In vitro cytotoxicity of lipopolysaccharides for chinese hamster ovary cells

Abstract From our survey of various lipopolysaccharide (LPS) preparations, we demonstrated that three out of five commercial LPS preparations of Salmonella typhimurium were not cytotoxic for Chinese hamster ovary (CHO) cell monolayers at a concentration of 1000 μg/ml. One commercial LPS preparation produced cellular damage at a concentration of 1000 μg/ml and another at 400 μg/ml. Two S. typhimurium LPS preparations made in our laboratory were also cytotoxic at a concentration of 1000 μg/ml but not at lower concentrations. Cell-free sonic lysates of S. typhimurium TML R66 were cytotoxic when tested undiluted and up to a dilution of 1:20. Based on the 2-Keto-3-deoxyoctonate (KDO) content of all preparations, sonic lysateas were cytotoxic at KDO concentrations of 0.42 μg/ml while the KDO content of the most cytotoxic LPS preparation was 15.2 μg/ml. There was no apparent correlations between KDO content of the LPS preparations and cell detachment, leading to the conclusion that cell detachment activity of Salmonella cell lysates cannot be attributed to their LPS content.     

Heat shock inactivates cellular antioxidant defenses against hydrogen peroxide: protection by glucose

Hyperthermia is used in cancer treatment and potentiates the cytotoxicity of radiation and certain chemotherapy drugs. The mechanism(s) of heat killing and those involved in heat potentiation of cytotoxic modalities are not understood. This study examines whether heat shock causes a redox imbalance, leading to oxidative changes in Chinese hamster ovary cells. Decreases in the GSH/GSSG ratio reflected an oxidative imbalance in heated (42 degrees C) and in H(2)O(2)-challenged cells. Glucose provided protection against these changes. Glucose also protected cells against cytotoxicity of H(2)O(2) and/or hyperthermia (42 to 43 degrees C). Glucose appears to protect cells against H(2)O(2) and heat shock by providing NADPH through its metabolism via the pentose phosphate cycle (PC). When cells were deprived of glucose, there was a marked decrease in the GSH/GSSG ratio and in NADPH levels, indicating a severe redox imbalance. Glucose deprivation caused cell death, which was consistent with increased accumulation of H(2)O(2), since three distinct H(2)O(2)-detoxifying systems (N-acetyl-L-cysteine, sodium pyruvate, and catalase) rescued cells against cytotoxicity. Nontoxic levels of H(2)O(2) stimulated a corresponding increase in both PC activity and NADPH levels. NADPH levels and basal activity of the PC increased at 42 degrees C. However, the oxidant-stimulated increases in PC activity and NADPH levels were lost in heated cells. Therefore, heat shock inactivates an important cellular defense mechanism against oxidants. These findings suggest that heat shock may enhance the cytotoxicity of oxidants by inhibiting increases in PC activity following oxidative stress. These data are potentially relevant to understanding the potentiation of cytotoxicity of radiation and oxidant-generating drugs by heat shock, used in combined modality cancer treatment.     

Selection and characterization of chinese hamster ovary cells resistant to the cytotoxicity of lectins

Summary Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Pha^R) phenotype is stable in the absence of selection; its frequency in a sensitive population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminalα-mannose residues is dramatically reduced (⩽5% of the activity detected in wild-type CHO cells) in several independent Pha^R clones. These clones also exhibit (a) a decreased ability to bind [¹²⁵I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) andLens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytotoxicity of concanavalin A (Con A); (d) an increased ability to bind¹²⁵I-Con A; and (e) decreased surface galactose residues—all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistant clones from each of two complementary CHO auxotrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC or LCA, and 4–5 “lectin-resistance” phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con A^R mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Inhibition of Chinese hamster ovary cell DNA synthesis by hydrogen peroxide

