Modification of SR 2508 sensitization in hypoxic V79 cells by manipulation of glutathione levels

This series of experiments employed the hypoxic cell sensitizer SR 2508 in concentrations ranging from 0.1 to 10 mM and V-79 cells irradiated in air or made hypoxic in glass syringes, then irradiated with 15 MV X rays. Using a series of survival curves measured at the various concentrations, K curves relating sensitizer enhancement ratio (SER) to SR 2508 concentration were calculated with normal GSH levels or with depletion of GSH to 0% using 1 mM buthionine sulfoximine (BSO) or elevation to 200% of normal using 1 mM oxothiazolidine carboxylate (OTZ). Survival curves were fitted by computer, allowing calculation of standard errors for the SER values. The depletion of GSH by BSO sensitized hypoxic and aerated cells significantly and caused more than additive enhancement of SR 2508 sensitization in hypoxic cells. Elevation of GSH with OTZ protects cells irradiated in air or hypoxia and reduces the SER obtained with SR 2508. The results further support the importance of GSH levels in influencing sensitization by nitroimidazoles.     

Potentiation of CCNU toxicity by AF-2 in V79 spheroids: implications for mechanisms of chemosensitization

Spheroids of Chinese hamster V79-171 cells were incubated for 1.5 hr. with 0-10 micrograms/ml of the nitrofuran AF-2 (a potent radiosensitizer and hypoxic cell cytotoxin) under 1-10% oxygen, then exposed to 0-3 micrograms/ml CCNU for a further 30 minutes in the presence of the AF-2 and a fluorescent bisbenzimide stain. Single cells from the disaggregated spheroids were then sorted by a dual laser cell sorter for subsequent analyses of clonogenicity. Two sorting criteria were intercompared: sorting on the basis of cell position in the spheroid (defined by the bisbenzimide uptake), or on the basis of the bound AF-2 (which is itself fluorescent). These studies indicated chemosensitization was not observed in the absence of AF-2 toxicity, cellular oxygen content modified AF-2 toxicity more than AF-2 potentiation of CCNU toxicity, and cellular susceptibility to intracellular AF-2 dominated the net response.     

Chemosensitivity testing in V79 spheroids: drug delivery and cellular microenvironment

Chinese hamster V79 multicell spheroids growing in tissue culture exhibit many of the same properties as solid tumors outgrowing their blood supply, including the spontaneous development of both noncycling and hypoxic cell populations expected to be resistant to many chemotherapeutic agents. Cell-sorting techniques were used to select cells as a function of their position (depth) within the spheroid to test this prediction. "Sensitivity profiles" of cells from various regions within spheroids after treatment with doxorubicin, bleomycin, 5-fluorouracil, carmustine, cisplatin, chlorambucil, and mitomycin are presented. Additionally, exposure of preseparated cells was used to distinguish inherent sensitivity from environmental or locational factors. For these drugs, penetration was a problem only for doxorubicin; in contrast, the microenvironment in the intact spheroid had a much greater influence on cell sensitivity.     

Microsome-mediated mutagenesis in V79 Chinese hamster cells by various nitrosamines.

A microsome-mediated mutagenesis system has been established with the V79 Chinese hamster cell line. The cells, grown in monolayer, were treated with various nitrosamines in the presence of a postmitochondrial fraction ((S15) from rat liver and a reduced nicotinamide adenine dinucleotide-generating system for 1 hr, washed and incubated for 2 to 3 hr in fresh culture medium, and then plated for toxicity and mutagenicity assays. Mutation was determined by resistance to 20mug 8-azaguanine per ml. In this assay system, the S15 fraction and cofactors by themselves were not toxic to the cells; dose-related mutagenicity and cytotoxicity were induced by N-nitrosodimethylamine (DMN) only in the presence of the S15 fraction and cofactors. Pretreatment of rats with phenobarbitone led to an approximately 2-fold increase in the mutation rate over that with tissues from untreated rats with concentrations of DMN from 10 to 50 mM, while aminoacetonitrile pretreatment reduced the mutagenic effect. Methylcholanthrene pretreatment resulted in an increase in the mutation frequency with a higher concentration of DMN (50mM). Various other nitrosamines were also assayed in the presence or absence of the S15 fraction from phenobarbitone-pretreated rats and a reduced nicotinamide adenine dinucleotide phosphate-generating system. With the exception of N-nitrosomethylphenylamine, the carcinogenic nitrosamines (DMN, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, N-nitrosodi-n-pentylamine, N-nitrosomethyl-n-propylamine, N-nitrosomorpholine, N-nitrosopyrrolidine, N-nitroso-N'-methylpiperazine, and N-nitrosomethylphenylamine) were mutagenic to the V79 Chinese hamster cells in the presence of the S15 fraction and cofactors. Neither N-nitrosodiphenylamine nor N-nitrosomethyl-tert-butylamine had a mutagenic effect. These findings show that chemical carcinogens can be tested for mutagenicity in cultured mammalian cells in the presence of a metabolic activation system. The results are discussed in relation to the carcinogenicity and mutagenicity of these compounds in other test systems.     

