The use of Zn-desferrioxamine for radioprotection in mice, tissue culture, and isolated DNA.

Redox-active metals mediate oxidative injury and might also potentiate radiation damage. The iron chelator desferrioxamine (DFO), which diminishes oxidative damage in many chemical and biological systems as well as in human subjects, has a controversial role in radiobiology and reportedly acts both as a radiosensitizer and a radioprotector. The present research focused on the radioprotective activity of its zinc complex. Zn-DFO was studied using three test systems differing by their complexities: isolated DNA from pUC 19 plasmid, cultured V79 Chinese hamster cells, and C3H mice. Zn-DFO (0.5-2 mM) protected isolated DNA against gamma-radiation better than each of its components alone; however, neither Zn-DFO nor DFO (50-100 microM) alone affected the radiation sensitivity of cultured cells. With total body irradiation, Zn-DFO, but not DFO alone at 100 micromol/kg body weight, administered to mice 30 min before irradiation provided significant radioprotection (P < 0.01). Zn-DFO had an LD(50/30) of 10.3 Gy, whereas DFO and vehicle alone had LD(50/30) of 8.03 Gy and 7.91 Gy, respectively. The effect of Zn-DFO on the hemodynamic parameters in mice did not differ from that of the vehicle (saline) alone. This excludes the explanation that the radioprotective activity of Zn-DFO results from its effect on oxygen levels. In addition to the possible direct effect of Zn, other potential modes of action underlying the radioprotective activity of Zn-DFO might involve a displacement of iron and its substitution by zinc, a greater proximity of the drug to DNA, and less likely an improved penetration of the drug into cells because of its structure. The failure of Zn-DFO to protect cells in tissue cultures indicates that it has some systemic role in the whole animal, possibly due to a prolonged half-life in the animal's circulation.

The cytotoxicity of nitroxyl: possible implications for the pathophysiological role of NO.

In addition to the broad repertoire of regulatory functions nitric oxide (NO) serves in mammalian physiology, the L-arginine:NO pathway is also involved in numerous pathophysiological mechanisms. While NO itself may actually protect cells from the toxicity of reactive oxygen radicals in some cases, it has been suggested that reactive nitrogen oxide species formed from nitric oxide synthase (NOS) can be cytotoxic. In addition to NO, the one electron reduction product NO- has been proposed to be formed from NOS. We investigated the potential cytotoxic role of nitroxyl (NO-), using the nitroxyl donor Angelis's salt, (AS; sodium trioxodinitrate, Na2N2O3) as the source of NO-. As was found to be cytotoxic to Chinese hamster V79 lung fibroblast cells over a concentration range of 2-4 mM. The presence of equimolar ferricyanide (Fe(III)-(CN6)3-), which converts NO- to NO, afforded dramatic protection against AS-mediated cytotoxicity. Treatment of V79 cells with L-buthionine sulfoximine to reduce intracellular glutathione markedly enhanced AS cytotoxicity, which suggests that GSH is critical for cellular protection against the toxicity of NO-. Further experiments showed that low molecular weight transition metal complexes associated with the formation of reactive oxygen species are not involved in AS-mediated cytotoxicity since metal chelators had no effect. However, under aerobic conditions, AS was more toxic than under hypoxic conditions, suggesting that oxygen dramatically enhanced AS-mediated cytotoxicity. At a molecular level, AS exposure resulted in DNA double strand breaks in whole cells, and this effect was completely prevented by coincubation of cells with ferricyanide or Tempol. The data in this study suggest that nitroxyl may contribute to the cytotoxicity associated with an enhanced expression of the L-arginine:NO pathway under different biological conditions.

Protection from radiation-induced chromosomal aberrations by the nitroxide Tempol.

