Role of Hypoxia in Anticancer Drug-Induced Cytotoxicity for Ehrlich Ascites Cells1

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[CANCER RESEARCH 47, 2407-2412, May 1, 1987] Role of Hypoxia in Anticancer Drug-induced Cytotoxicity for Ehrlich Ascites Cells1

Vicram Gupta,2 and John J. Costanzi

Division of Hematology-Oncology, Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas 77550

tained by i.p. injection of 1 x IO7cells weekly in ICR white mice. Cells ABSTRACT from animals bearing 6-8-day old ascites tumors were used. A review of the literature on the effect of hypoxia on in vitro drug In vitro studies on the effect of oxygen on drug-induced cell survival sensitivity had suggested that there was consistently more cytotoxicity were performed as follows. EAT3 cells (1-5 x IO6)were suspended in under hypoxic conditions for the drugs misonidazole and mitomycin C 10 ml of Hams FIO media in 25-ml Erlynmyer glass flasks. The flasks while there was much conflicting data for the drugs Adriamycin and were sealed with rubber sleeve stoppers and fitted with two 21-gauge bleomycin. We have examined the effect of oxygen on the cellular needles for an inlet and outlet port. The flasks were gassed with oxygen response of Ehrlich's ascites tumor cells to the drugs mitomycin C, mixture of interest at flow rates of 1 liter/min for 1.5 h at 37*C on an misonidazole, Adriamycin, and bleomycin. Significant differences were Orbital Shaker, model 3520 (Lab-Line Instruments, Inc.) at 1800 rpm. observed when we compared the cytotoxicity of mitomycin C and mison This is similar to the method reported by Mohindra and Rauth (15). idazole as a function of oxygen concentration. For the drugs Adriamycin Gas mixtures used contained 0-20% O2, 5% CO2, and balance N2 and bleomycin no differential effects of oxygen were noted for a 1-h drug (Linde Corp., Houston, TX). Following this prÃ©Ã©quilibraiionof oxygen, exposure with hypoxia while some differences were noted only for bleo drugs were added directly without opening the flask by using a 21- mycin for an 8-h drug exposure time. Because differences dependent on gauge needle in a 0.1 -ml volume. The effect of both short and prolonged oxygen concentration were observed for some drugs but not others at the drug exposure time during hypoxia on drug cytotoxicity was examined same experimental conditions, and as indicated by a review of the in some cases. For the 1-h drug exposures a cell density of 1 x 105/ml literature, it is suggested that some of the conflicting data in the literature was used without humidification. Measurement of volume of media with respect to some of these drugs may be cell-line dependent. Other failed to reveal any significant effect of evaporation. For the 8-h drug variables which may also be of importance were the duration of drug exposure under hypoxia, a cell density of 5 x KlVnil was used with exposure time in hypoxia and cell density. The observed oxygen concen humidification as this time period would have resulted in significant tration-dependent changes in cell survival for the Ehrlich cells with drugs evaporation. In some instances cells were exposed to a humidified examined could not be explained on the basis of changes in drug-induced preincubation atmosphere of 5% O2 (oxic) or N2 (hypoxic) for 16.5 h cellular oxygen consumption. before drug incubation for l h at the stated oxygen atmosphere in order to determine whether the duration of hypoxia before drug exposure influences cytotoxicity. After drug incubation with continuous gassing, INTRODUCTION the cells were centrifuged, washed with 10 ml of media, and resuspended Oxygen microenvironment in tumors is heterogeneous (1,2). in fresh Hams FIO media for plating. The colony assay for the EAT cells has been described previously (32). Tumor cells were suspended It is known that cell killing by radiation is dependent upon in Hams FIO media containing 15% fetal calf serum, 1% penicillin- oxygen concentration and that hypoxic cells are resistant to streptomycin, and 0.3% agar and plated in 35-mm l'etri dishes in a 2- radiation (3). Hypoxic cells may also be resistant to drugs ml volume. The dishes were then incubated in humidified Modulator because of drug delivery problems secondary to inadequate Incubator Chambers (Billup-Rothenberg Inc., Del Mar, CA) and vasculature (4) and/or intrinsic effects of hypoxia on cell re flushed with 5% O2, 5% CO2, and 90% N2. A 5% O2 atmosphere was sponse to drugs (5). There is some evidence that oxygen con used for the plating because we had previously found there was no centration may play a significant role in drug-induced cell growth of the EAT cells at 20% ( ).-in the absence of antioxidants (32). killing (5). Drugs such as mitomycin C and misonidazole are Seven to 8 days later, plates were scored for colony formation (>50 known to be selectively metabolized under hypoxic conditions cells). to active compounds (6, 7) while drugs such as Adriamycin and Oxygen measurements in solution were performed using a Diamond bleomycin may be cytotoxic through oxygen radical-mediated Electrotech, Inc. (Ann Arbor, MI) oxygen measuring system which mechanisms (8, 9). Pre-, during, and postdrug treatment influ consisted of an amplifier unit (no. 1201), calibration cell (no. 1251), and Clarke type oxygen electrode. Oxygen measurements were made ences of varying oxygen concentrations have all been described in separate experiments with and without cells similar to the drug in the literature (10,11). The effect of oxygen concentration on experiments except that phosphate buffered saline was used. For each the cytotoxicity of misonidazole has been established (12, 13) experiment a separate calibration curve using N2 and 0.1, 1, 5, 10, and and similar data with mitomycin C have been recently published 21% O2 was performed. The current was recorded as a function of time (14). However, the in vitro data are conflicting with the excep and the oxygen concentration calculated from the calibration curve. tion of misonidazole. Because of the conflicting data summa Using this system, oxygen level below 0.1 % cannot be measured accu rized in Table 1 we have examined the effect of oxygen concen rately. The data was plotted according to an equation (Equation A) tration on cell survival for the anticancer agents mitomycin C, described by Whillans and Rauth (33): misonidazole, Adriamycin, and bleomycin. (Cm - CJ = (Cw - C.)e-*" (A)