The mutagenicity of capsaicin and dihydrocapsaicin in V79 cells

A crude mixture of capsaicinoids (CAPs) from Capsicum frutrescens, capsaicin (C) and dihydrocapsaicin (DC) but not 4-methyl-dihydrocapsaicin (MC), a synthetic homolog of DC, were mutagenic in the V79 assay. Oxidative metabolism of C and DC by microsomes or ferricyanide yielded a dimer of C or DC. Based on the lack of mutagenicity of MC, the mutagenicities of C and DC and the supporting chemical data, a mechanism of action involving an intermediate phenoxy radical is proposed.     

DNA single-strand breaks by nitropyrenes and related compounds in Chinese hamster V79 cells

The DNA single-strand breaks (SSB) induced by nitropyrenes and related compounds were studied in Chinese hamster V79 cells. 1-Nitropyrene (1-NP), 1,6-dinitropyrene (1,6-DNP), 1,8-dinitropyrene (1,8-DNP), 1-nitrosopyrene (1-NOP) and 1-aminopyrene (1-AP) caused DNA-SSB in the cells. 1-NP induced SSB most effectively among the compounds tested, though the mutagenic activity of 1-NP towards mammalian cells has been reported to be negative or marginal. 1-NOP induced SSB less effectively than 1-NP, though 1-NOP is assumed to be the intermediate to the ultimate metabolite. From in vitro experiments, this result could be explained, at least partly, by the detoxicating function of reduced glutathione (GSH) or other sulfhydryl materials.     

Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8- hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)- 1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.      

Effects of D,L-buthionine-S,R-sulfoximine on cellular thiol levels and the oxygen effect in Chinese hamster V79 cells

The role of glutathione (GSH) and total non-protein thiols (NPSH) in repairing radiation-induced free radical damage incurred under aerated and hypoxic conditions was investigated using Chinese hamster V79 cells cultured in vitro. GSH and NPSH levels were depleted in V79 cells of varying cell densities using the gamma-glutamyl-cysteine-synthetase inhibitor, D,L-Buthionine-S,R-sulfoximine (BSO). A small change in hypoxic cell radiosensitivity could be attributed to the loss of GSH while depletion of thiols to lower levels affected both aerated and hypoxic cell radiosensitivity, resulting in no change in the OER. Only a long term incubation with BSO produced a large change in the OER, by which time many other biochemical pathways using GSH and amino acids are likely to be affected.     

Enhancement of thermal killing by polyamines. II. Uptake and metabolism of exogenous polyamines in hyperthermic chinese hamster cells

The uptake and metabolism of the polyamines spermine, spermidine, cadaverine and putrescine, previously shown to potentiate heat sensitivity, were studied in cultured Chinese hamster cells. Heat (42°C) causes enhanced uptake of exogenously supplied polyamines into the acid-soluble fraction of the cells. Putrescine is taken up exceptionally fast at 37°C, about 10 times faster than its homologue, cadaverine. This uptake is slower at 42°C. The polyamines taken up were metabolized to some extent and the metabolites were similar at 37°C and 42°C except in the case of putrescine. These results suggest that potentiation of heat-sensitivity is probably mediated by the polyamines as such and not by their metabolites. Polyamines slightly protect the cells against the inhibitory effect of heat on RNA and protein synthesis. It is suggested that exogenous polyamines interact with nucleic acids inside the cell, and this interaction may underlie their synergism with heat. The exact nature of this interaction and the way it leads to enhanced thermal sensitivity are still obscure.     

