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The aim of this research was to assess the total antioxidant activity (TAA) of lipophilic (Lextr) and hydrophilic (Hextr) tomato extracts using in vitro chemical tests and cell-based assays, focusing on possible synergistic actions between tomato antioxidants. Both Hextr and Lextr were HPLC analysed for their carotenoids, phenolic compounds, and ascorbic acid contents. For the evaluation of TAA, extracts were assayed alone or in combination using in vitro chemical tests (TEAC, FRAP) and cell-based (CAA) assays using human hepatoma (HepG2) and human histiocytic lymphoma (U937) cells. The only carotenoid detected in Lextr was lycopene, while a mixture of phenolic compounds (chlorogenic acid, caffeic acid, and rutin) was identified in Hextr. Ascorbic acid was not found either in Hextr or in Lextr. Upon extract combination (1:1, v/v), the FRAP assay revealed additive action between Lextr and Hextr, whilst a slight synergistic action was observed in TAA as measured by the TEAC assay. Synergistic action was better revealed when TAA was analysed using either U937 or HepG2 cells. This could be explained by the presence of a multiphase media (cell membrane and extra- and intracellular media) that might facilitate the distribution and interaction of antioxidants with different polarities and different mechanisms of action.

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Cytolethal distending toxins (CDT) are considered the prototype of inhibitory cyclomodulins, and are produced by a wide range of Gram-negative pathogenic bacteria, including Escherichia coli strains of various sero- and pathotypes. CDT is a heterotripartite toxin consisting of three protein subunits, CdtA, CdtB and CdtC. The active subunit, CdtB has DNase activity and causes DNA damage and cell cycle arrest in the target cell. However, several studies have highlighted different roles for CdtA and CdtC subunits. In order to reveal the necessity of CdtA and CdtC subunit proteins in the CDT-specific phenotype, expression clones containing the cdt-V subunit genes were constructed. Using cell culture assays, we demonstrated that clones expressing only the CdtB subunit or in combination with only CdtA or CdtC were unable to trigger the specific cell cycle arrest and changes in cell morphology in HeLa cells. At the same time, the recombinant clone harbouring the whole cdt-V operon caused all the CDT-associated characteristic phenotypes. All these results verify that all the three CDT subunit proteins are necessary for the genotoxic effect caused by CDT-V.

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