Untersuchungen zur Zytotoxizität und zum Wirkmechanismus einiger neuer Lösungsmittel (Ionischer Flüssigkeiten) und ihrer möglichen Kombinationswirkungen mit dem Schwermetall Cadmium.

Abstract

The heavy metal Cadmium is a extensively researched toxic and carcinogenic environmental pollutant, while Ionic Liquids, although they were first described in the 1920?s, started to catch the interest of the chemical industry only a couple of years ago.In this work on the one hand the toxicity of four imidazolium derives from a homologue order with increasing side chain length ((C4MIM)(BF4), (C6MIM)(BF4), (C8MIM)(BF4) and (C10MIM)(BF4)) on C6 rat glioma cells was analysed, the type of cell death (necrosis versus apoptosis) was investigated and its mechanism illuminated.On the other hand the present study was focused on the effects on mammalian cells in combinations of Ionic Liquids and Cadmium (CdCl2) regarding to the concentrations or exposition term.It was shown, that the toxicity of Ionic Liquids increases with the length of the side chain. (C4MIM)(BF4) and (C6MIM)(BF4) were hardly or weakly toxic, so that no EC50 could be determined up to a concentration of 3 mM, where as (C8MIM)(BF4) had an EC50 of 350 Ã ?Ã µM and (C10MIM)(BF4) of 30 Ã ?Ã µM after 24 hours incubation in the MTT viability assay. For Cadmium an EC50 of 9 Ã ?Ã µM was determined. Further analyses of effects on mitochondria have shown that either treatment with (C8MIM)(BF4) or with (C10MIM)(BF4) caused mitochondrial membrane depolarisation. The level of reactive oxygen species (ROS) did not increase either after short-term incubation with (C8MIM)(BF4) up to 3 hours, nor after treatment up to 24 hours. H2O2 mediated ROS increase was also not enhanced by Ionic Liquids. Thus damage of the cell membrane that would cause permeability for molecules of the size of the tripeptide Glutathione (GSH) was not found. On the other side, cells that were treated with Ionic Liquids had an decreasing ability to keep neutral red inside. Illuminating the possibility of cells to detoxify H2O2, it was found, that the speed of H2O2-detoxification decreased after preincubation with (C8MIM)(BF4) for 1 hour. Looking at the type of cell death it was shown, that cells treated with toxic concentrations of (C8MIM)(BF4) caused phospatidylserine translocation in cell membranes (treatment for four hours) as well as chromatin condensation (treatment for 12 and 24 hours) could be observed. Furthermore, toxic concentrations of (C8MIM)(BF4) and (C10MIM)(BF4) caused DNA fragmentation (after 24 hours). Additionally it could be shown, that (C8MIM)(BF4) in concentrations above 500 Ã ?Ã µM activated caspase-3 and caspase-9. In concentration dependent combination studies one substrate extended from subtoxic to highly toxic concentrations while the other one was kept at a weakly toxic concentration. A possible influence of the time of exposure was tested with concentrations slightly below the EC50 and preincubation times between 15 minutes and one hour for short terms and 12 hours for long terms studies. The results on combination effects of (C8MIM)(BF4) and (C10MIM)(BF4) with CdCl2 have shown, that either in combinations regarding to concentration changes as well as in time dependence combinations the effects were slightly lower than the calculated data for independent action (IA-data). For that reason it can be concluded, that cadmium and the tested Ionic Liquids might act at least in some ways antagonistic.