Toxicological Assays

The toxicity of compounds that may be present in our food varies not only in strength, but also in it’s nature of expression. We have a set of methods aimed to determine the cytotoxicity, genotoxicity or endocrine disruption of food constituents and contaminants and to elucidate the underlying mechanisms at the molecular-biological level. Our experiments are typically carried out in vitro on human carcinoma or extended primary cell lines, but might – in cooperation with our partners - also involve animal studies or human intervention studies.

The following methods are used to assess cytotoxicity at different end points:

• sulforhodamine B (SRB) assay: total protein content, cell growth

• water soluble tetrazolium 1 (WST-1) assay: mitochondrial activity, cell viability

• lactate dehydrogenase (LDH) assay: cell membrane integrity, cell viability

• live cell imaging

For the observation of genotoxic effects, our most common assay is the single cell gel electrophoresis (“comet assay”), where the induction of DNA strand breaks can be measured. Additional use of formamidopyrimidine-DNA-glycosylase (FPG) allows for the discrimination of oxidative damages and other mechanisms.

Endocrine disruption is monitored using the alkaline phosphatase reporter gene assay, where an activation of estrogen receptors can be detected, and E-screen assays, where the estrogenic impact of a compound manifests in enhanced cell proliferation.

RT-PCR is used to assess the influence of toxins on gene expression and SDS-PAGE/Western Blotting to assess protein expression or activation as well as the inhibition of signalling pathways. Also, we investigate effects on oxidative stress with dichlorofluorescein (DCF) assays and GSSG/GSH depletion assays, and the cellular uptake of compounds with LC-MS based methods.

One special field of expertise of our group is the inhibition of topoisomerases, enzymes that are crucial for DNA maintenance, which often results in genotoxic effects of active compounds. To assess these, we conduct both cell-free assays like the decatenation and relaxation assay as well as cell-based assays like the “in vivo complex of enzyme” (ICE) assay.

Additional methods include the use of fluorescence-activated cell sorting (FACS) and confocal microscopy, where various toxicological endpoints can be assessed.