Harriet L. Zuckerbraun

Research Strategies in the Study of the Pro-Oxidant Nature of Polyphenol Nutraceuticals

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.

Benzoyl peroxide cytotoxicity evaluated in vitro with the human keratinocyte cell line, RHEK-1

The human keratinocyte cell line, RHEK-1, was used to evaluate the cytotoxicity of benzoyl peroxide (BZP). As determined with the neutral red (NR) cytotoxicity assay, the 24-h midpoint (NR50) toxicity values, in mM, were 0.11 for BZP and 29.5 for benzoic acid, the stable metabolite of BZP. Irreversible cytotoxicity occurred after a 1-h exposure to 0.15 mM BZP and greater. When exposed to BZP for 7 days, a lag in growth kinetics was first observed at 0.06 mM BZP. Damage to the integrity of the plasma membrane was evident, as leakage of lactic acid dehydrogenase occurred during a 4-h exposure to BZP at 0.05 mM and greater. Intracellular membranes were also affected, as extensive vacuolization, initially perinuclear but then spreading throughout the cytoplasm, was noted in BZP-stressed cells. The generation of reactive free radicals from BZP was suggested by the following: the intracellular content of glutathione was lowered in cells exposed to BZP; cells pretreated with the glutathione-depleting agent, chlorodinitrobenzene, were hypersensitive to a subsequent challenge with BZP; lipid peroxidation by BZP was inducible in the presence of Fe2+; and cells previously maintained in a medium amended with vitamin E, an antioxidant, were more resistant to BZP, showed less lipid peroxidation in the presence of BZP+Fe2+ and did not develop the extensive intracellular vacuolization as compared to non-vitamin E maintained cells.

In vitro cytotoxicity of glyco-S-nitrosothiols. a novel class of nitric oxide donors.

The cytotoxicities of the nitric oxide (NO) donors, S-nitroso-N-acetylpencillamine (SNAP) and three glyco-SNAPs, glucose-1-SNAP, glucose-2-SNAP, and fructose-1-SNAP, towards the human gingival epithelioid S-G cell line and three human carcinoma cell lines derived from tissues of the oral cavity were compared using the neutral red (NR) assay. In general, the glucose-SNAPs were more cytotoxic than SNAP, which, in turn, was more cytotoxic than fructose-1-SNAP. Further studies focused on the response of S-G cells to glucose-2-SNAP. The cytotoxicity of glucose-2-SNAP was attributed to NO, as glucose-2-SNAP (t1/2=20 h at 28 degrees C) aged for 4 days was nontoxic, toxicity was eliminated in the presence of hydroxocobalamin, a specific NO scavenger, and toxicity was not noted with glucose-2-AP (the parent compound used to construct glucose-2-SNAP). Exposure of cells to glucose-2-SNAP resulted in a lessening of the intracellular level of glutathione and cells pretreated with the glutathione-depleter, 1,3-bis-(chloroethyl)-1-nitrosourea, were more sensitive to a subsequent challenge with glucose-2-SNAP. Cytotoxicity of glucose-2-SNAP was lessened upon coexposure with the antioxidants, myricetin, N-acetyl-L-cysteine, and L-ascorbic acid. S-G cells exposed to glucose-2-SNAP exhibited bi- and multinucleation. Death of S-G cells exposed to glucose-2-SNAP apparently occurred by apoptosis, as demonstrated with fluorescence microscopy by the appearance of brightly stained, hypercondensed chromatin in spherical cells and of membrane blebbing and by the DNA-ladder of oligonucleosome-length fragments noted with gel electrophoresis. In comparison with other classes of NO donors the sequence of toxicity towards S-G cells was S-nitrosoglutathione>glucose-SNAPs>SNAP, sodium nitroprusside>spermine NONOate>DPTA NONOate>DETA NONOate>fructose-1-SNAP>>SIN-1.

Pomegranate Extract, A Prooxidant with Antiproliferative and Proapoptotic Activities Preferentially Towards Carcinoma Cells

