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Metabolism induced binding of 4C-misonidazole to hypoxic cells: Kinetic dependence on oxygen concentration and misonidazole concentration
0 Session II
METABOLISM INDUCED BINDING OF “C-MISONIDAZOLE TO HYPOXIC CELLS: KINETIC DEPENDENCE ON OXYGEN CONCENTRATION AND MISONIDAZOLE CONCENTRATION CAMERON
J. KOCH, PH.D., CORINNE C. STOBBE, B.Sc. AND KATHY A. BAER, B.Sc.
CrossCancer Institute. Radiation Oncology, I 1560 University Avenue. Edmonton. Alberta. Canada T6G 122 Under conditions of extreme hypoxia, metabolic products of the metabolism of misonidazole bind to cellular molecules at a rate which is linear with time and proportional to the square root of misonidazole concentration. Very small amounts of oxygen reduce the overall rate of binding and cause a change in the dependence on misonidazole concentration from square root (half order) to linear (first order). Because of the known electron affinity of misonidazole, a model is presented whereby the nitro-group is reduced to a radical in a first order reaction. This radical binds to cellular molecules in a slow first order reaction and either disproportionates or dimerizes in a fast second order reaction. Based on the overall effect of oxygen on the kinetics of the rate of binding, the radical is tentatively assumed to be the 3 electron reduction product. Nitroheterocyclics, cells, Metabolism
Chemical reduction and metabolism; and identification.
Oxygen
dependent reactions; Electron
transfer;
Hypoxic
.
METHODS AND MATERIALS
INTRODUCTION
methods used in these have been described previously.7-‘o Briefly. V79-WNRE cells were maintained in exponential growth phase using Eagle’s minimal essential medium supplemented with 13.5% V/V fetal calf serum and antibiotics (MEM; cell medium components from Gibco). The afternoon before an experiment, cells were inoculated onto the central area (approximately 100,000 cells per square cm) of glass petri dishes and incubated overnight at 37°C in a water jacketed incubator with gas phase 95% air, 5% CO2 (100% relative humidity). On the morning of the experiment, drugs (if required) were added to MEM containing 20 mM HEPES buffer (pH 7.4) and ‘/5 normal bicarbonate (4.5 mM). The medium on the dishes was aspirated and then the experimental medium was added, first as a 0.5 ml rinse which was also aspirated and then as a final 1 ml to be used for the experiment. The dishes were placed in aluminum leak-proof chambers and the air in the chambers was replaced with gas containing that of the desired oxygen content via a series of gas changes. Using these techniques the cells on the petri dishes were covered by a uniform layer of medium about 100 microns
The cytotoxic action towards mammalian cells of nitroheterocyclics like misonidazole is inhibited by some conditions and chemicals but the most potent of these is oxygen.9.‘o.‘” Concentrations of only a few micromolar have been shown to be effective. Although the mechanism for this protection is thought to involve an effective decrease in the reductive metabolism of the nitro-containing a comprehensive model for this metabolism drt&.‘,” has not been presented. However, one phenomenon associated with the metabolism of misonidazole by mammalian cells has been studied in a detailed kinetic manner. That is the binding of products of the metabolism of radioactively-labelled misonidazole (BPMM) to hypoxic cells incubated at 37°C.3.‘5-‘7 Chapman et al. showed that the binding rate was proportional to the square root of the concentration of misonidazole over a considerable range of concentrations.3 We have studied the effect of oxygen concentration on the rate of BPMM to mammalian cells. Our expectation was that information obtained on the oxygen dependency of various metabolic effects of misonidazole would lead to a greater understanding of its overall metabolism.
The culturing and experimental
experiments
Also, we wish to thank Dr. Jim Raleigh for many helpful discussions on radical and other chemical reactions and for preliminary information on the stoichiometry of electron consumption during misonidazole reduction. Accepted for publication 22 March 1984.
