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 investi- gated in Chinese hamster V79 cells. The cells were pretreated with agents that either lower (buthionine sulfoximine or diethyl maleate) or elevate (oxothiazo- lidine carboxylate) intracellular glutathione levels. These cells were then exposed to 1-6 pg/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 neo- carzinostatin toxicity. High performance liquid chro-matography analysis of cysteine in untreated and glu-tathione-depleted cells showed cysteine levels lower than 0.2 m , indicating that cysteine does not play a major role in the reductive activation of neocarzinos- tatin in untreated OT glutathione-deplet~ cells. When intracellular cysteine levels were artificially elevated by oxothiazolidine carboxylate treatment of glutathi-one-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 neces- sary for the activation of neocarzinostatin normal tissues to a greater extent than in the tumor tissue. Our work suggests that before

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 1-6 pg/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 m , indicating that cysteine does not play a major role in the reductive activation of neocarzinostatin in untreated OT glutathione-deplet~ 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 n~arzinostatin in a mammalian cell line.
NCS' is a polypeptide antibiotic that has shown some antitumor activity in clinical trials (1,2) and which continues to be the subject of clinical investigation (3,4). NCS contains a nonprotein chromophore (5), which is responsible for the biological activities of the parent compound (6). NCS induces single-strand breaks in DNA by damaging the deoxyribose moiety of a nucleotide (7). These breaks occur preferentially at thymine residues and occasionally at adenine residues (8); the base is released intact from the DNA molecule (9, 101, leaving a gap bounded on the 3' side by a phosphate group and on the 5' side by the deoxyribose cleavage moiety attached to a phosphate group (7, 11). The reaction of high concentrations (100 pg/ml) of the NCS chromophore with DNA occurs ' The abbreviations used are: NCS, n e~~i n o s~t i n ; GSH, glutathione (~-~-glutamy~-~-cysteinylglycine~; BSO, m-buthionine S,Rsulfoximine; OTZ, oxothiazolidine 4-carboxylate; DEM, diethyl maleate; SSA, sulfosalicylic acid; HPLC, high performance liquid chromatography. in cell-free systems in the absence of any reducing agent, but sulfhydryl group donors such as 2-mercaptoethanol stimulate the DNA strand scission activity at least 1000-fold (12). DNA strand scission has been shown to be oxygen-dependent (13). Recently, it has been shown that 2 mol eq of sulfhydryl and 1 mo1 eq of 0 2 are consumed for each mole equivalent of NCS chromophore reacting with DNA (14).
GSH is usually the major nonprotein thiol in the mammalian cell (15) and is a potential source of sulfhydryl groups for the bioactivation of NCS in viuo or in cell culture. Based on this fact alone, one might expect that depletion of the cellular GSH pool would lower the toxicity of NCS, since there would be fewer s u l~y~l groups available to react with the NCS chromophore. On the other hand, GSH is important in protecting cells from free radical damage (15), and there is evidence that free radical production is important in the activity of NCS (16,17). Favaudon (18) has proposed that oxygen is necessary in the thioi-mediated activation of the NCS chromophore because in the absence of oxygen, a third thiol group would react with the methylene radical produced at the C-5' position of deoxyribose by activated NCS, terminating the radical, and preventing DNA damage. Thus, it is theoretically possible that depletion of cellular GSH could actually increase NCS toxicity.
It is possible to deplete GSH to levels of less than 5% of control values in V79 cells by exposing the cells to BSO, a specific inhibitor of y-glutamylcysteine synthetase (19). GSH levels can also be depleted by treatment of the cells with DEM (20), an agent that binds GSH in a reaction catalyzed by GSH S-transferase (21,22). Treatment of V79 cells with OTZ increases ~ntracellular GSH levels to 150-300% of control values (23). Using these three compounds, we have investigated the effect of reduced and elevated GSH levels on the cytotoxicity of NCS in V79 cells.
Another thiol compound that could have a role in the bioactivation of NCS is cysteine. To investigate this possibility, high levels of intracellular cysteine were induced, without a concurrent increase in GSH levels, by simultaneous BSO and OTZ treatment. Cysteine levels were monitored by HPLC in untreated, BSO-treated, and ~SO/OTZ-treated cells.