The DNA synthesis inhibitory effect of hydrogen peroxide has been examined under a number of experimental conditions. Results have indicated that the effect of the oxidant is more pronounced when the treatment is performed at 37 degrees C than at 4 degrees C and in low density as compared to high density cultures. In addition, similar levels of inhibition were achieved by measuring the incorporation of radiolabelled thymidine in the presence of, or following treatment with, the oxidant. Although early events seem to be responsible for the decreased rate of DNA synthesis, it would appear that hydrogen peroxide does not alter thymidine extracellularly and/or decrease the transport of the nucleoside across the plasma membrane, which may actually be slightly augmented. Thus, the previously illustrated results may represent an underestimate of the actual capacity of the oxidant to reduce DNA synthesis. This inference is further supported by the fact that the effect of hydrogen peroxide appears markedly enhanced in cells preloaded with the radiolabelled precursor. A temporal relationship seems to exist between the steady state level of DNA single strand breaks and the extent of DNA synthesis inhibition by hydrogen peroxide. The oxidant has no effect on DNA chain elongation. In conclusion, data presented in this paper suggest that early events, involving selective effects on replicon initiation, mediate the DNA synthesis inhibitory effect of hydrogen peroxide.     

Endogenous defenses against the cytotoxicity of hydrogen peroxide in cultured rat hepatocytes

The catalase activity of cultured rat hepatocytes was inhibited by 90% pretreatment with 20 mM aminotriazole without effect on the activities of glutathione peroxidase or glutathione reductase, or on the viability of the cells over the subsequent 24 h. Glutathione reductase was inhibited by 85% by pretreatment with 300 microM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) without effect on glutathione peroxidase, catalase, or on viability. Both pretreatments sensitized the hepatocytes to the cytotoxicity of H2O2 generated either by glucose oxidase (0.05-0.5 units/ml) or by the autoxidation of the one-electron-reduced state of menadione (50-250 microM). Aminotriazole pretreatment had no effect on the GSH content of the hepatocytes. BCNU reduced GSH levels by 50%. Depletion of GSH levels to less than 20% of control by treatment with diethyl maleate, however, did not sensitize the cells to either glucose oxidase or menadione, indicating that the effect of BCNU is related to inhibition of the GSH-GSSG redox cycle rather than to the depletion of GSH. With glucose oxidase, most of the cell killing in hepatocytes pretreated with either aminotriazole or BCNU occurred between 1 and 3 h. The antioxidant diphenylphenylenediamine (DPPD) had no effect on viability at 3 h. Catalase added to the culture medium 1 h after the addition of glucose oxidase prevented the cell killing measured at 3 h. The sulfhydryl reagents dithiothreitol (200 microM), N-acetyl-L-cysteine (4 mM), and alpha-mercaptopropionyl-L-glycine (2.5 mM) prevented the cell killing with exogenous H2O2 in hepatocytes sensitized by the inhibition of catalase or glutathione reductase. With menadione, there was no killing of nonpretreated hepatocytes at 1 h, and DPPD did not prevent the cell death after 3 h. Aminotriazole pretreatment enhanced the cell killing at 3 h but not at 1 h, and DPPD was not protective. Catalase added to the medium at 1 h inhibited the cell death measured at 3 h. In contrast, menadione killed hepatocytes pretreated with BCNU within 1 h. DPPD prevented cell death at 1 h, and there was evidence of lipid peroxidation in the accumulation of malondialdehyde in the culture medium. Catalase added with menadione did not prevent the cell killing at 1 h but did prevent it at 3 h. These data indicate that catalase and the GSH-GSSG cycle are active in the defense of hepatocytes against the toxicity of H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic characterization of methotrexate-resistant chinese hamster ovary cells

Summary In a previous report, we described the selection and partial characterization of three methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained an apparent structural alteration in dihydrofolate reductase. Class II cells had an alteration affecting the permeability of the drug. Class III cells, selected from Class I cells, had an increased activity of the altered enzyme. In the work described here, it has been shown that the spontaneous mutation rate to Class I resistance is in the order of 2 × 10⁻⁹ mutations per locus per generation and that in single-step mutagenized selections the number of resistant colonies of Classes I and II are about equal. Class I and Class III resistance is expressed codominantly in somatic cell hybrids, whereas the Class II resistant marker is a recessive trait. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. This research was supported by the Medical Research Council of Canada, the National Cancer Institute of Canada and the National Institutes of Health of the United States. W. F. was a Postdoctoral Fellow of the Medical Research Council of Canada.     