Reprint requests to: Dr. Cameron Koch. .~ckno~ledgemenrs-Work supported by the National Cancer Institute of Canada. The authors would like to thank Dr. Don Chapman for generating much enthusiasm in the development of this project and for sharing his radioactive misonidazole.
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thick which equilibrated very rapidly with the gas phase of the chamber.6 At a cell density of lo5 cells/cm’, and an oxygen consumption rate of 3 X IO-” M/cell/set the maximum oxygen gradient across the thin medium layer would be 1.5 PM.* 6 However, at these low oxygen concentrations. cellular oxygen consumption is reduced.’ Since the precise gradient across the medium layer is unknown, all oxygen values quoted in this paper are for gas-phase equilibrium. Radioactive misonidazole (14CZ)was obtained by Dr. J. D. Chapman through the generous auspices of Dr. W. E. Scott at Hoffman-La Roche Inc. (Nutley, NJ). The specific activity was 230 $Zi/mg. Radioactive purity was greater than 90% (determined by Dr. J. Raleigh using thin layer chromatography) and contaminants were not responsible for the binding reported herein, since one could directly compete against binding by purified unlabelled misonidazole. Combinations of radioactive and non-radioactive misonidazole were mixed in the experimental medium. After the gas phase of the chambers had reached the desired oxygen level (30 minutes at room temperature), the chambers containing the dishes with cells were incubated at 37°C in a forced air incubator. Temperature equilibrium was established after 30 min. At various times the chambers were removed from the incubator, the oxygen content of the chamber was tested6 and the chambers were opened and dishes removed. The radioactive medium was removed, the dishes were rinsed twice with 3 ml of medium and than an additional 3 ml (chase medium) was added. The dishes were incubated at 37°C for 15 min in air to allow all non-metabolized radioactive misonidazole to leave the cells. It was established that this rinsing procedure left only background amounts of radioactivity on dishes which contained cells and radioactive medium but without any incubation at 37°C. The dishes were cooled on ice and the chase medium was removed. The dishes were rinsed with ice cold PBS and the cells were scraped into 1.5 ml of 5% TCA. This suspension was added to a centrifuge tube and the dish was rinsed with 1.O ml of TCA which was also added to the centrifuge tube. The tubes were spun at 2500 RPM at 0°C for 20 min. and the supematant was added to a liquid scintillation vial containing 10 ml of Scintiverse (Fisher). This sample was considered to contain acid soluble bound products of the metabolism of misonidazole (ASBPMM). The TCA insoluble pellet was solubilized by incubating at room temperature in 0.3 ml of 1N NaOH for 60 min with vortexing at 30 min. The base was neutralized and the sample centrifuged as above and added to another scintillation vial containing Scintiverse. This
* At the altitude of Edmonton (atmospheric pressure = 700 mm/Hg = 94 kPa) air equilibrated medium at 37T contains approximately 2 IO PM O2 so a direct correspondence exists between the concentration of oxygen in the medium (PM) and
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1984.
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8
sample was considered to contain acid precipitable bound products of the metabolism of misonidazole (APBPMM). The samples were countedt with correction for quenching. Each experiment contained a set of dishes which were incubated in extreme hypoxia with 20 1M of ‘“C-misonidazole. The rate of binding (approximately 3.000 cpm/ IO6 cells/hr) for this standard condition was taken to be unity and the rate of binding for all other concentrations and conditions was compared with this standard. It was found that the radioactive misonidazole behaved identically to non-radioactive misonidazole. That is. the absolute counts of BPMM for 20 PM “C-misonidazole plus 180 PM non-radioactive misonidazole was the expected ‘/,. of the counts of BPMM for 200 KM “C-misonidazole. Therefore, the normalized rate for any condition and total concentration of misonidazole was computed as: observed rate/relative R=
fraction of radioactive: non-radioactive misonidazole
rate for extremely hypoxic cells at 20 PM all rates being expressed as cpm/ lo6 cells/hr of incubation. It was found in all of the many experiments performed that one could equally compare rates of binding for the ASBPMM as for the APBPMM. (That is there were no differential effects of oxygen concentration or misonidazole concentration on the acid soluble versus insoluble fraction of bound material). Since the acid soluble compartment contained roughly 2-fold more radioactive bound products, the data shown in this paper were computed from this soluble fraction of bound material.