BACKGROUND: The nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable, free radical that exhibits protection from ionizing radiation damage and from oxidative stress mediated through exposure of cells to superoxide or hydrogen peroxide. Radiation protection has been observed in both in vivo and in vitro models. To understand the mechanism of Tempol-mediated radioprotection better, the production of radiation-induced chromosome aberrations was evaluated. This study analyzed Tempol-mediated radioprotection of human peripheral blood lymphocytes (PBLs). METHODS: Peripheral blood lymphocytes were exposed to control (0mM), 10 mM (Tp10), and 50 mM (Tp50) concentrations of Tempol for 20 minutes before irradiation with 0, 150, 300, and 450 cGy. One quarter ml whole blood was cultured in F12 medium and phytohemagglutinin at 37 degrees C for 49, 54, 59, and 64 hours. Colcemide was added to each sample for the last 5 hours before harvest. Cells were harvested, treated with hypotonic solution, and fixed before dropping on cold clean slides. Mitotic indices and frequency of dicentric, ring, and triradial chromosomal aberrations were determined at 1000x magnification for each treatment group at each collection point. RESULTS: Treatment of cells with Tempol alone did not induce the chromosomal aberration frequency above that for unirradiated controls. Radiation dose response curves for total chromosome aberration production revealed radioprotection for Tempol treatment for both 10 and 50 mM exposures. Tempol protection factors (assessed at 0.2 aberrations/cell level) for Tp 10 and Tp 50 were 2.2 and 2.8, respectively. CONCLUSIONS: Tempol protects against radiation-induced chromosome aberrations in human PBLs. This finding is consistent with and lends support to previous studies in which Tempol was reported to enhance cell survival and reduce radiation-induced DNA double strand breaks.

Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage.

The stable free radical Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) has been shown to protect against X-ray-induced cytotoxicity and hydrogen peroxide- or xanthine oxidase-induced cytotoxicity and mutagenicity. The ability of Tempol to protect against X-ray- or neocarzinostatin (NCS)-induced mutagenicity or DNA double-strand breaks (dsb) was studied in Chinese hamster cells. Tempol (50 mM) provided a protection factor of 2.7 against X-ray-induced mutagenicity in Chinese hamster ovary (CHO) AS52 cells, with a protection factor against cytotoxicity of 3.5. Using the field inversion gel electrophoresis technique of measuring DNA dsb, 50 mM Tempol provides a threefold reduction in DNA damage at an X-ray dose of 40 Gy. For NCS-induced damage, Tempol increased survival from 9% to 80% at 60 ng/mL NCS and reduced mutation induction by a factor of approximately 3. DNA dsb were reduced by a factor of approximately 7 at 500 ng/mL NCS. Tempol is representative of a class of stable nitroxide free radical compounds that have superoxide dismutase-mimetic activity, can oxidize metal ions such as ferrous iron that are complexed to DNA, and may also detoxify radiation-induced organoperoxide radicals by competitive scvenging. The NCS chromophore is reduced by sulfhydryls to an active form. Electron spin resonance (ESR) spectroscopy shows that 2-mercaptoethanol-activated NCS reacts with Tempol 3.5 times faster than does unactivated NCS. Thus, Tempol appears to inactivate the NCS chromophore before a substantial amount of DNA damage occurs.

4-Fluorobenzylamine and phenylalanine methyl ester conjugates of 2-nitroimidazole: evaluation as hypoxic cell radiosensitizers.

We have synthesized two 2-nitroimidazole derivatives and evaluated their hypoxic radiosensitization properties. The first, a 4-fluorobenzylamine conjugate of 2-nitroimidazole (PK-110), was designed so that it could also be labeled with the F-18 and used for positron emission tomographic imaging of hypoxia. The second, the L-phenylalanine methyl ester conjugate of 2-nitroimidazole (PK-130), was designed in an attempt to exploit amino acid transport channels to enhance drug transport into the tumor. The effects of these drugs (and SR-2508, for comparison) in vitro on the aerobic and hypoxic radiosensitivity of Chinese hamster V79 cells were evaluated using clonogenic assays. PK-130 and PK-110 at 0.1 and 1.0 mM were more efficient hypoxic cell radiosensitizers than obtained with 1.0 mM SR-2508. Marginal aerobic radiosensitization was observed for 1.0 mM treatment with PK-130 and PK-110, however, no aerobic radiosensitization was observed at 0.1 mM. Glutathione (GSH) depletion (less than 5% of control levels) by L-buthionine sulfoximine (BSO) further enhanced the SER for both PK-130 and PK-110 at 0.1 mM to 3.2 +/- 0.63 and 2.4 +/- 0.16, respectively. The results of this study encourage the in vivo tumor radiosensitization evaluation of PK-130 and PK-110.

