Cytotoxicity of RSU 1069 in spheroids and murine tumors

Hypoxia following treatment with the alkylating nitroimidazole, RSU 1069, greatly enhanced cell killing in the Lewis lung tumor and Chinese hamster V79 spheroids. When mice were injected with RSU-1069 and tumors were excised after 3 hr to measure colony formation in soft agar, significant cell killing was observed. However, if tumors were excised 18 hr after drug injection, viability was increased, and cell killing was confined to cells distant from the blood supply. In subsequent experiments, viability observed at 3 hr could be greatly increased if the tumors were cooled to 4 degrees C immediately after excision, and were then rapidly disaggregated. This suggested that the hypoxia which occurred after animal sacrifice and during the tumor disaggregation procedure was sufficient to account for the additional cell killing at early times after drug injection. Results using V79 spheroids similarly suggest that tumor excision soon after injection of RSU 1069 can give false information on RSU 1069 toxicity if efforts are not made to prevent tumor hypoxia during processing. In spheroids, hypoxia-induced toxicity after aerobic exposure decreased as the time between RSU 1069 exposure and hypoxic incubation increased; spheroid cells exposed to RSU 1069 under air lost sensitivity to subsequent hypoxic incubation with a half-time of about 10 hr, representing the time for cell turnover and/or repair from damage produced under aerobic conditions.     

Hypoxic toxicity of misonidazole in a glucose-6-phosphate dehydrogenase deficient mutant Chinese hamster ovary cell line

The metabolic activation of misonidazole (MISO) and its effects on the hexose monophosphate pathway (HMP) and on cell viability were studied in hypoxic mutant Chinese hamster ovary (CHO) cells deficient in glucose-6-phosphate dehydrogenase and their parent wildtype cells. The metabolic activation of MISO was similar in both cell lines as indicated by the binding of 14C-MISO to the acid-insoluble fraction of these cells; it was decreased by the absence of glucose. In the wildtype CHO cells, MISO caused a significant stimulation of the activity of the HMP while in the mutant CHO cells no HMP activity was measurable, even in the presence of MISO. In both cell lines clonogenicity began to decline after 2 hr and trypan blue exclusion after 4 hr of hypoxic incubation. The effect of MISO on both parameters of cell viability was somewhat more pronounced in the wildtype CHO cells. This difference became especially significant at the longer incubation times. The results indicate that reducing equivalents for the metabolic activation of MISO are provided not only by the HMP but that pathways other than the HMP, such as glycolysis or pathways starting from mitochondrial tricarboxylates, are of similar or even greater importance in this respect.     

Cytological effects of 1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol (misonidazole) on hypoxic mammalian cells in vitro

Hypoxic Chinese hamster V-79 cells were examined for light-microscope morphology, progression through the cell cycle, chromosomal aberrations, and viability, after incubation with the 2-nitroimidazole, misonidazole [1-(2-nitro-1-imidazolyl)-3 methoxy-2-propanol]. Cytological examination of cells up to 42 hr after incubation with the drug at 37 degrees indicated that increasing contact time and increasing drug concentrations interfered with cell attachment and progressively slowed cell progression through the cell cycle. Forty-two hr after a 5.5-hr treatment with 5 mM misonidazole, the majority of cells contained heteropyknotic nuclei, whereas less than 3% had progressed into mitosis. Of the few cells that reached mitosis by 42 hr, the level of chromosomal aberrations was 6 times that due to hypoxia alone. However, the majority of metaphases (70%) were unaltered; thus about 2% of the treated cell population passed into mitosis unaltered. After a 5.5-hr incubation with 5 mM misonidazole, 98% of the cells also had lost their ability to produce clones. It is suggested that the cytotoxic effect of this drug on hypoxic cells is that the cytotoxic effect of this drug on hypoxic cells is largely mediated via an interphase cell death, with a minor effect due to chromosome aberrations and cell death from genetic inequality of progeny cells. The ability of misonidazole to kill hypoxic, noncycline cells, which may limit the curability of some tumors with conventional X-rays or chemotherapy agents, makes it of considerable potential interest.     

