The endocrine disruptors : effects and action mechanisms on estrogen-induced cell functions

Abstract

Aim of this project was to study the impact of natural or synthetic chemicals on 17β-estradiol (E2)-dependent cell functions evaluating their effects, action mechanisms, and the involvement of estrogen receptors (ERs). Both in the first and the second year our work has been focused on natural phytochemicals and in particular on flavonoids. In the last 20 years scientific interest has been focused on flavonoids, plant secondary metabolites identified in edible plants and regularly consumed with the human diet [1], because of their protective effects on several human degenerative pathologies[2-5]. According to their beneficial effects a huge number of preparations are commercially available on the market in the form of plant extracts or mixtures. The commercial success of these preparations is evident, even though several activities and action mechanisms of flavonoids are still unclear. Depending on their chemical structure and concentration, flavonoids have been described as estrogen mimetics or antiestrogens, as antioxidants or as kinase inhibitors [6]. We have previously demonstrated that the flavanone naringenin (Nar) and the flavonol quercetin (Q), well known for their antioxidant activity, impairs ERα activities [7]. Particularly, Nar selectively decouples ERα from plasma membrane impairing the activation of specific rapid signals important for E2-induced cell cycle progression [5]. On the other hand, upon binding to ERα naringenin and quercetin activates a pro-apoptotic cascade via p38/MAPK pathway [5]. Interestingly, Nar mimics the E2 effects in ERβ containing cells leading cells to apoptosis via p38/MAPK pathway [5]. First of all we evaluated the possibility that the flavonoid-induced cancer cell apoptosis was ER dependent without any contribution of other action mechanisms (i.e., antioxidant or kinase inhibition). For this purpose we choose quercetin as flavonoids because of its well known antioxidant properties. The obtained data confirmed that quercetin prevents, in a dose-response manner, H2O2-induced ROS production both in the presence that in the absence of ERα; whereas no kinase inhibition was present at the Q concentration tested. However, Q activated a pro-apoptotic cascade in cancer cells only in the presence of ERα [8] sustaining the dependence from ER of this flavonoid effect. On the other hand, Q, as well as Nar, mimics E2 effects, preserving ERβ transcriptional activity and leading cells to apoptosis via p38/MAPK pathway. To evaluate if these similar effects were also present in non tumoral cells we performed experiments by stimulating rat skeletal muscle cells (L6) with Nar. These cells represent an E2 target tissue in which the hormone induces differentiation via ERα-dependent signals [9]. Nar stimulation did not alter the rate of proliferation in these cells, but it decouples ERα mechanism of action impeding the E2-dependent differentiative effect in skeletal muscle [10], exerting, like E2, a protective role against H2O2-induced ROS damage via ERβ. Besides E2, androgens (i.e., testosterone and dihydrotestosterone) and other hormones (insulin-like growth factor, IGF-1) promote myoblast differentiation trough their cognate receptors. This prompted us to evaluate if Nar could also impair the effect of these hormones. Our results demonstrated that Nar only affect E2-induced differentiation raising the existence of a greater susceptibility of female sex steroid hormanes to flavonoids. Mammalians are exposed to several EDs, rather than a single compounds resulting in systemic circulation of ED and flavonoid mixtures in the body. In addition, flavonoid ability to maintain their protective effects against cancer growth even in the presence of E2 is completely unknown. This latter point is particular intriguing in that the final outcome of the exposure to a single or a mixture of these compounds, is strictly dependent on the interaction of the flavonoid-activated and hormone-activated signals [12]. Thus we also evaluated Nar effects on cancer cell survival and proliferation in the presence of a background of E2 or Bisphenol A (BPA). Data obtained show that ERα-mediated rapid/non-genomic signaling pathways are necessary for BPA, Nar, and E2 effects in cancer cell lines. In particular, Nar specifically antagonize the E2- and BPA-induced ERα-dependent rapid signals, with the net effect to reduce the proliferative actions of both potent endogenous hormone and endocrine disruptors in promoting cellular proliferation [11, 13]. In the 2 presence of ERβ BPA did not affect cell proliferation, whereas, in presence of E2 background, BPA prevented both E2-induced ERβ transcriptional activity and E2-induced rapid signals activation (p38 pathway) important for E2-propoptotic effect [11, 14]. However, when ERβ expressing cancer cell line were stimulated with a mixture of BPA and Nar, Nar preserved its ability to act as an E2-mimetic, leading to the activation of a pro-apoptotic cascade (i.e. p38 activation, and in turn caspase-3 and PARP cleavage), thus completely abrogating BPA antiestrogenic effect in ERβ-expressing cancer cells [13]. As a whole, these data enlarge the knowledge on the endocrine disruptor action mechanisms underlying the ER dependence of protective effects of dietary compounds such as naringenin and quercetin. In particular, the impact of flanonoids on ERα activities highlight the importance of investigating the effect of these natural compounds in different stages of life that could be characterized by a different level and role of this receptor isoform. As a consequence age and sex of individuals must be take in account before the administration of flavonoids as dietary supplements and healthy products. Furthermore, these data bring to light a new view of E2 effects as balance of the relative expression of ER isoform and as the balance between the signals originated by each isoform [9, 10, 13, 14], increasing the complexity of E2 action. Therefore, also ED action is more complex than originally considered, since different ligands induce ERs to assume different conformations responsible for specific signaling pathway activation.