Differential responses of nascent DNA synthesis and chain elongation in V79 and V79/79 cells exposed to u.v. light and chemical mutagens

DNA repair after u.v., N-methyl-N-nitrosourea (MNU) and ethylmethanesulphonate (EMS) in Chinese hamster V79 cells and the mutagensensitive derivative V79/79 was investigated by measurementof five parameters: production of strand breaks in templateDNA, incorporation of [³H]TdR, semi-conservative and repairsynthesis, molecular weights of pulse labelled DNA after mutagenexposure (nascent synthesis) and molecular weights of DNA pulselabelled and chased after mutagen exposure (elongation and ligation).Equal template strand breakage was evident in both cell linesimmediately after MNU and EMS exposure and by 4–5 h afterMNU the extent of fragmentation was greater in V79/79 cells.After u.v. irradiation template fragmentation was evident inV79/79 but not in V79 cells, even though V79/79 cells failedto excise cyclobutane dimers and repair synthesis was demonstablein V79 cells but not in V79/79 cells after exposure to all threemutagens. The rate of incorporation of [³H]TdR during semi-conservativeDNA synthesis was inhibited equally in a dose dependent mannerafter u.v. and MNU exposure; incorporation by V79/79 cells wasinhibited to a greater extent than by V79 cells after EMS exposure.Nascent DNA synthesis was suppressed more in V79/79 cells thanin V79 cells after u.v. but to similar extents in both celllines after MNU and EMS treatment. Pulse chase experiments indicateda lower rate of elongation of nascent DNA in V79/79 cells afterMNU and u.v. exposure but little difference was detectable afterEMS.     

Detoxification of optically active bay- and fjord-region polycyclic aromatic hydrocarbon dihydrodiol epoxides by human glutathione transferase P1-1 expressed in Chinese hamster V79 cells

Dihydrodiol epoxides (DEs) are important carcinogenic metabolites of polycyclic aromatic hydrocarbons (PAHs). The metabolic formation of four stereoisomeric DEs (a pair of optically active diastereomers termed as syn- and anti-form) is possible. Glutathione tranferases (GSTs) have been demonstrated to catalyze the detoxification of DEs. Purified GSTs display remarkable differences in catalytic efficiencies towards bay- and fjord-region DEs along with a high degree of regio- and stereoselectivity. Here we determined to which extent heterologously expressed human GSTP1-1, a major GST isoform in lung, affects the mutagenicity of stereoisomeric bay-region DEs of benzo[a]pyrene in Chinese hamster V79 cells. To evaluate the influence of sterical crowding in the substrate on the activity of GSTP-1, the study was extended to the strongly mutagenic fjord-region (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene. GSTP1-1,reduced preferentially the mutagenicity (studied at the hprt locus) of (+)-anti and (+)-syn-DEs of benzo[a]pyrene (by 66 and 67%) as compared with the corresponding (-)-anti- and (-)-syn-enantiomers (by 15 and 13%). These results are in line with previous studies on the enantioselectivity of purified GSTP1-1 towards the DE isomers of benzo[a]pyrene and benzo[c]phenanthrene showing that enantiomers with (R)-configuration at the benzylic oxiranyl carbon are better substrates than those with (S)- configuration. Interestingly, the (-)-anti-DEs of benzo[c]phenanthrene and dibenzo[a,l]pyrene were efficiently detoxified by GSTP-1-1 in the constructed cell line (reduction of mutagenicity by 66 and 64%). This study demonstrates that differences in the caalytic activity seen for purified GST towards individual mutagens do not necessarily reflect the detoxification of DEs by the same enzyme in a living cell and provides further evidence that specific human GSTs play a role in the detoxification of DEs of PAHs.      

Binding of 14C-misonidazole to hypoxic cells in V79 spheroids.

