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Polychlorinated dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls are persistent environmental pollutants which ubiquitously occur as complex mixtures and accumulate in the food and feed chain due to their high lipophilic properties. Of the 419 possible congeners, only 29 share a common mechanism of action and cause similar effects, the so called dioxin-like compounds. Dioxin-like compounds evoke a broad spectrum of biochemical and toxic responses, i.e. enzyme induction, dermal toxicity, hepatotoxicity, immunotoxicity, carcinogenicity as well as adverse effects on reproduction, development, and the endocrine system in laboratory animals and in humans. Most, if not all, of the aforementioned responses, are mediated by the aryl hydrocarbon receptor. In the present work, the elicited biochemical effects of a selection of dioxin-like compounds and the non dioxin-like PCB 153 were examined in mouse (in vivo) and in human liver cell models (in vitro). Emphasis was given to the main contributors to the total toxic equivalents in human blood and tissues TCDD, 1-PnCDD, 4-PnCDF, PCB 118, PCB 126, and PCB 156, which likewise contribute about 90 % to the dioxin-like activity in the human food chain.
Three mouse in vivo studies were carried out aiming to characterize the alterations in hepatic gene expression as well as the induction of hepatic xenobiotic metabolizing enzymes after single oral dose. Based on the results obtained from mouse 3-day and 14-day studies, the seven test compounds can be categorized into three classes; the ones which are 'pure' AhR ligands (TCDD, 1-PnCDD, 4-PnCDF, and PCB 126) or solely CAR inducers (PCB 153), and the ones which are AhR/CAR mixed-type inducers (PCB 118, PCB 156). Moreover, the analysis of hepatic gene expression patterns after a single oral dose of either TCDD or PCB 153 revealed that the altered genes fundamentally differed. Profiling of significantly altered genes led to the conclusion that changes in gene expression were associated with different signalling pathways, in fact by AhR and CAR.
For investigating the role of the AhR in mediating biological responses, several experimental approaches were carried out, such as the analysis of blood plasma metabolites in Ahr knockout and wild-type mice. Genotype specifics and similarities were determined by HPLC-MS/MS analysis. Several plasma metabolites could be identified in both genotypes, but also differences were detected. Furthermore, an in vivo experiment was performed aiming to characterize AhR-dependent and -independent effects in female Ahr knockout and wild-type mice. For this purpose, mice received a single oral dose of TCDD and were killed 96 h later. Microarray analysis of mouse livers revealed that although the Ahr gene was knocked out in Ahr-/- mice, the quantity of affected genes were in the same order of magnitude as for Ahr+/+ mice, but the pattern of altered genes distinctly differed. In addition, the relative liver weights of TCDD-treated Ahr+/+ mice were significantly increased which led to the conclusion, that TCDD induced the development of hepatic steatosis in female Ahr wild-type.
The performed in vitro experiments aimed to characterize the effects elicited by selected DLCs and PCB 153 in human liver cell models by the use of HepG2 cells and primary human hepatocytes. In general, primary human hepatocytes were less responsive than HepG2 cells. This was not only observed in EC values derived from EROD assay, but also regarding microarray analysis in terms of differently regulated genes. In vitro REPs gained from both liver cell models widely confirmed the current TEFs, but some deviations occurred. The comparison of the TCDD-altered genes in both human cell types revealed that only a considerably small number of genes was in common up regulated by both human liver cell models, such as the established AhR-regulated highly inducible cytochrome P450s 1A1, 1A2, and 1B1 as well as other AhR target genes. Although the overlap was rather small, the TCDD-induced genes could be consistently associated with the broad spectrum of established dioxin-related biological responses. The gene expression pattern in primary human hepatocytes after treatment with selected DLCs (TCDD, 1-PnCDD, 4-PnCDF, and PCB 126) and PCB 153 was additionally characterized by microarray analysis. The highest response in terms of significantly altered genes was determined for TCDD, followed by 4-PnCDF, 1-PnCDD, and PCB 126, whereas exposure to PCB 153 did not evoke any significant changes in gene expression. The pattern of significantly altered genes was very homogenous among the four congeners. Genes associated with well-established DLC-related biological responses as well as novel dioxin-inducible target genes were identified, whereby an extensive overlap in terms of up regulated genes by all four DLCs occurred. In conclusion, the results from the in vitro experiments performed in primary human hepatocytes provided fundamental insight into the congeners' potencies and caused alterations in gene expression patterns. The obtained findings implicate that although the extent of enzyme inducibilities varied, the gene expression patterns are coincidental. Microarray analysis identified species-specific (mouse vs. human) as well as model-specific (in vitro vs. in vivo and transformed cells vs. untransformed cells) differences. In order to identify novel biomarkers for AhR activation due to treatment with dioxin-like compounds, five candidates were selected based on the microarray results i.e. ALDH3A1, TIPARP, HSD17B2, CD36, and AhRR. Eventually, ALDH3A1 turned out to be the most reliable and suitable marker for exposure to DLCs in both human liver cell models eliciting the highest mRNA inducibility among the five chosen candidates. In which way these species- and cell type-specific markers are involved in the dioxin-elicited toxic responses should be further characterized in vivo and in vitro.