Proteomics as an alternative approach for hazard characterization and the identification of specific chemical targets : elucidation of potential biomarkers for differentiating endocrine disruption from hepatotoxicity

Ayobahan, Steve Uwa; Hollert, Henner (Thesis advisor); Schäfers, Christoph (Thesis advisor); Schäffer, Andreas (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Ecotoxicological testing of the chemical impact on reproductive endocrine function for regulatory purposes is currently focused on the characterization of apical endpoints rather than identifying the molecular mechanisms accountable for the observed adverse effects. Specifically, alterations in endpoints and biomarkers related to reproduction such as vitellogenin (VTG) content, gonadal morphology and fecundity may also be affected by disruption being unconnected to a perturbed endocrine system. As the liver is the site of VTG production, also hepatotoxicants can induce similar changes on this biomarker. Since regulation of chemicals is different when they are identified as endocrine disruptors, there is a growing demand for the development of novel endpoints to be included in testing strategies. This dissertation aimed at demonstrating the sensitivity and the applicability of integrating proteomics into the conventional fish short term reproduction assay (FSTRA, OECD 229), not only for linking changes in apical endpoints with the corresponding underlying molecular alterations but also to detect candidate makers for discriminating hepatotoxicity from endocrine mediated toxicity. In order to ensure consistency in substance evaluation and to avert further extensive testing, the development of an additional weight-of-evidence approach to differentiate between effects caused by hepatotoxicity and endocrine modulated actions is of significant importance for industry and regulatory authorities. To this end, an FSTRA according to OECD TG 229 was performed with one known hepatotoxic substance (acetaminophen, APAP) and an endocrine disrupting chemical (EDC, fadrozole). Fadrozole is an anti-breast cancer drug, which acts as a non-steroidal aromatase inhibitor that specifically inhibits estrogen synthesis. APAP, a non-steroidal anti-inflammatory drug, is one of the commonly used over the counter medication for pain and fever when taken at the recommended therapeutic dose. At high concentrations, the substance has been identified to inhibit cyclo-oxygenase enzymes and cause severe hepatic injury. The effects of these two substances were monitored separately in exposed male and female zebrafish. During the 21-day conduct of the assay, substance impacts on fish reproduction was measured daily and quantitatively across all the conditions used in these studies. At the termination of the assay, histopathological examination of gonads and livers, evaluation of hyaluronic acid for the detection of liver toxicity and the assessment of VTG a known indicator of reproductive endocrine disruption were measured in both sexes of zebrafish. In addition, this study focused on the interrelationship between hepatic distortion and reproductive endocrine disorders. In view of this, proteomic techniques were applied for identifying organ-specific protein expression changes. The two substances investigated in this study demonstrated a strong impact on the reproductive fitness of zebrafish. For fadrozole treatment, a concentration-dependent decrease in egg number, a reduction in plasma VTG concentrations and a mild oocyte atresia with oocyte membrane folding in females were detected. Consistent with these apical measures, proteome responses revealed a significant deficit in estrogen synthesis and perturbed binding of sperm to zona pellucida in the ovary. The exposure to APAP triggered a significant decrease in egg number, an increase in plasma hyaluronic acid, and the presence of single cell necrosis in the hepatocyte. Interestingly, functional enrichment analysis revealed an intense distortion in liver function ranging from stress induction in endoplasmic reticulum, dysfunctional mitochondria, and glutathione depletion to excessive accumulation of reactive oxygen species. Mitochondria disorders attributed to a dysfunctional oxidative phosphorylation system was commonly detected in the livers and gonads of exposed male and female zebrafish, denoting liver damage. This indicated that APAP-induced alterations of zebrafish reproductive functions could be attributed to the observed distortion in the hepatocytes and not to a function of estrogen deficiency. In comparison with exposure to fadrozole, three potential biomarkers for identifying liver toxicity, i.e. cahz, c3a.1 and atp5f1b, were identified. These results show that the integration of quantitative proteomics in the weight-of-evidence approach could significantly improve the differentiation of hepatotoxic and endocrine disrupting substances. The observed potential biomarkers can be further assessed and utilized as starting points for detecting and prioritizing hepatotoxic substances. In practical terms, this approach will promote the development of a biomarker database, necessary for hazard identification in EU-legislation and to decide upon the option for risk assessment. Taken together, this study demonstrates the sensitivity and feasibility of integrating sex-specific proteome responses for the identification of distinct chemical targets in fish. The results presented in this thesis provide significant evidence to support the utilization of a proteomics approach as a hazard characterisation strategy for linking chemical-induced molecular alterations with phenotypic changes in EDCs testing.