Influence of heat on the intracellular uptake and radiosensitization of 2-nitroimidazole hypoxic cell sensitizers in vitro

The effect of elevated temperature (44 degrees) on the intracellular uptake of the 2-nitroimidazole hypoxic cell radiosensitizer, misonidazole (MIS), and analogues more hydrophilic than MIS was studied in Chinese hamster ovary cells. It was found that the intracellular uptake of these compounds which enter cells by restricted passive diffusion can be enhanced approximately 4-fold when incubated at 44 degrees compared to the uptake at 37 degrees. Peak intracellular uptake (expressed as the ratio of intracellular concentration to extracellular concentration) following incubation of cells in 2 mM MIS was 100% at 44 degrees but only 25% at 37 degrees. Furthermore, a short-term nonlethal heat pulse (44 degrees for 15 min) with MIS present caused a 2-fold enhancement in uptake which was sustained for an additional 45 min at 37 degrees. This same nonlethal heat pulse was found to induce a similar enhancement in uptake even when MIS was added at subsequent time intervals at 37 degrees. The heat pulse induced a time-related enhancement of uptake at 37 degrees which increased for 1 hr and persisted for at least 6 hr. Finally, in vitro radiosensitization studies of hypoxic Chinese hamster ovary cells showed that the nonlethal heat pulse of 44 degrees for 15 min could greatly enhance the sensitization by low concentrations (0.5 mM) of MIS added after heating due to increased intracellular concentrations of the drug. MIS (0.5 mM) alone achieved a radiosensitization enhancement ratio of 1.29 (compared to irradiated hypoxic cells alone), while the addition of the short-term heat pulse, which had only a minor effect itself, achieved an enhancement ratio of 1.78.     

Cell killing, radiosensitization and cell cycle redistribution induced by chronic hypoxia

Some of the biological changes associated with extreme hypoxia at 37 degrees C (less than 10 ppm pO2) were examined in Chinese hamster V79 cells. Specifically, extreme hypoxia caused an initial decrease in plating efficiency to 55% in 4 hr after the onset of hypoxia. Beyond this time, the decline in plating efficiency was more gradual reaching 35% of control at 20 hr. Flow microfluorimetry (FMF) studies, in which cells are sorted on the basis of DNA content and then assayed for viability, demonstrated that mid S phase cells were most sensitive to chronic hypoxia, with surviving fraction equal to 2.5% at 20 hr. Furthermore, the viability of G1 and G2/M cells, after 20 hr of hypoxic storage, was also reduced to 20 and 7.6%, respectively. Hypoxia also caused alterations in the cell cycle distribution of initially asynchronous cells, as determined by dual parameter FMF measurements of both cellular DNA content and incorporated BudR. In particular, G2/M cells completed mitosis, while G1 cells showed little or no movement. Lastly, cells stored in chronic hypoxia displayed an enhanced radiosensitivity when compared to acutely hypoxic cells. Possible reasons for these observations are discussed.     

Cytotoxicity of metronidazole (Flagyl) and misonidazole (Ro-07-0582): enhancement by lactate.

The cytotoxic activity of metronidazole (Flagyl) and misonidazole (MISO) to hypoxic Chinese hamster ovary (CHO) cells suspended in standard medium in sealed vials and in gassed spinner flasks has been investigated. Flagyl (5 mM) was only cytotoxic at high initial cell densities. However, when lactate (20 mM) was included in the medium the cytotoxicity of Flagyl at low cell densities was considerable, and similar to that of misonidazole under the same conditions. The relevance of this "lactate effect" to in vivo systems, and the possible mechanisms involved, are discussed.     

