- Email: [email protected]
Enhanced radiation-sensitivity by preincubation with nitroimidazoles: Effect of glutathione depletion
Int. J. Radiation
Oncology
Pergamon
Biol. Phys., Vol. 29, No. 2. pp. 345-349, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0360-3016/94 $6.00 + .OO
0360-3016(93)E0012-U
??Bioreductive
Cytotoxins: Mechanism
and Tumor Response
ENHANCED RADIATION-SENSITIVITY BY PREINCUBATION WITH NITROIMIDAZOLES: EFFECT OF GLUTATHIONE DEPLETION CAMERON *Radiation
Oncology,
University
J. KOCH, of Pennsylvania, Cancer Research
PH.D.*
AND KIRSTEN
A.
SKOV, PH.D.+
Philadelphia, PA 19104-6072; and ‘Medical Centre, Vancouver, BC V5Z 1L3 Canada
Biophysics,
British Columbia
Purpose: The mechanism of enhanced radiosensitization by nitroheterocyclics after a preincubation period under hypoxic conditions was investigated. The hypothesis that this phenomenon was caused by glutathione depletion was tested. Methods and Materials: The phenomenon of enhanced radiosensitization by nitroheterocyclics after a preincubation period under hypoxic conditions is potentially of importance therapeutically because essentially nonlethal preradiation exposures to the electron affinic drugs cause a much larger radiation sensitization than would otherwise be expected. We have investigated this interesting property of several 2-nitroimidazoles to determine its possible cause and to test various hypotheses about maximizing its possible therapeutic benefit. In view of many observations that thiols are depleted by incubation of cells with nitroimidazoles under hypoxic conditions, we have specifically investigated this aspect of the preincubation effect. Depletion of glutathione was either enhanced by an overnight incubation with buthionine sulfoximine or minimized by preincubation with a 2-nitroimidazole which is sterically inhibited from causing thiol depletion. Results: When conditions were chosen which minimized variations in cellular glutathione content during the preincubation period, no preincubation effect was observed. At low, therapeutically relevant radiation doses, where 2nitroimidazoles are less efficient sensitizers, the preincubation effect may be even more important, but thiol depletion still minimizes its impact in this region of the dose-response curve. Conclusion: These results suggest that the preincubation effect is caused by a “self-sensitization” involving the known enhancement of radiation sensitization by thiol depletion. 2-nitroimidazoles,
Radiation sensitizing agents, Preincubation effect, Thiol depletion.
INTRODUCTION
Hypoxic cells are resistant to radiation and some chemotherapy drugs and extensive efforts have been undertaken in the past decades to eliminate these cells, often believed to limit the effectiveness of therapy in certain tumor types. Nitroimidazoles like misonidazole and etanidazole can kill hypoxic cells through bioreductive activation and can increase the radiation sensitivity of such cells via both radiochemical and biochemical means. The latter has been described as “the preincubation effect,” and was discovered independently by Hall and Biaglow (5) and Wong et al. (16). These investigators found that incubation of hypoxic cells with nontoxic concentrations of misonidazole led to an enhanced radiosensitization by
the same drug (preincubation effect). The cause of this interesting effect is at present poorly understood, but several suggestions have been made including an interaction of sublethal drug damage with radiation sensitivity, inactivation of a key enzyme, inhibition of repair of potentially lethal radiation damage, changes in intracellular drug concentration, depletion of an important protective molecule such as glutathione (GSH), etc. The latter process, known to occur with most nitroaromatics (1) would cause a “self-sensitization” (3) since thiol depletion is well known to cause radiation sensitizing drugs to become more efficient (more active at the same concentration, or as active at a lower concentration) (4, 7). We wondered if the preincubation effect was affected or even specifically mediated by changes in cellular GSH and/or other thiols. The present data support this hypothesis.