Chromosomal aberrations caused by sesquiterpene lactones in chinese hamster ovary cells

The cytotoxic behaviour of 20 sesquiterpene lactones toward Chinese hamster ovary cells was examined. The structural pre-requisite for cytotoxicity was the α-methylene γ-lactone moiety. Certain sesquiterpene lactones caused chromosomal aberrations suggesting that DNA was the cellular target. The cellular target for most of these compounds, however, is probably not the nucleus and the cytotoxicity may be accounted for by Michael-type additions with sulphydryl groups of enzymes and other proteins.     

Enhancement of ricin cytotoxicity in Chinese hamster ovary cells by depletion of intracellular K+: evidence for an Na+/H+ exchange system in Chinese hamster ovary cells

Depletion of intracellular K+ has been reported to result in an arrest of the formation of coated pits in human fibroblasts (Larkin, J.M., M.S. Brown, J.L. Goldstein, and R.G.W. Anderson, 1983, Cell, 33:273-285). We have studied the effects of K+ depletion on the cytotoxicities of ricin, Pseudomonas exotoxin A, and diphtheria toxin in Chinese hamster ovary (CHO) cells. The cytotoxicities of ricin and Pseudomonas toxin were enhanced in K+-depleted CHO cells whereas the cytotoxicity of diphtheria toxin was reduced by K+ depletion. The effects of NH4Cl on the cytotoxicities of ricin, Pseudomonas toxin, and diphtheria toxin were found to be similar to those of K+ depletion, and there were no additive or synergistic effects on ricin cytotoxicity by NH4Cl in K+-depleted medium. The enhancement of ricin cytotoxicity by K+ depletion could be completely reversed by the addition of K+, Rb+, and partially by the addition of Cs+, before the ricin treatment, whereas Li+ was ineffective. These protective effects of K+ or Rb+ requires a functional Na+/K+ ATPase. CHO cells grown in K+-depleted media were found to contain 6.3-fold increase in intracellular Na+ level, concomitant with a 10-fold reduction in intracellular K+ level. The enhanced cytotoxicity of ricin in K+-free medium and the increased uptake of Na+ could be abolished by amiloride or amiloride analogues, which are known to be potent inhibitors of the Na+/H+ antiport system. Our results suggest that a depletion of intracellular K+ results in an influx of Na+, which is accompanied by the extrusion of H+. Consequently, there is an alkalinization of the cytosol and the ricin-containing endosomes. As a result, ricin is more efficiently released from the endosomes in-K+-depleted cells. Results from the studies of the binding, internalization, and degradation of 125I-ricin, and the kinetics of inhibition of protein synthesis by ricin in K+-depleted cells are consistent with this working hypothesis.     

Sensitization to the cytotoxicity of melphalan by ethacrynic acid and hyperthermia in drug-sensitive and multidrug-resistant Chinese hamster ovary cells

