Toxicogenomics of Aryl Hydrocarbon- and Estrogen Receptor Interactions in Fish: Mechanisms and Profiling of Gene Expression Patterns in Chemical Mixture Exposure Scenarios

Abstract

Almost without exception, biological processes such as overt morphological changes, development (both reproductive and growth), toxicological responses and clinical manifestation to disease, have molecular basis. From our perspective (i.e. toxicological perspective), the evidence of receptor-mediated mechanisms of xenobiotic-induced effects is provided if the effect is tissue specific, predictable, if increases in the transactivation of specific genes can be demonstrated, transcriptional responses occur rapidly, compounds bind reversibly to intracellular macromolecules or compounds are stereo-specific. Thus, the primary objective of toxicological in vitro studies on cells and tissues is to characterize cellular and molecular substrates and pathways that contribute to adverse effects in an organism after toxicant exposure. The estrogenic and xenobiotic biotransformation gene expressions are receptor-mediated processes that are ligand structure-dependent interactions with estrogen-receptor (ER) and aryl hydrocarbon receptor (AhR). The anti-estrogenic activities of AhR agonists have been reported in vitro and in vivo studies. In teleost species, exposure to AhR agonists has been associated with reduced vitellogenin (Vtg) synthesis or impaired gonadal development. Recently, several studies have shown that AhR-agonists directly activate ERs and induce estrogenic responses in mammalian in vitro systems. The overall objective of this thesis was to develop diagnostic gene and protein response tools in the study of the molecular mechanisms of gene expression patterns of xenoestrogens and xenobiotic interactions in wildlife species. Contaminants known to be estrogenic (ethynylestradiol; EE2 and nonylphenol; NP) and/or anti-estrogenic (PCBs), either by direct ER or indirect AhR mechanistic pathways, were used as model xenobiotics and evaluated either singly or in combination using in vitro and in vivo test systems.

Suppressive subtractive hybridization (SSH) was used to create a cDNA library of clones containing differentially expressed genes from Atlantic salmon (Salmo salar) separately exposed to ER and AhR agonists. Based on differentially expressed genes from the library, a targeted cDNA array (SalArray) was developed. Cellular in vitro systems, like cell and tissue models, facilitate the investigation of the direct molecular mechanisms accounting for predictable adverse effects of xenobiotic compounds on wildlife and humans. Consequently, in the studies presented primary hepatocyte cultures were isolated from the liver of trout and salmon by the collagenase perfusion method. The targeted SalArray and quantitative real-time PCR (q-PCR) were used to demonstrate that exposure of salmon hepatocytes to the ER-agonist NP singly or in combination with the produced differential gene expression patterns in salmon liver.Exposure of hepatocytes to NP mainly altered genes involved in the estrogenic pathway, including genes involved in steroid hormone synthesis and metabolism. The anti-estrogenic properties of PCB77 were demonstrated in the array analysis as NP induced gene expressions decreased by exposure of hepatocytes to PCB77. Our data showed a reciprocal inhibitory interaction between ER- and AhR-agonists. PCB77 produced anti-estrogenic effects by decreasing the mRNA expression of ER-responsive genes, and NP produced anti-AhR mediated effects as inhibitor of AhRR, Arnt, CYP1A1 and UGT expression. In vivo exposure of salmon to EE2 produced a significant decrease of CYP1A1 expression and these effects paralleled EROD activity and AhRR mRNA, suggesting a direct role of EE2 in controlling the cellular detoxification machinery.

While a clear pattern of negative effects on ER-mediated gene expression was found in hepatocytes exposed to PCB77, exposure of cells to the more potent AhR-agonist and dioxinlike PCB126 induced transcriptional activation of ER signalling demonstrated by increased Vtg and ERα mRNA and ERα protein levels. The decreased levels of ERα and Vtg expression in cells treated with PCB126 in the presence of ICI is novel, indicating a possible, but not conclusive “ER-hijacking” not previously reported in any fish species or lower vertebrate. Different gene expression patterns were obtained at similar time-interval with fish from different seasons, demonstrating the complexity of AhR-ER interactions. Thus, the direct estrogenic actions of PCB126 observed contribute new insight on the complexity of the mechanisms involved in ER-AhR crosstalk, prompting a new wave of discussion on whether AhR-mediated anti-estrogenicity is an exception, rather than a rule of action. This thesis demonstrates a complex mode of interactions between two different classes of ligandactivated receptors and provides novel mechanistic insights on signalling pathways. Therefore, the degree of simultaneous interactions between the ER and AhR gene transcripts demonstrated support the concept of cross-talk between these signalling pathways, in addition to generating new hypotheses that need to be evaluated empirically. AhR-agonist PCB77