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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.

This study compared the in vitro responses of human gingival fibroblasts and of carcinoma cells derived from the tongue to theaflavin-3-gallate (TF-2A) and theaflavin-3'-gallate (TF-2B), polyphenols in black tea. The antiproliferative and cytotoxic effects of the theaflavin monomers were more pronounced to the carcinoma, than to the normal, cells. In phosphate buffer at pH 7.4, the theaflavins generated hydrogen peroxide and the superoxide anion, suggesting that their mode of toxicity may be due, in part, to the induction of oxidative stress. In a cell-free assay, TF-2A and TF-2B reacted directly with reduced glutathione (GSH), in a time- and concentration-dependent manner. Intracellular storages of GSH were depleted on treatment of the cells with the theaflavin monomers. Depletion of intracellular GSH was more extensive with TF-2A than with TF-2B and was more pronounced in the carcinoma, than in the normal, cells. The toxicities of the theaflavins were potentiated when the cells were cotreated with the GSH depleter, d,l-buthionine-[S,R]-sulfoximine. In the presence of catalase, pyruvate and divalent cobalt, all scavengers of reactive oxygen species, the cytotoxicities of the theaflavins were lessened. TF-2A and TF-2B induced lipid peroxidation in the carcinoma cells, whereas in the fibroblasts, peroxidation was evident upon exposure to TF-2A, but not to TF-2B. These studies demonstrated that the black tea theaflavin monomers, TF-2A and TF-2B, act as prooxidants and induce oxidative stress, with carcinoma cells more sensitive than normal fibroblasts.

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 effects of Co 2+ (as CoCl 2 ) on the cytotoxicity of green tea polyphenol (GTP) and black tea polyphenol (BTP) extracts towards proliferation of immortalized human gingival epithelial-like S-G cells were studied. The 24 h potencies of GTP and BTP extracts, as determined with the neutral red (NR) cell viability assay, were greatly reduced in the presence of 250, but not of 50, μM Co 2+ . The cytotoxicities of the GTP and BTP extracts were due, in part, to their generation of hydrogen peroxide (H 2 O 2 ) in the cell culture medium (DMEM). Progressively increasing the concentration of Co 2+ in the tea polyphenol-amended cell culture medium resulted in a lowering of the level of H 2 O 2 . The cytotoxicity of freshly added H 2 O 2 to S-G cells was abolished in the presence of 250 μM Co 2+ and the level of freshly added H 2 O 2 to cell culture medium was progressively lowered as the concentration of Co 2+ was increased. Apparently, under the conditions of these studies, the decreases in the cytotoxicity of GTP and BTP extracts in the presence of CoCl 2 were due to the rapid catalytic decomposition by Co 2+ of the H 2 O 2 generated in the tea polyphenol-amended cell culture medium.

This study compared the in vitro responses of malignant and normal cells from the human oral cavity to tea extracts and to its main polyphenolic component, (-)-epigallocatechin gallate (EGCG). The antiproliferative effects of tea polyphenolic extracts and EGCG were more pronounced towards immortalized, tumourigenic (CAL27, HSC-2, and HSG(1)) and non-tumourigenic (S-G) cells than towards normal (GN56 and HGF-1) fibroblasts and green tea was more toxic than black tea. As the addition of tea extract or EGCG to cell culture medium led to the formation of hydrogen peroxide (H(2)O(2)), the research then focused on EGCG as an inducer of oxidative stress, using CAL27, the cancerous cells most sensitive to EGCG, HSG(1), the cancerous cells least sensitive to EGCG, and GN56 cells. The toxicity of EGCG was decreased in the presence of catalase, an enzyme that degrades H(2)O(2), or of deferoxamine, a chelator of Fe(3+). Conversely, pretreatment of the cells with the glutathione depleters, 1-chloro-2,4-dinitrobenzene and 1,3-bis(2-chloroethyl)-N-nitrosourea, potentiated the toxicity of EGCG. A 4-hr exposure to EGCG lessened the intracellular level of reduced glutathione in the CAL27 and HSG(1) cells, but not in the GN56 fibroblasts. Whereas EGCG itself did not induce lipid peroxidation, Fe(2+)-induced lipid peroxidation was potentiated by EGCG. A 72-hr exposure to cytotoxic concentrations of EGCG induced significant cytoplasmic vacuolization in all cell types. The results presented herein are consistent with EGCG acting as a prooxidant, with the cancerous cells more sensitive to oxidative stress than the normal cells.