Antimutagenicity of a low molecular weight superoxide dismutase mimic against oxidative mutagens.

A set of stable nitroxide free radicals that are used as spin labels have been shown to possess metal-independent superoxide dismutase-like activity. Unlike superoxide dismutase (SOD), these compounds are low molecular weight, and readily penetrate into the cell. A representative nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (Tempol), was investigated for antimutagenic activity in the XPRT forward mutation assay in CHO AS52 cells. AS52 cells were exposed to hydrogen peroxide, or the hypoxanthine/xanthine oxidase superoxide generating system, in the presence or absence of 10 mM Tempol. Tempol itself was not mutagenic or toxic to AS52 cells. Tempol protected cells nearly completely from the cytotoxic and mutagenic effects of hydrogen peroxide and hypoxanthine/xanthine oxidase. We have previously shown that nitroxides do not alter the extracellular concentration of hydrogen peroxide, and that they are taken up by mammalian cells, suggesting that the antimutagenic activity of Tempol is an intracellular phenomenon.

In vitro photodynamic treatment of normal and human papilloma virus-transfected keratinocytes with photofrin II and red light.

Photodynamic therapy involves the use of light of appropriate wavelength to excite a photosensitizer to result in tissue destruction. The photosensitizer dihematoporphyrin ether and red light were used to treat normal and human papilloma virus type 18-transfected keratinocytes in vitro. Although both cell lines were sensitive to treatment, normal keratinocytes retained more dihematoporphyrin ether and were more sensitive to photodynamic therapy than were transfected cells. In vitro data fail to show the selective retention of dihematoporphyrin ether reported elsewhere in the literature in papillomas and tumors. We did not find selective uptake or retention of dihematoporphyrin ether by human papilloma virus-transfected cells, despite previously published in vivo data to the contrary. Dihematoporphyrin ether retention and thus selective photosensitivity of papillomas in vivo is not caused by individual cellular differences in the processing of dihematoporphyrin ether. In addition, because in vitro results may not parallel in vivo results, both in vivo and in vitro models must be investigated in the study of phototherapeutic agents.

Biologically active metal-independent superoxide dismutase mimics.

Superoxide dismutase (SOD) is an enzyme that detoxifies superoxide (O2.-), a potentially toxic oxygen-derived species. Attempts to increase intracellular concentrations of SOD by direct application are complicated because SOD, being a relatively large molecule, does not readily cross cell membranes. We have identified a set of stable nitroxides that possess SOD-like activity, have the advantage of being low molecular weight, membrane permeable, and metal independent, and at pH 7.0 have reaction rate constants with O2.- ranging from 1.1 x 10(3) to 1.3 x 10(6) M-1 s-1. These SOD mimics protect mammalian cells from damage induced by hypoxanthine/xanthine oxidase and H2O2, although they exhibit no catalase-like activity. In addition, the nitroxide SOD mimics rapidly oxidize DNA-FeII and thus may interrupt the Fenton reaction and prevent formation of deleterious OH radicals and/or higher oxidation states of metal ions. Whether by SOD-like activity and/or interception of an electron from redox-active metal ions they protect cells from oxidative stress and may have use in basic and applied biological studies.

Misonidazole hypoxic cytotoxicity and chemosensitization in two cell lines with different intracellular glutathione levels.

