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A cranberry juice extract (CJE), rich in proanthocyanidins, had weak prooxidant properties, generating low levels of hydrogen peroxide (H2O2) and superoxide. Generation of H2O2 was pH dependent, increasing at alkaline pH, and was lowered in the presence of catalase and, to a lesser extent, of superoxide dismutase (SOD). Growth inhibition and cytotoxicity were noted towards human oral carcinoma HSC-2 cells, with midpoint cytotoxicity at 200 µg/mL CJE, but not towards human gingival HF-1 fibroblasts. Being a mild prooxidant, CJE toxicity was unaffected by exogenous catalase and pyruvate, scavengers of H2O2, but triggered intracellular synthesis of reduced glutathione, as confirmed by cell staining with Cell Tracker™ Green. The presence of exogenous SOD potentiated the toxicity of CJE, possibly by stabilizing the CJE phenols and hindering their degradative autooxidation. Conversely, ‘spent’ CJE, i.e. CJE added to cell culture medium and incubated for 24 h at 37 °C prior to use, was much less toxic to HSC-2 cells than was freshly prepared CJE. These differences in toxicity between SOD-stabilized CJE, freshly prepared CJE, and ‘spent’ CJE were confirmed in HSC-2 cells stained with aceto-orcein, which also indicated that the mode of cell death was by the induction of apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.

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 polyphenolper sefrom 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 andN-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.

There is much interest in the positive health effects of nutraceuticals, in particular, polyphenols, which have both antioxidant and prooxidant characteristics. Pyruvate, a scavenger of hydrogen peroxide, is a component in some, but not in all, commercial formulations of cell culture media, Dulbecco’s modified Eagle’s medium in particular. This study showed that the cytotoxicities to human fibroblasts of hydrogen peroxide, tert-butyl hydroperoxide, and various prooxidant nutraceuticals were lessened in Dulbecco’s modified Eagle’s medium formulated with pyruvate, as compared to the same medium but formulated without pyruvate. Intracellular glutathione was unaffected in cells treated with hydrogen peroxide in Dulbecco’s modified Eagle’s medium formulated with pyruvate, as compared to medium formulated without pyruvate. In these studies, intracellular glutathione was analyzed in acid-soluble cell extracts by determining the oxidation of reduced glutathione by 5,5′-dithiobis(2-nitrobenzoic acid) to glutathione disulfide, with the formation of the yellow chromagen, 5-thio-2-nitrobenzoic acid, measured spectrophotometrically at 412 nm and by the visualization of reduced glutathione in cells stained with the fluorescent dye, Cell Tracker™ Green 5-chloromethylfluorescein diacetate. A survey of various cell culture media, formulated with and without pyruvate, confirmed that the level of added hydrogen peroxide was greatly lessened in those media formulated with pyruvate. This study suggested that the pyruvate status of Dulbecco’s modified Eagle’s medium be specified in the experimental design, especially in studies involving oxidative stress.

Treatment of human oral squamous carcinoma HSC-2 cells and normal GN46 fibroblasts with theaflavin-3,3'-digallate (TF-3), a polyphenol in black tea, showed a concentration and time dependent inhibition of growth, with the tumor cells more sensitive than the fibroblasts. In buffer and in cell culture medium, TF-3 generated reactive oxygen species, with lower levels detected in buffer amended with catalase and superoxide dismutase, indicating the generation of hydrogen peroxide and superoxide, respectively, and suggesting that TF-3 may be an inducer of oxidative stress. The toxicity of TF-3 was decreased in the presence of catalase, pyruvate, and divalent cobalt, all scavengers of reactive oxygen species, but was potentiated in the presence of diethyldithiocarbamate, an inhibitor of superoxide dismutase. The intracellular level of glutathione in HSC-2 cells was lessened after a 4-h exposure to 250 and 500 microM TF-3. However, for GN46 fibroblasts, a 4-h exposure to 250 microM TF-3 stimulated, but to 500 microM TF-3 lessened, intracellular glutathione. Treatment of the cells with the glutathione depleters, 1,3-bis(2-chloroethyl)-N-nitrosourea, 1-chloro-2,4-dinitrobenzene, and d,l-buthionine-[S,R]-sulfoximine potentiated the toxicity of TF-3. Induction of apoptotic cell death in HSC-2 cells treated with TF-3 was noted by apoptotic cell morphologies, by TUNEL staining, by PARP cleavage, and by elevated activity of caspase-3. Apoptosis was not noted in GN46 fibroblasts treated with TF-3.