Data on the biologic activity of protocatechuic acid are contradictory; some studies have shown that it acts as an antioxidant and suppresses chemical-induced carcinogenesis and others that it induces oxidative stress and promotes tumour formation. The anticarcinogenicity of protocatechuic acid was postulated to be related, in part, to its specific suppression of neoplastic hyperproliferation. To determine whether protocatechuic acid was preferentially antiproliferative to malignant cells, non-malignant and carcinoma cells were exposed for 24 hr to protocatechuic acid (2.5 to 25 mM) and viability was assessed with the neutral red assay. The cell lines were derived from tissues of the human oral cavity, the initial site of exposure upon ingestion of dietary protocatechuic acid, and included normal GN61 gingival fibroblasts, immortalized, non-tumorigenic S-G gingival epithelial cells, and malignant HSG1 cells derived from the salivary gland, HSC-2 cells from the floor of the oral cavity, and CAL27 cells from the tongue. Selective toxicity of protocatechuic acid to malignant cells was not observed. Furthermore, using a total cellular protein determination to quantitate cell growth, no differences in comparative sensitivities of S-G epithelial cells and HSG1 carcinoma cells were noted in a 3 day continuous exposure to 2.5 to 12.5 mM protocatechuic acid and in recovery from a 24 hr exposure to 3 to 15 mM protocatechuic acid. The S-G and HSG1 cells were then used to study the effects of elevated concentrations of protocatechuic acid on oxidative stress. For both cell types, protocatechuic acid induced oxidative stress, presumably through its bioactivation by a tyrosinase pathway. A brief exposure to 25 mM protocatechuic acid lowered the levels of intracellular glutathione and potentiated Fe2+-induced lipid peroxidation of the cells. As determined with the neutral red assay, S-G and HSG1 cells exposed briefly to a non-toxic level (0.5 mM) of the glutathione depleter, 1,3-bis(2-chloroethyl)-N-nitrosourea, were hypersensitive to a subsequent challenge with 10 mM protocatechuic acid and preexposure of the S-G and HSG1 cells to a nontoxic level of protocatechuic acid (2.5 mM) enhanced their sensitivity to a subsequent exposure to tert-butyl hydroperoxide. These findings were consistent with protocatechuic acid, at high levels (≥10 mM), acting as an inducer of oxidative stress.

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.

A modification of the Transwell insert methodology was evaluated by using the neutral red uptake (NRU) assay in a cytotoxicity test. The Transwell insert methodology was developed to assess the biocompatibility of solid materials used in dentistry and, when initially designed, used the release of radiochromium (51Cr) in the cytotoxicity assay. Another aim of this study was to evaluate different exposure regimes with which to assess cytotoxicity. The exposure regimes included: a 1-hour exposure in buffer followed by a 24-hour incubation in growth medium; a 2-hour exposure in buffer followed by a 24-hour incubation in growth medium; a 24hour exposure in serum-limited medium; and a 24-hour exposure in a serum-sufficient medium. The bioindicator target was the Smulow-Glickman (S-G) human gingival cell line and the biomaterials were dental restoratives. The Transwell insert methodology with the NRU cytotoxicity assay as the cytotoxicity endpoint was effective in differentiating the potencies of the dental restoratives; a 2-hour exposure in buffer and a 24-hour exposure in serum-limited medium were the exposure regimes that most clearly differentiated the test agents according to their potencies. The sequence of cytotoxicity of the dental restoratives to the S-G cells was Vitremer > Ketac-Molar Aplicap > Flow-It.

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.

