Genotoxicity and endocrine effects – population relevant impacts of steroids

The first part of the present dissertation assesses genotoxic effects of environmental samples (e.g. sediment, fish blood) from the River Danube and the Tietê River in Brazil. The genotoxic potentials of both rivers were determined by means of the comet assay and the micronucleus test. The results, regarding the ecological status of the Danube River, reveal an overall moderate to severe genotoxic potential with a highly differential localization. Regarding the Tietê River, strong hazard potentials could especially be detected in samples from Billings reservoir, indicating a major impact of discharges of São Paulo. Overall, however, genotoxicity decreases downstream. In situ observations (i.e. the micronucleus test with fish erythrocytes) on the River Danube and the Tietê River showed excellent correlation with corresponding in vitro tests (i.e. the comet assay and the micronucleus test with RTL-W1 cells) and document the ecological relevance of in vitro studies with sediment extracts. In order to extend the usage of the micronucleus test, modified versions were tested. Results demonstrated that the micronucleus assay with fixed (liver) tissues and the modified micronucleus test with homogenized zebrafish embryos represent valuable tools for the in vivo identification of the mutagenic potentials of pure substances, environmental samples and field locations. As a next step, the genotoxic hazard potential of trenbolone, a model androgen, was tested in vitro in the permanent fish cell line RTL-W1 as well as in primary cell cultures derived from zebrafish (Danio rerio) embryos after in vivo exposure. Results confirm the conclusion that the steroid trenbolone may act as a genotoxic substance. Once the genotoxicity of trenbolone was assessed in the micronucleus test and the comet assay in vitro (with RTL-W1 cells) and in vivo (with zebrafish embryo cell suspensions), the uptake pattern of trenbolone in zebrafish embryos was investigated in order to assess if the observed genotoxicity is linked to a certain tissue in the zebrafish embryos. The results demonstrate that trenbolone has easy access to all tissues of the embryonic and larval stages of zebrafish and thus no masking of genotoxic effects is expected. Knowing that trenbolone is ubiquitous in the body of the zebrafish embryo, the question emerges if this substance might have further impact on organisms than the genotoxicity observed and the known endocrine effect. Thus, a two generation test with trenbolone was performed, which revealed that the major effect was the masculinisation in the F1 generation. The significant changes in length were the only effects observed, which could not exclusively be related to an endocrine effect. The last part of this thesis was a validation of a definite fish test (full life-cycle/two generation) with zebrafish. For this end, beside trenbolone, the anti-androgen flutamide and the anti-estrogen tamoxifen citrate were tested in multi-generation tests. Currently available fish screening assays cover only mechanisms of endocrine action related to sexual endocrine disrupting chemicals. For substances other than sexual endocrine disruptors, there is no possibility to apply a tiered testing strategy. In such cases, a fish life-cycle and/or multigenerational test, as definitive test, is directly required to eliminate reasonable suspicion of an endocrine mode of action. Therefore, it is advisable that future studies focus on harmonization and validation of several elements of the available guidance for fish full lifecycle tests and 2-generation tests