MATERIALS AND METHODS where C\,\ is the oxygen tension in solution at time t. f H is the initial oxygen tension in solution, C, is the oxygen concentration at equilib Tumor line used for the experiments was a tetraploid Ehrlich's ascites rium, and *i (s ') is a physical constant independent of oxygen concen tumor (Mason Research Institute, Worcester, MA) which was main- tration and depletion rate. From the measured values of oxygen con Received4/9/86; revised 12/2/86; accepted2/4/87. sumption rate (R) and k,, one can calculate the effect of oxygen The costs of publication of this article were defrayed in part by the payment depletion on the oxygen concentration at equilibrium using the rela of page charges. This article must therefore be hereby marked advertisement in tionship C. = CM - R/ki (33) where C[girepresents oxygen concentra accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by USPHS Grant CA-32938, National Cancer Institute tion in the gas phase. (NCI), and Cancer Center Grant CA-17701, NCI. Oxygen consumption studies were performed on a Gilson Oxygraph 1To whom requests for reprints should be addressed, at Division of Hematol- located in the Division of Radiation Research, University of Texas ogy, Oncology/Internal Medicine Department, Clay Hall, H82, University of Texas Medical Branch, Galveston, TX 77550. 1The abbreviation used is: EAT, Ehrlich ascites tumor cells. 2407 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1987 American Association for Cancer Research. HYPOXIA AND DRUG CYTOTOXICITY