Nitroimidazoles such as misonidazole (Miso) or SR-2508 are known to be cytotoxic to hypoxic cells and, with preincubation under hypoxic conditions, to sensitize cells to certain chemotherapy drugs, notably melphalan. In addition, these nitroimidazoles afford hypoxic radiosensitization; however, high intracellular glutathione (GSH) levels have been shown to significantly reduce radiosensitization by some nitroimidazoles. Using two cell lines that have an 8-fold difference in cellular GSH content, we have investigated whether inherent GSH levels influence Miso-induced hypoxic cytotoxicity, hypoxic GSH depletion, or chemosensitization to melphalan. Hypoxic incubation with varying concentrations of Miso resulted in greater cytotoxicity in Chinese hamster V79 cells than in A549 human lung adenocarcinoma cells, which have much higher GSH levels. However, the rate of GSH depletion for three concentrations of Miso was the same in the two cell lines, despite the large difference in inherent GSH levels. While the inherent sensitivity to melphalan was markedly different between the cell lines, hypoxic preincubation with 2 mM Miso with subsequent aerobic exposure to melphalan resulted in similar levels of sensitization. These results indicate that the potentiation of melphalan cytotoxicity by hypoxic Miso preincubation can occur independent of intracellular GSH levels.

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.

Evaluation of nitroimidazole hypoxic cell radiosensitizers in a human tumor cell line high in intracellular glutathione.

Five nitroimidazole hypoxic cell radiosensitizers were evaluated in a human lung adenocarcinoma cell line (A549) whose GSH level was 8-fold higher than Chinese hamster V79 cells. One millimolar concentrations of Misonidazole (MISO), SR-2508, RSU-1164, RSU-1172, and Ro-03-8799 sensitized hypoxic A549 cells to radiation, with Ro-03-8799 giving the highest sensitizer enhancement ration (SER) (2.3). However, MISO, SR-2508 and Ro-03-8799 were less effective in this cell line than in V79 cells, presumably due to higher GSH content of the A549 cells. Increased hypoxic radiosensitization was seen with 0.1 mM Ro-03-8799 after GSH depletion by BSO as compared to 0.1 mM Ro-03-8799 alone (SER-1.8 vs 1.3). The combination of GSH depletion and 0.1 mM Ro-03-8799 was considerably more toxic than 0.1 mM or 1.0 mM Ro-03-8799 alone. This sensitivity was much greater than has been observed for SR-2508. These data show that Ro-03-8799 was the most efficient hypoxic cell radiosensitizer in a human tumor cell line considerably higher in GSH than the rodent cell lines often used in hypoxic radiosensitization studies. Thus, Ro-03-8799 may be a more effective hypoxic cell sensitizer in human tumors that are high in GSH.

Radiation sensitivity of human lung cancer cell lines.

X-Ray survival curves were determined using a panel of 17 human lung cancer cell lines, with emphasis on non-small cell lung cancer (NSCLC). In contrast to classic small cell lung cancer (SCLC) cell lines, NSCLC cell lines were generally less sensitive to radiation as evidenced by higher radiation survival curve extrapolation numbers, surviving fraction values following a 2 Gy dose (SF2) and the mean inactivation dose values (D) values. The spectrum of in vitro radiation responses observed was similar to that expected in clinical practice, although mesothelioma was unexpectedly sensitive in vitro. Differences in radiosensitivity were best distinguished by comparison of SF2 values. Some NSCLC lines were relatively sensitive, and in view of this demonstrable variability in radiation sensitivity, the SF2 value may be useful for in vitro predictive assay testing of clinical specimens.

Halogenated pyrimidines as radiosensitizers for high-LET radiation.

The incorporation of iododeoxyuridine (IdUrd) into Chinese hamster cells was examined as a possible radiosensitizer for fission spectrum neutrons. Dose-response curves comparing both X rays and neutrons in the same cell line with the same IdUrd replacement showed a similar radiation enhancement for IdUrd incorporation. Enhancement ratios at the 1% survival level were 1.8 for X rays and 1.5 for fission spectrum neutrons. While the mechanism of this enhancement in the response for fission neutron radiation is unclear, these positive data should support further exploration to determine if halogenated pyrimidine incorporation results in sensitization for neutron energies employed in therapy.

Radiation sensitivity and study of glutathione and related enzymes in human colorectal cancer cell lines.

