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Selective Cytotoxicity of a Grape Seed Proanthocyanidin Extract to Human Oral Carcinoma HSC-2 Cells

Alyssa G. Schuck, Weisburg JH, Greenbaum RE, Golfeiz MD, Segal JR, Weiss RA, Liebman EC, Zuckerbraun HL and Babich H

Grape seed extract (GSE), a nutraceutical rich in polyphenol proanthocyanidins, was evaluated for its toxicity to human oral cells. Using the neutral red cytotoxicity assay, human squamous carcinoma (HSC-2) cells were shown to be more sensitive to GSE than were gingival fibroblasts. Polyphenols undergo auto-oxidation reactions in cell culture medium, to generate reactive oxygen species, in particular, hydrogen peroxide. Using the FOX assay to quantify hydrogen peroxide, GSE, when added to cell culture medium, generated hydrogen peroxide, albeit at relatively low levels. No hydrogen peroxide was detected in the presence of catalase, which catalyzes the decomposition of hydrogen peroxide, and minor levels were detected in the presence of superoxide dismutase, which stabilizes polyphenols. Superoxide free radical, detected with nitroblue tetrazolium, was identified in GSE-amended medium. Focusing on carcinoma HSC-2 cells, the 24-hr toxicity of GSE was unaffected by the hydrogen peroxide scavengers, catalase and pyruvate, indicating that hydrogen peroxide played no role in toxicity. For HSC-2 cells cotreated with GSE and D,L-buthionine-[S,R]-sulfoximine, a depleter of intracellular glutathione, no potentiation of toxicity occurred. Over the 24 hr toxicity range, GSE did not affect the level of intracellular glutathione; however, some depletion occurred, but only at elevated GSE concentrations. GSE toxicity, apparently, was independent of oxidative stress. The 24-hr cytotoxicity of GSE to HSC-2 cells was potentiated in the presence of SOD, indicating that the proanthocyanidins per se, rather than their auto-oxidation products, accounted for toxicity. To confirm the instability of GSE in cell culture medium with a concomitant lowering of potency, studies compared medium freshly-amended with GSE to “spent” medium, i.e., GSE-amended medium left in the incubator for 24 hr prior to usage. The potency of GSE to HSC-2 cells was significantly lowered following incubated in “spent” medium for 24 hours.