The antiproliferative and proapoptotic effects of pomegranate extract (PE), as correlated with its prooxidant activity, were studied. PE exerted greater antiproliferative effects towards cancer, than to normal, cells, isolated from the human oral cavity. In cell-free systems, PE generated hydrogen peroxide (H(2)O(2)) in cell culture media and in phosphate buffered saline, with prooxidant activity increasing from acidic to alkaline pH, and oxidized glutathione (GSH) in an alkaline, phosphate buffer. Detection of PE-generated H(2)O(2) was greatly lessened in medium amended with N-acetyl-L-cysteine. Using HSC-2 carcinoma cells as the bioindicator, the cytotoxicity of PE was potentiated towards cells pretreated with the GSH depleter, 1-chloro-2,4-dinitrobenzene, and attenuated in cells co-treated with the H(2)O(2) scavengers, catalase, pyruvate, and divalent cobalt ion. Intracellular GSH was lessened in cells treated with PE; GSH depletion in PE-treated cells was confirmed visually with the fluorescent dye, Cell Tracker™ Green 5-chloromethylfluorescein diacetate. These studies demonstrated that the antiproliferative mechanism of PE was, in part, by induction of oxidative stress. The mode of cell death was by apoptosis, as shown by flow cytometry, activation of caspase-3, and cleavage of PARP. Lessening of caspase-3 activation and of PARP cleavage in cells co-treated with PE and either cobalt or pyruvate, respectively, as compared to PE alone, indicated that apoptosis was through the prooxidant nature of PE.

Gingko biloba leaf extract induces oxidative stress in carcinoma HSC-2 cells.

The antiproliferative effects of a Gingko biloba leaf extract to cells from tissues of the human oral cavity were studied. Toxicity to carcinoma HSC-2 cells was correlated with the prooxidative nature of the extract. G. biloba leaf extract generated reactive oxygen species (ROS) in cell culture medium and, albeit to a lesser extent, in buffer, with higher levels detected at alkaline pH. Lowered levels of ROS were detected in culture medium coamended with the extract and with either catalase or superoxide dismutase, indicating the generation of hydrogen peroxide and superoxide anion, respectively. Biological activity of the extract was through oxidative stress. Toxicity to the HSC-2 cells was lessened by the ROS scavengers, divalent cobalt and pyruvate, by catalase, and by the antioxidant, N-acetyl-L-cysteine, and was potentiated by the glutathione depleters, DL-buthionine-[S,R]-sulfoximine, 1-chloro-2,4-dinitrobenzene, and bis(2-chloroethyl)-N-nitrosourea. G. biloba reacted directly with authentic glutathione and lowered the intracellular glutathione content in HSC-2 cells. Induction of apoptosis upon exposure of HSC-2 cells to G. biloba extract was noted by apoptotic cell morphologies, by TUNEL staining, and by PARP cleavage. The data strongly suggest that the prooxidative nature of the G. biloba extract was the cause of apoptotic cell death.

In vitro toxicity of sodium nitroprusside to human endothelial ECV304 cells

The cytotoxicity of sodium nitroprusside (SNP) to the human endothelial cell line, ECV304, was studied. The cytotoxicity of SNP was primarily related to the liberation of nitric oxide (NO). S-nitroso-N-acetyl-d-penicillamine (SNAP), an NO donor, was highly toxic. Other degradation products of SNP either exerted much less toxicity (i.e. cyanide and nitrite) or were non-toxic (i.e. ferricyanide and ferrocyanide). SNP induced multinucleation, inhibited cell proliferation, lowered the endogenous level of reduced glutathione (GSH), and induced apoptotic cell death. The plasma membrane was not the prime site of toxic action, as leakage of lactic acid dehydrogenase (LDH) occurred only at a relatively high concentration of SNP. Cells treated with non-toxic levels of the glutathione-depleting agents, 1-chloro-2,4-dinitrobenzene (CDNB), dl-buthionine-[S,R]-sulfoximine (BSO), and 1,3-bis-(chloroethyl)-1-nitrosourea (BCNU), were hypersensitive to subsequent exposure to SNP. The GSH status of the cells was, therefore, a key factor in determining the cytotoxicity of SNP.

In Vitro Studies on the Responses of Healthy and Cancerous Cells Derived from Tissues of the Human Oral Cavity to Tea Theaflavins and Catechins

In vitro studies with carcinoma cells from tumors of the human oral cavity indicate that theaflavins and catechins in teas have chemopreventive properties. In addition to their anti-oxidant properties, theaflavins and catechins behave as pro-oxidants, generating reactive oxygen species in cell culture media and elevating intracellular levels of reactive oxygen species. The pro-oxidant toxicity of tea polyphenols was indicated by their lowering of intracellular levels of reduced glutathione, by the lessening of their cytotoxicities in the presence of catalase and scavengers of reactive oxygen species, and by their elevated toxicities to cells pretreated with glutathione depleters. The more compromised antioxidant defense systems in cancerous cells, than in normal gingival fibroblasts, may explain their greater sensitivities to the oxidative stress induced by tea pro-oxidant polyphenols. Other chemopreventive properties of tea theaflavins and catechins include cancer cell arrest in cell cycle progression, induction of apoptosis, and inhibition of cancer cell mobility and metastasis, processes all involving molecular signaling transduction pathways.

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

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.