Reprint requests to: Dr. Cameron J. Koch, University of Pennsylvania, Radiation Oncology, 195 John Morgan Bldg., Philadelphia, PA 19 104-6072. Acknowledgements-Dimethylmisonidazole was provided by Dr. Jerry Born, University of New Mexico; etanidazole and Lbuthionine S/R sulfoximine were provided by MS Nancita Lo-
max, Drug Synthesis and Chemistry Branch, National Cancer Institute. The authors wish to thank Yimei Caio, Haibo Zhou, and Kathleen Finn for expert technical assistance. Supported by grant # CA-49498 from the NIH, U.S.A. (CJK) and grants from the NC1 and MRC of Canada (KAS). Accepted for publication 14 October 1993. 345
346
I. J. Radiation Oncology 0 Biology 0 Physics
METHODS
AND
MATERIALS
Experiments were performed in control vs. GSH-depleted V-79 Chinese hamster fibroblasts using two different procedures. In the first (suitable for relatively high levels of killing), they were grown on glass Petri dishes. The medium on the dishes was exchanged with medium for the experiment (+ BSO, 2-nitroimidazole) and the dishes were placed into leakproof aluminum chambers which were deoxygenated using a series of gas exchanges (nitrogen, less than 10 ppm oxygen). The chambers were then held at 4” or 37” for O-2 h, cooled to 4”, if necessary, and then irradiated. Following irradiation, the dishes were removed from the chambers, and the cells trypsinized and plated for colony formation (7). The second procedure was developed to allow the assessment of cell survival at very low levels of killing, using a dynamic microscope image processing system (DMIPS) (11, 14). Cells were irradiated in suspension, with vs. without preincubation with 2-nitroimidazole, and were subsequently plated into tissue culture flasks for analysis by the DMIPS. For both procedures, GSH was depleted by overnight incubation in the presence of 50-100 PM L-buthionine sulfoximine (BSO). In some experiments, the use of BSO was continued throughout the preincubation period (in the presence of 2-nitroimidazole at 37” under hypoxic conditions). Cellular thiols were measured using two techniques; the Tietze assay (for GSH) and high performance liquid chromatography’ (HPLC) using electrochemical detection ( 13, 15). For HPLC analysis, cells were lysed in 50 mM 5-sulfosalicylic acid, and the precipitate removed by centrifugation. The acid was supplemented with the copper chelator diethyldithiocarbamate (100 PM) to prevent autooxidation of thiols (data not shown). Samples were stored at 4” and analyzed on a reverse phase column’ within 24 h. It was found that the solvent system previously described ( 13) was unable to resolve low levels of cysteine, which co-elute with acids and salts because of its extreme polarity. Therefore, the pH of the mobile phase (flow rate 0.9 ml/min, helium sparged) was decreased to partially uncharge the acid group of cysteine, using 100 mM phosphoric acid (pH 2.0). Methanol was added at 5- 10% as was the detergent and ion pairing molecule I-heptanesulfonic acid (3.3 mM) (6). The concentrations of sensitizer used were chosen using several criteria; for nonGSH depleted (control) cells, the drug concentration used was low enough that there would be no (or minimal) toxicity for the preincubations under hypoxia, but high enough that a reasonable efficiency of sensitization would be found. Both criteria were met using a drug concentration sufficient to provide a level of radiation resistance (dose to produce same effect) ( 12) which was roughly intermediate between air and nitrogen-2
’ HPLC pumps, autoinjector were from Waters.
and electrochemical
detector
Volume 29, Number 2, I994
mM for etanidazole and 6 mM for dimethylmisonidazole (DMM) (2). For GSH-depleted cells, it was necessary to determine a drug concentration that would provide similar levels of radiation resistance, and corresponding degree of toxicity. We have found that GSH-depleted cells, compared with control cells, are sensitive to the toxic effects of nitroaromatics at 4-6-fold lower drug concentrations (data not shown). Thus, use of 0.5 mM etanidazole in glutathione-depleted cells provided similar levels of absolute radiation resistance (see results) to 2.0 mM drug with control cells. Based on toxicity data at higher drug concentrations, 0.5 mM etanidazole would be somewhat more toxic in glutathione-depleted cells than would 2.0 mM drug in control cells. RESULTS
AND
DISCUSSION
The Tietze assay, because of its limited sensitivity, is not very practical for use in experiments involving extensive thiol depletion. With the cell concentrations typically used (lOO,OOO-500,000 per ml), glutathione depletion can only be monitored at low resolution and accuracy with this method. If cell density is increased, then the effects of BSO and bioreductive thiol depletion often change substantially. Thus, it was our initial impression that overnight incubation of V79 cells with 100 PM BSO led to glutathione levels which were - 10% of controls. The recent demonstration by Fahey et al. (9) that there can be artifactual influences of thiols other than glutathione in the Tietze assay reduced substantially our confidence in the reliability of this value. Additionally, we have found that many substances can change the apparent efficiency of the glutathione reductase in the Tietze assay. It was felt therefore, that a more direct assay would be highly beneficial, and we chose the electrochemical method because it measures the actual substances of interest, without any intermediate steps of derivatization. An inspection of the thiol levels in BSO pretreated vs. control cells indicates two unexpected results: first, the glutathione level is about 1% of control levels (not 10%) and secondly, the cysteine level is about 5 times higher than controls (Fig. 1). For the conventional (high radiation dose) assays, preincubation (2 h at 37”) of control cells with 2 mM etanidazole provided a very substantial increase in radiosensitizing efficiency, without toxicity (Fig. 2). In this and all succeeding figures, the survival data were not normalized to zero-dose plating efficiency so that any changes in plating efficiency would be apparent. An even larger relative effect was seen in the low radiation dose assays, because etanidazole (or other nitroimidazoles) are less efficient in this region ofthe survival curve (data not shown). When cells were pretreated with BSO, only a minimal increase in radiosensitizing efficiency by 0.5 mM etani-
* Alltech Adsorbosphere HS Cl8 7U; 250 mm by 4.6 mm.
