Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD.

Endometriosis is a debilitating disease found in 10-15% of reproductive-age women and is characterized by the presence of endometrial tissue outside of the uterus. The present study characterizes the expression of AhR and ARNT mRNA in a human endometrial explant culture model in the absence and presence of TCDD exposure. In a parallel, companion study using this model, TCDD exposure was shown to induce CYP1A1 mRNA, CYP1B1 mRNA, EROD (7-ethoxyresorufin-O-deethylase) activity, and CYP1B1 protein in human endometrial explants. Explants were prepared from specimens obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. These specimens were a subset of the specimens used in the parallel study. The explants were cultured in medium containing 10 nM estradiol (E(2)) or 1 nM estradiol plus 500 nM progesterone (E(2) + P(4)) with or without TCDD (first 24 h). After culture, AhR and ARNT mRNA expression were quantified by RT-PCR. TCDD treatment significantly increased the expression of AhR mRNA, but not ARNT mRNA. The expression of both genes was similar for all individual explants and the ratio of AhR:ARNT mRNA expression across all samples was 1.7 to 1.8. Constitutive AhR mRNA expression was donor age dependent (increasing with age), while ARNT mRNA expression was donor age and tissue phase dependent (increased in older and proliferative phase specimens). Similar to results in the parallel study on expression of CYP1A1 mRNA, CYP1B1 mRNA, EROD activity, and CYP1B1 protein, the presence of endometriosis did not affect the expression of AhR or ARNT mRNA, either constitutively or following TCDD exposure. However, the detection of disease-specific change was limited by small sample size and variability in tissue cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase, hormonal exposure, and donor age.

Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium.

Endometriosis is a debilitating disease estimated to affect 10% of reproductive-age women and characterized by the growth of endometrial tissue outside of the uterus. The present study characterizes a human endometrial explant culture model for studying the direct effects of TCDD exposure by assessing the expression of CYP1A1 and CYP1B1 mRNA (Northern blotting), protein (Western blotting), and activity (7-ethoxyresorufin-O-deethylase; EROD) in explants cultured with and without TCDD. Explants were obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. The explants were cultured with 10 nM estradiol (E(2)) or 1 nM E(2) plus 500 nM progesterone (P(4)) with or without TCDD (first 24 h). The expression of CYP1A1 and CYP1B1 mRNA was greatest with 10 nM TCDD and increased up to 72 h after initial exposure. EROD activity increased up to 120 h. Explants from a secretory phase biopsy became reorganized in culture and formed a new epithelial membrane, while maintaining basic endometrial morphology and viability for up to 120 h. At 24 h, TCDD significantly increased CYP1A1 and CYP1B1 mRNA, and at 72 h, TCDD significantly increased EROD activity and CYP1B1 protein compared to explants cultured without TCDD for similar times. CYP1B1 protein also exhibited substantial constitutive expression that was similar in uncultured biopsies, where CYP1B1 protein was immunolocalized in the cytoplasm of epithelial glands, with only occasional patches of protein in the surface epithelial membrane. In explants cultured with and without TCDD exposure, CYP1B1 protein was localized in the cytoplasm of the new surface epithelial membrane and glands closest to the surface. CYP1A1 protein was not detected in uncultured biopsies or explants. Both younger age (age 30 and under) and proliferative phase were associated with higher TCDD-induced EROD activity in specimens treated with E(2):P(4). No significant endometriosis-related differences were observed for any of the biomarkers, but the detection of disease-specific change was limited by small sample size and variability in tissue-cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase and hormonal exposure.