Table 1 Summary of literature on the effect of oxygen concentration on drug cytotoxicily Preferentially toxic to Preferentially toxic Drug oxic cells to hypoxic cells No difference Misonidazole CHO"(12)EMT6(13) Metronidazole CHO and HeLa (15) Mitomycin C EMT6 (10, 16-19), CHO (14, 20), V79, CHO (16) KHT, HeLa (20), S180 (17), EAT*, WiDR and HEC-1A (19) and P3884 HTC (19) Procarbazine EMT6 (10) Actinomycin D EMT6 (10, 18) EMT6 (21) Vincristine EMT6 (10) EMT6 (21) Streptonigrin EMT6 (17) Bleomycin EMT6 (10) EMT6 (24) V79 (22) CHO (23) EAT* Adriamycin B14 FAF28 (25) EMT6(10, 17) EMT6(21) V79 (26) S180(I7) EAT* CHO (23) WHF1B(27) Bisantrene L1210, HTC (28) DTIC CHO (29) Cyclophosphamide V79 (30) CHO (23) BCNU CHO (23) EMT6(10, 21) S 1X0(17) Cisplatin EMT6 (18) EMT6 (10, 21) CHO (23) 5-Fluorouracil EMT6 (10, 21) Methotrexate EMT6(10, 21) Etoposide EMT6 (31) Alkeran EMT608) Nitrogen mustard EMT6 (18) Â°CHO, Chinese hamster ovary; DTIC, 5-(3,3-dimethyl-l-triereno)imidazole-4-carboxamide; BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea; EMT6, mouse mammary tumor cells; V79, B14 FAF28, Chinese hamster, S180, KHT, mouse sarcoma cells; P388 and 1.1210. mouse leukemic cells; HeLa, WiDR, HEC-1 A, human epithelial cancer cell lines; HTC, human tumor biopsy cells. * This study.

Medical Branch, Galveston, TX, through the courtesy of Dr. Neil 1.0 Iloud I. Oxygen consumption rates at 21% O2 were determined with and without the drugs Adriamycin, bleomycin, and mitomycin C at SO Mg/ml directly inside the oxygen consumption chamber. In addition, mitomycin C (3 /ig/ml)- and misonidazole (1000 Â¿tg/ml)-treatedcells at 0.01% (>.,for l h were washed free of drug, resuspended in media, and the <>,consumption rates were determined.

RESULTS 0.1 Measurements of oxygen tension in solution without cells o with time were used to determine the physical constant kÂ¡ W according to data in Fig. 1. A kt value of 0.55 min"' with a u half-time of 1.26 min for equilibration was obtained. The for mer value is considerably higher than previously reported sys tems (33, 34) and reflects the high degree of gas exchange occurring in our system. This can be explained on the basis of geometry of vessel used (33, 34), and the high rate of shaking causing sufficient dispersion of the fluid over the glass surface and thereby decreasing the depth of the fluid. 0.01 The respiratory rate of the EAT cells at 37Â°Cwas 1.312 Â± 234 0.179 nmol O2/min/106 cells. This is similar to the previously TIME (MIN) quoted respiratory rate of about 0.1% O2/min/106 cells for Fig. 1. Data shown is a semilogarithmic plot of Equation A from three separate these cells (34, 35). experiments. From the experimental values of k\ and R, the theoretical oxygen level in solution at equilibrium (Câ€ž)wascalculated for mentally in Figs. 2 and 4, we were unable to detect any signifi a cell density of 1 x 105/ml. Calculated CÂ«,valuesfor C{g]of 20, cant differences in the measured oxygen levels with or without 10, 5, 1, 0.1, and 0.01% O2 were 19.985, 9.985, 4.985, 4.985, cells for a 1% O2 atmosphere. Similar measurements at 0.1% 0.985,0.085, and 0% O2, respectively. The latter value may not O2 were not attempted because this represents the limit of our be correct as the oxygen consumption rate changes in the 0.01- oxygen measuring system. 0.1% O2 range (12, 35, 36). At 0.01% O2 the oxygen consump The effect of oxygen concentration on the cytotoxicity of tion rate for EAT cells decreases to about 25% of initial value mitomycin C for the EAT cells is shown in Fig. 2. There was (35). This would result in a C. value of 0.0064% O2 for a 0.01 % an increase in the cytotoxicity of mitomycin C as the oxygen O2 atmosphere. At the cell density of 1 x 105/ml used experi- concentration was decreased to 0.01% O2. At the higher drug concentrations of mitomycin C (2 and 3 ng/ml) there was no 4 V. Gupta, unpublished observations. apparent difference in the cytotoxicity at 0.1% or 0.01% O2. 2408 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1987 American Association for Cancer Research. HYPOXIA AND DRUG CYTOTOXICITY

o 6< oc u. 0.01 - O

ce. co 0.001

0.0001 01 23456 /ng/ml MITOMYCIN-C Fig. 2. Surviving fraction of 1 x 10*/ml EAT cells treated with mitomycin C 0.0001 for l h after preincubation with different oxygen concentrations for 1.5 h. Ban, O 100 500 750 1000 mean Â±SE of three to five experiments. Â¿ig/mlMISONIDAZOLE