The ability of physical and pharmacological modulators to increase the cytotoxicity of melphalan was investigated in Chinese hamster ovary cells using a clonogenic cell survival assay. Hyperthermia has potential for use in cancer treatment, particularly as an adjuvant to chemotherapy or radiotherapy. Ethacrynic acid is a glutathione S-transferase inhibitor and also undergoes conjugation with glutathione. Interactions between hyperthermia (41-43 degrees C), ethacrynic acid and melphalan were evaluated in multidrug-resistant (CH(R)C5) cells with overexpression of P-glycoprotein (33.69-fold), and in drug-sensitive (AuxB1) cells. GST alpha was expressed at a higher level (3.65-fold) in CH(R)C5 cells than in sensitive cells, whereas levels of isoforms pi and mu were the same. GST pi was the most highly expressed isoform in the two cell populations. Ethacrynic acid was cytotoxic at elevated temperatures, while it caused little or no cytotoxicity at 37 degrees C. This effect occurred in drug-resistant and drug-sensitive cells, and attributes thermosensitizing properties to ethacrynic acid. Ethacrynic acid (20 microM) alone did not alter the cytotoxicity of melphalan at 37 degrees C. Hyperthermia potentiated drug cytotoxicity in cells, both with and without ethacrynic acid treatment. Ethacrynic acid could be useful in cancer treatment by acting as a thermosensitizer when combined with heat and by enhancing the cytotoxicity of melphalan at elevated temperatures. A major advantage arising from the use of regional hyperthermia is the ability to target drug cytotoxicity to the tumor volume. A useful finding is that ethacrynic acid, heat and/or melphalan are also effective against multidrug-resistant cells with overexpression of P-glycoprotein.     

Genetic characterization of methotrexate-resistant chinese hamster ovary cells.

In a previous report, we described the selection and partial characterization of three methotrexate (Mtx)-resistant Chinese hamster ovary cells (CHO) (1). Class I cells contained an apparent structural alteration in dihydrofolate reductase. Class II cells had an alteration affecting the permeability of the drug. Class III cells, selected from Class I cells, had an increased activity of the altered enzyme. In the work described here, it has been shown that the spontaneous mutation rate to Class I resistance is in the order of 2 X 10-9 mutations per locus per generation and that in single-step mutagenized selections the number of resistant colonies of Class I and II are about equal. Class I and Class III resistance is expressed codominantly in somatic cell hybrids, whereas the Class II resistant marker is a recessive trait.     

Inhibition of antioxidants and hyperthermia enhance bleomycin-induced cytotoxicity and lipid peroxidation in Chinese hamster ovary cells.

Regional hyperthermia has potential for human cancer treatment, particularly in combination with systemic chemotherapy or radiotherapy. Heat enhances the cytotoxic effect of certain anticancer agents such as bleomycin, but the mechanisms involved in cell killing are currently unknown. Bleomycin generates reactive oxygen species. It is likely that hyperthermia itself also increases oxidative stress in cells. We evaluate whether oxidative stress has a role in the mechanism of cell death caused by bleomycin and heat in Chinese hamster ovary cells. Heat (41 to 44 degrees C) increased cytotoxicity of bleomycin, evaluated by clonogenic cell survival. Decreased levels of cellular antioxidants should create an imbalance between prooxidant and antioxidant systems, thus enhancing cytotoxic responses to heat and to oxidant-generating drugs. We determine the involvement of four major cellular antioxidant defenses, superoxide dismutase (SOD), the glutathione redox cycle (GSH cycle), catalase, and glutathione S-transferase (GST), in cellular sensitivity to bleomycin, alone or combined with hyperthermia. These cellular defenses were inhibited by diethyldithiocarbamate, l-buthionine sulfoximine, aminotriazole, and ethacrynic acid, respectively. We show that levels of antioxidants (SOD, GSH cycle, and GST) affect cellular cytotoxic responses to bleomycin, at normal and elevated temperatures (41 to 44 degrees C), suggesting the involvement of oxidative stress. Bleomycin and iron caused oxidative damage to membrane lipids in intact cells, at 37 and 43 degrees C. Lipid peroxidation was evaluated by fluorescence detection of thiobarbituric acid-reactive products. There was an increase in damage to membrane lipids when the antioxidant defenses, SOD and catalase, were inhibited. The differing effects of antioxidant inhibitors on bleomycin-induced cytotoxicity and membrane lipid damage suggest that different mechanisms are involved in these two processes. However, free radicals appear to be involved in both cases. The marked sensitization of cells by diethyldithiocarbamate, to both bleomycin-induced cytotoxicity and lipid peroxidation, suggests that superoxide could be involved in both of these processes.     

