ENDOCRINE DISRUPTING ACTION OF TRIBUTYLTIN IN REPRODUCTIVE AND METABOLIC FUNCTIONS

Abstract

A large number of investigations during the last two decades describe adverse trends in male and female reproductive health, which have been proposed to be caused by environmental factors with endocrine disrupting properties. In contrast to many other toxicants, endocrine disruptors (EDs) often do not show linear dose-response relationships typical of those found in traditional toxicological studies. For many compounds, low-dose exposure causes effects opposite to the ones seen after high-dose exposure. In addition, the timing of exposure has been found to be critical factor. Hence, to correctly assess the impact of endocrine disruptors on reproductive health requires in-depth knowledge of their mechanisms of action. This thesis aimed at identifying the mechanisms underlying the effects of a well studied persistent and ubiquitous environmental contaminant Tributyltin chloride (TBTCl) on reproductive and metabolic disorders. At the beginning, Chapter 1 introduces briefly about the endocrine system and the endocrine disrupting chemicals (EDCs). It also deals with the key receptors and the major signaling pathway targeted by these EDCs. This introductory chapter provides an overview of the levels and sites at which EDs affect steroid actions. We tried to point to more recent findings and opened questions. Finally detailed objective to be attained in this study is specified here. Followed by this, Chapter 2 presents (i) a detailed review on steroidogenesis and factors governing it; (ii) genomic and non genomic action of steroid hormones; (iii) a brief description about EDCs, with special emphasis on their mode of action and screening strategies involved; (iii) understanding the effects of different classes of EDCs on reproductive system and steroidogenesis; (iv) recent research detailing endocrine disrupting effect of TBTCl and finally (v) the hypothetic idea behind the present thesis to unravel the harmful effects of low dose TBTCl treatment. Each of these hypotheses was further explored in subsequent chapters of the thesis. The main objective of the present work is to decipher the endocrine disrupting effect of TBTCl in reproductive and metabolic disorders followed by understanding its mode of action. There are various in vitro and in vivo assays which are already established to confirm a chemical to be ED. All these parameters are elaborated in Chapter 3 of this thesis. In this concern, this chapter contains the overall principles and methodology of all the experimental assays that were performed to identify a molecule to be EDs. The stage I includes ____________________________________________________________________Abstract biochemical parameters like E-screen, T-screen, Oil-O-Red staining and receptor specific bioassays which were used initially to check for (anti)/estrogenic and (anti)/thyrogenic and (anti)/adipogenic effect; and in stage II, the principles of the biological mechanisms are studied at transcriptional and translational levels of various genes to understand how a chemical acts intracellularly, mainly the assays which show the basic determination of the underlying pathways. Further several cell lines used for the study have been discussed in detail. An increasing number of chemical compounds from the environment have been identified as EDs utilizing various in vitro and in vivo bioassays. These include pesticides, industrial chemicals, waste water effluents, house utilities, cosmetics and pharmaceuticals that behave like ligands for the steroid or aryl hydrocarbon receptors producing effects that mimic the natural hormone. In Chapter 4 few of these chemicals belonging to the above group were screened using the cell proliferation and reporter based cell bioassays with a major focus on (anti)/estrogenic, (anti)/thyrogenic and (anti)/adipogenic activities. Among all the compound studied TBTCl was found to be more potent causing endocrine disruption at very low dose. Next, in Chapter 5, after the initial screening the most potent chemical (TBTCl) was tested for its estrogenic potential in vitro in estrogenic receptor (+) breast adenocarcinoma, MCF-7 cell line. Our result showed that low dose treatment of TBTCl had a proliferative effect on MCF-7 cells and resulted in up-regulation of aromatase enzyme activity and enhanced estradiol (E2) production. TBTCl was found to mimic estrogenic actions at a low dose and its mechanism involved both classical and non-classical membrane mediated pathways through activating MAP-Kinase (MAPK) signaling. In Chapter 6, we focused to elaborate the estrogenic effect of TBTCl on the female reproductive system. For this the endocrine disrupting effect of TBTCl was checked on primary cultured mouse granulosa cells and on both intact and ovariectomized mice. Our data showed an accelerating effect of TBTCl on estrogen production in ovarian granulosa cells as well as in blood serum of TBTCl treated mice. Further, TBTCl up regulated estrogen receptor (ER) interaction as checked in ER transactivation assay and altered expression of steroidogenic and steroid related proteins. The importance of TBTCl as an EDC in different animal models is well known; however, its adverse effects on the thyroid gland are less understood. Hence, in the next section (Chapter 7), we aimed to evaluate the thyroid-disrupting effects of this chemical using both in vitro (HepG2) and in vivo (swiss albino male mice) models. Our results showed ____________________________________________________________________Abstract that TBTCl disrupts thyroid functions through various mechanisms. This chemical acts as an antagonist to thyroid receptor (TR) and inhibits T3-mediated transcriptional activity in vitro through the recruitment of NCoR. In addition in the in vivo study, some of the genes linked to thyroid glands were down-regulated in response to this chemical. Together, in vitro, in utero and in vivo studies have reported the adipogenic or obesogenic potential of TBTCl in several species, including humans, making it the most studied obesogen. However to our knowledge, no comprehensive mechanistic study has been performed so far to unravel the full mode of action of this obesogenic compound both in vitro and in vivo. Therefore in Chapter 8, we sought to determine if biologically relevant levels of TBTCl effect the development, endocrine function, and metabolism of adipose tissue in a way that it promotes obesity. Since inflammation plays an important role in the development of obesity in different experimental models an attempt was made to understand whether TBTCl induced inflammatory change contributes to obesity. Our result demonstrates the obesogenic potential of TBTCl in vitro and in vivo through multiple mechanism including increased adipogenesis and inflammation via SIRT 1 inactivation. Finally, the Chapter 9 summarizes the major findings of current thesis and provides suggestions for future work in this area. Results of the individual experiments are discussed chapter wise in detail which provides an overview of the entire thesis and message delivered in terms of various targets and different modes of action of chemicals tested in the present study. The scientific findings dealt within this thesis may be of use to the future researchers working in this area. Last but not the least, the Chapter 10, listed the bibliography which was consulted in course of the present work.