Biology of cell killing by 1-beta-D-arabinofuranosylcytosine and its relevance to molecular mechanisms of cytotoxicity

Cells of the Chinese hamster ovary cell line were used to study the process of cell death induced by pulse treatment with 1-beta-D-arabinofuranosylcytosine (ara-C). Cells were synchronized by mitotic selection and pulse treated in early S phase with a concentration of ara-C (1 mM) which was sufficient to reduce plating efficiency to a few percentages of the control. The process of when and how the lethally damaged cells die was studied using a series of techniques in parallel. These included time-lapse microcinematography, flow microfluorimetry, and chromosome morphology in both anaphases/telophases and Colcemid-arrested metaphases. Most of the lethally damaged Chinese hamster ovary cells progressed through one, and many through two, cell cycles before death occurred. The cell death and abnormal divisions can be accounted for by the chromosome aberrations observed in Colcemid metaphases and anaphases/telophases. Death without any attempted division occurred between 3 and 9 normal cell cycle times after ara-C treatment. Chinese hamster ovary cells were also treated continuously with 1 mM ara-C. Under these conditions, cell death was still primarily division related. We argue that these data are not consistent with the actual incorporation of ara-C moieties into DNA being the primary cause of cell death. The data are discussed in relation to the postulated molecular mechanisms of toxicity of this drug.     

Influence of hypoxia and an acidic environment on the metabolism and viability of cultured cells: potential implications for cell death in tumors

Hypoxia and an acidic environment are known to occur in regions of solid tumors and might be involved in the causation of necrosis. The viability and energy metabolism of cells in tissue culture were therefore investigated under hypoxic and/or acidic conditions. Acute exposure of Chinese hamster ovary (CHO) cells or human bladder cancer MGH-U1 cells to hypoxia plus low pH (6.5 to 6.0) was cytotoxic in a time- and pH-dependent manner; surviving fraction was reduced to approximately 10(-4) following a 6-h exposure to hypoxia at pH 6.0. There was no effect on viability when aerobic CHO cells were exposed for 6 h at pH 6.0, or when either cell line was rendered hypoxic for 6 h at pH 7.0; MGH-U1 cells showed slight sensitivity to acidic pH in air. Decrease in viability of CHO cells incubated under acid conditions was observed over the range of oxygen concentrations from 0.2 to 0.05%, similar to the range which causes change in cellular sensitivity to radiation. Glucose consumption and lactate production by both cell lines were inhibited at low pH under both aerobic and hypoxic conditions. Cellular adenosine triphosphate (ATP) levels and the energy charge [(ATP + 1/2 adenosine diphosphate)/(adenosine monophosphate + adenosine diphosphate + ATP)] of CHO cells were reduced by about 85 and 25%, respectively, after a 6-h exposure to hypoxia at pH 6.0 but were not influenced by hypoxia or acid pH alone. Inhibition of glycolysis by incubation of CHO cells under hypoxic conditions in the absence of glucose (at pH 7.0) led to a larger fall in cellular ATP and energy charge, but cell survival fell to only approximately 10(-2) at 6 h. These results demonstrate that hypoxia and an acid environment interact to cause marked toxicity. A decrease in energy charge of the cells may contribute to loss of viability, but additional mechanisms appear to be involved.     

Hypoxic cell sensitization to radiation damage by a new radiosensitizer: cis-dichloro-bis(1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole-N3)platinum(II) (flap)

A new, stable platinum coordination complex (FLAP) containing the 5-nitroimidazole, metronidazole, has been prepared and characterized. The square-planar platinum(II) complex has two metronidazole molecules and two chlorine atoms in the cis configuration. The properties of FLAP differ significantly from metronidazole alone or other platinum complexes tested in the same system. It has a low toxicity towards Chinese hamster ovary cells and is a very effective radiosensitizer toward hypoxic cells in vitro: a one-h pretreatment with a non-toxic dose of 50 microM gave an enhancement ratio of 2.4. No potentiation of aerated cells to X-irradiation damage was observed after a similar schedule of pretreatment at the higher dose of 100 microM FLAP.     