Triclosan and sodium lauryl sulfate (SLS) are antimicrobial agents used, both singularly and in combination, in dentifrices and mouth-rinses. Studies by Waaler et al. (Scand. J. Dent. Res. 101 (1993) 192–195) with human volunteers showed that the adverse side-effects induced by SLS in mouth-rinses i.e. desquamation of oral epithelium and a burning sensation, were lessened by the addition of triclosan. However, Baert et al. (Int. J. Exp. Pathol. 77 (1996) 73–78) showed that triclosan did not protect the hamster cheek pouch mucosa from irritation caused by SLS. The studies presented herein further evaluated, using a cell culture system, the triclosan-SLS interaction. The in vitro cytotoxicities of triclosan and SLS, alone and in combination, were determined with human gingival S-G epithelial cells and GF fibroblasts. The 24-h midpoint (NR50) cytotoxicity values towards the S-G cells were 0.052 mM triclosan and 0.0075% SLS and for the GF fibroblasts the respective values were 0.095 mM triclosan and 0.0127% SLS. Both agents at their NR50 values induced vacuolization. Coexposures of triclosan and SLS were additive in their cytotoxicities towards the S-G epithelial cells and GF fibroblasts. Pretreatment with triclosan potentiated the toxicity of a subsequent exposure of SLS to the S-G cells; a similar pretreatment of the GF fibroblasts with triclosan had no effect on a subsequent challenge with SLS.

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.

Chlorhexidine digluconate is the active ingredient in mouthrinses used to prevent dental plaque and gingivitis. The in vitro cytotoxicity of chlorhexidine was evaluated with the Smulow-Glickman (S-G) gingival epithelial cell line. The potency of chlorhexidine was dependent on the length of exposure and composition of the exposure medium. The midpoint cytotoxicity values for 1-, 24-, and 72-h exposures were 0.106, 0.011, and 0.0045 mmol/L, respectively. S-G cells exposed for 2 h to chlorhexidine and then maintained for 48 h in chlorhexidine-free medium were unable to recover from the initial insult. The adverse effects of chlorhexidine on the plasma membrane were suggested by the leakage of lactic acid dehydrogenase from chlorhexidine-treated S-G cells and by the increased permeability of chlorhexidine-treated liposomes to Ca2+. The toxicity of a 24-h exposure to chlorhexidine to the S-G cells was progressively lessened as the content of fetal bovine serum (FBS) in the exposure medium was increased from 2% to 8%. The potency of a 1-h exposure to chlorhexidine was reduced in medium amended with albumin, lecithin, and heat-killed Escherichia coli. These reductions in toxicity were presumably due to the binding of the cationic chlorhexidine to the negatively charged chemical moieties of the components of FBS and of albumin and lecithin and of sites on the surfaces of bacteria. Combinations of chlorhexidine and carbamide peroxide were additive in their cytotoxicities.

The comparative acute cytotoxicities were determined for a varied spectrum of minor dietary non-nutrients that have been implicated as chemopreventive agents. Cytotoxicity was determined with the neutral red (NR) assay, using BALB/c mouse 3T3 fibroblasts as the bioindicators. Based on midpoint cytotoxicity (NR50) values, the range of cytotoxicity for the different chemicals varied by 1000 times. The sequence of potency was tannic acid, tamoxifen citrate, quercetin, benzyl and phenethyl isothiocyanate>glycyrrhetinic acid>indole-3-carbinol>caffeic acid>phytic acid>vanillin>ellagic acid> d -saccharic acid 1,4-lactone. Vanillin, at slight to moderately toxic concentrations, was the only test agent that induced multinucleation in the 3T3 fibroblasts.

The cytotoxicity of eugenol to replicating cells, as mediated by the intracellular level of glutathione and by metabolic activation, was evaluated with the neutral red (NR) assay. The cytotoxicity of eugenol to human HFF fibroblasts and human HepG2 hepatoma cells was increased somewhat in the presence of a hepatic S-9 microsomal fraction from Aroclor-induced rats or hamsters. Exposure of human HepG2 hepatoma cells to eugenol depleted the level of intracellular glutathione. Cells treated with 1-chloro-2,4-dinitrobenzene (CDNB) or buthionine sulphoximine (BSO), agents that deplete intracellular glutathione, were hypersensitive to eugenol. A 1-hr pretreatment with CDNB enhanced the cytotoxicity of eugenol, as did a 24-hr pretreatment with BSO. Intracellular glutathione levels were, apparently, significant in mediating the toxicity of eugenol.

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.

