Abstract
The increase in the production of man-made chemicals in recent decades and their subsequent release into the environment has raised concern as some of them are capable of affecting both humans and wildlife. Some of these environmental chemicals can act as endocrine disrupting chemicals (EDCs), that is, substances that can interfere with the endogenous hormone pathways of living organisms and cause adverse health effects. One of the mechanisms by which EDCs exert their action is by interacting with nuclear receptors (NRs), ligand-activated transcription factors that control a wide range of processes such as development, homeostasis, and metabolism in living organisms through gene regulation. The link between environmental exposure to EDCs and adverse health effects has been investigated by epidemiological, clinical and laboratory studies but nevertheless, the assessment of environmental exposure to EDCs is often very complicated, both because it is difficult to determine the time and duration of exposure and because we are exposed to a mixture of substances whose effects are unknown. To try to better understand the effects that EDCs can have on living organisms, in vitro experiments using cells with NR-expressing reporter genes have been developed with the aim of considerably reducing animal testing, which is especially important because of the large number of molecules suspected to be EDCs. Among NRs, members of the steroid receptor (SR) subfamily, such as estrogen receptors (ERs) and androgen receptor (AR), have been extensively studied as targets of EDCs, but other SRs, such as progesterone receptor (PR), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), have been less so. Therefore, we developed selective stable cell lines expressing human SRs to test environmental chemicals for their endocrine disrupting activities. In addition, because the OECD (Organization for Economic Cooperation and Development) guideline for testing EDCs requires the use of in vitro tests expressing only human SRs (level 2 of the conceptual framework), in many cases mammalian toxicological data are extrapolated to other species. Therefore, we set out to develop stable reporter gene cell lines expressing zebrafish SRs to study possible interspecies differences. My doctoral research project aimed to study the structural and functional interactions between human and zebrafish SRs with environmental chemicals in order to investigate possible interspecies differences between humans and zebrafish in EDC risk assessment and, consequently, to refine the extrapolation of toxicological data between the species. My research group had already pointed out that the ERs show slight differences in selectivity between human and zebrafish subtypes and that many progestins tested on PR-selective cell lines proved to be agonists on the human receptor and antagonists on the zebrafish receptor. The results of my experiments showed that AR and GR revealed few interspecies differences when tested with steroids on the respective SR-selective cell lines. On the contrary, PR and MR show great interspecies differences between human and zebrafish. The second objective of my thesis was to study how SRs can be differently targeted by environmental chemicals. The results showed that not only ERs and AR can be targeted by chemicals in the environment, but also MR and PR, while GR does not seem to be affected. The results of my thesis work complemented other studies on the interactions of EDCs with human and zebrafish NRs and demonstrated the value of reporter cell lines in this context.