Assessing anti-estrogenic effects of AHR ligands in primary human and rat endometrial epithelial cells.

Unopposed estrogenic action in the uterus can lead to the development of endometrial cancer in both humans and rats. Aryl hydrocarbon receptor (AHR) activation gives rise to anti-estrogenic actions and may consequently reduce the development of endometrial cancer. In this study, the anti-estrogenic potential of the AHR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and DELAQ, a metabolite of the pharmaceutical laquinimod, was assessed in in primary human and rat endometrial epithelial cells (EECs) with and without co-exposure to endogenous hormones. In human EECs, estradiol and progesterone did not affect AHR gene expression, but in rat EECs, progesterone decreased Ahre xpression (1.4-fold). In accordance, AHR-mediated induction of Cyp1a1/1b1 expression by DELAQ and TCDD decreased in hormone-treated rat EECs. DELAQ was 22-fold more potent than TCDD in human EECs in inducing CYP1A1/1B1 gene expression, while DELAQ was approximately 16-33-fold less potent than TCDD in rat EECs. In human EECs, 10 nM DELAQ decreased estradiol-induced expression of growth-regulated estrogen receptor binding 1 (GREB1) by 1.8-fold. In rat EECs, both DELAQ and TCDD did not affect the expression of estradiol-induced genes. This study shows that AHR ligand DELAQ, but not TCDD, causes anti-estrogenic effects in primary human EECs. Furthermore, although AHR-mediated CYP1A1/1B1/Cyp1a1/1b1 induction by DELAQ and TCDD was stronger in rat EECs than human EECs, this did not result in apparent anti-estrogenic effects in the rat cells. This study shows that primary human and rat endometrial cells respond differently towards hormones and AHR ligands. This should be considered in human risk assessment based on rodent studies.

Primary endometrial 3D co-cultures: A comparison between human and rat endometrium.

Human and rat reproductive systems differ significantly with respect to hormonal cyclicity and endometrial cell behavior. However, species-differences in endometrial cell responses upon hormonal stimulation and exposure to potentially toxic compounds are poorly characterized. In this study, human and rat endometrial hormonal responses were assessed in vitro using a 3D co-culture model of primary human and rat endometrial cells. The models were exposed to the aryl hydrocarbon receptor (AHR) ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), laquinimod, and its AHR active metabolite DELAQ. In both the human and rat endometrial models, estrogen receptor and progesterone receptor gene expression was modulated by the hormonal treatments, comparable to the in vivo situation. AHR gene expression in the human endometrial model did not change when exposed to hormones. In contrast, AHR expression decreased 2-fold in the rat model when exposed to predominantly progesterone, which resulted in a 2.8-fold attenuation of gene expression induction of cytochrome P450 1A1 (CYP1A1) by TCDD. TCDD and DELAQ, but not laquinimod, concentration-dependently induced CYP1A1 gene expression in both human and rat endometrial models. Interestingly, the relative degree of DELAQ to induce CYP1A1 was higher than that of TCDD in the human model, while it was lower in the rat model. These data clearly show species-differences in response to hormones and AHR ligands between human and rat endometrial cells in vitro, which might greatly affect the applicability of the rat as translational model for human endometrial effects. This warrants further development of human relevant, endometrium-specific test methods for risk assessment purposes.

GH3 and RC-4BC cell lines are not suitable as in vitro models to study prolactin modulation and AHR responsiveness in rat pituitary.

Some environmental contaminants and pharmaceuticals increase the incidence of uterine tumors in toxicological studies with rats. These tumors can result from a hormonal imbalance due to rat-specific disrupted pituitary prolactin regulation, and are therefore of questionable relevance for humans. In this study we compared in vitro prolactin regulation in rat primary pituitary cells to that in pituitary cell lines, GH3 and RC-4BC. Moreover, we assessed the potential effects of aryl hydrocarbon receptor (AHR) activation on prolactin regulation by using two different AHR agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and DELAQ, the N-deethylated minor metabolite of the pharmaceutical laquinimod. In rat primary pituitary cells, known prolactin stimulant thyrotropin-releasing hormone (TRH) marginally increased prolactin secretion (1.2-fold) and gene expression (1.3-fold). In contrast, synthetic dopamine receptor agonist quinpirole, a known inhibitor of prolactin release, significantly inhibited prolactin secretion (2.6-fold) and gene expression (3.6-fold). In GH3 cells, TRH strongly increased prolactin secretion (6.8-fold) and gene expression (30.8-fold), whereas quinpirole did not affect prolactin secretion nor gene expression. In RC-4BC cells, both TRH and quinpirole did not modulate prolactin secretion nor gene expression. Prolactin secretion and gene expression did not significantly change upon exposure to TCDD or DELAQ. However, DELAQ, but not TCDD, attenuated quinpirole-inhibited prolactin gene expression by 51% in primary pituitary cells. This study shows that pituitary prolactin regulation in rat primary pituitary cells in vitro is distinctly different from rat pituitary cell lines GH3 and RC-4BC. Therefore, effects on pituitary prolactin regulation in vitro should best be performed using rat primary pituitary cells. Additionally, AHR ligands may interact with rat pituitary prolactin regulation, but this appears to depend on the ligand and constitutive prolactin secretion. However, interpretation of the in vitro results with respect to occurrence of uterine tumors in rats should take the complex regulation of prolactin release in the pituitary into account as well as the in vivo hypothalamus-pituitary-gonadal (HPG) axis and its feedback loops.