MATERIALS AND METHODS
Exponentially growing cultures of Chinese hamster V79 cells were grown in F-12 medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Plating efficiencies ranged from 85 to

95%.
For drug studies, 100-mm Petri dishes were seeded with 10' cells/ dish and incubated overnight. These cultures were then exposed to NCS (National Cancer Institute Drug Synthesis and Chemistry Branch) by removing the medium and replacing it with fresh medium containing varying concentrations of NCS and incubating at 37 "C for 1 h. Following drug exposure, the cultures were rinsed twice, trypsinized, counted, and plated for colony formation. All work was done in dim yellow light to minimize photoactivation or deactivation of NCS. After 6-8 days of incubation, the colonies were fixed, stained, and counted. All experiments were done twice. Cellular GSH was depleted by pretreatment with 1 mM BSO (Chemical Dynamics c o p . ) for 8 h or 0.5 mM DEM (Aldrich) for 2 h and elevated by 10 mM OTZ for 2 h. All of these treatments were done in complete medium at 37 "C. Pretreatment with these drugs did not affect plating efficiency. After BSO, DEM, or OTZ pretreatment, the medium was changed to medium containing various concentrations of NCS plus the respective pretreatment drug. NCS exposure was then done as described above. In some studies, cells were pretreated with BSO, then rinsed with phosphate-buffered saline and treated with NCS as described above, but without BSO present during NCS exposure.
For studies investigating the possible role of cysteine, cells were treated with 1 mM BSO for 8 h, and then 10 mM OTZ was added for 0.5 h prior to NCS exposure. For HPLC analysis of intracellular cysteine levels, cells were grown in 150-mm Petri dishes to a density of 50 X IO6 cells/dish and then treated with BSO and OTZ as described above. The cell monolayer was rinsed three times with cold phosphate buffered saline and then extracted with 3 ml of cold SSA. Thiols were measured by HPLC after derivatization with monobromobimane as described by Fahey et al. (24) and modified by Anderson and Meister (25) for use with an automatic injection system (Waters corp.). In addition to stabilizing the monobromobimane derivative by the method of Anderson and Meister (25), argon was layered over the cell monolayer just prior to the addition of the SSA, to lessen autoxidation. Argon was also used to saturate the SSA and phosphatebuffered saline solutions, as well as all of the reagents used in the monobromobimane derivatization process.
GSH determinations were made for each survival experiment. A number of additional plates were seeded with the same number of cells as for the drug studies. Pretreatment of these cultures with BSO, DEM, or OTZ was identical to that described above. Following pretreatment, each plate was rinsed with cold phosphate-buffered saline, after which 2.5 ml of cold 0.6% SSA was added to each plate and left on for 10 min at 4 'C. The SSA was then removed and assayed for total GSH content by the GSH reductase procedure (26).
Treated samples were compared to control samples.

RESULTS
Pretreatment with BSO or DEM decreased total GSH levels to less than 5% of control values. OTZ pretreatment increased GSH levels to 250-260% of control values. Combined BSO and OTZ treatment also reduced total GSH to less than 5% of control values. All GSH measurements were made at the time NCS was added. In one experiment, GSH determinations were repeated after the 1-h NCS exposure (at 5 pg/ml) and showed no change from pre-NCS exposure levels. NCS treatment alone ( 5 pg/ml for 1 h) caused no detectable change in total GSH. Control levels of GSH for these experiments were from 2 to 5 mM, as measured by the GSH reductase procedure, and 5 mM as measured by HPLC in one experiment.
GSH depletion by either BSO or DEM reduced the toxicity of NCS by a factor of 1000 or greater at 5 pg/ml (Figs. 1 and 2), indicating that GSH plays a major role in the activation of NCS in mammalian cells. Another way to compare these data is to calculate Do values, the amount of NCS needed to reduce the surviving fraction to 0.37 along the linear portion of the curve (27) for the survival curves. The average Do for NCS in these experiments was 0.57 pg, while in BSO-treated cells, the Do for NCS was 6 pg, and in DEM-treated cells, it was 10 pg. To rule out that a reaction between NCS and BSO in the medium could be causing these results, GSH depletion by BSO was done as described above, but the BSO was removed before NCS exposure. GSH levels remained low for several hours following the removal of BSO from V79 cells (data not shown). These data are also shown in Fig. 1 and are nearly identical to the data from experiments where BSO was left on during NCS exposure.
Increasing intracellular cysteine levels in the absence of GSH resulted in intermediate NCS toxicity (Fig. 3). HPLC analysis of untreated and BSO-treated cells showed no detectable cysteine (Fig. 4). The limit of detection for cysteine in this assay was 50 pmol, which is equivalent to 0.2 PM when 50 X lo6 cells are assayed. Cysteine and GSH standards for HPLC analysis were prepared by placing micromolar concen- trations of the compounds directly into the monobromobimane mixture system; the resultant sensitivity, particularly for cysteine, is less than that usually attained (1-pmol range) by using millimolar concentrations, yet this variation in method may better simulate experimental conditions found in the in vitro system under study. Treatment of cells with OTZ and BSO increased cysteine levels to approximately 40 p~, a 200-fold increase over normal cellular cysteine levels. Raising intracellular GSH levels by pretreatment with OTZ altered NCS toxicity only slightly in one of the two experiments (Fig. 5).