Bead-to-bead transfer of chinese hamster ovary cells using macroporous microcarriers

Chinese hamster ovary cells (CHO-K1) were cultivated in macroporous gelatin microcarriers (CultiSpher G and CultiSpher S) in spinner flasks and a 5 1 bioreactor. Near-to-confluent cultures were harvested by bead-to-bead transfer where intact microcarriers with cells were transferred from a spinner flask to another spinner flask or to the bioreactor with naked microcarrier beads. Successful bead-to-bead transfer was achieved in various split ratios. The duration of attachment seemed to be important where the direct contact of beads to each other can be achieved by intermittent stirring. Repeated transfers were performed and at least four transfers in spinner flasks were achieved.Two variations of bead-to-bead transfer were performed in the 5 1 bioreactor either by seeding the bioreactor with near-to-confluent beads cultivated in spinner flasks orin situ transfer by adding fresh beads to the bioreactor. As in the spinner case, attachment was achieved by intermittent stirring where donor beads were in close proximity to the acceptor beads. Again successful transfers were obtained as evidenced by the good growth on acceptor beads where cell yields were in the range of 3100–4500 cells/bead.The results suggest that bead-to-bead transfer of CHO-K1 cells can be easily performed and do provide an alternative route to applications where dissolution techniques may not offer an efficient solution.     

Effect of hyperthermia on intracellular pH in Chinese hamster ovary cells

Chinese hamster ovary cells were heated at 45.5 or 43.0 degrees C at acidic pH (6.7) or normal physiological pH (7.4) to have a survival of 10(-3). The weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C), was used to measure the intracellular pH (pHi) both during and following hyperthermia. Tritiated water and a Particle Data machine were used to measure cellular volume as well. With 99.9% of the cell population destined to die clonogenically, the physiologically alive cells, as determined by the exclusion of trypan blue dye, maintained their pH differential between pHe and pHi as well as unheated cells. Furthermore, the cell's ability to regulate its pHi in response to changes in pHe was not affected by the same hyperthermic treatment. However, cellular volume decreased by 15-30% by 5 h after the onset of heat treatment. We conclude that heat does not perturb the cellular regulation of intracellular H+ concentration. Therefore, there is no thermal damage to the pHi-regulatory mechanism that could be responsible for either heat-induced reproductive cell death or low pH sensitization of heat killing.     

Arsenite induces apoptosis in chinese hamster ovary cells by generation of reactive oxygen species

Arsenic, a human carcinogen, possesses a serious environmental threat but the mechanism of its toxicity remains unclear. Knowledge of how arsenic induces cell death and how cells escape the death path may help to understand arsenic carcinogenesis. We have investigated the nature of sodium arsenite-induced cell death in Chinese hamster ovary K1 cells. Following phosphate-citric acid buffer extraction, apoptotic cells with lower DNA content than the G1 cells were detected by flow cytometry. Immediately after 4 h of 40 μM arsenite treatment, no appreciable fraction of cells with sub-G1 DNA content was detected; however, the sub-G1 cell fraction increased with postarsenite incubation time, and detectable increase started at 8 h of incubation, whereas the intracellular peroxide level as measured by the fluorescent intensity of 2′,7′-dichlorofluorescein increased immediately following a 4-h arsenite treatment. Simultaneous treatment with arsenite plus antioxidant (N-acetyl-cysteine, Trolox, and Tempo); copper ion chelator (neocuproine); protein kinase inhibitor (H-7) or protein synthesis inhibitor (cycloheximide) reduced the fraction of sub-G1 cell and internucleosomal DNA degradation. Trolox, neocuproine, or cycloheximide given after arsenite treatment also effectively reduced apoptosis. These results lead to a working hypothesis that arsenite-induced apoptosis in CHO-K1 cells is triggered by the generation of hydrogen peroxide, followed by a copper-mediated Fenton reaction that catalyzes the production of hydroxyl radicals, which selectively activates protein kinase through de novo synthesis of macromolecules. © 1996 Wiley-Liss, Inc.     