RESULTS The counts of ASBPMM and APBPMM were found (Fig. 1) to increase linearly with time (Fig. 1) over the course of a typical experiment involving incubation for up to 8 hours at 37°C. To determine a rate of binding, the slopes of curves such as those in Figure 1 containing 4-8 points were fit via a linear regression forced through time zero. When the logarithm of the rate of binding was plotted against the logarithm of misonidazole concentration an extremely interesting observation was made. Whereas the slope of the plot was 4 for extremely hypoxic cells, in precise agreement with the results of Chapman ef al.,’ it changed to 1 in the presence of oxygen. That is, the rate of binding changed from a half order to a first order dependence on misonidazole concentration when small amounts of oxygen were made available to the cell. For the Chinese hamster cells used in this study, the
the oxygen partial pressure of the gas phase (thousands of parts/ million = kppm). t Beckman Scintillation Counter.
Binding of ‘C-misonidazole
to hypoxic cells 0 C.
16
0” “Q 8
et al.
1329
dioactive misonidazole at these low binding rates, we have not yet determined the extent of the second component of oxygen-concentration-dependent change in the rate of BPMM to the cells.
.
m 5 12
J. KOCH
. .
t
DK3CUSSION
I
2
3
TIME
(HOURS)
4
5
6
Fig. 1. Rate of binding of “C2-misonidazole (thousands of counts per minute per million cells) as a function of time of incubation for extreme hypoxia at 20 PM drug concentration (m, 0) and for moderate hypoxia (2.1 PM 02) at 1,000 PM drug concentration (100 PM “C2 + 900 PM non radioactive (A, A). The absolute ratios of counts between the two conditions (not corrected for concentration) is 1.80 using the ASBPMM (B, A) and 1.76 using the APBPMM (Cl, A) and these are not signif-
icantly different. kinetic change in the rate of binding was present at only 2 PM oxygen (Fig. 2). As the oxygen concentration was increased further, the overall rate of binding decreased but with the maintenance of first order kinetics. In the range of 7-20 PM oxygen there was essentially no additional inhibition of binding by the increase in oxygen concentration. However, at even higher oxygen concentrations approaching air saturation levels there was yet another increase in the order of the kinetic dependence of binding on misonidazole concentration (Fig. 2). Because of the requirement for very large amounts of ra-
1
kI
RN02 + e- -
A’- c* A’. H’
(1)
The radical may be negative or neutral depending on the pH. The fate of the radical then follows 2 paths, a first order reaction leading to binding, and a second order loss via dismutation or dimerization. k?
i.e. A + Substrate -
Product (covalently bound)
A’ + A’ 2 A + Arcduced(or A - A dimer)
(2) (3)
Since A’ is the agent directly responsible for binding we determine its net rate of change and under steady state conditions let this equal zero:
I
1
The observation that the rate of binding of misonidazole to extremely hypoxic cells varies as the square root of misonidazole concentration over a very broad concentration range suggested a relatively simple chemical kinetic mechanism was operating. Although we were unable to find a description of this type of kinetics in standard chemical texts, there appeared to be a precise analogue of the present results with those of the radiochemical peroxidation of lipids. I3 In the case of lipid peroxidation, a lipid peroxy radical is produced which either propagates in a slow first order reaction with other lipids or disappe.ares in a fast second order reaction. In the case of misonidazole metabolism, assume that misonidazole is reduced in a one electron step by a first order competition for electrons from other metabolic redox-reactions: ‘.I’
0 . f
??
.
. .
-d[A’] dt
= 2k3[A.]’ + kz[A’] - k,[RNOZ] = 0.