Enhanced
15 TIME
with nitroimidazoles
0 C. J. KOCH
AND
K. A.
347
SKOV
h
-
0
radiation-sensitivity
30
AFTER
INJECTION
-2 0
15
AT 0 AND
30
0
15 MINUTES
5
IO
15
RADIATION
Fig. 1. High performance liquid chromatograms of glutathione and cysteine. In the left panel, a standard (50 pL, 0 time) consisting of 5 PM cysteine and 10 FM glutathione is compared with the same volume of an acid lysis of control cells (1 O6 cells per ml), injected at 15 min. Cysteine and glutathione are resolved at 6 and 10 min postinjection, respectively. The cellular cysteine peak is almost invisible on this scale. In the right panel, a standard (50 FL, 0 time) consisting of 1 KM cysteine and 2 PM glutathione is compared with the same volume of an acid lysis of BSO pretreated cells ( IO6 cells per ml), injected at I5 min. In this case (note expanded current axis), cysteine is by far the dominant cellular thiol, and is (on an absolute basis) about 5-fold greater than in control cells.
dazole was observed, and there was no difference in effect if fresh BSO was added during the preincubation period (Fig. 3). Using the low-dose assay a similar result was observed-radiosensitization by 0.5 mM etanidazole was only minimally enhanced by BSO pretreatment (Fig. 4). Again, it should be emphasized that no corrections have been made for plating efficiency, which was minimally
20
DOSE
25
30
(Gy)
Fig. 3. The preincubation effect after BSO pretreatment. BSOpretreatment sensitizes nondrug treated controls (0) (compare Fig. 2) but again there is no effect of preincubation at 37” (U). In the presence of 0.5 mM drug, absolute radiation resistance of nonpreincubated cells (A) is very comparable to control cells with 2 mM drug (Fig. 2) but almost no preincubation effect is seen (A) even if fresh BSO is added to the drug-containing medium (e).
decreased by either the BSO pretreatment, or by the preincubation treatment (Fig. 4). Detailed analysis (to be published elsewhere) of thiol decreases during the preincubation period indicated that there were still substantial effects of the sensitizer in BSO pretreated cells-both on the small residual levels of glutathione and on the cysteine levels which presumably rise to partially compensate for the cellular loss of glutathione. Thus, a final set of experiments was done using DMM, an analogue of misonidazole, which had been ring methylated at the 4 and 5 position (2). This compound was synthesized by Dr. Jerry Born, University of New Mexico, with the specific aim of eliminating the nucleophillic at-
0 . \
A
. \
.OI’,“““““““““““.“‘..’ 0
5
\ \ \
4 10
RADIATION
15
20 DOSE
25
(Gy)
.l 0
10
5 RADIATION
Fig. 2. The preincubation effect with control cells. In the absence of sensitizer, there is no effect of preincubation (0) on the radiation response at 0” (0) or room temperature (data not shown). In contrast, 2 mM etanidazole, after 2 h incubation under hypoxic conditions at 37” (A) causes much greater sensitization than 2 mM drug alone (A).
\ ’ A
30
DOSE
(Gy)
Fig. 4. The preincubation effect after BSO pretreatment-low dose assay. As above, in the presence of 0.5 mM drug, nonpreincubated cells (A) are minimally sensitized by a 2 h preincubation period at 37” (A)-BSO was added to the drug-containing medium.