A panel of 13 human colorectal cell lines was studied, with these lines exhibiting a histological profile similar to that observed in clinical practice. In the five lines tested, variable sensitivity to radiation was observed, from the relatively sensitive NCI-H716 to the highly resistant line NCI-H630, with the latter cell line derived from a patient who had previously received radiation treatment. Glutathione levels and glutathione related enzyme activity varied widely between all 13 cell lines, showing no relationship to radiation sensitivity. The variability observed suggests that some colonic tumours may be responsive to radiation, although their identification remains difficult. However, this may prove possible by incorporation of recently developed cell adhesive matrix assays using survival following a 2 Gy radiation dose as a parameter of radiation sensitivity. This panel of human cancer cell lines offers an ideal model for the study of parameters affecting the radiosensitivity and chemosensitivity pattern of colorectal cancer cells.

Chemosensitivity testing of human lung cancer cell lines using the MTT assay.

Thirty human lung cancer cell lines were tested for chemosensitivity using the semi-automated, non-clonogenic MTT assay. The tumour cell lines came from three major categories of patients: untreated small cell lung cancer (SCLC); SCLC relapsing on chemotherapy; and non-SCLC predominantly from untreated patients. From these data IC50 values were derived for each drug in each cell line. While some inter-experimental variability was observed, the rank order of chemosensitivity of each cell line within this panel was significantly correlated between experiments. These results show that tumour cell lines derived from untreated small cell lung cancer patients were the most chemosensitive for adriamycin, melphalan, vincristine and VP16 compared to the other cell types. In addition, untreated SCLC was more sensitive than non-SCLC to BCNU and cis-platin, while vincristine was the only drug to which treated SCLC was more sensitive compared to the non-SCLC lines. In contrast, no significant differences between the lung cancer types were observed for vinblastine. Thus, this panel of lung cancer cells exhibited a drug sensitivity profile paralleling that observed in clinical practice. These results suggest that this lung cancer cell line panel in combination with a relatively simple but reproducible chemosensitivity assay, such as the MTT assay, has potential for the testing of drug combinations and evaluating new anti-cancer agents in vitro.

Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.

Drug sensitivity assays were performed using a variation of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay on V79, CHO-AuxB1, CHRC5, NCI-H460, and NCI-H249 cell lines following optimization of experimental conditions for each cell line. Results from this assay were compared with data assimilated simultaneously by clonogenic assay and by dye exclusion assay. Good correlation was observed using the CHO-AuxB1 cell line and the pleiotropic drug-resistant mutant CHRC5, with similar degrees of relative resistance observed with both the MTT and clonogenic assays. Good correlation was observed between the clonogenic and MTT assays for 1-h drug exposures, although the MTT assay was more sensitive to vinblastine. In general, the clonogenic assay was more sensitive when continuous drug exposures were utilized, although this was primarily related to the increased drug exposure time. While the use of the MTT assay in drug sensitivity testing of primary tumor samples is limited, since contaminating normal cells may also reduce the tetrazolium, the MTT assay can be semiautomated, and therefore it offers a valid, simple method of assessing chemosensitivity in established cell lines.

Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity.

Radiation survival curves were generated for V79 Chinese hamster and two human lung cancer cell lines (NCI-H460 and NCI-H249) with doubling times of 10, 20, and 85 h, respectively, using a standard clonogenic assay, a dye exclusion assay, and a semiautomated colorimetric assay utilizing a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide. Comparable results for D0 and extrapolation number (n) were observed for all assays in the lines with doubling times of 10 and 20 h. In these instances the tumor cell lines had undergone seven or more doublings after radiation. For the tumor line (H249) with an 80-h doubling time the D0S were comparable between the assays while the extrapolation number was increased in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay, a result probably related to the lower number of doublings (less than 4) after radiation. We then tested the ability of the assays to detect radiation protection and sensitization using known agents. We found that cysteamine treatment resulted in radioprotection (by a factor of 8 at 8 Gy) while 5-bromo-2-deoxyuridine incorporation caused enhancement of radiation sensitivity in all three assays. We conclude that, while optimal conditions for each cell line (cell number plated and doubling time) must be established, using characterized tumor cell lines, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay could be automated and thus be of great value in screening large numbers of potential radiosensitizers or protectors.

Glutathione dependence of neocarzinostatin cytotoxicity and mutagenicity in Chinese hamster V-79 cells.

