Toxicity Assessment of Food and By-Products: In Vitro Methods

The in vitro toxicity assessment using cell-based models is a powerful tool to determine key mechanisms of toxicity induced by food and by-products. The present chapter focuses on cyto- and genotoxicity in vitro methods. For the cytotoxicity assessment, herein are depicted the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) colorimetric assays. While MTT evaluates the cellular mitochondrial activity by the conversion of tetrazolium salt into formazan crystals, the LDH assay is based on a coupled enzymatic reaction where the LDH released from membrane-damaged cells oxidates lactate to generate NADH, which in turn reduces a tetrazolium salt into formazan. Moreover, the 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay, commonly used to assess the in vitro antioxidant potential, is also explored. Regarding the genotoxicity assessment, this chapter describes the histone gamma-H2AX phosphorylation (γ-H2AX) quantification by flow cytometry and comet assay (alkaline and enzymatic modified versions) methodologies. Both assays are biomarkers of DNA damage (DNA strand breaks): the first one measures the phosphorylation of the Ser-139 residue of the histone variant H2AX (γ-H2AX) by staining live cells with an γ-H2AX monoclonal antibody; the second assay is based on a single cell electrophoresis, where the fragmented and negatively charged DNA migrates out of the nucleoid body (also known as the “comet head”) and forms a DNA stain in the agarose gel (i.e., “comet tail”) when subjected to an electric field.