(4)
Solving the quadratic equation: [A’] = $?
+
[ RNO?].
3
-6 LOG
-5
CONCENTRATION
-4 MISONIDAZOLE
(5)
If k1 e k, -3 (MOLAR)
Fig. 2. The rate of binding of “Cz-misonidazole to V79-WNRE Chinese hamster cells as a function of misonidazole and oxygen concentration (all relative lo the rate of extremely hypoxic cells at 20 PM). Kinetic analysis ofthe results gives a l/zorder reaction for extreme hypoxia (0) but a first order reaction at low oxygen levels (0. 2 PM: 0. 7 PM). There is no difference in absolute rate of binding between 7 PM Oz (0) and 20 PM O2 (A) and there is an additional change in the kinetic order of the reaction at 0: levels approaching air (B-180 PM).
r
(6) The simplest interpretation of [A’] from this analysis suggests that the nitro-radical is the important species for binding. However. the reaction mechanism is completely general as long as “some” radical is formed in a reaction which is first order in misonidazole concentration and that radical reacts either in a relatively slow first order
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Radiation Oncology 0 Biology 0 Physics
reaction to produce binding or a fast second order reaction: i.e. RNOz -,-+-+A’-HA’. k, effect’ve
(7) H+
Since there is considerable evidence in the literature that the reduction of agents like misonidazole occurs in three or more electron steps (see refs. 1 & I 1 for review) a more likely candidate for A’ is the nitroso radical (anion?). Indeed our own data suggests that there are more steps in the sequence than reactions l-6 would indicate because oxygen has 2 distinct effects; at very low levels ( 1- 10 PM) and at much higher levels (approximately 100 MM). The absolute number of electrons per reduced misonidazole equivalent would depend on the fate of the second-order loss of A’. If this loss were by dismutation one would expect a 4 electron reduction. However, if this loss were by dimerization one would expect a 3 electron reduction.‘4 The reaction scheme proposed correctly predicts the consequences for the addition of oxygen, since electrons will be transferred rapidly from the radical anion to oxygen leading to the production of superoxide and then hydrogen peroxide.’ kl
i.e. RN02 + e- -
A’- -
A’
(8)
H+ kz
A’- + Subst. -
covalent binding
(9)
kr
A’- + O2 -
A + Oz-
(10)
The steady state concentration of A’- (or A’) will now be first order in RNOz concentration since there will be much less of this radical and the second order loss reaction will become insignificant. We are presently measuring the ratio of binding of metabolic products of “C-misonidazole to another cell line (EMT-6 ed) and preliminary data indicates good agreement with the results found for the V79 cell line
August 1984. Volume 10. Number 8
used in the present experiments. We are also attempting to ascribe kinetic parameters to the rates of killing of moderately hypoxic and extremely hypoxic cells by high concentrations of misonidazole. The dependence of toxicity towards extremely hypoxic cells on misonidazole concentration reported previously’ would be consistent with a square root relationship. However. we have not yet determined whether this relationship changes at low oxygen levels. Previous attempts to measure the oxygen dependence of the metabolism and/or binding of sensitizer molecules’2.‘6 have possibly over-estimated the oxygen requirement for inhibition because of the difficulties involved with maintaining a suspension culture of cells at low oxygen concentrations.4.6,‘8 Similarly, previous measurements of the relative binding rates in N2 vs. air have not found the large differentials reported here because of the low specific activities of “C-Misonidazole used.‘5-‘7.‘9 It is clear from the results shown in Figure 2 that high drug concentrations tend to minimize the differences between aerobic and hypoxic rates of binding. Our results also have some implications for the use of radioactive nitroheterocycles as markers for hypoxic cells and as radiosensitizers,2,3.‘5-‘7.‘9 Since oxygen not only reduces the overall rate of binding of APBPMM and ASBPMM but also changes the kinetics of the reaction(s) it is clear that the differential rate of binding for aerobic versus hypoxic cells increases as the misonidazole concentration decreases (Fig. 2). Thus, if a gamma-emitting isotope were to be used in a nuclear medicine type assay for hypoxic cells one would need a very high specific activity of isotope to achieve the best contrast. Finally, it is crucial to determine if other nitroimidazoles (metronidazole, Sri-2508) have the same type of kinetic behavior of metabolism. The present results demonstrate the feasibility of using the kinetic dependencies of drug metabolism on oxygen concentration to identify the (radical?) species which are important for toxicity and for binding.