A concerted effort is currently in progress to develop alternatives to the use of live animals for the acute toxicity testing of xenobiotics. To this end, the neutral red in vitro cytotoxicity assay was developed which, although initially based on the use of mammalian cells in culture, has also been adapted for ecotoxicity studies using fish cells in culture. The neutral red assay is based on the binding of neutral red, a weakly cationic supravital dye, to the lysosomal matrix of viable cells after their incubation with toxic agents. Spectrophotometric quantitation of the extracted dye with a scanning microtitre well reader at 540nm was found to be linear with the number of surviving, viable cells. The assay has been used to determine the relative acute cytotoxicities of a broad spectrum of chemical test agents, to establish structure-toxicity relationships for series of related chemicals, to study metabolism-mediated cytotoxicity, to evaluate interactions between combinations of test agents, to evaluate differential and selective toxicities of cancer chemotherapeutics and other pharmaceuticals, and to study temperature-toxicity interactions.

Neutral red assay, as an index of cytotoxicity, has been applied to predictive screening of chemotherapeutic agents. Human hepatoma and melanoma tumor cells and normal melanocytes, keratinocytes, and fibroblasts were incubated for 2, 24, and 48 h with graded concentrations of cis-platinum (0.1 to 80 microM), doxorubicin (0.01 to 100 microM), and 5-fluorouracil (1 to 1000 microM). Cells were most sensitive after 48 h. Tumor cells, based on 50% toxicity values, were 2-4 times more sensitive than the normal cells, except for cis-platinum, where only melanoma cells, as compared to normal melanocytes, showed a marked difference in cytotoxic response. Methotrexate (1 to 10 microM) toxicity could be reversed in the presence of 100 microM of leucovorin. This sensitive, rapid, and economical assay is suitable for preclinical screening and drug development.

The sequence of cytotoxic effects for a series of mercury compounds to the BG/F epithelioid cells derived from fin tissue of bluegill sunfish was phenyl mercuric chloride greater than methyl mercuric chloride greater than ethyl mercuric chloride much greater than mercuric chloride. This sequence of in vitro cytotoxicity was similar to that observed in a 48-h LC50 in vivo acute toxicity assay with rainbow trout. Using induction of micronuclei as an indicator of genetic damage, the organomercurials, but not mercuric chloride, were noted to be clastogenic to the BG/F cells.

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.

Laboratory experiments on the transfer of a plasmid to a bacterium, whether by conjugation, transformation or transduction, are available for use in the teaching laboratory. However, the transfer of plasmid from a bacterium (prokaryote) to a plant cell (eukaryote) has received little attention. The following is an experiment for studying this latter type of genetic transformation. The Gram negative soil bacterium, Agrobacterium tumefaciens L., is the causative agent of crown gall disease, a malignant tumor, occurring on the stems and leaves of infected dicotyledonous plants. Induction of this tumor is mediated by the presence in the bacterium of the large (200 kbp in length) tumor-inducing (Ti) plasmid. Upon bacterial infection of a wounded plant, phenolic defense chemicals released from the plant activate virulence genes on the Ti plasmid, leading to the processing and transferring of a small (20-23 kbp) segment of the Ti plasmid into the host plant cell. The bacterium remains outside the plant cell, living in the intercellular spaces of the plant. By transference through a conjugation tube initiated by the bacterium, this small segment of the Ti plasmid, designated transferred DNA or T-DNA, penetrates the host plant cell and subsequently incorporates into the plant's genome. T-DNA contains genes for the syntheses of opines, chemicals used as sources of carbon and nitrogen by the infecting bacterium, and phytohormones. Overproduction of these phytohormones (i.e. auxin and cytokinin) results in uncontrolled plant cell growth and hence the development of a tumor (Gelvin & Karcher 1996; Russell 1996). In this exercise, we induce crown

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.