Differential cytotoxicity of diaziquone toward Chinese hamster ovary cells under hypoxic and aerobic exposure conditions

Diaziquone [AZQ, 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone] has been investigated for its toxicity toward Chinese hamster ovary cells AA8-4 under both aerobic and hypoxic conditions. Under acute (1-5 h) exposures to 2.5-10 microM AZQ in alpha-medium plus 10% fetal calf serum, AZQ showed an approximately linear concentration x time dependency for cell killing which was 3-4 times less under hypoxic compared to aerobic conditions. This selective toxicity toward hypoxic cells was prevented by low levels of oxygen. Under aerobic exposure conditions the toxicity of 2.5 microM AZQ was greatly increased by addition of 1-2 mM ascorbate. This ascorbate mediated toxicity of AZQ, presumably extracellular, could be prevented by the simultaneous addition of catalase. Under hypoxic exposure conditions there was no enhancement of AZQ toxicity by ascorbate or protection by catalase. The present results are consistent with two mechanisms for AZQ toxicity proposed earlier by others: toxicity due to (a) redox cycling and increased levels of oxidative stress and (b) reduction of the quinone leading to enhanced reactivity of the aziridines. The relative potency of AZQ as a hypoxic or aerobic cell selective toxin is determined by the balance between these two mechanisms.     

Hydroethidine: a fluorescent redox probe for locating hypoxic cells in spheroids and murine tumours

The fluorescent redox probe hydroethidine was accumulated and metabolised about five times faster in aerobic than in hypoxic mammalian cells. Patterns of fluorescence in Chinese hamster V79 spheroids also indicated that internal hypoxic cells were less able to metabolise the drug; toxicity was observed in cells only when cell fluorescence exceeded about 500 times background. In medium equilibrated with air or nitrogen, cell accumulation of the stain was rapid, and began to plateau after 30 min; loss of ethidium was initially rapid, with a slower component after 30 min, and transfer of the metabolite ethidium between stained and unstained cells was observed after 2 h co-incubation. Sorting cells from irradiated spheroids on the basis of ethidium fluorescence provided good separation of aerobic radiosensitive and hypoxic radioresistant cells, although separation using the perfusion probe, Hoechst 33342, was superior. Similar experiments with the murine SCCVII squamous cell carcinoma suggested that hydroethidine might be a useful indirect stain for locating hypoxic cells in experimental tumours when used in combination with a perfusion probe such as Hoechst 33342.     

Inhibition of DNA synthesis by nitroheterocycles. II. Mechanisms of cytotoxicity.

Nitroheterocycles have been shown to inhibit the incorporation of 3H-TdR by cultured L-929 cells, and the degree of inhibition is related to their electron-affinity. On the basis of their chemical reactivity and potential clinical utility, nitrofurazone, misonidazole and metronidazole were selected for more detailed studies of the mechanism of inhibition of DNA synthesis. Double-isotope labelling in conjunction with hydroxyapatite chromatography allowed the evaluation of drug effects on initiation of DNA replicons, DNA chain elongation and DNA damage (single-strand breaks), and their correlation with eventual cell viability. Partial inhibition of initiation of DNA synthesis generally preceded other measurable effects, and was not reversed by incubation in the absence of drug. In the absence of DNA strand breaks (at low drug doses or after a repair interval) the rate of elongation was similar in both treated and untreated cell populations. Measurable DNA damage (strand breaks) was predictive for cytotoxicity. At lower drug doses, or under aerobic conditions, DNA synthesis was not always associated with a decrease in plating efficiency (cytotoxicity) but was reflected in decreased colony size (growth rate) of the cells. Thus the aerobic "toxicity" previously reported for chronic exposure to these agents may be better described as a "cytostatic" effect. Under anaerobic conditions (where cell killing is much greater) inhibition of initiation plays a less important role, and the nitroheterocycles are metabolically reduced to intermediates which are truly cytotoxic.     

