A bioassay for the simultaneous measurement of metabolic activity, membrane integrity, and lysosomal activity in cell cultures

The aim of this study was the development of an in vitro bioassay that combines several endpoints of general cytotoxicity for the screening of compounds or mixtures of compounds with potential bioactivity. The Alamar Blue assay was employed to assess metabolic activity, the Neutral Red assay was used for the assessment of membrane function and lysosomal activity, and the lactate dehydrogenase leakage assay was employed for the assessment of membrane integrity. Each assay was performed separately and in combination using a human fibroblast cell line (MRC-5). Three fungal secondary metabolites of different chemistry that affect different cellular targets were tested as model compounds: deoxynivalenol, enniatin B1, and 2-amino-14,16-dimethyloctadecan-3-ol. The obtained inhibitive compound concentrations for the assays performed separately and in combination were not significantly different (P<0.05, n=9). The combination of several cytotoxicity endpoints in a single assay increases the chance that potential bioactive/cytotoxic compounds are discovered during the screening of mixtures of natural compounds (e.g., extracts from fungal cultures or plants) when one endpoint fails and, at the same time, might give some basic information on the cellular target.