The metabolism-induced binding of 14C-labelled misonidazole (MISO) to hypoxic V79 cells in multicell spheroids has been quantitated using autoradiography. Hypoxia was shown to be the major determinant of the rate of binding. Maximally hypoxic cells bound MISO several times more rapidly than necrotic material in the centre of the spheroids, and up to 50 times more rapidly than well oxygenated cells. The rate of binding to chronically hypoxic cells at the edge of the necrotic centre was 20 times less than to similar cells in other spheroids made maximally hypoxic with N2. This difference is consistent with the greater radio-sensitivity of the chronically hypoxic cells, which is a consequence of their intermediate level of oxygenation. The results indicated that the ability to bind MISO might have considerable potential as a marker for hypoxic cells in tumours. However, some binding patterns cannot be explained by the simplest model of O2 diffusion. It may be necessary to invoke more complex models of O2 diffusion or metabolic gradients within the spheroid which affect the rate of binding.     

Frequency and molecular analysis of hprt mutations induced by estradiol in Chinese hamster V79 cells

The natural hormone estradiol (E2) induces tumors in rodents and various types of DNA damage in vitro and in vivo, but has not been mutagenic in bacterial or mammalian assays. Recent reports of chromosomal and genetic lesions induced by E2 has led us to re-examine the mutation frequency and molecular alterations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in Chinese hamster V79 cells. E2 at both physiological and pharmacological concentrations (10-11, 10-10, and 10-7, 10-6 M) significantly increased the mutation frequency of the hprt gene by 2. 57-, 3.45-, 2.63-, and 8.78-fold, respectively, compared to the controls, while 10-13, 10-12, 10-9, or 10-8 M E2 induced little change (< or =0.93-fold). PCR and a molecular analysis of the hprt coding sequence identified genetic lesions in the cDNA and/or genomic DNA in 15 of the 21 picked E2-induced mutants (71%). Simple base substitutions, such as Tright curved arrow G or Tright curved arrow A transversions, were the most common mutations (8/21 or 38%) and frequently occurred at 122 bp or 407 bp of the hprt coding sequence. Deletion mutations were detected in 6 of the 21 clones (29%). An Aright curved arrow G and a Cright curved arrow T transition and a four-base insertion (TATT) were identified each in one mutant clone. A RT-PCR analysis demonstrated an abundant expression of the estrogen receptor-alpha (ERalpha). However, ICI 182,780, an antagonist of ERalpha, acted in an additive manner with E2 and increased the hprt mutation frequency. In conclusion, E2 induces a low frequency of mutations (deletions and point mutations) in V79 cells, which is consistent with the weak carcinogenic activity of this hormone. The mutagenic effects of E2 in V79 cells are not mediated by the ERalpha.     

Depletion of cellular glutathione by N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea as a determinant of sensitivity of K562 human leukemia cells to 4-hydroperoxycyclophosphamide

N,N'-Bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea (BHCNU) is a nitrosourea which has carbamoylating but not alkylating activity. It has been shown to carbamoylate and inactivate glutathione reductase thereby reducing the intracellular levels of glutathione (GSH). Since GSH depletion by buthionine-S,R-sulfoximine potentiates the cytotoxicity of cyclophosphamide, with a corresponding increase in DNA cross-linking, we have investigated the potential interaction between BHCNU and cyclophosphamide. Treatment of K562 human leukemia cells with 15 microM BHCNU for 1 h resulted in depletion of glutathione to 40% of control values, without significant reduction of cell viability. Subsequent treatment with 10 microM 4-hydroperoxycyclophosphamide (4-HC), a self-activating derivative of cyclophosphamide, reduced the level of glutathione to less than 20% of control values. BHCNU pretreatment enhanced the cytotoxicity of 4-HC resulting in a dose modification factor of 2.5. Alkaline elution analysis of cellular DNA demonstrated that the level of interstrand cross-linking was 2-fold higher in GSH-depleted cells than in nondepleted cells, and the induction of single strand breaks was markedly increased. These findings demonstrate that BHCNU potentiates the cytotoxicity of 4-HC and suggest that this is due to the increased formation of DNA interstrand cross-links caused by a reduced intracellular conjugation of 4-HC with glutathione which results in an increased binding of 4-HC to DNA targets.     