Fig. 4. Same as in Fig. 2 except that misonidazole was used. Points, mean of quadruplicate plates from one experiment. 5 9 Â§S 1.0 SIE 20% 02

0.1 1 g 0.01 0.1 1.0 10 100 G % OXYGEN CONCENTRATION lice ci 001 Fig. 3. Relative drug concentration of mitomycin C necessary as a function of oxygen concentration to achieve equivalent cell killing. Data extrapolated from Fig. 2. The data are expressed as a ratio of drug concentration necessary to reduce Ioc cell survival to 0.31(0,,) for the various oxygen levels when compared to 0.01% tn O,. 0001

There was also no difference in survival curves between 0.01% O: or N2 atmosphere (data not shown). The relative increase in drug concentration of mitomycin C 0.0001 necessary to achieve equivalent reduction of cell survival is 0.5 1.0 1.5 2.0 3.0 plotted in Fig. 3 based on the data in Fig. 2. The half-maximal Â¿/g/mlADRIAMYCIN increase in drug concentration to achieve equivalent cell killing Fig. 5. Surviving fraction of l x KI'm I EAT cells treated with Adriamycin occurs at 4.0% O2. This is different than what is observed with for 1 h after preincubation in 20% ( >..or N.. atmosphere for 1.5 h. Points, mean radiation-induced cell killing where cell survival decreases as Â±SE of three to six experiments. the oxygen concentration is increased with a half-maximal value of approximately 0.5% O2 (3). 16.5 h (Table 2) differences in the plating efficiency of drug- These data with mitomycin C are in contrast to the influence untreated cells, in cell cycle distribution by flow cytometry, or of oxygen concentration on the cytotoxic effects of misonida- in uptake of initiated thymidine were not observed (data not zole in Fig. 4. There is an apparent stringent requirement for shown). extreme hypoxia for the induction of the cytotoxicity of mison- The effects of cell density and pH on bleomycin cytotoxicity idazole. Differences between survival curves of misonidazole were determined because these variables may affect the cytotox and mitomycin C are apparent for N2 and 0.01 and 0.1% ().- icity of bleomycin if different cell densities are used under atmosphere (Figs. 2 and 4). Similar experiments with Adria- hypoxic and oxic conditions. As can be seen from Table 3, there mycin and bleomycin (Figs. 5 and 6) failed to reveal significant were significant differences with the relative survival fraction differences in the cytotoxicity between the N2 and 20% O2 increasing with cell density. We were unable to demonstrate a atmosphere for a 1-h drug exposure. However, significant dif significant effect of pH on bleomycin cytotoxicity over a range ferences in cell survival were observed for a more prolonged of 6.49-7.22 (data not shown). drug exposure (8 h) during hypoxia for bleomycin (Fig. 7) but In order to determine whether some of the differences in the not Adriamycin (Fig. 8) under similar experimental conditions. cell survival curves as a function of oxygen concentration with EAT cells exposed for 16.5 h to a 5% O2 or N2 atmosphere the drugs mitomycin C and misonidazole were due to changes before treatment with 1 itg/ml of Adriamycin for 1 h under in drug-induced cell oxygen consumption rates, measurements hypoxic and oxic conditions failed to reveal significant differ of oxygen consumption were performed under conditions sim ences in cell survival (Table 2). The cell survival values obtained ilar to that utilized in the experiments. We were unable to are comparable to that shown in Fig. 5 for the same drug demonstrate any significant effects. In addition, we were also concentration but for a much shorter duration of preincubation unable to demonstrate significant changes in cellular oxygen hypoxia. For cells exposed to a 5% O2 or N2 atmosphere for consumption after the addition of extremely high drug concen- 2409

20% Oj 20% 02 N,

0.01 0.01 20 40 60 80 100 4 6 Â¿jg/mlBLEOMYCIN EXPOSURE TIME (MRS) Fig. 6. Same as in Fig. S except bleomycin was used. Points, mean Â±SE of Fig. 8. Same as in Fig. 7 except that 0.1 Mg/ml of Adriamycin was used. three experiments. Points, mean Â±SE of four experiments.

