Christiane Hindrichs

• The thyroid hormone (TH) synthesis is regulated by negative feedback mechanism involving the hypothalamus, pituitary gland and thyroid gland which is called the hypothalamic-pituitary-thyroid (HPT)-axis. This HPT-axis flexible adjusts to the T4 and T3 concentrations in blood to either stimulate or repress TH synthesis. THs are important regulators for various processes such as metabolic rate and thermogenesis but also play an important role during development in particular of the neuronal system. Within this TH homeostasis TH-related disruption might occur through various mode of actions (MOAs) by e.g. industrial chemicals, drugs, or pesticides. The effect on TH concentrations in rat plasma as well as brain by different TH-related MOAs caused by representative substances were investigated during the scope of this project. To assess these alterations, first method development was conducted to detect THs (T4, T3, rT3, T2, and T1) in the given matrices using an online-SPE-LC-MS/MS system. The investigated MOAs were divided by either directly interfering with the TH system through e.g. inhibition of the thyroid peroxidase (TPO) directly affecting TH synthesis or indirectly such as by competitively binding to plasma TH-binding proteins. The shown results indicated the strongest decline of TH concentrations after exposure to substances directly interfering with the TH synthesis which was evident in most of the analyses investigated. Additionally, the free fraction of TH in blood was assessed for which first different technical devices were tested from which one was further methodically improved. As a final step the free fraction in plasma samples from rats exposed to different TH disrupting substances were determined. TH analysis in rat plasma was further expanded to TH detection in rat whole brain as well as in specific brain regions. In rat plasma and rat brain the course of TH concentration with increasing age (post-natal day (PND) 4, 21, and 97-99 day-old) was investigated and contrasted between the matrices. This revealed for example peak concentrations of analyte T3 in both matrices and of T2 in brain on PND21. Moreover, in-vitro analysis of rT3 to T2 conversion was assessed in human liver microsomes via online-SPE-LC-MS/MS analysis. The potential interference by exemplary substances with the responsible deiodinase enzyme (DIO1) was evaluated by calculating the concentration at which half of the enzyme is inhibited (IC50). These results were compared to the initial method evaluating the release of Iodide by a colorimetric reaction. The assessment of rT3 and T2 concentrations by online-SPE-LC-MS/MS analysis was advantageous especially when assessing the influence of substances structurally containing Iodide. Furthermore, it has been identified that T2 concentrations were a superior readout parameter than rT3 because it demonstrated to be independent of handling errors that were visible in rT3 concentrations. Besides TH analysis in rats, the developed method was further adjusted to assess TH whole embryo content in zebrafish (Danio rerio). Zebrafish embryos are alternative models because below 120 hours post fertilization (hpf), they do not account to animal welfare regulations and current research showed that until this age they seem to be a suitable model to assess thyroid disruption. Thus, after primarily analyzing THs in control zebrafish samples to determine the number of embryos needed as well as secondly to assess the course of TH concentrations over time. An exemplary study was performed exposing the embryos to the positive substance 6-Propy-2-thiouracil (PTU) which is a thyreostatic drug directly inhibiting TPO demonstrated reduced T4 and T3 concentrations even after beginning of TH synthesis (~ 70 to 80 hpf). All in all, an easily adjustable online-SPE-LC-MS/MS method was developed which was easily modified for TH analysis of various tissues in-vivo and in-vitro. This expansion to various matrices of different origin enables a TH modality assessment of various MOAs within the TH system.