15
I. J. Radiation
348
Oncology
0 Biology 0 Physics
A\
0
5
10 RADIATION
15
20 DOSE
25
30
(Gy)
Fig. 5. The preincubation effect with control cells. As above, in the absence of sensitizer, there is no effect of preincubation (0) on the radiation response of cells (0). The presence of 6 mM dimethlmisonidazole (A) causes about the same sensitization as 2 mM etanidazole (see above). A 2-h incubation under hypoxic conditions at 37” either before (A) or after (+) radiation causes no change in response.
tack by thiols on the reduced nitroaromatic ( 10). Dimethylmisonidazole has a lower electron affinity than most 2nitroimidazoles (2), and therefore, was used at 6 mM to provide similar levels of radiosensitization to 2 mM etanidazole. No preincubation effect was seen with this compound (Fig. 5). Our results show that, over the whole survival curve range, the preincubation effect is minimized under conditions where either (a) there is no depletion of cellular glutathione or (b) glutathione depletion is already extensive due to inhibition of its synthesis by BSO. A reasonable explanation for our findings, consistent with suggestions made previously by Bump and Brown (3), is that the preincubation effect is caused by an “autosensitization,” involving metabolic depletion of the dominant intracellular radioprotective agent, glutathione. The role of glutathione (or other thiols) in modifying the relative effi-
Volume
29, Number
2. 1994
ciency of radiation sensitizers can be described in terms of a “competition model” whereby radiation produced radicals on key cellular molecules (such as DNA) are either oxidized by agents such oxygen or the nitroaromatics, or reduced by agents like the aminothiols. Thus, efficiency of sensitization is enhanced by nitroaromatic induced metabolic depletion of thiols. or BSO-induced depletion of glutathione, but both depletion mechanisms cause the same net effect. Our experimental results cast doubt on other proposed mechanisms of the preincubation effect. For example, it seems very unlikely that an interaction with sublethal drug damage is involved, since 0.5 mM etanidazole would be more toxic to glutathione depleted cells than 2.0 mM drug to control cells. Similarly, it seems unlikely that inactivation of a key enzyme, involved in either metabolism of repair. is involved. It is known that bioreductively activated nitroaromatics interact predominantly with protein thiols (12) and this reaction would be enhanced, not diminished. in the absence of low molecular weight thiols. Nevertheless. there are many quantitative aspects of the preincubation effect that should be pursued. The role of cysteine in the above processes is at present poorly defined. We are unaware of previous reports showing enhanced cysteine levels after BSO preincubation, but perhaps this should not be considered an unexpected finding. Since cysteine has consistently been shown to be a better radioprotector than glutathione. our results may shed some light on the nature of enhanced radiosensitization after glutathione depletion. We and others have always found sensitizer enhancement ratios to be much less than the factoral changes in glutathione. On the one hand, our present HPLC results, showing that BSO pretreatment leads to substantially less glutathione than previous estimates using the Tietze assay, enhance this discrepancy. On the other hand, our finding that BSO pretreatment causes substantial increases in cysteine may lead to its explanation. Clearly, much more work needs to be done in this interesting area.
REFERENCES 1. Biaglow, J. E.; Varnes,
2.
3.
4.
5.
M. E.; Koch, C. J.; Sridhar, R. Metabolic activation ofcarcinogenic nitro-compounds to oxygen reactive intermediates. In: Floyd, R. E., ed. Free radicals and cancer. New York: Marcel Deckker Inc.: 1982:441502. Born, J. L.; Smith, B. R.; Harper, N.; Koch, C. J. Metabolism and radiosensitization of 4,5-dimethylmisonidazole. a ring substituted analog of misonidazole. Biochem. Pharmacol. 43:1337-1344; 1992. Bump, E. A.; Brown, J. M. Role of glutathione in the radiation response of mammalian cells in vitro and in viva. Pharmac. Ther. 47: 117- 136; 1990. Bump, E. A.; Yu, N. Y.; Brown, J. M. Radiosensitization of hypoxic tumor cells by depletion of intracellular glutathione. Science 2 17:544-545; 1982. Hall, E. J.; Biaglow, J. E. Ro-07-0582 as a radiosensitizer and cytotoxic agent. Int. J. Radiat. Biol. Oncol. Phys. 2: 521-530: 1977.