Table 2 Effect of prolonged hypoxia on Adriamycin cytotoxicity EAT cells were exposed to the stated O2 atmosphere for 16.5 h with continuous gassing and then treated with Adriamycin (1 Mg/ml) l h at the same atmosphere.

Experiment1rOxygenconcentration5%O2 fraction0.010

N2 6.49 0.031 5%O2N2pH6.83 6.69 0.042 6.50Surviving 0.036 " No changes in cell cycle distribution by flow cytometry or uptake of initiated t(umiliine at end of experiment for control cells.

Table 3 Effect of cell density on bleomycin cytotoxicity Cells were treated with 50 Mg/rnl bleomycin for l h at 20% O2. At cell densities of 1 x lO'-lO" and 1 x IO1 the pH was 7.12 and 6.86, respectively, at end of experiment. Relative increase in Cell density survival fraction 1 x IO4 1 1 x IO5 1.21 Â±0.40Â« 1 x 10* 1.68 Â±0.08 001 1 x IO7 246 4.35 Â±2.00 " Mean Â±SE of three to four experiments. EXPOSURE TIME (MRS) Fig. 7. Surviving fraction of 5 x lOVml EAT cells treated with 10 jig/ml bleomycin for 2-8 h drug exposure time after initial l.S-h preincubation time at 20% ( ), or Nj. Points, mean Â±SE of four experiments. pH measurements under cient to demonstrate biological differences in the oxygen con oxic and hypoxic conditions for the 8-h time period varied from 7.13 to 7.22 and centration-dependent cytotoxicity of anticancer agents. 7.13 to 7.09, respectively. Differences at 8-h point only were significant (/' < The data in Figs. 2 and 3 clearly demonstrate that the O.OS3)by analysis of variance. cytotoxicity of mitomycin C for the EAT cells is highly oxygen concentration dependent, being more toxic to hypoxic than trations (50 Mg/ml) of Adriamycin, bleomycin, and mitomycin well-oxygenated cells. For the EAT cells there was very little C. These O2 cell consumption rates were 12.78 Â±2.14, 13.00 difference in the cytotoxicity of mitomycin C in the 0.0-0.1% Â±2.12, and 14.30 Â±2.65 nmol O2/min/107 cells, respectively, O2 range. These data are in contrast to recently published data when compared to a control respiratory rate of 13.12 Â±1.79 by Marshall and Rauth (14). These investigators observed that nmol O2/min/107 cells. most of the change in toxicity of mitomycin C under hypoxic conditions occurred in the 0.001-1% O2 range for Chinese hamster ovary cells. Moreover, these differences were apparent DISCUSSION after a 5-h drug exposure with very little difference observed at The drug misonidazole was first studied because there are 1 h. in contrast to our data with a 1-h drug exposure time. An consistent data in the literature demonstrating its selective examination of cell survival curves from other studies would toxicity towards hypoxic cells in vitro. In addition, the oxygen also suggest that quantitative differences in the effect of mito concentration-dependent cytotoxicity of misonidazole had been mycin C under hypoxic and oxic conditions are in some in established and had indicated that it is most toxic at oxygen stances a function of duration of drug exposure time (16, 20). concentrations less than 0.1% O2 (12, 13). The data presented In this regard it has previously been shown that the duration of in Fig. 4 contimi these observations and indicate that the hypoxia before drug exposure is not an important variable for experimental conditions used in the present study were suffi- mitomycin C cytotoxicity (37). Some work published by Ludwig 2410 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1987 American Association for Cancer Research. HYPOXIA AND DRUG CYTOTOXICITY et al. (19) would suggest that the enhanced cytotoxicity of for the expression of cytotoxic effects of bleomycin. Such cells mitomydn C under hypoxic conditions may be cell line de were more resistant than oxygenated cells with time. We found pendent. These workers were unable to demonstrate a signifi that the EAT cells were also more resistant to bleomycin as the cant effect of mitomycin C under hypoxic conditions at clini cell density increased. This is similar to the effects of cell density cally achievable drug concentrations for human tumor biopsy on Adriamycin cytotoxicity (25, 43). Since some investigators specimens, WiDR and HEC-1A human tumor cell lines, while have used