A new approach for the assessment of the toxicity of polyphenol-rich compounds with the use of high content screening analysis

Abstract

The toxicity of in vitro tested compounds is usually evaluated based on AC50 values calculated from dose-response curves. However, there is a large group of compounds for which a standard four-parametric sigmoid curve fitting may be inappropriate for estimating AC50. In the present study, 22 polyphenol-rich compounds were prioritized from the least to the most toxic based on the total area under and over the dose-response curves (AUOC) in relation to baselines. The studied compounds were ranked across three key cell indicators (mitochondrial membrane potential, cell membrane integrity and nuclear size) in a panel of five cell lines (HepG2, Caco-2, A549, HMEC-1, and 3T3), using a high-content screening (HCS) assay. Regarding AUOC score values, naringin (negative control) was the least toxic phenolic compound. Aronox, spent hop extract and kale leaf extract had very low cytotoxicity with regard to mitochondrial membrane potential and cell membrane integrity, as well as nuclear morphology (nuclear area). Kaempferol (positive control) exerted strong cytotoxic effects on the mitochondrial and nuclear compartments. Extracts from buckthorn bark, walnut husk and hollyhock flower were highly cytotoxic with regard to the mitochondrion and cell membrane, but not the nucleus. We propose an alternative algorithm for the screening of a large number of agents and for identifying those with adverse cellular effects at an early stage of drug discovery, using high content screening analysis. This approach should be recommended for series of compounds producing a non-sigmoidal cell response, and for agents with unknown toxicity or mechanisms of action.

Differentiated mitochondrial function in mouse 3T3 fibroblasts and human epithelial or endothelial cells in response to chemical exposure

“…It is important to note that glycerol and isopropyl alcohol were chosen as potentially harmless substances, and these were used at very low concentrations compared to their IC 50 values (Table ), so that the observed effect could be considered unspecific. In summary, our findings indicate that AUOC score is a useful tool for the evaluation of the dose‐dependent effects of drugs and that it has successfully been validated using AC 50 as a conventional end‐point …”

“…In our previous in vitro experiments performed on five cell lines, including human epithelial and endothelial cells, 3T3 mouse fibroblasts were found to be the most sensitive to the influence of polyphenol‐rich compounds in the assessment of mitochondrial membrane potential (MMP): a sensitive marker of cytotoxicity . The present study uses the same assay and the same cell lines to examine cell sensitivity following exposure to eight well‐described chemicals known to exhibit pro‐apoptotic activity.…”

“…In some cases, the applied cells responded linearly to chemical compounds and sometimes cell response was biphasic (Figure ). As a result, the sensitivity of 3T3 fibroblasts was evaluated using the AUOC value (Figure S1), as previously reported …”

“…These cell lines had previously been used in a similar study . Changes in the MMP profiles of the treated 3T3 fibroblasts were compared to those observed in epithelial and endothelial cell lines.…”

“…Thus, the differences in mitochondria from different organs or species may be relevant during mitochondrial toxicity testing. Indeed, a recent study of the influence of polyphenol‐rich compounds on mitochondrial membrane potential found 3T3 mouse fibroblasts to be more sensitive to the tested compounds than human epithelial (A549, Caco‐2 and HepG2) and endothelial cells (HMEC‐1) . To determine whether the high sensitivity of 3T3 cell line revealed by the MMP assay is part of more general phenomenon referred to other potentially toxic agents, the present study compares changes in MMP value in mouse 3T3 fibroblasts with those of the four human cell lines mentioned above following exposure to eight different chemicals, most of which are known for their pro‐apoptotic activity.…”