6. Koch. C. J.; Giandomenico, A. R.: lyengar, C. W. L. Bioreductive metabolism of AF-2 [2(2-furyl)-3-(5-nitro2furyl)acrylamideJ combined with 2-nitroimidazoles: Implications for use as hypoxic cell markers. Biochem. Pharmacol. 46:1029-1036; 1993. Koch, C. J.; Skov, K. A. Comparisons of cellular radiation response using absolute rather than relative parameters. Radiat. Res. 132:40-49; 1992. Koch, C. J.; Stobbe, C. C.; Bump, E. A. The effect on the Km for radiosensitization at 0°C of thiol depletion by diethylmaleate pretreatment: Quantitative differences found using the radiation sensitizing agent misonidazole or oxygen. Radiat. Res. 98:141-153; 1984. Loh, S. N.; Dethlefsen, L. A.: Newton, G. L.; Aguilera, J. A.: Fahey, R. C. Nuclear thiols: Technical limitations on the determination of endogenous nuclear glutathione and the potential importance of sulfhydryl proteins. Radiat. Res. 121:98-106; 1990.
Enhanced radiation-sensitivity with nitroimidazoles 0 C. R. A. Molecular interactions and biological 10. McClelland, effects of the products of reduction of nitroimidazoles. In: Adams, G. E., Breccia, A., Fielden, E. M., Wardman, P., eds. Selective activation of drugs by redox processes. New York: Plenum Press; 1990: 125% 136.
I I. Palcic, B.; Skarsgard,
L. D. Reduced oxygen enhancement ratio at low doses of ionizing radiation. Radiat. Res. 100: 328-339; 1984.
12. Raleigh, J. A.; Koch, C. J. The importance of thiols in the reductive binding of 2-nitroimidazoles to macromolecules. Biochem. Pharmacol. 40:2457-2464; 1990. 13. Shaw. L. M.; Bonner,
H. S.; Turrisi, A. T.; Norfleet,
A. L.:
J. KOCH AND K. A. SKOV
349
Kligerman, M. Measurement ofS-2(3-ammopropylammo) ethanethiol (WR-1065) in blood and tissue. J. Liqu. Chromatog. 9:845-859; 1986. 14. Skov, K. A.; MacPhail, H. S. Effect of BSO on the radiation response at low (O-4 Gy) doses. Int. J. Radiat. Biol. Oncol. Phys. 22:533-537; 1992. 15. Tietze, F. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal. Biochem. 27:502-522; 1969. 16. Wong, T. W.; Whitmore, G. F.; Gulyas, S. Studies on the toxicity and radiosensitizing ability of misonidazole under conditions of prolonged incubation. Radiat. Res. 75:541555: 1978.
Oncology
Pergamon
Biol. Phys., Vol. 29, No. 2. pp. 345-349, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0360-3016/94 $6.00 + .OO
0360-3016(93)E0012-U
??Bioreductive
Cytotoxins: Mechanism
and Tumor Response
ENHANCED RADIATION-SENSITIVITY BY PREINCUBATION WITH NITROIMIDAZOLES: EFFECT OF GLUTATHIONE DEPLETION CAMERON *Radiation
Oncology,
University
J. KOCH, of Pennsylvania, Cancer Research
PH.D.*
AND KIRSTEN
A.
SKOV, PH.D.+
Philadelphia, PA 19104-6072; and ‘Medical Centre, Vancouver, BC V5Z 1L3 Canada
Biophysics,
British Columbia
Purpose: The mechanism of enhanced radiosensitization by nitroheterocyclics after a preincubation period under hypoxic conditions was investigated. The hypothesis that this phenomenon was caused by glutathione depletion was tested. Methods and Materials: The phenomenon of enhanced radiosensitization by nitroheterocyclics after a preincubation period under hypoxic conditions is potentially of importance therapeutically because essentially nonlethal preradiation exposures to the electron affinic drugs cause a much larger radiation sensitization than would otherwise be expected. We have investigated this interesting property of several 2-nitroimidazoles to determine its possible cause and to test various hypotheses about maximizing its possible therapeutic benefit. In view of many observations that thiols are depleted by incubation of cells with nitroimidazoles under hypoxic conditions, we have specifically investigated this aspect of the preincubation effect. Depletion of glutathione was either enhanced by an overnight incubation with buthionine sulfoximine or minimized by preincubation with a 2-nitroimidazole which is sterically inhibited from causing thiol depletion. Results: When conditions were chosen which minimized variations in cellular glutathione content during the preincubation period, no preincubation effect was observed. At low, therapeutically relevant radiation doses, where 2nitroimidazoles are less efficient sensitizers, the preincubation effect may be even more important, but thiol depletion still minimizes its impact in this region of the dose-response curve. Conclusion: These results suggest that the preincubation effect is caused by a “self-sensitization” involving the known enhancement of radiation sensitization by thiol depletion. 2-nitroimidazoles,
Radiation sensitizing agents, Preincubation effect, Thiol depletion.