Limited scientific studies suggest that myrrh (Commiphora molmol) has antibacterial and anti-inflammatory activities. This study determined myrrh oil (MO) cytotoxicity to human gingival fibroblasts and epithelial cells and its effect, measured by ELISA, on interleukin (IL)-1beta-stimulated IL-6 and IL-8 production. Cell viability and cytotoxicity were determined by metabolic reduction of a tetrazolium salt to a formazan dye (MTT assay) and by release of lactate dehydrogenase (LDH) from membrane damaged (LDH release assay) cells, respectively. Based on the MTT assay, 24- and 48-h exposures to/=0.001% MO had little effect on fibroblast and epithelial cell (24-h only) viability. At 48 h, 0.0005-0.001% MO decreased epithelial cell viability 30-50%. After 24 and 48 h, MO, at/=0.005%, maximally decreased viability of all cell lines. In the LDH release assay, exposure to/=0.0001% MO caused10% cytotoxicity to all cells. At 24 h,/=0.0025% MO caused maximal cytotoxicity;/=0.001% MO caused 10-70% cytotoxicity. At longer exposure times, epithelial cells were more susceptible to cytotoxic effects of MO. There was little or no detectable IL-1beta-stimulated production of IL-6 or IL-8 by cells exposed to/=0.0025% MO, probably reflective of loss of viability. At subtoxic MO levels (0.00001-0.001%), there was a significant reduction of IL-1beta-stimulated IL-6 and IL-8 production by fibroblasts, but not by epithelial cells.

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.

Bluegill sunfish BF-2 fibroblasts were used to evaluate the in vitro cytotoxicities of 1,4-naphthoquinone (NQ), 5,8-dihydroxy-1,4-NQ, and 2,3-dichloro-1,4-NQ (dichlone); comparisons were made with previously obtained data on the response of human hepatoma HepG2 cells. For both cell types, the sequence of potency was 5,8-dihydroxy-1,4-NQ>1,4-NQ>dichlone. Dichlone, and, although to a lesser extent, 1,4-NQ and 5,8-dihydroxy-1-4-NQ, induced endoreduplication in the BF-2 cells; for the HepG2 cells, endoreduplication was induced only with dichlone. Exposures to the three NQs reduced intracellular glutathione levels in both cell types. For the BF-2 and HepG2 cells, pretreatments with buthionine sulfoximine (BSO), a glutathione-depleting agent, potentiated the cytotoxicity of 5,8-hydroxy-1,4-NQ and dichlone; pretreatment with dicoumarol, an inhibitor of DT-diaphorase, had no effect on toxicity of these two NQs. Apparently, for these two quinones the predominant metabolic pathway in both the BF-2 and HepG2 cells involved redox cycling via a one-electron reduction reaction, generating reactive oxygen intermediates that consumed intracellular glutathione. Pretreatment of the BF-2 cells with BSO, but not with dicoumarol, potentiated the toxicity of 1,4-NQ, again indicating that metabolism occurred via one electron reduction. However, for the HepG2 cells, pretreatment with dicoumarol, but not with BSO, potentiated the cytotoxicity of 1,4-NQ. Apparently, in the HepG2, as compared to the BF-2, cells, 1,4-NQ was metabolized by DT-diaphorase in a reaction involving a two electron reduction.

The cytotoxicity of the pro-oxidant fungicide dichlone (2,3-dichloro-1,4-naphthoquinone), to the human endothelial cell line, ECV304, was evaluated. The sensitivity of these cells to dichlone was intermediate between that of human hepatoblastoma HepG2 cells (least sensitive) and that of human GMO5757 fibroblasts. The midpoint cytotoxicity values for a 24-hr exposure to dichlone was about 0.02 m m when evaluated with the neutral red, acid phosphatase, and XTT tetrazolium assays. Lactic acid dehydrogenase leakage, after a 4-hr exposure, occurred initially at 0.05 m m dichlone. As with other naphthoquinones, cellular metabolism of dichlone presumably could proceed either by a one- or a two-electron reduction reaction. The enhancement of potency of dichlone towards ECV304 cells pretreated with the glutathione-depleting agents, dl -buthionine-[S,R]-sulfoximine, 1-chloro-2,4-dinitrobenzene, and 1,3-bis(chloroethyl)-1-nitrosourea; the reduction in potency of dichlone to cells pretreated with (−)-2-oxo-4-thiazolidine carboxylic acid; the decrease in intracellular glutathione on exposure to dichlone; the subtle damage to the plasma membrane of dichlone-treated cells (as detected by the leakage of lactate dehydrogenase from these cells); and the lack of potentiation of dichlone toxicity by pretreatment with dicoumarol, are all consistent with the one-electron reduction reaction as the dominant pathway and with the subsequent generation of reactive oxygen molecules. The ECV304 cell line proved to be a useful research tool to study cytotoxic injury to endothelial cells.

