A new approach to the study of toxicity of polyphenol-rich compounds

Raw data gathered by Proteon Pharmaceuticals were further analyzed by co-workers of the Medical University of Lodz.Proteon Pharmaceuticals and one of the co-authors (Magdalena Lukasiak) were responsible mainly for cell culture and for performing of HCS analysis. Raw data gathered by Proteon Pharmaceuticals were further analyzed by co-workers of the Medical University of Lodz).The toxicity of in vitro tested compounds is usually evaluated based on EC50 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 EC50. 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.Proteon Pharmaceuticalsinfo:eu-repo/grantAgreement/[European Regional Development Fund Innovative Economy Operational Programme]/UDA-POIG.01.03.01-10-129/08-00/[Production of polyphenol extracts of plant origin with antiplatelet and cardioprotective properties – FLAWOPIRYNA

Associations between cell lines used for estimating toxicity of polyphenol-rich compounds.

<p>Associations between cell lines used for estimating toxicity of polyphenol-rich compounds.</p

Typical HCS images showing the effects of kaempferol on mitochondrial membrane potential, cell membrane permeability and nuclear area in HepG2 cells.

<p>Collagen-coated wells of 96-well microplates were inoculated with 3,500 HepG2 cells. Following 24-hour incubation, the cells were treated with 175 μM kaempferol or vehicle (culture medium with DMSO) and incubated for the next 24 hours. Images of cells stained with YO-PRO 1, MitoTracker, and Hoechst 33342 were acquired with a Cellomics ArrayScan HCS Reader in order to assess the changes in mitochondrial membrane potential, cell membrane permeability and nuclear area.</p

The toxicity rank of polyphenol-rich compounds according to global AUOC-based normal score, non-adjusted averaged AUOC-based normal score and double-adjusted averaged AUOC-based normal score.

<p>The examined polyphenol-rich compounds were ranked in ascending order of toxicity through the cell lines and assays (increasing global AUOC-based normal scores).</p

Hierarchical clustering of polyphenol-rich compounds for the estimation of plant extract toxicity in the analysis of cell membrane integrity.

<p>The toxicity of polyphenol-rich compounds was examined in five cell lines: HepG2 (A), Caco-2 (B), A549 (C), HMEC-1 (D), and 3T3 cells (E), based on the AUOC scores obtained in a test of cell membrane permeability. The examined polyphenols were ranked in ascending order of toxicity towards the cells (increasing AUOC value). Naringin and kaempferol served as negative and positive controls, respectively.</p