umbreit TH (1986) Interactive effects of estradiol and 2,3,7,8-tetrachlordibenzo-p-dioxin on hepatic cytochrome P-450 and mouse uterus. Toxicol Lett 32:123–132CrossRefPubMedGoogle Scholar
  14. Gierthy JF, Lincoln DW, Gillespie MB, Seeger JI, Martinez HL, Dickerman HW, Kumar SA (1987) Suppression of estrogen-related extracellular tissue plasminogen activator activity of MCF-7 cells by 2,3,7,8-tetrachlordibenzo-p-dioxin. Cancer Res 47:6198–6203PubMedGoogle Scholar
  15. Hankinson O (1995) The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol 35:307–340Google Scholar
  16. Hayes CL, Spink DC, Spink BC, Cao JQ, Walker NJ, Sutter TR (1996) 17β-Estradiol hydroxylation catalyzed by human cytochrome P4501B1. Proc Natl Acad Sci USA 93:9776–9781PubMedGoogle Scholar
  17. Jellinck PH, Newcombe A, Keeping HS (1979) Peroxidase as a marker enzyme in estrogen-responsive tissues. Adv Enzyme Regul 17:325–331CrossRefGoogle Scholar
  18. Kharat I, Saatcioglu F (1996) Antiestrogenic effects of 2,3,7,8-tetrachlordibenzo-p-dioxin are mediated by direct transcriptional interference with the liganded estrogen receptor. Cross-talk between aryl hydrocarbon- and estrogen-mediated signaling. J Biol Chem 271:10533–10537CrossRefPubMedGoogle Scholar
  19. Krishnan V, Porter W, Santostefano M, Wang X, Safe S (1995) Molecular mechanism of inhibition of estrogen-induced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. Mol Cell Biol 15:6710–6719PubMedGoogle Scholar
  20. Lee AJ, Cai MX, Thomas PE, Conney AH, Zhu BT (2003) Characterization of the oxidative metabolites of 17β-estradiol and estrone formed by 15 selectively expressed human cytochrome P450 isoforms. Endocrinology 144:3382–3398CrossRefPubMedGoogle Scholar
  21. Lyttle CR, DeSombre ER (1977a) Generality of oestrogen stimulation of peroxidase activity in growth responsive tissues. Nature 268:337–339PubMedGoogle Scholar
  22. Lyttle CR, DeSombre ER (1977b) Uterine peroxidase as a marker for estrogen action. Proc Natl Acad Sci USA 74:3162–3166PubMedGoogle Scholar
  23. Nutter LM, Wu YY, Ngo EO, Sierra EE, Gutierrez PL, Abul-Hajj YJ (1994) An o-quinone form of estrogen produces free radicals in human breast cancer cells: correlation with DNA damage. Chem Res Toxicol 7:23–38PubMedGoogle Scholar
  24. Ohtake F, Takeyama K, Matsumono T, Kitagawa H, Yamamoto Y, Nohara K, Tohyama C, Krust A, Mimura J, Chambon P, Yanagisawa J, Fujii-Kuriyama Y, Kato S (2003) Modulation of oestrogen receptor signaling by association with the activated dioxin receptor. Nature 423:545–550CrossRefPubMedGoogle Scholar
  25. Pineau T, Fernandez-Salguero P, Lee SS, McPhail T, Ward JM, Gonzalez FJ (1995) Neonatal lethality associated with respiratory distress in mice lacking cytochrome P450 1A2. Proc Natl Acad Sci USA 92:5134–5138PubMedGoogle Scholar
  26. Poland A, Knutson JC (1987) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22:517–554CrossRefGoogle Scholar
  27. Safe SH (1986) Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu Rev Pharmacol Toxicol 26:371–399Google Scholar
  28. Safe SH (1995) Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlordibenzo-p-dioxin and related compounds. Pharmacol Ther 67:247–281PubMedGoogle Scholar
  29. Safe S, Krishnan V (1995) Chlorinated hydrocarbons: estrogens and antiestrogens. Toxicol Lett 82–83:731–736Google Scholar
  30. Safe S, Astroff B, Harris M, Zacharewski T, Dickerson R, Romkes M, Biegel L (1991) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds as antiestrogens: characterization and mechanism of action. Pharmacol Toxicol 69:400–409PubMedGoogle Scholar
  31. Savas U, Christou M, Jefcoate CR (1993) Mouse endometrium stromal cells express a polycyclic aromatic hydrocarbon-inducible cytochrome P450 that closely resembles the novel P450 in mouse embryo fibroblasts (P450EF). Carcinogenesis 14:2013–2018PubMedGoogle Scholar
  32. Savas U, Bhattacharyya KK, Christou M, Alexander DL, Jefcoate CR (1994) Mouse cytochrome P-450EF, representative of a new 1B subfamily of cytochrome P-450s. Cloning, sequence determination, and tissue expression. J Biol Chem 269:14905–14911PubMedGoogle Scholar
  33. Sesardic D, Pasanen M, Pelkonen O, Boobis AR (1990) Differential expression and regulation of the cytochrome P450IA gene subfamily in human tissues. Carcinogenesis 11:1183–1188PubMedGoogle Scholar
  34. Shimada T, Inoue K, Suzuki Y, Kawai T, Azuma E, Nakajima T, Shindo M, Kurose K, Sugie A, Yamagishi Y, Fujii-Kuriyama Y, Hashimoto M (2002) Arylhydrocarbon receptor-dependent induction of liver and lung cytochrome P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6 J mice. Carcinogenesis 23:1199–1207CrossRefPubMedGoogle Scholar
  35. Simpson E, Rubin G, Clyne C, Robertson K, O’Donnell L, Davis S, Jones M (1999) Local estrogen biosynthesis in males and females. Endocr Relat Cancer 6:131–137PubMedGoogle Scholar
  36. Umbreit TH, Hesse EJ, Macdonald GJ, Gallo MA (1988) Effects of TCDD–estradiol interactions in three strains of mice. Toxicol Lett 40:1–9CrossRefPubMedGoogle Scholar
  37. White TE, Gasiewics TA (1993) The human estrogen receptor structural gene contains a DNA sequence that binds activated mouse and human Ah receptors: a possible mechanism of estrogen receptor regulation by 2,3,7,8-tetrachlordibenzo-p-dioxin. Biochem Biophys Res Commun 193:956–962CrossRefPubMedGoogle Scholar
  38. Wormke M, Stoner M, Saville B, Safe S (2000) Crosstalk between estrogen receptor α and the aryl hydrocarbon receptor in breast cancer cells involves unidirectional activation of proteasomes. FEBS Lett 478:109–112CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Kei Takemoto
    • 1
  • Miki Nakajima
    • 1
  • Yuto Fujiki
    • 1
  • Miki Katoh
    • 1
  • Frank J. Gonzalez
    • 2
  • Tsuyoshi Yokoi
    • 1
  1. 1.Division of Drug Metabolism, Faculty of Pharmaceutical SciencesKanazawa UniversityKanazawa Japan
  2. 2.National Cancer InstituteBethesdaUSA

Personalised recommendations