Use of a cell sorter for assays of cell clonogenicity

A fluorescence-activated cell sorter has the capability of recognizing, categorizing, counting, and sorting cells, and it thus provides a convenient and rapid method for "micromanipulation" of known numbers of cells into appropriate vessels for cell viability or clonogenicity studies. Subsequent microscopic observation of the sorted cells allows discrimination between the separate processes of cell attachment (plating efficiency) and clonogenic growth (viability). Examples are presented showing the power of these techniques for studying the low-dose regions of radiation survival curves for Chinese hamster V79 cells grown as monolayers or spheroids, for cells irradiated under aerobic versus hypoxic conditions, and for cells of large spheroids exposed to Adriamycin, where the slow penetration of the drug results in a differential exposure to the various cell subpopulations.     

Cytotoxicity and DNA lesions produced by mitomycin C and porfiromycin in hypoxic and aerobic EMT6 and Chinese hamster ovary cells

Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which form a therapeutically resistant cell population within the tumor that is difficult to eradicate by ionizing irradiation and most existing chemotherapeutic agents. As an approach to the therapeutic attack of hypoxic cells, we have measured the cytotoxicity and DNA lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin, two structurally similar antibiotics, in oxygen-deficient and aerobic cells. Mitomycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 cells in culture, with porfiromycin producing a greater differential kill of hypoxic EMT6 cells relative to their oxygenated counterparts than did mitomycin C. Chinese hamster ovary cells were more resistant to these quinone antibiotics; although in this cell line, porfiromycin was significantly more cytotoxic to hypoxic cells than to aerobic cells, and the degree of oxygenation did not affect the toxicity of mitomycin C. Alkaline elution methodology was utilized to study the formation of DNA single-strand breaks and DNA interstrand cross-links produced by mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary cells. A negligible quantity of DNA single-strand breaks and DNA interstrand cross-links were produced in hypoxic and aerobic Chinese hamster ovary cells by exposure to mitomycin C or porfiromycin, a finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared to be produced by these antitumor antibiotics under both hypoxic and aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with substantially more DNA interstrand cross-linking being produced under hypoxic conditions. Mitomycin C and porfiromycin caused the same amount of cross-linking under conditions of oxygen deficiency; however, mitomycin C produced considerably more DNA cross-linking than did porfiromycin in oxygenated cells. DNA interstrand cross-links were observed in hypoxic EMT6 cells throughout a 24-h period following removal of mitomycin C and porfiromycin, with a decrease in DNA interstrand cross-links observed at 24 h. An increase in DNA interstrand cross-links occurred in aerobic EMT6 cells treated with mitomycin C and porfiromycin at 6 h after drug removal, with a decrease in these lesions being observed by 24 h, suggesting that the rate of formation of the cross-links may be slower and the removal of cross-links more rapid under aerobic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo interaction of anti-cancer drugs with misonidazole or metronidazole: cyclophosphamide and BCNU.

The addition of misonidazole (MISO) or metronidazole (METRO) to treatment with cyclophosphamide (CY) increased delay to regrowth of 2 experimental tumours. The effect was observed for large an small tumours, was present for doses of MISO that are ineffective for killing hypoxic cells, and required that it be given with, or shortly before CY. Mice receiving combined treatment had more weight loss and myelosuppression than those receiving CY alone, and the Therapeutic Index was lower. MISO caused a marked increase in growth delay when combined with BCNU to treat the KHT sarcoma. This effect was observed for small and large tumours, required simultaneous administration of drugs, and also led to increased host toxicity. There was no therapeutic advantage from combined treatment. Survival of aerobic or anoxic Chinese hamster ovary (CHO) cells was assessed after exposure in vitro to serum from mice that had received CY or BCNU alone. MISO alone, or combined treatment. Results of these experiments suggest that (1) MISO delays the excretion or breakdown of active metabolites of CY, and (2) at a dose that does not kill hypoxic cells, it may selectively "sensitize" hypoxic cells (but not aerobic cells) to the action of BCNU. The presence of other undetermined interactions of BCNU and MISO is inferred from the increased toxicity to (aerobic) normal tissue. Misonidazole or metronidazole should be used with caution in patients who are receiving BCNU or cyclophosphamide.     

