Research strategies in the study of the pro-oxidant nature of polyphenol nutraceuticals

Abstract

Polyphenols of phytochemicals are thought to exhibit chemopreventive effects against cancer. These plant-derived antioxidant polyphenols have a dual nature, also acting as pro-oxidants, generating reactive oxygen species (ROS), and causing oxidative stress. When studying the overall cytotoxicity of polyphenols, research strategies need to distinguish the cytotoxic component derived from the polyphenol per se from that derived from the generated ROS. Such strategies include (a) identifying hallmarks of oxidative damage, such as depletion of intracellular glutathione and lipid peroxidation, (b) classical manipulations, such as polyphenol exposures in the absence and presence of antioxidant enzymes (i.e., catalase and superoxide dismutase) and of antioxidants (e.g., glutathione and N-acetylcysteine) and cotreatments with glutathione depleters, and (c) more recent manipulations, such as divalent cobalt and pyruvate to scavenge ROS. Attention also must be directed to the influence of iron and copper ions and to the level of polyphenols, which mediate oxidative stress.

Figure 1

Comparative toxicities of pomegranate extract and olive fruit extract towards proliferation of human oral HSC-2 carcinoma cells as quantified with the neutral red assay, a cell viability assay. Data are presented as the mean percent of control ± S.E.M. Data for the pomegranate study are from Weisburg et al. [14] and for the olive fruit extract from Schuck (unpublished).

Figure 2

Comparative generation of hydrogen peroxide (H₂O₂) in phosphate buffer, maintained at different pH levels, and in cell culture medium supplemented with Gingko biloba extract. The Dulbecco's modified Eagle medium (DMEM) in this study was amended 10% Serum Plus, 2% fetal bovine serum, and antimicrobial agents and was the medium in which the cells were exposed to the test agents. H₂O₂, generated by the plant extract, was quantified with the FOX assay, after a 2-hour incubation at room temperature. Data, from Babich et al. [13], are presented as the mean percent of H₂O₂  ± S.E.M.

Figure 3

Decreases in intracellular reduced glutathione (GSH) following a 4-hour exposure of HSC-2 carcinoma cells in Dulbecco's modified Eagle medium amended with pomegranate extract [14] and with Ginkgo biloba extract [13]. The data are expressed as the arithmetic mean ± S.E.M.; in unexposed cells, the basal level was approximately 32 nmoles GSH/10⁶ cells.

Figure 4

Comparative toxicities of theaflavin-3,3′-digallate (TFdiG) and Ginkgo biloba extract towards human oral HSC-2 carcinoma cells, untreated (control) and treated with glutathione depleters. Cell proliferation was quantified with the neutral red assay, a cell viability assay. Data are presented as the mean percent of control ± S.E.M. Data for the TFdiG study are from Schuck et al. [19] and for G. biloba from Babich et al. [13].

Figure 5

Comparative toxicities of epigallocatechin gallate (EGCG), theaflavin-3-digallate, and theaflavin-3′-gallate extract towards human oral HSC-2 carcinoma cells in the absence and presence of exogenously added catalase. Cell proliferation was quantified with the neutral red assay, a cell viability assay. Data are presented as the mean percent of control ± S.E.M. Data for EGCG are from Weisburg et al. [12] and for the black tea theaflavin monogallates are from Babich et al. [20].

Figure 6

Comparative toxicities of pomegranate extract (PE) and olive fruit extract (OFE) towards human oral HSC-2 carcinoma cells, in Dulbecco's modified Eagle medium (DMEM) commercially formulated without pyruvate (−pyr) and with pyruvate (+pyr). Cell proliferation was quantified with the neutral red assay, a cell viability assay. Data are presented as the mean percent of control ± S.E.M. Data for pomegranate extract are from Weisburg et al. [14] and for olive fruit extract from Schuck (unpublished).

Figure 7

Comparative toxicities of theaflavin-3,3′-digallate, Ginkgo biloba extract, and pomegranate extract towards human oral HSC-2 carcinoma cells, untreated (control) and treated with cobalt chloride. Cell proliferation was quantified with the neutral red assay, a cell viability assay. Data are presented as the mean percent of control ± S.E.M. Data for the theaflavin-3,3′-gallate are from Schuck et al. [19], for G. biloba extract from Babich et al. [13], and for pomegranate extract from Weisburg et al. [14].