The aryl-hydrocarbon receptor pathway during early development of the zebrafish : assessing transcription, formation and activity of receptor-related proteins after exposure to four compounds with dioxin-like mode of action

In standardized chemical risk assessment, test systems representing an alternative to conventional animal experiments have become increasingly important during the last decade. Besides other approaches, the fish embryo toxicity test (FET) using early life stages was an attempt to substitute the standard fish toxicity test with adult individuals. In 2013, the FET was implemented in an OECD guideline (no. 236) and serves as the preferred procedure for testing the environmental risk of a substance. The correlation between embryonic and adult toxicity data has been proven in many former studies. However, not all types of compounds were equally predictable depending on their properties. At the same time, the embryo represents a dynamic system whose toxic response might not yet correspond to the adult reaction to a chemical. Molecular signaling pathways are probably not yet completely developed and functional. Involved enzymes become the limiting factors in the competition for cofactors necessary for the toxic response or normal development. The present PhD thesis focuses on the signaling pathway of the aryl-hydrocarbon receptor (AHR) in embryonic zebrafish (Danio rerio). The AHR functions as a transcription factor for many different genes, including P450 cytochromes (CYP) that contribute to the metabolism of xenobiotic sub-stances. Accordingly, the present experiments focused on the questions (1) whether gene transcription is altered depending on the type of the applied chemical, (2) whether the effects on the DNA level translate onto the higher biological levels of protein formation and enzymatic activity, and (3) how the embryonic development influences these processes. Ligands of the AHR belong to the group of dioxins and compounds with dioxin-like potential including environmentally relevant chemicals like polyaromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and NSO-heterocyclic molecules (NSO-het). Four representatives of these types of chemicals have been selected for the experiments at hand: beta-naphthoflavone (BNF) and ben-zo[a]pyrene (BaP) as PAHs, PCB126 as the most potent PCB, and 2,3-benzofurane (2,3-BF) as NSO-het. Zebrafish embryos were exposed to these substances from 6 to 118 hours after fertilization (hpf). All cofactors involved in the AHR signaling pathway (AHR itself, AIP, ARNT, and HSP90β), the cytochromes CYP1a, CYP1b, CYP1c1, and CYP1c2, as well as the repressor of the AHR (AHR-R), were examined with regard to their transcript amounts in very short intervals (every 4 hours) during embryogenesis. In untreated embryos, a very distinct pattern of expression was detected for all AHR-dependent genes. The time course of the chemically induced expression varied depending on the applied chemical and its properties (hydrophobicity, planarity, biodegradability). None of the cofactors displayed impaired transcript amounts after exposition to any of the compounds. Since cyp1a was induced to the highest extent by all selected chemicals, its amount of protein was assessed, next to its transcription, in a specifically designed ELISA. Significant changes were not detected in any exposure scenario or at any time point during embryogenesis. The EROD assay captures the activity of all enzymes with deacetylase character (e. g. cyp1a) and represents the next biological level of response to xenobiotic exposure. In this test system, activity was found to be up-regulated only in embryos treated with PCB126 and after the moment of hatch (62 hpf). Experiments conducted with the same substance at similar conditions in a liver cell-line of the zebrafish (ZF-L) revealed low comparability between the two test systems with regard to the AHR- induced genes. The cofactors of the signaling pathways were not affected here either. An analysis of the whole organisms’ transcriptome was conducted for the time points of 12, 48, and 96 hpf to identify pathways that are regulated in a developmental, stage-dependent manner. Interactions with the AHR and its pathway represent potential mechanisms of toxicity after chemical exposure. The transcriptomic analysis of PCB126 treated embryos revealed several processes that were modified by the substance next to xenobiotic metabolism, even at the very early time point of 12 hpf. In addition to basic cell functions, pathways involved in the development of the muscles, the spine, the nervous system, and the eye were especially affected. Their dysregulation by PCB126 can cause the multiple malformations that have been observed in former studies after PCB126 exposure. However, direct linkage to the AHR was in most cases not evident. Taken together, the results of this PhD thesis lead to the conclusion that the binding of a ligand by the AHR is not the only decisive factor for the toxic response in embryonic zebrafish. The strong reaction in transcription could be transferred neither to the level of protein nor to enzymatic activity. Additionally, the pattern of induction differed depending on the gene and substance. Conclusively, it can be assumed that the applied substances not only impair biotransformation enzymes like the CYPs in their formation but also other important regulating molecules, e. g. kinases, in their activity. The involvement of the AHR in these processes remains to be subject of future studies. The hatch was furthermore identified as an essential cesura during embryogenesis. Therefore, the findings at hand illustrate, that standardized test systems using early life stages (FET) or cell cultures must be revised with regard to their reliability. The zebrafish should not be used earlier than 48 hpf, the initiation of the hatch, in order to obtain high comparability to results from adult fish. Furthermore, endpoints like sublethal malformations, AHR dependent gene induction, and EROD activity must be reconsidered regarding their explanatory power. Results of the present PhD thesis show that none of these endpoints alone is suitable to evaluate the toxicity of a sub-stance with a dioxin-like mode of action reliably. For an appropriate chemical risk assessment, the molecular mechanisms of the AHR signaling pathways, as well as the mutual interactions with other pathways and especially developmental processes, must be understood in detail and stay subject to intensive research so that embryos become a better tool when elucidating chemical modes of action and predicting the toxic potential of a substance