Amine oxidase, spermine, and hyperthermia induce cytotoxicity in P-glycoprotein overexpressing multidrug resistant Chinese hamster ovary cells.

Multidrug resistance is a major obstacle for the successful use of chemotherapy. The multidrug resistance phenotype is often attributed to overexpression of P-glycoprotein, which is an energy-dependent drug efflux pump. We investigated a new strategy to overcome multidrug resistance, using purified bovine serum amine oxidase, which generates two major toxic products from the polyamine spermine. The cytotoxicity of the aldehyde(s) and H2O2, produced by the enzymatic oxidation of micromolar concentrations of spermine, was evaluated in multidrug resistant Chinese hamster ovary cells CHRC5 with overexpression of P-glycoprotein, using a clonogenic cell survival assay. We examined the ability of hyperthermia (42 degrees C), and inhibition of cellular detoxification systems, to sensitize multidrug resistant cells to spermine oxidation products. Severe depletion of intracellular glutathione was achieved using L-buthionine sulfoximine and inhibition of glutathione S-transferase by ethacrynic acid. CH(R)C5 cells showed no resistance to the toxic oxidation products of spermine, relative to drug-sensitive AuxB1 cells. Exogenous catalase protected cells against cytotoxicity of H2O2, but spermine-derived aldehyde(s) still caused some cytotoxicity. Hyperthermia (42 degrees C) enhanced cytotoxicity of spermine oxidation products. Cytotoxic responses in CH(R)C5 cells were compared to the drug-sensitive cells, to determine whether there are differential responses. CH(R)C5 cells were more sensitive to the cytotoxic effect of spermine oxidation products under more extreme conditions (higher temperature, higher spermine concentration, and longer exposure time). Glutathione depletion or glutathione S-transferase inhibition also led to enhanced cytotoxicity of spermine oxidation products in CH(R)C5 and AuxB1 cells. Our findings suggest that hyperthermia, combined with toxic oxidation products generated from spermine and amine oxidase, could be useful for eliminating drug-sensitive and multidrug resistant cells.     

Mutagenicity of hydrogen peroxide in V79 Chinese hamster cells

Hydrogen peroxide (H2O2) was investigated for its potential to induce gene mutations in V79 Chinese hamster cells. Exposure of 2-3 X 10(6) cells/100-mm dish to 0.5-4.0 mM H2O2 for 1 h resulted in a concentration-dependent increase in the frequency of 6-thioguanine-resistant clones. At 4 mM H2O2 the mutation frequency was increased about 6-fold above that in controls and survival of the cells was reduced by 50%. Cytotoxicity was markedly increased at lower cell densities. When only 100-200 cells/100-mm dish were exposed to H2O2 for 1 h, 50% were killed at an H2O2 concentration as low as 60 microM. The results show that mutagenicity of H2O2 in mammalian cells in vitro has escaped attention previously because the concentrations tested were too low, presumably because the likely toxicity of H2O2 to V79 cells treated at high cell densities was overestimated.     