The acute cytotoxicities of a series of alkyl-l,4-naphthoquinones (NQ) and of 2-hydroxy-3-alkyl-l,4-NQs, as well as some amino derivatives, were evaluated with the neutral red cytotoxicity assay, using BALB/c mouse 3T3 fibroblasts. As compared to the unsubstituted 1,4-NQ: (i) Substitution at the 2 position reduced toxicity, with the extent of reduction following the sequence, hydroxyl ⪢ dimethylamino ⪢ C1–C5alkyl group, (ii) Substitution with C2–C5 alkyl groups at position 3 enhanced toxicity. As noted with the n-alkyl-l,4-NQs, increasing the chain length of the 2-hydroxy-3-alkyl-l,4-NQs did not appreciably change potency of the test agent, (iii) Substitution with amino groups at position 3 had little effect on cytotoxicity. Some differences in cytotoxicity of specific test agent were noted between the 3T3 fibroblasts and isolated rat hepatocytes, as reported in the literature.

The acute cytotoxicities of methylated phenylenediamines (PDs) were evaluated with the neutral red assay, using BALB/c 3T3 mouse fibroblasts as the bioindicators. When the test agents were grouped according to their degree of methylation, good correlations were noted between their in vitro cytotoxicity and their in vivo myotoxicity to experimental animals, as well as to their in vitro autoxidation rates. For test agents of comparable methylation, the sequence of potency was ring-methylated p-PD > N-methylated p-PD >> N-methylated o-PD > N-methylated m-PD.

The neutral red cytotoxicity assay was used in vitro to evaluate the potencies of phenylpropanolamine (PPA), nicotine, caffeine, some of their metabolites, and related chemicals. The human cell types used as targets included fibroblast (HFF), melanoma (SK-Mel/27), and hepatoma (HepG2) cell lines and early passage endothelial (ENDO) cells and keratinocytes (NHEK). For all of these cells, nicotine was more cytotoxic than cotinine, its major metabolite; in turn, cotinine was more cytotoxic than chemically related compounds such as nicotinic acid and nicotinamide. Nicotine, but neither cotinine, nicotinic acid, nor nicotinamide, induced cytoplasmic vacuolization in all the cell types tested. Except for the ENDO cells, caffeine and its metabolite, theophylline, showed approximately equivalent cytotoxic potencies. However, for the ENDO cells, caffeine was more cytotoxic than theophylline. Furthermore, the ENDO cells were 2–3 times more sensitive to caffeine and theophylline than were the other cell types. The HFF, SK-Mel/27, and HepG2 cells were more sensitive than the ENDO and NHEK cells to PPA. Phenylpropanolamine induced cytoplasmic vacuolization only in the ENDO cells. Combinations of caffeine + PPA interacted synergistically in their cytotoxicity towards the HepG2 cells; a similar synergistic interaction was not noted with the ENDO cells.

Cultured fish cells can be used in a variety of cytotoxicity and genotoxicity assays for the preliminary testing of environmental chemical hazards that may be hazardous to the aquatic biota. Such assays can also be used to evaluate synergistic and antagonistic interactions between combinations of test agents and to establish structure-activity relationships for series of related chemicals. A range of fish cell lines are available for use in such assays and a variety of endpoints may be used. To detect toxicants that require bioactivation the chosen cell line must have significant P-450 activity, or a metabolizing component must be incorporated into the assay. Fish cells in culture respond to the same chemical mutagens and clastogens that are genotoxic to mammalian cells in culture. However, since fish cells in culture are eurythermic, they represent a unique system for studying temperature as a parameter in mediating the genotoxicity and the cytotoxicity of a test agent.

Studies were conducted to determine whether the differential toxicities of the two valence states of Cr (+3 and +6) occur with fungi as they do with bacteria as noted in previous studies. It was found that mycelial growth rates were inhibited more by Cr/sup 6 +/ than by Cr/sup 3 +/. Hexavalent Cr was also more toxic than equivalent trivalent Cr to spore formation and spore germination of the fungi tested. The greater toxicity of hexavalent Cr than of trivalent Cr to fungi is in agreement with the results observed with the bacterial studies. (JMT)