REFERENCES Biaglow.J.E., Varnes. M.E.. Koch. C.J.. Sridhar, R.: Metabolic activation of carcinogenic nitro-compounds to oxygen reactive intermediates. In Free Radicals and Cancer, R.E. Floyd (Ed.). New York. Marcel Dekker. 1983, pp. 441502. 2. Chapman, J.D.: Hypoxic sensitizers-Implications for radiation therapy. New Eng. J. Med. 301: 1429-1432, 1979. 3. Chapman, J.D.. Baer. K.. Lee, J.: Characteristics of the metabolism-induced binding of misonidazole to hypoxic mammalian cells. Cuncer Res. 43: 1523-1528. 1983. 4. Franko, A.J., Koch. C.J.: The oxygen dependence ofbinding of ‘%Z-misonidazole to multi-cell spheroids and rodent tumors. Inf. J. Radial. Oncol. Biol. Phys. 10: (In press) 1984. 5. Holtzman, J.L.. Crankshaw. D.L.. Peterson. F.J.. Palnaszek, C.F.: The kinetics of the aerobic reduction of nitrofurantoin I.
by NADPH Cytochrome P-450 (c) reductase. Molec. Phurm. 20: 669-673. 1981. 6. Koch, C.J.: A “thin-film” culturing technique allowing rapid gas-liquid equilibration (6 seconds) with no toxicity to mammalian cells. Rudiut. Res. 97: 434-442, 1984. Koch. C.J., Biaglow, J.E.: Respiration of mammalian cells at low concentrations of oxygen: I. Effect of hypoxic cell radiosensitizing drugs. Br. J. Cancer 37(Suppl. III): l63167. 1978. Koch, C.J., Howell, R.L.: Combined radiation-protective and radiation-sensitizing agents: 11. Radiosensitivity of hypoxic or aerobic Chinese hamster fibroblasts in the presence of cysteamine and misonidazole: Implications for the “oxygen effect.” Rudiur. Res. 87: 265-283. 1981. Koch. C.J., Howell, R.L.: Misonidazole: Inter-related factors
Binding of “C-misontdazole affecting 693-696.
cytotoxicity.
In/. J. Radial.
Oncol.
Biol.
Phy.
to hypoxtc cells 0 C. J. 8:
1982.
IO. Koch.
C.J.. Howell. R.L.. Biaglow, J.E.: Ascorbate anion potentiates cytotoxicity of nitro-aromatic compounds under hypoxic and anoxic conditions. Br. J. Cancer 39: 32 l-329. 1979.
1I. Mason.
R.P.: Free radical intermediates of foreign comand their toxicological significance. In Review in Biochemical Toxicologic. Vol. I. E. Hodgson. J.R. Bend and R.M. Philpot (Eds.). Amsterdam. Elsevier North Holland. 1973. pp. 151-200. 12. Olive. P.L.. Durand. R.E.: Fluorescent nitroheterocycles for identifying hypoxic cells. Cancer Res 43: 3276-3280. pounds
1983. 13. Raleigh.
oxidation rubidium
J.A., Kremers. W.: Promotion of radiation perin models of lipid membranes by caesium and counter-ions: micellar linoleic and linolenic acids.
Int. J. Radial. 14. Raleigh. J.A..
troimidazoles: Biol.
Biol.
34: 439-447.
Liu. SF.:
Reductive
1978.
fragmentation
of 2-ni-
1331
KOCH e/ ul
Amines and aldehydes.