INTRODUCTION
Hypoxic cells are resistant to radiation and some chemotherapy drugs and extensive efforts have been undertaken in the past decades to eliminate these cells, often believed to limit the effectiveness of therapy in certain tumor types. Nitroimidazoles like misonidazole and etanidazole can kill hypoxic cells through bioreductive activation and can increase the radiation sensitivity of such cells via both radiochemical and biochemical means. The latter has been described as “the preincubation effect,” and was discovered independently by Hall and Biaglow (5) and Wong et al. (16). These investigators found that incubation of hypoxic cells with nontoxic concentrations of misonidazole led to an enhanced radiosensitization by
the same drug (preincubation effect). The cause of this interesting effect is at present poorly understood, but several suggestions have been made including an interaction of sublethal drug damage with radiation sensitivity, inactivation of a key enzyme, inhibition of repair of potentially lethal radiation damage, changes in intracellular drug concentration, depletion of an important protective molecule such as glutathione (GSH), etc. The latter process, known to occur with most nitroaromatics (1) would cause a “self-sensitization” (3) since thiol depletion is well known to cause radiation sensitizing drugs to become more efficient (more active at the same concentration, or as active at a lower concentration) (4, 7). We wondered if the preincubation effect was affected or even specifically mediated by changes in cellular GSH and/or other thiols. The present data support this hypothesis.
Reprint requests to: Dr. Cameron J. Koch, University of Pennsylvania, Radiation Oncology, 195 John Morgan Bldg., Philadelphia, PA 19 104-6072. Acknowledgements-Dimethylmisonidazole was provided by Dr. Jerry Born, University of New Mexico; etanidazole and Lbuthionine S/R sulfoximine were provided by MS Nancita Lo-
max, Drug Synthesis and Chemistry Branch, National Cancer Institute. The authors wish to thank Yimei Caio, Haibo Zhou, and Kathleen Finn for expert technical assistance. Supported by grant # CA-49498 from the NIH, U.S.A. (CJK) and grants from the NC1 and MRC of Canada (KAS). Accepted for publication 14 October 1993. 345
346
I. J. Radiation Oncology 0 Biology 0 Physics
METHODS
AND
MATERIALS
Experiments were performed in control vs. GSH-depleted V-79 Chinese hamster fibroblasts using two different procedures. In the first (suitable for relatively high levels of killing), they were grown on glass Petri dishes. The medium on the dishes was exchanged with medium for the experiment (+ BSO, 2-nitroimidazole) and the dishes were placed into leakproof aluminum chambers which were deoxygenated using a series of gas exchanges (nitrogen, less than 10 ppm oxygen). The chambers were then held at 4” or 37” for O-2 h, cooled to 4”, if necessary, and then irradiated. Following irradiation, the dishes were removed from the chambers, and the cells trypsinized and plated for colony formation (7). The second procedure was developed to allow the assessment of cell survival at very low levels of killing, using a dynamic microscope image processing system (DMIPS) (11, 14). Cells were irradiated in suspension, with vs. without preincubation with 2-nitroimidazole, and were subsequently plated into tissue culture flasks for analysis by the DMIPS. For both procedures, GSH was depleted by overnight incubation in the presence of 50-100 PM L-buthionine sulfoximine (BSO). In some experiments, the use of BSO was continued throughout the preincubation period (in the presence of 2-nitroimidazole at 37” under hypoxic conditions). Cellular thiols were measured using two techniques; the Tietze assay (for GSH) and high performance liquid chromatography’ (HPLC) using electrochemical detection ( 13, 15). For HPLC analysis, cells were lysed in 50 mM 5-sulfosalicylic acid, and the precipitate removed by centrifugation. The acid was supplemented with the copper chelator diethyldithiocarbamate (100 PM) to prevent autooxidation of thiols (data not shown). Samples were stored at 4” and analyzed on a reverse phase column’ within 24 h. It was found that the solvent system previously described ( 13) was unable to resolve low levels of cysteine, which co-elute with acids and salts because of its extreme polarity. Therefore, the pH of the mobile phase (flow rate 0.9 ml/min, helium sparged) was decreased to partially uncharge the acid group of cysteine, using 100 mM phosphoric acid (pH 2.0). Methanol was added at 5- 10% as was the detergent and ion pairing molecule I-heptanesulfonic acid (3.3 mM) (6). The concentrations of sensitizer used were chosen using several criteria; for nonGSH depleted (control) cells, the drug concentration used was low enough that there would be no (or minimal) toxicity for the preincubations under hypoxia, but high enough that a reasonable efficiency of sensitization would be found. Both criteria were met using a drug concentration sufficient to provide a level of radiation resistance (dose to produce same effect) ( 12) which was roughly intermediate between air and nitrogen-2
’ HPLC pumps, autoinjector were from Waters.