Factors involved in depletion of glutathione from A549 human lung carcinoma cells: implications for radiotherapy

We have measured the rate of GSH resynthesis in plateau phase cultures of A549 human lung carcinoma cells subjected to a fresh medium change. Buthionine sulfoximine (BSO) blocks this resynthesis. Diethyl maleate (DEM) causes a decrease in accumulation of GSH. If DEM is added concurrently with BSO there is a rapid decline in GSH that is maximal in the presence of 0.5 mM DEM. GSH depletion rapidly occurs when BSO is added to log phase cultures which initially are higher in GSH content. Twenty-four hr treatment of A549 cells with BSO results in cells that are more radiosensitive in air and show a slight hypoxic radiation response. A 2 hr treatment with either 0.25 mM or 0.5 mM DEM results in some hypoxic sensitization and little increase in the aerobic radiation response. The 24 hr BSO + 2 hr DEM treatment sensitizes hypoxic cells to a greater degree than either agent alone but does not increase the aerobic response more than is seen with BSO alone. Cells treated simultaneously with BSO + DEM show little increase in the hypoxic radiation response, compared to DEM alone, but are more sensitive under aerobic conditions. Decreased cell survival for aerobically irradiated log phase A549 cells occurs within minutes after addition of a mixture of BSO + DEM. The decreased cell survival following aerobic irradiation, after prolonged treatment with BSO or acute exposure to BSO + DEM, may be in part due to inhibition of glutathione peroxidases. For example, glutathione-S-transferase, known to have glutathione peroxidase activity (non-selenium), is nearly completely inhibited by the BSO treatments. In addition, cellular capacity to react with peroxide (glutathione peroxidase, selenium containing) was also inhibited. We suggest that the enhanced aerobic radiation response is related to an inability of GSH depleted cells to inactivate either peroxy radicals or hydroperoxides that may be produced during irradiation of BSO treated cells. Furthermore, enhancement of the aerobic radiation response may be useful in vivo if normal tissue responses are not also increased.     

Superoxide anion production and toxicity in V79 cells of six hydroxy-anthraquinones.

This study investigates the cytotoxic and genotoxic effects of various carboxy AQ, 1,4-dihydroxy 6-carboxy AQ, 1,8-dihydroxy 3-carboxy AQ, 1,4-dihydroxy AQ, 1,5-dihydroxy AQ, 1,8-dihydroxy AQ and 2,6-dihydroxy AQ in V79 Chinese hamster cells. The V79 cells were used since, as they contain flavoproteins but not cytochrome P-450, they can bioactive xenobiotics only through the reductive pathway excluding the oxidative one. In addition, the abilities of AQs to stimulate O2-production using both purified flavoproteins (NADH-dehydrogenase, NADPH-cytochrome P-450 reductase) and V79 subcellular fractions (homogenate and microsomes) were assayed. The NADH and NADPH consumption stimulated by AQs in V79 microsomes was also determined. The results showed that the carboxylic-containing drugs and the 1,4-dihydroxy AQ were weak sister chromatid exchange inducers and the most toxic among the six anthraquinones examined. Dicumarol, a potent inhibitor of DT-diaphorase, reduced, rather than potentiated, both the cytotoxicity and genotoxicity caused by these AQs. Thus, the higher superoxide formation rates stimulated by the carboxylic-containing AQs compared to those of the other quinones with all the in vitro systems used, suggested, except for the 1,4-dihydroxy AQ, a possible relationship between cytotoxicity and O2-production. For the 1,4-dihydroxy AQ toxicity, a specific bioactivation route was hypothesized.     

Radioprotective effects of troxerutin against gamma irradiation in V79 cells and mice

The purpose of this study was to determine radioprotective effects of troxerutin. Cell experiments were carried out to test the cytotoxicity of troxerutin on V79 cells and to observe effects on apoptosis caused by 60CO γ rays. A model of 8 Gy ray-caused damage of mice was established to observe the effect that troxerutin has on the physical symptom of irradiated mice and to calculate the 30-day survival rate. It showed that troxerutin had no obvious cytotoxicity at the level of less than 20 μg/ml; but had a redioprotective effect in dose-dependence on viability of V79 cells at the range of 0.2-5 μg/mL irradiated by 5 Gy ray of 60CO γ ray. After the 8 Gy irradiation, the mice lost some weight, were dried up in fur and feather, low spirit, awkward in movement, shrinking in body and handicapped in sight, while mice with troxerutin were much better. So it was clear that troxerutin could increase the 30-day survival rates of irradiated mice dramatically. These results collectively indicate that troxerutin is an effective radioprotective agent.