The growth inhibitory effects of a theaflavin mixture from black tea were more pronounced to malignant (CAL27; HSC-2; HSG1) and immortalized (S-G; GT1) cells than to normal (HGF-2) cells from the human oral cavity. Studies with malignant carcinoma CAL27 cells and immortalized GT1 fibroblasts showed that cytotoxicity of the theaflavin mixture was enhanced as the exposure time was increased, with the tumor CAL27 cells more sensitive than the GT1 cells. Hydrogen peroxide (H(2)O(2)) was detected in cell culture medium amended with the theaflavin mixture. The level of H(2)O(2) in cell culture medium amended with the theaflavin mixture was lessened in the presence of catalase and CoCl(2); the level of authentic H(2)O(2) was also lessened in the presence of CoCl(2), suggesting that Co(2+) led to the rapid catalytic decomposition of H(2)O(2). The cytotoxicity of the theaflavin mixture was due, in part, to the generation in the cell culture medium of H(2)O(2), which lessened the intracellular levels of glutathione in the CAL27 cells and, to a lesser extent, in the GT1 cells. For both cell types, coexposures of the theaflavin mixture with catalase or CoCl(2) afforded protection.

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.

The in vitro cytotoxicology of triclosan, the active ingredient in some mouthrinses and dentifrices used in the prevention and treatment of gingivitis and plaque, was studied using the Smulow-Glickman (S-G) human gingival epithelial cell line. The 24 h midpoint cytotoxicity value was 0.05-0.06 mM triclosan as assessed with the neutral red (NR) assay. Triclosan is used in dentifrices in combination with either zinc citrate or sodium fluoride (NaF). The sequence of potencies of these test agents, as assessed with the NR assay, was triclosan>zinc citrate>>NaF; combinations of triclosan + zinc citrate and triclosan + NaF were additive in their toxicities. Damage to the integrity of the plasma membrane, as assessed by the leakage of lactic acid dehydrogenase during a 3-h exposure, was initially evident with 0.1 mM triclosan. When exposed to triclosan for 3 d, a lag in the growth kinetics of the S-G cells was first observed at 0.01 mM triclosan. A reduction in attachment of S-G cells to dentin chips, previously exposed to triclosan for 1 h, was noted at 0.25 mM triclosan and greater. Triclosan-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis of extracted oligonucleosomal fragments.

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.

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.

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 (H2O2) 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 H2O2 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 cotreated with the H2O2 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 TrackerTM 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 casapase-3, and cleavage of PARP. Lessening of caspase-3 activation and of PARP cleavage in cells cotreated with PE and either cobalt or pyruvate, respectively, as compared to PE alone, indicated that apoptosis was through the prooxidant nature of PE.

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.

The cytotoxicity of (-)-catechin gallate (CG), a minor polyphenolic constituent in green tea, towards cells derived from tissues of the human oral cavity was studied. The sequence of sensitivity to CG was: immortalized epithelioid gingival S-G cells>tongue squamous carcinoma CAL27 cells>salivary gland squamous carcinoma HSG cells>>normal gingival HGF-1 fibroblasts. Further studies focused on S-G cells, the cells most sensitive to CG. The response of the S-G cells to CG was dependent on the length of exposure, with midpoint cytotoxicity values of 127, 67 and 58muM CG for 1-, 2- and 3-day exposures, respectively. The sequence of sensitivity of the S-G cells to various green tea catechins was characterized as follows: CG, epicatechin gallate (ECG)>epigallocatechin gallate (EGCG)>epigallocatechin (EGC)>>epicatechin (EC), catechin (C). The cytotoxicity of CG, apparently, was not due to oxidative stress as it was a poor generator of H(2)O(2) in tissue culture medium, had no effect on the intracellular glutathione level, its cytotoxicity was unaffected by catalase, and it did not induce lipid peroxidation. However, CG did enhance Fe(2+)-induced, lipid peroxidation. CG-induced apoptosis was detected by nuclear staining, both with acridine orange and by the more specific TUNEL procedure. The lack of caspase-3 activity in cells exposed to CG and the detection of a DNA smear, rather than of discrete internucleosomal DNA fragmentation, upon agarose gel electrophoresis, suggest, possibly, that the mode of cell death was by a caspase-independent apoptotic pathway. The overall cytotoxicity of CG was similar to its epimer, ECG and both exhibited antiproliferative effects equivalent to, or stronger than, EGCG, the most abundant catechin in green tea.