Phenol, a waste product of industrial processes that is introduced into aquatic ecosystems, adversely affects the indigenous biota, including algae, protozoa, invertebrates, and vertebrates. In addition to overt toxicities, phenol causes many subtle effects to the biota, such as reduced fertility, decreased survival of the young, and inhibition of growth. The toxicity of phenol to the aquatic biota is modified by several abiotic (e.g., dissolved oxygen, salinity, water hardness, temperature) and biotic (e.g., age, size, nutritional status) factors. Phenol is also produced commercially and has many uses which bring it into direct contact with human beings. It is rapidly absorbed through the skin and by inhalation through the lungs. Upon absorption of moderate amounts of phenol, it is detoxified by conjugation with sulfuric and glucuronic acids and excreted in the urine. High exposures to phenol may be fatal to human beings; infants appear to be hypersusceptible to phenol. Although phenol is a tumor promoter, it is not a carcinogen, cocarcinogen, or teratogen and, most probably, is not a mutagen.

The toxicity of lead (Pb) to fungi in pure culture was influenced by several abiotic factors: pH, inorganic anions, clay minerals, and particulate (humic acid) and soluble organic matter. The toxicity of Pb was potentiated under acidic conditions (pH 5 and 6), and phosphate or carbonate anions reduced the toxicity, apparently as a result of the formation of sparingly soluble Pb salts. Clay minerals (montmorillonite greater than attapulgite greater than kaolinite) and particulate humic acid protected against the toxicity of Pb, presumably as the result of sorption, by cation exchange of the Pb to the exchange complexes, which reduced its availability for uptake by the fungi. Soluble organics, such as tryptone, yeast extract, cysteine, succinic acid, and increasing concentrations of neopeptone, also reduced the toxicity of Pb.

An epithelioid cell line derived from fin tissue of bluegill sunfish (designated BG/F) exhibited early indications of cell transformation upon exposure to methylazoxymethanol acetate (MAM acetate). Such changes included the induction of polyploidy, increased colony-forming efficiency, loss of contact inhibition, and formation of transformed foci. Unlike later transformation characteristics observed with mammalian cells, the MAM acetate-treated BG/F cells could not be propagated under conditions of anchorage independence in soft agar. Incubation of BG/F cells with N-methyl-N'-nitro-N-nitrosoguanidine, followed by exposure to 12-O-tetradecanoylphorbol-13-acetate, was not observed to cause cell transformation under the experimental conditions. The controls of a fibroblastic cell culture derived from gill tissue of bluegill sunfish showed spontaneous transformation after 6 months of passage, similar to the transformation observed in the experimental MAM acetate treated gill cultures.

Epithelioid cells derived from fin tissues of bluegill sunfish fingerlings were used at early passage with the neutral red assay to assess the relative cytotoxicities of organochlorine pesticides. These fin cells were able to differentiate between the cytotoxicities of various test agents, although they were less sensitive than were the BF-2 cells, an established fibroblastic cell-line derived from the caudal trunk of bluegill fry. Both the fin epithelioid and the BF-2 fibroblast cells lacked significant xenobiotic metabolising capacity, based on determinations of 7-ethoxycoumarin O-deethylase as the indicator of monooxygenase activities. Such enzymatic activity could not be induced by exposure of the cultures to Arochlor 1254.

The first of a three-part series explores the causes and consequences of the acid-rain phenomenon which is spreading rapidly across the US. Sulfur and nitrogen oxides from industrial and automotive emissions combine with atmospheric water to form dilute acids that can be transported long distances until they fall as rain. The effects on the abiotic and biotic components of both the terrestrial and aquatic ecosystems are being monitored to determine the amount of toxicity that results from changing the pH effect and to determine how widespread the geographical effects range. Reduced fossil-fuel combustion and higher emission standards will not prevent acid rain from becoming an international problem, according to Environmental Protection Agency forecasts. 74 references. (DCK)

The rainbow trout hepatoma cell line, RTH-149, was evaluated for use as a bioindicator cell type in the neutral red cytotoxicity assay. The cells were exposed for six days to various polycyclic aromatic hydrocarbons, including chemicals that are direct-acting toxicants and chemicals that require enzymatic biotransformation to cytotoxic metabolites. Whereas benzo[a]pyrene was only slightly cytotoxic, its metabolites — (±)trans-7,8-diol-benzo[a]pyrene and 3-hydroxy-benzo[a]pyrene — were highly cytotoxic. 7,12-Dimethylbenz[a]anthracene was cytotoxic, but cytotoxicity did not occur with benzo[a]anthracene, benzo[b]fluoranthene and benzo[k]fluoranthene. This cell line appears to lack sufficient xenobiotic metabolising capacity to biotransform many of these polycyclic aromatic hydrocarbons to activated cytotoxic metabolites.

