Molecular mechanisms of AhR mediated endocrine disruption of estrogen and retinoic acid signalling pathways

Abstract: Dioxins and similar compounds are toxic substances ubiquitously present in the environment. The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is considered to be one of the most toxic compounds known to man. Dioxins have been shown to act as potent endocrine disruptor compounds (EDCs) by inducing dysregulation of several hormone signaling pathways, which include, but are not limited to, estrogens, retinoids and thyroid hormone signaling. Most, if not all, of the effects of dioxins are mediated through ligand binding and subsequent transcriptional activation of the intracellular aryl hydrocarbon receptor (AhR). The present study aimed at investigating the mechanisms behind the endocrine disrupting effects of dioxins on the retinoid- and estrogen-hormone signaling pathways, utilizing mainly in vitro model systems. In addition, we have investigated the biological activity of a recently discovered retinoid metabolite. In paper I, a microarray study was performed in order to screen for novel genomic effects of dioxin exposure. Male Sprague-Dawley rats were exposed with a single low or high dose of TCDD. After 6h-7days the animals were sacrificed and hepatic samples were analyzed using a global microarray chip. Approximately 185 genes were found to be differentially regulated >=2-fold by dioxins. Analysis of the differentially altered genes revealed that dioxin-AhR is directly or indirectly involved in regulating widespread cellular functions such as metabolism and excretion of endo- and xenobiotics (CYP1A1, 1A2, UGT1A6/7), cell cycle regulation (Cyclin D1), cellular signaling (RXRgamma), steroid metabolism (Srd5a1), circadian regulation (Per2) and cellular differentiation (IGF1), to mention a few. In particular, decreased expression was seen for CYP7A1, SHP, FXR, Ntcp and Oatp2. Decreased expressions of this network of genes imply major deregulation of cholesterol metabolism, bile acid synthesis and transport. Several mechanisms have been proposed to explain the antiestrogenic action of dioxins, including alterations of estrogen receptor (ER) levels, induction of estradiol-metabolizing enzymes that alter tissue levels of estrogens and competition for common DNA-binding sites. Recently, it was found that ARNT, which is an obligatory partner protein for AhR, acts as a potent coactivator for the ERs. In paper II, mechanisms of ARNT in ER dependent transcription was further characterized. These studies showed that reducing the levels of available ARNT by activation of the AhR- or HIF-pathways or targeted downregulation of ARNT using siRNA coincided with a decrease in ER transcriptional activity, especially of the ERbeta subtype. These findings demonstrate that competition for ARNT may contribute to the antiestrogenic effects of dioxins. One of the most sensitive and early signs of dioxin exposure in vivo is disturbed retinoid homeostasis with hepatic depletion of retinoid stores and increased levels of all trans-retinoic acid (at-RA), the biological ligand for the nuclear retinoid receptors (RARs and RXRs), In paper III, the effects of cotreatment with dioxin on at-RA induced transcription in vitro was investigated. We found that TCDD repressed at-RA induced mRNA levels of the at-RA target genes RARbeta2 and CYP26a1, using Realtime-PCR. Interestingly, at-RA was found to reciprocally repress TCDD induced transcription of AhR target genes AhRR and CYP1A1. In addition, a novel finding was that RXRbeta and ARNT can interact in the presence of both TCDD and at-RA. The importance of this interaction needs to be further investigated. In paper IV, the recently identified retinoid metabolite S-4o9cDH-RA was found to be a biologically active retinoid with the ability to regulate RAR-dependent transcription, both in vitro and vivo. We suggest that S-4o9cDH-RA is a novel endogenous ligand for at least RARalpha and RARbeta.