Constitutive and TCDD-induced expression of Ah receptor responsive genes with special focus on the brain and pituitary

Abstract: The 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and related substances are ubiquitous environmental pollutants causing a number of pathological alterations, the most severe being progressive anorexia and body weight loss. The effects on body weight homeostasis suggest a possible involvement of the nervous system and neuroendocrine areas including the pituitary gland. The studies described in this thesis aimed at investigating the expression of aryl hydrocarbon receptor (AHR) responsive genes in different brain regions and pituitaries after treatment with the AHR ligands - TCDD and beta- naphthoflavone (betaNF). The diurnal expression of AHR related genes was also studied. Male Sprague-Dawley rats and 129/SV/C57BL/6 mice, as well as male mice deficient in the cellular retinol-binding protein (CRBP-I) were used as in vivo experimental models. The mouse pituitary AtT-20 cell line was selected as an in vitro model. A single oral dose of TCDD (10 µg/kg or 15 µg/kg in rats; 50 µg/kg or 250 µg/kg in mice) was administrated and the animals were sacrificed 1, 3, 6, 7 or 28 days after treatment. The results revealed that basal mRNAs levels of AHR and aryl hydrocarbon receptor translocator (ARNT) were present ubiquitously in the different brain regions and pituitaries, which provided anatomical and biochemical basis for a possible direct action of AHR ligands on these regions. The expression of cytochrome P450 1A1 (CYP1A1) was dramatically increased after TCDD treatment and the pituitary was found the most sensitive among the tissues investigated. In contrast to CYP1A1, the expression changes of AHR and ARNT mRNAs were limited. The expression of AHR repressor (AHRR) was induced by TCDD or betaNF in vivo and in vitro. To shed some light on central mechanisms involved in the wasting syndrome induced by TCDD, we investigated the expression of several hypothalamic neuropeptides regulating food intake. Both body weight stimulatory (neuropeptide Y, NPY; melanin concentrating hormone, MCH), and inhibitory (cocaineand ampbetamine-regulated transcript, CART; proopiomelanocortin, POMC) neuropeptides were affected by TCDD, which indicated that the central peptidergic circuitries had been dysregulated by TCDD. Results from immunohistochemical double staining analyses showed that AHRR co-localized with NPY-, CART-, MCH- and orexin- immunopositive neurons. Xenobiotic responsive elements (XREs) were found in these neuropeptide genes with the exception of MCH. Furthermore, the proteins encoded by the POMC gene, the adrenocorticotrophic hormone (ACTH) and beta-endorphin, were increased in AtT-20 cells after TCDD exposure. These data suggest that body weight loss and wasting syndrome after TCDD treatment may be induced by AHR-mediated regulation of neuropeptides in the brain and in the pituitary. The study on the mice that lacked the cellular retinol-binding protein (CRBP-I) showed that the CRBP- I -/- mice have a significantly higher basal expression of the AHRR gene in the pituitary compared to the wild type mice. The vitamin A homeostasis seems to play a role on the TCDD-induced activation of AHR- regulated gene transcription. The results of a 24-h expression study in rats showed a significant diurnal variation of CYP1A1 mRNA level in the anterior and posterior pituitary, as well as in the liver. A daily upregulation of CYP1A1 and period (PER2) mRNAs was observed in anti-phase to the AHR and ARNT mRNAs in the liver. The high inducibility of CYP1A1 and AHRR as well as hypothalamic neuropeptides changes that may explain the anorexia and body weight loss in response to TCDD exposure, indicate that the brain and pituitary gland may be direct targets for TCDD.