Decreased hypoxic toxicity and binding of misonidazole by low glucose concentration

The modulation of the hypoxic toxicity and binding of Misonidazole (MISO) by glucose and lactate was studied by exposing exponential EMT6/Ro cells to 5 mM MISO under hypoxic conditions. The concentrations of glucose used were 0.015 mM and 5 mM, and the concentrations of lactate were 0, 3 and 10 mM. There was no significant hypoxic toxicity due to MISO in the absence of glucose. However, with 5 mM glucose, after a latent period of 0.5 hours, there was a rapid decrease in cell survival to less than 0.1% at 2.5 hours incubation in 5 mM MISO. The binding of MISO was also increased by glucose. The amount of MISO bound to cells in 0.015 mM glucose leveled off at 2 nmoles MISO/7 X 10(5) cells at 1 hour, whereas the binding in 5 mM glucose continued to increase to more than 5 nmoles MISO/7 X 10(5) cells after 3 hours incubation. There was no detectable effect of lactate on the binding of MISO to the cells either in 0.015 mM or 5 mM glucose. The high affinity of this binding was indicated by the lack of exchange of radioactive MISO with non-radioactive MISO even after 2 hours of incubation. These data showed that glucose concentrations could modify the toxicity and binding of MISO to hypoxic cells.     

Mechanism of melphalan crosslink enhancement by misonidazole pretreatment

Sensitization of Chinese hamster ovary cells to melphalan (L-PAM) toxicity by prior treatment with misonidazole (MISO, 5 mM, 2 hr, hypoxic conditions, 37 degrees C) is associated with increased levels of DNA crosslinks believed to be the critical lesion for bifunctional alkylating agent toxicity. Enhanced L-PAM crosslinking of DNA could occur by a variety of mechanisms in MISO-pretreated cells including: (1) increased transport or binding of L-PAM, (2) decreased repair of L-PAM monoadducts which would allow more time for their conversion to crosslinks, (3) decreased crosslink repair (unhooking of one arm), or (4) chemical modification of the DNA structure, presumably by bound MISO derivatives, such that crosslink formation is facilitated. Previous studies have eliminated mechanisms (1) and (3). Mechanism (4) was investigated by following MISO-pretreatments of whole cells with L-PAM treatments of the isolated DNA from these cells. This was accomplished by using a modification of the alkaline elution assay for DNA crosslink measurement in which a 1 hr treatment with L-PAM (0-12 micrograms/ml) was inserted between the cell lysis steps and DNA elution procedure. Treatment of bare DNA with L-PAM modeled very well the crosslinking behavior in whole cells although it was somewhat more efficient (more crosslinks at a given L-PAM dose). In the presence of double stranded DNA and absence of repair systems during and after the L-PAM exposure, it was determined that MISO-pretreatments did not increase the crosslinking efficiency of L-PAM (mechanism [4] above). Inhibition of repair of L-PAM monoadducts (mechanism [2] above) still remains as a possible means for crosslink enhancement by MISO-pretreatment.     

Pre- and post-irradiation radiosensitization by SR 4233

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a bioreductive agent which exhibits highly selective killing of hypoxic cells in a variety of mammalian cell lines in vitro and in murine tumors in vivo. The selective toxicity of the drug results from its one-electron reduction under hypoxic conditions to form a free radical intermediate capable of damaging DNA, through the formation of strand breaks. Using the neutral filter elution assay, SR 4233 was found to be more efficient at producing DNA double strand breaks in Chinese hamster ovary (CHO) cells than an equitoxic dose of gamma-rays. Drug and radiation sequencing experiments were also performed, with both cell survival and DNA strand break rejoining used as endpoints. As a result of these studies, we now describe two additional properties of SR 4233: (a) radiosensitization of aerobic cells in culture produced by hypoxic incubation with drug either before or after irradiation, and (b) the inhibition of subsequent rejoining of radiation-induced DNA double strand breaks after hypoxic pretreatment with drug. The magnitude of the radiosensitization produced did not vary for drug treatments which, when given alone, reduced cell survival over a range from 30% to 2%. The extent of DNA repair inhibition increased with increasing severity of the SR 4233 pretreatment, but was quite small for non-lethal drug exposures.     

