Harvey Babich

Cytotoxicity and genotoxicity assays with cultured fish cells: A review

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.

In vitro cytotoxic and anti-inflammatory effects of myrrh oil on human gingival fibroblasts and epithelial cells

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 caused <10% 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.

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.

Anin vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells

Chlorhexidine digluconate is the active ingredient in mouthrinses used to prevent dental plaque and gingivitis. Thein 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-killedEscherichia coli. These reductions in toxicity were presumably due to the binding of the cat onic 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.

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.

Eugenol cytotoxicity evaluated with continuous cell lines.

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.

Cytotoxic and morphological effects of phenylpropanolamine, caffeine, nicotine, and some of their metabolites studied In vitro.

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.

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.