Effects of N-acetylcysteine amide (NACA), a thiol antioxidant on radiation-induced cytotoxicity in Chinese hamster ovary cells

Ionizing radiation is known to cause tissue damage in biological systems, mainly due to its ability to produce reactive oxygen species (ROS) in cells. Many thiol antioxidants have been used previously as radioprotectors, but their application has been limited by their toxicity. In this investigation, we have explored the possible radioprotective effects of a newly synthesized thiol antioxidant, N-acetylcysteine amide (NACA), in comparison with N-acetylcysteine (NAC), a commonly used antioxidant. Protective effects of NACA and NAC were assessed using Chinese hamster ovary (CHO) cells, irradiated with 6 gray (Gy) radiation. Oxidative stress parameters, including levels of reduced glutathione (GSH), cysteine, malondialdehyde (MDA), and activities of antioxidant enzymes like glutathione peroxidase, glutathione reductase, and catalase, were measured. Results indicate that NACA was capable of restoring GSH levels in irradiated cells in a dose dependent manner. In addition, NACA prevented radiation-induced loss in cell viability. NACA further restored levels of malondialdehyde, caspase-3 activity, and antioxidant enzyme activities to control levels. Although NAC affected cells in a similar manner to NACA, its effects were not as significant. Further, NAC was also found to be cytotoxic to cells at higher concentrations, whereas NACA was non-toxic at similar concentrations. These results suggest that NACA may be able to attenuate radiation-induced cytotoxicity, possibly by its ability to provide thiols to cells.     

The protective role of polyphenols in cytotoxicity of hydrogen peroxide.

A variety of synthetic and natural polyphenols protect mammalian cells from hydrogen peroxide (H2O2). Cytotoxicity of H2O2 on Chinese hamster V79 cells was assessed with a colony formation assay, and several polyphenols prevented the decrease in the number of colonies caused by H2O2. A study of the structure-activity relationship revealed that affinity of the polyphenols for the cell membrane and the presence of an ortho-dihydroxy moiety in their structure proved essential to this protection.     

Control of passive permeability of chinese hamster ovary cells by external and intracellular ATP

External ATP causes passive permeability change in several transformed cells, but not in untransformed cells. We studied the effect of external ATP on the passive permeability of CHO-K1 cells, a transformed clone of Chinese hamster ovary cells. Treatment of the cells with external ATP alone did not produce a permeability change, and this was observed only when a mitochondrial inhibitor, such as rotenone or oligomycin, was present together with ATP. These inhibitors reduced the concentration of intracellular ATP and a permeability change by external ATP was observed when intracellular ATP was decreased more than 70%. This requirement for permeability change of CHO-K1 cells was quite unique, since passive permeability change of other transformed cells so far tested was induced by ATP alone. Treatment of CHO-K1 cells with cyclic AMP analogues increased their sensitivity to external ATP about 2-fold. The roles of external and intracellular ATP in controlling passive permeability are discussed.     

Changes in bleb formation following hyperthermia treatment of Chinese hamster ovary cells

Chinese hamster ovary cells in suspension cultures were heated for various times at 41.5, 43.5, and 45.5 degrees C, and quantitative determinations of microblebbing and macroblebbing of the cell membrane were performed for cells maintained at 4, 25, and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating at 45.5 degrees C was dependent upon the duration of heating with increases from 40% for 5 min to 90% for 30 min. Cells exposed to lower temperatures exhibited less blebbing which was not quantifiable. The changes in bleb formation following 45.5 degrees C were dependent upon the posthyperthermia temperature: a slight decrease of macroblebbing at 25 degrees C, a decrease to 50% by 2 h at 37 degrees C, and a sharp decrease of macroblebbing to less than 10% by 1 h at 4 degrees C. Microblebbing increased slightly at 37 degrees C. When cells were transferred rapidly from the 4 degrees C posthyperthermia incubation to 37 degrees C, the bleb formation percentages returned rapidly to the higher levels which existed before posthyperthermia incubation at the lower temperatures. Gamma irradiation of 20 and 50 Gy produced only a small increase in microblebbing at longer periods (5 to 6 h) but no increase in macroblebbing. The survival of cells heated for 20 min at 45.5 degrees C was decreased 40% for suspension cells maintained at 4 degrees C for 2 to 3 h before incubation at 37 degrees C for colony formation compared to cells immediately incubated at 37 degrees C after heating. The survival of cells maintained at 25 degrees C after heating was not altered in comparison.