different cell densities under oxic and hypoxic con demonstrating a significant effect for EMT6 and S180 murine ditions, it is possible that this may explain some of the quanti tumor cells. These data, if confirmed, may have significant tative differences in the literature. Our data and that of the clinical implications for the use of mitomycin C to selectively literature does not explain why hypoxic cells should be more kill hypoxic tumor cells. resistant to bleomycin than oxygenated cells with time. The The increased metabolism of mitomycin C under hypoxic observed differences are difficult to explain on the basis of a conditions to alkylating intermediates is well known and forms cellular oxygen radical-mediated mechanism. This is supported the basis for the increased cytotoxicity under hypoxic conditions by the fact that differences in the cytotoxic effects of bleomycin (5, 6). Although one can demonstrate that rate of formation of for a 1-h drug exposure time or significant changes in cellular alkylating intermediates is higher under hypoxic than aerobic oxygen consumption rates at rather high drug concentrations conditions, there is no quantitative correlation between these of bleomycin were not observed. The cell survival differences intermediates and the extent of cell killing under hypoxic with bleomycin after 8-h exposure to hypoxia cannot also be conditions (16). These data would suggest that factors other explained on the basis of changes in proliferative characteristics than, or in addition to, the formation of alkylating intermedi since EAT cells exposed to 16.5 h of hypoxia (Table 2) did not ates may influence cytotoxicity of mitomycin C under hypoxic display significant changes in cell cycle distribution or uptake conditions. Such reasons may explain the differences in the of tritiated thymidine. oxygen concentration dependency curves of mitomycin C be The data in the literature with respect to the effect of Adria tween our data and that of Marshall and Rauth (14). In fact, mycin under hypoxic or oxic conditions is most confusing. Ludwig et al. (19) in their study were unable to demonstrate Some data would suggest that chronically (23, 25, 26) but not the enhanced disappearance of mitomycin C under hypoxic acutely hypoxic cells (21, 23, 25, 26) are more resistant to conditions for the human cell line WiDR suggesting that the Adriamycin. However, other data would indicate that Adria enzymes responsible for enhanced metabolism under hypoxic mycin is more cytotoxic under oxic conditions (10) or that there conditions may not be present in some human tumor cell lines. are no significant differences in the effect of Adriamycin be Because hypoxic cells are radiation resistant, the use of tween acutely and chronically hypoxic cells (21). The reason mitomycin C as an adjunct to selectively kill hypoxic cells has for these differences in results is not clear. been proposed for clinical use (5). However, animal studies In contrast to our data with bleomycin, we were unable to have failed to demonstrate any significant selective cytotoxicity demonstrate any significant difference in the cytotoxicity of of mitomycin C for hypoxic cells in vivo (20, 38, 39). The Adriamycin between oxic and hypoxic conditions for 1-8 h of oxygen concentrations in normal and tumor tissues are heter drug exposure time or for cells previously exposed to a 16.5-h ogeneous and reported to be in the range of 2-5% O2 and 0.1- preincubation time in hypoxia (chronic hypoxia). It is possible 5% O2, respectively (1, 2,40). Estimates of the hypoxic fraction that some of the differences may be related to the induction of in tumors range from 0-100% (41). These observations and our oxygen-regulated proteins which may be of importance in mod data indicating significant cytotoxicity of mitomycin C at oxic ulating the response to Adriamycin under hypoxic conditions and intermediate levels of hypoxia may help to explain the (44, 45). In this regard it is interesting that prolonged exposure difficulty in trying to demonstrate a selective effect of mito to metabolic inhibitors such as 2-deoxyglucose and 2,4-dinitro- mycin C on hypoxic cells