Neocarzinostatin (NCS) is mutagenic in bacteria, yeast, fungi, and mammalian cells. In cell-free systems, DNA strand breakage induced by NCS requires a reducing agent like 2-mercaptoethanol, unless very high (greater than 100 micrograms/ml) concentrations of NCS are used. In this study, we have investigated the role of the sulfhydryl compound glutathione (GSH), which is usually the most common intracellular thiol, in the bioactivation of NCS to a toxic and mutagenic species. Chinese hamster V79 cells were pretreated with one of two GSH depleting agents, buthionine sulfoximine or diethyl maleate. These agents deplete GSH via different mechanisms, but both will lower GSH levels within the cell to less than 5% of control (untreated) values. GSH-depleted cells and control cells were then exposed to NCS concentrations of 0.5-2.5 micrograms/ml for 1 h, assayed for survival, and plated for expression of hypoxanthine-guanine phosphoribosyltransferase-negative (HGPRT-) mutants. After an expression period of 7 days, during which the cultures were subcultured twice, HGPRT- mutants were selected by plating in hypoxanthine-free medium containing 5 micrograms of 6-thioguanine per ml, at a density of 2 X 10(5) cells per 100 mm dish. NCS alone decreased the surviving fraction to about 1% at 2.5 micrograms/ml and produced dose-related increases in HGPRT-mutants that reached greater than 10 times the spontaneous mutation frequency at 2.5 micrograms NCS per ml. In GSH-depleted cells, however, NCS was only mildly cytotoxic (60-80% surviving fraction) and did not produce dose-related increases in HGPRT- mutants over cells treated only with diethyl maleate or buthionine sulfoximine. Thus, GSH appears to be the main reducing agent for the bioactivation of NCS to a toxic and mutagenic species in Chinese hamster V79 cells.

The effects of cellular glutathione elevation on the oxygen enhancement ratio.

The radiation responses at various oxygen tensions were evaluated in V79 Chinese hamster cells under conditions where their nonprotein thiols, primarily glutathione (GSH), were elevated by 2-oxothiazolidine-4-carboxylate (OTZ). OTZ, when cleaved by intracellular oxoprolinase, provides the cell with cysteine which stimulates GSH synthesis. A 2-hr pretreatment with 10 mM OTZ elevated GSH to 200% of controls. This elevation in GSH offered no protection to aerated cells; however, for O2 tensions less than or equal to 40,000 ppm modest protection was observed as evidenced by an increase in oxygen enhancement ratio. GSH elevation afforded maximal protection between 1000 and 10,000 ppm O2; however, the extent of protection was relatively small (protection factor = 1.3).

Glutathione depletion greatly reduces neocarzinostatin cytotoxicity in Chinese hamster V79 cells.

The role of the intracellular thiol glutathione in the reductive activation of neocarzinostatin was investigated in Chinese hamster V79 cells. The cells were pretreated with agents that either lower (buthionine sulfoximine or diethyl maleate) or elevate (oxothiazolidine carboxylate) intracellular glutathione levels. These cells were then exposed to 1-5 micrograms/ml neocarzinostatin for 1 h and assayed for survival. Depletion of glutathione to levels at or below the limit of detection resulted in a marked reduction in neocarzinostatin cytotoxicity, while increasing glutathione levels to 250% of control values had little or no effect on neocarzinostatin toxicity. High performance liquid chromatography analysis of cysteine in untreated and glutathione-depleted cells showed cysteine levels lower than 0.2 microM, indicating that cysteine does not play a major role in the reductive activation of neocarzinostatin in untreated or glutathione-depleted cells. When intracellular cysteine levels were artificially elevated by oxothiazolidine carboxylate treatment of glutathione-depleted cells, neocarzinostatin toxicity was about two-thirds that seen in cells with normal glutathione levels. In cell-free systems, others have shown that reducing agents such as 2-mercaptoethanol are necessary for the activation of neocarzinostatin to a species that will cleave DNA. In this study, we have identified glutathione as the major cellular reducing agent for the activation of neocarzinostatin in a mammalian cell line.