and electrochemical
detector
Volume 29, Number 2, I994
mM for etanidazole and 6 mM for dimethylmisonidazole (DMM) (2). For GSH-depleted cells, it was necessary to determine a drug concentration that would provide similar levels of radiation resistance, and corresponding degree of toxicity. We have found that GSH-depleted cells, compared with control cells, are sensitive to the toxic effects of nitroaromatics at 4-6-fold lower drug concentrations (data not shown). Thus, use of 0.5 mM etanidazole in glutathione-depleted cells provided similar levels of absolute radiation resistance (see results) to 2.0 mM drug with control cells. Based on toxicity data at higher drug concentrations, 0.5 mM etanidazole would be somewhat more toxic in glutathione-depleted cells than would 2.0 mM drug in control cells. RESULTS
AND
DISCUSSION
The Tietze assay, because of its limited sensitivity, is not very practical for use in experiments involving extensive thiol depletion. With the cell concentrations typically used (lOO,OOO-500,000 per ml), glutathione depletion can only be monitored at low resolution and accuracy with this method. If cell density is increased, then the effects of BSO and bioreductive thiol depletion often change substantially. Thus, it was our initial impression that overnight incubation of V79 cells with 100 PM BSO led to glutathione levels which were - 10% of controls. The recent demonstration by Fahey et al. (9) that there can be artifactual influences of thiols other than glutathione in the Tietze assay reduced substantially our confidence in the reliability of this value. Additionally, we have found that many substances can change the apparent efficiency of the glutathione reductase in the Tietze assay. It was felt therefore, that a more direct assay would be highly beneficial, and we chose the electrochemical method because it measures the actual substances of interest, without any intermediate steps of derivatization. An inspection of the thiol levels in BSO pretreated vs. control cells indicates two unexpected results: first, the glutathione level is about 1% of control levels (not 10%) and secondly, the cysteine level is about 5 times higher than controls (Fig. 1). For the conventional (high radiation dose) assays, preincubation (2 h at 37”) of control cells with 2 mM etanidazole provided a very substantial increase in radiosensitizing efficiency, without toxicity (Fig. 2). In this and all succeeding figures, the survival data were not normalized to zero-dose plating efficiency so that any changes in plating efficiency would be apparent. An even larger relative effect was seen in the low radiation dose assays, because etanidazole (or other nitroimidazoles) are less efficient in this region ofthe survival curve (data not shown). When cells were pretreated with BSO, only a minimal increase in radiosensitizing efficiency by 0.5 mM etani-
* Alltech Adsorbosphere HS Cl8 7U; 250 mm by 4.6 mm.
Enhanced
15 TIME
with nitroimidazoles
0 C. J. KOCH
AND
K. A.
347
SKOV
h
-
0
radiation-sensitivity
30
AFTER
INJECTION
-2 0
15
AT 0 AND
30
0
15 MINUTES
5
IO
15
RADIATION
Fig. 1. High performance liquid chromatograms of glutathione and cysteine. In the left panel, a standard (50 pL, 0 time) consisting of 5 PM cysteine and 10 FM glutathione is compared with the same volume of an acid lysis of control cells (1 O6 cells per ml), injected at 15 min. Cysteine and glutathione are resolved at 6 and 10 min postinjection, respectively. The cellular cysteine peak is almost invisible on this scale. In the right panel, a standard (50 FL, 0 time) consisting of 1 KM cysteine and 2 PM glutathione is compared with the same volume of an acid lysis of BSO pretreated cells ( IO6 cells per ml), injected at I5 min. In this case (note expanded current axis), cysteine is by far the dominant cellular thiol, and is (on an absolute basis) about 5-fold greater than in control cells.
dazole was observed, and there was no difference in effect if fresh BSO was added during the preincubation period (Fig. 3). Using the low-dose assay a similar result was observed-radiosensitization by 0.5 mM etanidazole was only minimally enhanced by BSO pretreatment (Fig. 4). Again, it should be emphasized that no corrections have been made for plating efficiency, which was minimally
20
DOSE
25
30
(Gy)
Fig. 3. The preincubation effect after BSO pretreatment. BSOpretreatment sensitizes nondrug treated controls (0) (compare Fig. 2) but again there is no effect of preincubation at 37” (U). In the presence of 0.5 mM drug, absolute radiation resistance of nonpreincubated cells (A) is very comparable to control cells with 2 mM drug (Fig. 2) but almost no preincubation effect is seen (A) even if fresh BSO is added to the drug-containing medium (e).