This study evaluated the biologic activity of epicatechin gallate (ECG), a polyphenol in tea, to carcinoma HSC-2 cells and normal HGF-2 fibroblasts cells from the human oral cavity. The relative cytotoxicity of ECG, as compared to five other polyphenols in tea, was evaluated. For the HSC-2 carcinoma cells, ECG, catechin gallate (CG), and epigallocatechin gallate (EGCG) grouped as highly toxic, epigallocatechin (EGC) as moderately toxic, and catechin (C) and epicatechin (EC) as least toxic. For the HGF-2 fibroblasts, ECG and CG grouped as highly toxic, EGCG as moderately toxic, and EGC, C, and EC as least toxic. The cytotoxic effects of the polyphenols were more pronounced to the carcinoma, than to the normal, cells. The addition of ECG to cell culture medium led to the generation of hydrogen peroxide (H2O2). However, ECG, as compared to EGCG, was a poor generator of H2O2 and, hence, the cytotoxicity of ECG was unaffected by the presence of the antioxidants, N-acetyl cysteine and glutathione, and catalase. The cytotoxicity of ECG was unaffected by a metabolic activating system, i.e., a hepatic microsomal S-9 mix. DNA fragmentation, caspase-3 activity, and nuclear staining, both with acridine orange and the TUNEL procedure, were used to assess ECG-induced apoptosis. ECG induced apoptosis in the carcinoma HSC-2 cells, but not in the normal HGF-2 fibroblasts. This research supports those studies suggesting that tea green is an effective chemopreventive agent of oral carcinoma.

Resveratrol, a natural hydroxystilbene found in grapes and wine, may play an active role in the prevention of heart disease and cancer. Little is known of the biological activity of this molecule to cells of the oral cavity. The data presented herein provide an in vitro cytotoxicity profile of resveratrol to human gingival epithelial S-G cells. For comparative purposes, initial studies were performed with various human cell types. Based on midpoint cytotoxicity values with the neutral red (NR) assay, the sequence of sensitivity to resveratrol was: tongue squamous carcinoma SCC-25 cells>S-G cells>RHEK-1 keratinocytes≫fibroblasts (gingiva, periodontal ligament, and pulp). The neoplastic (SCC-25) and immortal, nonmalignant (S-G and RHEK-1) cells were one to three times more sensitive to resveratrol than were the low-passaged normal human fibroblasts. For the short-term assays, the sequence of sensitivity of the S-G cells to resveratrol was: BrdU ELISA (DNA synthesis)>NR (lysosomal integrity)>alamarBlue (redox status of the cell)>WST-1 (mitochondrial dehydrogenase activities). Arrest of cell growth, due to inhibition of DNA synthesis, may explain the leveling of toxicity between day 2 and 3 for a 3-day continuous exposure to resveratrol. Irreversible damage to cell proliferation was noted in S-G cells exposed to 75–150 μM resveratrol for 2 days and then subsequently maintained for another 3 days in resveratrol-free medium. The cytotoxicity of resveratrol was neither potentiated nor ameliorated in the presence of an hepatic S9 microsomal fraction. The cytotoxicity of hydrogen peroxide to S-G cells was lessened by N-acetyl- l -cysteine and quercetin, but not by resveratrol. For nitric oxide, only N-acetyl- l -cysteine reduced toxicity. The ability of resveratrol to function as an antioxidant was, therefore, not noted under these test conditions.

The cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), towards cultured human cells from oral tissue was evaluated. The toxicity of SNAP to Smulow-Glickman gingival epithelial cells was correlated with the liberation of nitric oxide, as N-acetyl-D, L-penicilIamine, the SNAP metabolites, N-acetyl-D, L-penicillamine disulfide and nitrite, and preincubated (denilrosylated) SNAP did not affect viability. Comparing equimolar concentrations of various nitric oxide donors, cytotoxicity appeared to be inversely related to the relative stability (i.e., half-life) of the test compound; the sequence of cytotoxicity for a 4 hr exposure was S-nitrosoglutathione>>spermine NONOate> SNAP>DPTA NONOate>>DETA NONOate. Intracellular reduced glutathione (GSH) was lowered in S-G cells exposed to SNAP. Pretreatment of the cells with the GSH depleter, 1, 3-bis-(chloroethyl)-1-nitrosourea (BCNU), enhanced the toxicity of SNAP. Similar findings of enhanced sensitivity to SNAP were noted with gingival fibroblasts and periodontal ligament cells pretreated with BCNU. The toxicity of SNAP towards the gingival epithelial cells was decreased by cotreatment with the antioxidants, N-acetyl-L-cysteine, L-ascorbic acid, and (+)-catechin. Cells exposed to SNAP exhibited nuclear aberrations, including multilobed nuclei and multinucleation. SNAP-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis.

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.

Human carcinoma HSC-2 cells were more sensitive to a 24 h exposure to gallic acid than were normal human gingival fibroblasts. Acting as a prooxidant, gallic acid generated hydrogen peroxide in cell culture medium. The potency of gallic acid to HSC-2 cells was significantly lessened in the presence of scavengers of hydrogen peroxide, including catalase, pyruvate, and divalent cobalt cations, and was potentiated in the presence of the intracellular glutathione depleters, 1-chloro-2,4-dinitrobenzene, bis(2-chloroethyl)-N-nitrosourea, and DL-buthionine-[S,R]-sulfoximine. Exposure of HSC-2 cells to gallic acid decreased the level of intracellular reduced glutathione, caused lipid peroxidation, and increased the level of intracellular reactive oxygen species. Flow cytometric analyses of gallic acid-treated HSC-2 cells indicated a concentration-dependent response for the induction of apoptosis, which was reversed in the presence of divalent cobalt. Immunoblot analyses of gallic acid-treated cells showed proteolytic inactivation of poly(ADP-ribose) polymerase, an indication of apoptosis, which was also reversed in the presence of divalent cobalt cations. These studies demonstrated that the cytotoxic activities of gallic acid to HSC-2 cells were mediated through autooxidation of the polyphenol, leading to the induction of oxidative stress and thereby apoptotic cell death.

The in vitro cytotoxicity of sanguinarine chloride, a dental product used in the treatment of gingivitis and plaque, was compared using cell lines and primary cells from oral human tissues. For the established cell lines, sanguinarine chloride exhibited similar potencies to S-G gingival epithelial cells and to KB carcinoma cells, whereas HGF-1 gingival fibroblasts were more tolerant. However, a gingival primary cell culture was more sensitive to sanguinarine chloride than were the established cell lines. Detailed studies were performed with the S-G cells. The 24-hr midpoint (NR50) cytotoxicity value towards the S-G cells was 7.6 microM, based on the neutral red cytotoxicity assay; vacuolization and multinucleation were noted. When exposed to sanguinarine chloride for 3 days, a lag in growth kinetics was first observed at 1.7 microM. Damage to the integrity of the plasma membrane was evident, as leakage of lactic acid dehydrogenase occurred during a 3 hr exposure to sanguinarine chloride at 0.1275 mM and greater. The cytotoxicity of sanguinarine chloride to the S-G cells was lessened in the presence of an S9 hepatic microsomal fraction from Aroclor-induced rats or by including fetal bovine serum (15%) in the exposure medium. Progressively increasing the pH from 6.0 to 7.8 enhanced the potency of sanguinarine chloride, presumably due to the enhanced uptake of the lipophilic alkanolamine form, as compared to that of the cationic iminium form.

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.