Einfluss der Weichmachermetaboliten Monoethylhexyl-Phthalat (MEHP) und 4-Heptanon auf die Transkriptionsfaktoren PPAR alpha und PPAR gamma in humanen Leber- und Endothelzellen

Additional flexibility and softness in PVC can be achieved by adding plasticizers in order to broaden its usage. One commonly used plasticizer is DEHP, which belongs to the group of Phthalates. Since DEHP is not chemically bound to PVC-plastics it can easily be freed from plastic compounds. Several animal studies have shown evidence for toxic effects of DEHP. The focus of these effects has been its carcinogenicity observed in rodents. This effect could not be reproduced in humans. In humans the main effect of DEHP seems to be its toxicity on reproductivity. It is currently believed that DEHP is not only toxic by itself but also by its metabolites. There are several metabolites of DEHP, which can be found in humans, e.g. MEHP and 4-Heptanon. It has been shown, that several effects of DEHP and its metabolites are relayed by PPARs. These pathways involve different transcription factors, which are activated by several peroxisomeproliferators. These PPARs are divided into three different isoforms, PPAR, PPAR/ and PPAR. In early studies it has been shown, that MEHP is a ligand of PPARs but it remains unclear whether its metabolite 4-Heptanon can also lead to similar effects. Therefore, the aim of this thesis was to study the influence of MEHP and 4-Heptanon on the expression of the three PPAR-isoforms. All experiments were conducted on human liver or endothelium cell culture cells. Cells were stimulated with either MEHP or 4-Heptanon in different concentrations. 24 hours after stimulation, cells were lysated and RNA was extracted. After reverse transcription cDNA concentrations of the target genes was measured by quantitative Real-time PCR. All probes were normalised using GAPDH as housekeeping gene. This method, compared to photometric measurements RNA-concentrations, has the advantage that inaccuracies in RNA-measurements and different efficiency of reverse transcriptase does not lead to a falsification of results. MEHP is hardly water-soluble. Therefore, MEHP was dissolved using DMSO before the stimulation of livercells. By comparing cells treated with DMSO with cells completely unstimulated we found a diminishment of targetgene expression. Due to this observation we used cells treated with DMSO as controls. In experiments with endothelial cells DMSO was not used in order to prevent a falsification of data. As observed in earlier studies, MEHP induces an increase of expression of all PPAR isoforms in human liver and endothelial cells. However, the highest used concentration of MEHP was 1000 µmol/l lead to apoptosis of endothelial cells. In liver cells this concentration also did not lead to an increase of expression of PPAR isoforms, which could be regarded as a sign of cell damage. Stimulation of liver cells using 4-Heptanon did not lead to significant changes of expression. This is in contrast to the results of another study, which shows an increase of PPAR2 under similar experimental conditions. While expression of PPAR and PPAR2 was not significantly altered a slight increase of expression could be observed for PPAR. The highest used concentration of 4-Heptanon was 2000 µmol/l in liver cells. Because of the unclear results we used a 4-Heptanon-concentration of 10 mg/l as highest concentration for stimulation of endothelial cells. This concentration lead to a significant increase of expression of all PPAR isoforms. The results observed in this work imply, that the DEHP-metabolites MEHP and 4-Heptanon can effectively regulate expression of PPARs. Since PPARs as transcriptionfactors regulate a wide variety of processes in the human body it is likely that their metabolites might also play a role in the development of illness. However, their real health hazard can only be estimated until further knowledge about their deleterious effect can be gained.