decreased by either the BSO pretreatment, or by the preincubation treatment (Fig. 4). Detailed analysis (to be published elsewhere) of thiol decreases during the preincubation period indicated that there were still substantial effects of the sensitizer in BSO pretreated cells-both on the small residual levels of glutathione and on the cysteine levels which presumably rise to partially compensate for the cellular loss of glutathione. Thus, a final set of experiments was done using DMM, an analogue of misonidazole, which had been ring methylated at the 4 and 5 position (2). This compound was synthesized by Dr. Jerry Born, University of New Mexico, with the specific aim of eliminating the nucleophillic at-
0 . \
A
. \
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Fig. 2. The preincubation effect with control cells. In the absence of sensitizer, there is no effect of preincubation (0) on the radiation response at 0” (0) or room temperature (data not shown). In contrast, 2 mM etanidazole, after 2 h incubation under hypoxic conditions at 37” (A) causes much greater sensitization than 2 mM drug alone (A).
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Fig. 4. The preincubation effect after BSO pretreatment-low dose assay. As above, in the presence of 0.5 mM drug, nonpreincubated cells (A) are minimally sensitized by a 2 h preincubation period at 37” (A)-BSO was added to the drug-containing medium.
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A\
0
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10 RADIATION
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20 DOSE
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Fig. 5. The preincubation effect with control cells. As above, in the absence of sensitizer, there is no effect of preincubation (0) on the radiation response of cells (0). The presence of 6 mM dimethlmisonidazole (A) causes about the same sensitization as 2 mM etanidazole (see above). A 2-h incubation under hypoxic conditions at 37” either before (A) or after (+) radiation causes no change in response.
tack by thiols on the reduced nitroaromatic ( 10). Dimethylmisonidazole has a lower electron affinity than most 2nitroimidazoles (2), and therefore, was used at 6 mM to provide similar levels of radiosensitization to 2 mM etanidazole. No preincubation effect was seen with this compound (Fig. 5). Our results show that, over the whole survival curve range, the preincubation effect is minimized under conditions where either (a) there is no depletion of cellular glutathione or (b) glutathione depletion is already extensive due to inhibition of its synthesis by BSO. A reasonable explanation for our findings, consistent with suggestions made previously by Bump and Brown (3), is that the preincubation effect is caused by an “autosensitization,” involving metabolic depletion of the dominant intracellular radioprotective agent, glutathione. The role of glutathione (or other thiols) in modifying the relative effi-
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ciency of radiation sensitizers can be described in terms of a “competition model” whereby radiation produced radicals on key cellular molecules (such as DNA) are either oxidized by agents such oxygen or the nitroaromatics, or reduced by agents like the aminothiols. Thus, efficiency of sensitization is enhanced by nitroaromatic induced metabolic depletion of thiols. or BSO-induced depletion of glutathione, but both depletion mechanisms cause the same net effect. Our experimental results cast doubt on other proposed mechanisms of the preincubation effect. For example, it seems very unlikely that an interaction with sublethal drug damage is involved, since 0.5 mM etanidazole would be more toxic to glutathione depleted cells than 2.0 mM drug to control cells. Similarly, it seems unlikely that inactivation of a key enzyme, involved in either metabolism of repair. is involved. It is known that bioreductively activated nitroaromatics interact predominantly with protein thiols (12) and this reaction would be enhanced, not diminished. in the absence of low molecular weight thiols. Nevertheless. there are many quantitative aspects of the preincubation effect that should be pursued. The role of cysteine in the above processes is at present poorly defined. We are unaware of previous reports showing enhanced cysteine levels after BSO preincubation, but perhaps this should not be considered an unexpected finding. Since cysteine has consistently been shown to be a better radioprotector than glutathione. our results may shed some light on the nature of enhanced radiosensitization after glutathione depletion. We and others have always found sensitizer enhancement ratios to be much less than the factoral changes in glutathione. On the one hand, our present HPLC results, showing that BSO pretreatment leads to substantially less glutathione than previous estimates using the Tietze assay, enhance this discrepancy. On the other hand, our finding that BSO pretreatment causes substantial increases in cysteine may lead to its explanation. Clearly, much more work needs to be done in this interesting area.
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