Modification of the cytotoxic activity of mitomycin C by oxygen and ascorbic acid in Chinese hamster ovary cells and a repair-deficient mutant

The cellular and molecular damage produced by mitomycin C (MMC) in Chinese hamster ovary cells, AA8-4, and a repair deficient mutant of this line, UV-20, was studied by utilizing a system in which oxygen levels could be altered and monitored in solution during acute drug exposures. The cytotoxic activity of MMC decreased from hypoxic conditions to 1% oxygen in solution, while from 1 to 20% there was little change. The relative level of DNA cross-linking in cells was examined under these conditions by alkaline elution and found to increase as cell survival decreased. Utilizing a cell-free assay which detects formation of alkylating species it was confirmed that, while alkylation was observed under aerobic conditions, overall levels increased in the absence of oxygen. The presence of ascorbic acid in the exposure medium (0.284 mM) increased the aerobic but not the hypoxic cytotoxicity of MMC. This resulted in a diminished differential toxicity for cells exposed under aerobic versus hypoxic conditions in the presence of ascorbic acid. When ascorbic acid was present, net alkylation increased under aerobic conditions but was unchanged under hypoxic conditions. One interpretation of these results is that at least two mechanisms of activation of MMC to toxic intermediates may be present in these cells.     

Effects of glutathione depletion by buthionine sulfoximine on the sensitivity of EMT6/SF cells to chemotherapy agents or X radiation

The effects of depletion of cellular glutathione (GSH) on the sensitivity of cultured EMT6/SF cells to chemotherapy agents or x rays under hypoxic and aerated conditions were investigated. Buthionine sulfoximine (BSO), a potent inhibitor of the enzyme gamma-glutamyl-cysteine synthetase, was used to deplete cellular GSH. Addition of BSO (50 microM) to EMT6/SF cultures depleted cellular GSH with a half-time of approximately 2 hr. Cellular GSH reached very low levels within hours of addition of BSO. After removal of BSO, cellular GSH recovered with approximately the same kinetics as was seen for depletion. Incubation of EMT6/SF cells with BSO concentrations of up to 1 mM did not reduce the viability or inhibit growth when exposure was limited to times less than 24 hr. However, for longer exposure times, toxicity and growth inhibition were demonstrated in a dose dependent fashion. EMT6/SF cells were treated with chemotherapy agents under either aerated or extremely hypoxic conditions. Cells were more sensitive to cis-dichlorodiammino Pt(II) (DDP), mitomycin C (MitC), L-phenylalanine mustard (L-PAM), and nitrogen mustard (HN2) when treatment was under hypoxic conditions. The magnitude of this sensitization under hypoxic conditions ranged from a dose modifying factor (DMF) of 1.4 (HN2) to 4.1 (MitC), measured at the 0.1 level of cell survival. Hypoxic EMT6/SF cells were more resistant to the cytotoxic effects of actinomycin D (ActD) under hypoxic conditions (DMF = 10 at SF = 0.3). When cellular GSH was depleted to less than 5% of control by treatment with 50 microM BSO for 12-14 hr, cells were sensitized to DDP, L-PAM and HN2 under both aerated and hypoxic conditions. DMF's ranged from 1.4-6.5, depending on the agent. Hypoxic cell sensitization was never significantly greater than that seen in aerated cells, as was the case for X radiation (DMF = 1.3 for hypoxic cells only). GSH depletion also sensitized to MitC, but only under aerated conditions (DMF = 2.1). Hypoxic EMT6/SF cells were not sensitized to MitC by depletion of GSH. GSH depletion afforded slight protection against ActD toxicity under both aerated and hypoxic conditions. These studies suggest that cellular GSH plays an important role in modifying cellular response to cytotoxic drugs. GSH depletion may sensitize tumor cells to some chemotherapy agents, but differential sensitization of tumors compared to normal tissues, based on hypoxic tumor cells as targets, would not be expected based on these in vitro experiments.