in vivo. phenol which results in increased cell survival to Adriamycin Our data raise the question why there are differences in the (46) may also be associated with induction of new cell protein oxygen concentration dependency between misonidazole and synthesis (47-49). Because the EAT cells are already growing mitomycin C. Misonidazole is metabolized under hypoxic con in vivo under conditions of radiobiological hypoxia (50), it is ditions by a nitroreductase (5). The nitroreduction is highly possible that when these cells are used for in vitro drug re sensitive to inhibition by rather low concentrations of oxygen sponses that the relative resistance to Adriamycin reported and the extent of inhibition appears to be drug dependent (42). following a period of prolonged hypoxia may not be observed. The metabolism of mitomycin C to reactive alkylating inter In an assessment of in vivo response of hypoxic tumor cells to mediates is enhanced under hypoxic conditions (14, 16). These Adriamycin, Tannock (51) concluded that hypoxic cells were differences in drug metabolism between mitomycin C and mi spared by Adriamycin but that this was due to drug delivery sonidazole may help to explain the differences in the oxygen problems. concentration-dependent cytotoxicities observed with these We had previously demonstrated an effect of Superoxide drugs. Differences in the oxygen concentration dependency dismutase on overcoming the effect of excess oxygen toxicity between mitomycin C and misonidazole for the EAT cells on the growth of EAT cells (32). Therefore, the effect of oxygen cannot be explained on the basis of changes in drug-induced radical scavengers on Adriamycin-induced cell cytotoxicity was oxygen consumption since we did not observe significant studied. We were unable to demonstrate any significant effects changes in these measurements. on Adriamycin cytotoxicity with the oxygen radical scavengers The duration of hypoxia and drug exposure time (25, 26), Superoxide dismutase, catalase, dimethylsulfoxide, and vitamin cell density (25, 43), pH (25, 30), and proliferative status (44) E.5 Brox et al. have reported that they did not observe significant are some variables which may influence the response of cells to differences in DNA strand breakage due to Adriamycin under drugs under hypoxic conditions. The conflicting data published hypoxic or oxic conditions (52). We have presented data indi in the literature (Table 1) would suggest that the cytotoxicity cating that differences in Adriamycin-induced cytotoxicity un of bleomycin is enhanced under oxygenated conditions for 1-h (10), 4-h (22), and 4-8-h (23) drug exposure time. There are der hypoxic conditions or in cell oxygen consumption rates were not present for EAT cells. These observations suggest that two studies where no differences were observed for a 1-2-h oxygen radical-mediated mechanisms may not be important for drug exposure time (23, 24). Our data clearly indicate that for the EAT cells the drug exposure time in hypoxia was important 5V. Gupta, unpublished observations. 2411

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1987 American Association for Cancer Research. HYPOXIA AND DRUG CYTOTOXICITY some tumor cells for clinically achievable drug concentrations 24. Shrieve, D. C., and Harris, J. W. Effects of bleomycin and irradiation on euoxic and hypoxic cells. Int. J. RadiÃ¢t.Oncol. Biol. Phys. 5: 1495-1498, of Adriamycin at the cell level. 1979. 25. Born, R., and Eichholtz-Wirth, H. Effect of different physiological conditions on the action of adriamycin on Chinese hamster cells in vitro. Br. J. Cancer, ACKNOWLEDGMENTS Â¥Â¥.-241-246,1981. 26. Smith, E., Stratford, I. J., and Adams, G. E. Cytotoxicity of adriamycin on aerobic and hypoxic Chinese hamster V79 cells in vitro. Br. J. Cancer, 41: We thank Dr. Neil Howell for help with the oxygen consumption 568-573, 1980. measurements. Dr. James Hokanson, Biostatistical Services, Cancer 27. Martin, W. M. C., and McNally, N. J. 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2412

Vicram Gupta and John J. Costanzi

Cancer Res 1987;47:2407-2412.

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