Since Pb has no known biological function, elevated levels of Pb in soils and in other natural environments may adversely affect the indigenous biota, including the microbiota. Elevated levels of Pb in soil may also adversely affect microbemediated ecologic processes. There is, however, relatively little information on the mediating influence of the physicochemical factors of the recipient environment on the toxicity of Pb to microbe-mediated ecologic processes. This present study evaluated the influence of the clay minerals, kaolinite and montmorillonite, particulate humic acids, and compost on the degradation of glucose in soil.

The effects of Mn on mycelial proliferation of fungi and the effect of pH on Mn toxicity were evaluated. Results indicated that the fungi exhibited wide differences in their sensitivities to Mn. Incipient inhibition (i.e., the level of Mn at which growth inhibition was noted initially, P < 0.05) for Scopulariopsis brevicaulis and Aspergillus giganteus occurred at 100 ppM Mn; for Rhizopus stolonifer, Arthrobotrys conoides, Aspergillus niger, Aspergillus flavus, Trichoderma viride, and Penicillium vermiculatum at 500 ppM Mn; for Cephalosporium sp. at 1000 ppM Mn; and for Gliocladium sp. at 1000 to 1500 ppM Mn; growth of Aspergillus clavatus was not inhibited even at 2000 ppM Mn. No growth of S. brevicaulis occurred at 500 ppM Mn and of R. stolonifer at 1500 ppM Mn. The levels of Mn causing incipient and/or total inhibition of mycelial growth of the fungi studied were comparable to the levels reported to inhibit mycelial proliferation of some phylloplane fungi. Only A. conoides showed significant (P < 0.5) stimulation of mycelial growth by Mn; 10, 50, and 100 ppM Mn increased growth rates over control (0 ppM Mn) values. There was no consistent trend in the effect of pH on Mn toxicity to the fungi.more » However, each fungus showed a definitive response to Mn at the different pH levels. Thus, increasing the pH from 5.5 to 8.5 did not significantly affect the toxicity of Mn to Gliocladium sp., P. vermiculatum, or A. niger. The toxicity of Mn to R. stolonifer and T. viride was not different at pH 5.5 and 6.5, but increasing the pH to 7.5 or 8.5 significantly enhanced the toxicity.« less

Coliphage T1 was more sensitive than its host bacterium,Escherichia coli B, to nickel (Ni). A 5-h exposure to 100 ppm Ni in nutrient broth did not adversely affect T1, whereas 10 and 20 ppm Ni extended the lag phase of growth ofE. coli B, and no growth occurred with 40 or more ppm Ni. 5 ppm Ni enhanced the survival (after 4 wk) of T1 in sea or simulated estuarine water but was toxic (i.e., reduced viral infectivity) in lake water; 50 ppm Ni was not toxic to T1 in sea water, was moderately toxic in estuarine water, but was highly toxic in lake water; and 100 ppm Ni was toxic in all systems, with the sequence of loss in viral infectivity being lake > estuarine > sea water. 100 ppm Ni was not toxic to T1 in nutrient broth, even after 3 wk of exposure, probably because of the protective effect of the organic compounds in the broth.

A variety of microorganisms, including gram-negative and gram-positive eubacteria, actinomycetes, yeasts, and filamentous fungi, were tested for their sensitivity to cadmium (Cd). In general, the actinomycetes were more tolerant to Cd than were the eubacteria; gram-negative eubacteria were more tolerant to Cd than were gram-positive eubacteria. The period of exponential growth of the eubacteria and actinomycetes was extended in the presence of Cd. Wide extremes in sensitivity to Cd were noted among the fungi; there was no correlation between the class of fungus and tolerance to Cd. Fungal sporulation was more sensitive to Cd than was mycelial growth, as spore formation was inhibited at Cd concentrations that were noninhibitory to mycelial proliferation. The toxicity of Cd to the eubacteria, actinomycetes, and fungi appeared to be pH dependent, as toxicity was generally potentiated at pH 8 or 9.