DISCUSSION
In cell-free systems, the activation of NCS to a species capable of damaging DNA has been shown to require a reducing agent such as a sulfhydryl group donor of 2-mercaptoethanol, unless very high NCS concentrations are used. Because of the natural abundance of intracellular reducing equivalents, it is not necessary to add exogenous sulf'hydryl compounds for NCS to have toxic, mutagenic, or DNA strand scission activity in mammalian cells, bacteria, fungi, or yeast (28-32). In this study, we have investigated the role of intracellular GSH and cysteine in the toxicity of NCS to V79 cells. Reducing GSH to undetectable levels with BSO or DEM resulted in a marked reduction in NCS cytotoxicity, as evidenced by a 10-fold increase in the Do (a 10-fold increase in the slope of the survival curve).
Beerman et al. (12) have reported that NCS will cleave DNA in a cell-free system without the addition of a sulfhydryl compound, but such cleavage occurs slowly and at high NCS concentrations (100 pg/ml), compared to the same reaction when stimulated by 2-mercaptoethanol. They reported the stimulation by 2-mercaptoethanol to be at least lO~-fold. Thus, it is possible that the remaining toxic activity of NCS in GSH-depleted V79 cells (40-50% survival at 5 pg of NCS/ ml) results from a sulfhydryl-independent NCS-induced DNA cleavage. Alternately, reducing agents other than GSH may activate NCS. The HPLC analysis in the present study indicated that the concentration of cysteine in untreated and BSO-treated cells is less than 0.2 p~ (see Fig. 4 and "Results"). The concentrations of NCS used in these experiments were 0.1-0.5 pM. Since 2 mol of sulfhydryl groups is needed to activate each mole of NCS (14), normal intracellular levels of cysteine could, at most, only activate a small portion of the NCS molecules.
It is possible that there is some GSH left in the cell after depletion by BSO or DEM, but at a concentration too low to be detected by the assay used. The GSH assay used in these studies can detect a minimum of 1-5 ng of GSH (26), which corresponds to 0.8-4 p~ when 4 X lo6 cells are assayed (one 100-mm Petri dish seeded with lo6 cells 24 h prior to starting the experiment, with a doubling time of 10-12 h). Thus, it is possible that concentrations of GSH that are still 10-fold higher than the concentration of NCS could remain in the cell after BSO or DEM treatment. This concentration could be sufficient to activate a small portion of the NCS molecules.
The nearly identical results obtained with two different thiol depleters and the fact that BSO need not be present at the same time as NCS for reduction of NCS toxicity to occur indicate that the observed reduction in NCS toxicity is due to thiol depletion, and not to inactivation of NCS in the medium by reaction with the thiol-depleting compounds. Taken together, these results indicate the GSH is the major thiol used in the reductive activation of NCS in V79 cells and argue against a significant role for cysteine, when cysteine is present at normal levels.
GSH concentrations in untreated cells in these experiments were 2-5 mM, nearly 10,000-fold higher than the NCS concentrations used. This explains the lack of potentiation of NCS toxicity by pretreatment with OTZ. Even though 2 mol of sulfhydryl groups is required for each mole of NCS activated (13, there is still a considerable excess of GSH in an untreated cell, and raising the GSH concentration with OTZ would not he expected to increase the toxicity of NCS. Without the GSH biosynthetic pathway being blocked by BSO, OTZ treatment would also not be expected to significantly increase intracellular levels of cysteine. After combined BSO and OTZ treatment, higher levels of cysteine and very low levels of GSH are present in the cell, resulting in i n~r m~a t e NCS toxicity (see Fig. 3). The DO for NCS for BSO/OTZ-treated cells was 0.9 pg, higher than the 0.57-pg Do for NCS in untreated cells, indicating that even at chemically induced high levels, cysteine does not activate NCS to the same extent as does GSH.
BSO is currently being considered as an adjuvant in cancer radiation and chemotherapy. If this were done with a drug like NCS, which relies on intracellular GSH for activation, it would be counterproductive, unless some way is found of lowering GSH levels in normal tissues to a greater extent than in the tumor tissue. Our work suggests that before