Int
J. Radial.
Oncol.
Phys. 10: 1337-l 340. 1984.
15. Taylor. Y.C.. Rauth. A.M.: Differences in the toxicity and metabolism of the 2-nitroimidazole misonidazole (Ro-070582) in HeLa and Chinese hamster ovary cells. C’anwr Res. 38: 2745-2752. 1978. 16. Taylor. Y.C.. Rauth. A.M.: Sulphydryls. ascorbate and oxygen as modifiers of the toxicity and metabolism of misonidazole in vitro. Br. J. Cancer 41: 892-900. 1980. 17. Varghese. A.J., Whitmore. G.F.: Binding to cellular macromolecules as a possible mechanism for the cytotoxicity of misonidazole. Cancer Rex 40: 2 165-2 169. 1980. 18. Whillans. D.W., Rauth. A.M.: An experimental and analytical study ofoxygen depletion in stirred cell suspensions. Radial. Rex 84: 97-l 14. 1980. 19. Wong. T.W., Whitmore. G.F.. Gulyas. S.: Studies on the toxicity and radiosensitizing ability of Ro-07-0582 under Radtat. Rev. 75: 54 I conditions of prolonged incubation. 555, 1978.
METABOLISM INDUCED BINDING OF “C-MISONIDAZOLE TO HYPOXIC CELLS: KINETIC DEPENDENCE ON OXYGEN CONCENTRATION AND MISONIDAZOLE CONCENTRATION CAMERON
J. KOCH, PH.D., CORINNE C. STOBBE, B.Sc. AND KATHY A. BAER, B.Sc.
CrossCancer Institute. Radiation Oncology, I 1560 University Avenue. Edmonton. Alberta. Canada T6G 122 Under conditions of extreme hypoxia, metabolic products of the metabolism of misonidazole bind to cellular molecules at a rate which is linear with time and proportional to the square root of misonidazole concentration. Very small amounts of oxygen reduce the overall rate of binding and cause a change in the dependence on misonidazole concentration from square root (half order) to linear (first order). Because of the known electron affinity of misonidazole, a model is presented whereby the nitro-group is reduced to a radical in a first order reaction. This radical binds to cellular molecules in a slow first order reaction and either disproportionates or dimerizes in a fast second order reaction. Based on the overall effect of oxygen on the kinetics of the rate of binding, the radical is tentatively assumed to be the 3 electron reduction product. Nitroheterocyclics, cells, Metabolism
Chemical reduction and metabolism; and identification.
Oxygen
dependent reactions; Electron
transfer;
Hypoxic
.
METHODS AND MATERIALS
INTRODUCTION
methods used in these have been described previously.7-‘o Briefly. V79-WNRE cells were maintained in exponential growth phase using Eagle’s minimal essential medium supplemented with 13.5% V/V fetal calf serum and antibiotics (MEM; cell medium components from Gibco). The afternoon before an experiment, cells were inoculated onto the central area (approximately 100,000 cells per square cm) of glass petri dishes and incubated overnight at 37°C in a water jacketed incubator with gas phase 95% air, 5% CO2 (100% relative humidity). On the morning of the experiment, drugs (if required) were added to MEM containing 20 mM HEPES buffer (pH 7.4) and ‘/5 normal bicarbonate (4.5 mM). The medium on the dishes was aspirated and then the experimental medium was added, first as a 0.5 ml rinse which was also aspirated and then as a final 1 ml to be used for the experiment. The dishes were placed in aluminum leak-proof chambers and the air in the chambers was replaced with gas containing that of the desired oxygen content via a series of gas changes. Using these techniques the cells on the petri dishes were covered by a uniform layer of medium about 100 microns
The cytotoxic action towards mammalian cells of nitroheterocyclics like misonidazole is inhibited by some conditions and chemicals but the most potent of these is oxygen.9.‘o.‘” Concentrations of only a few micromolar have been shown to be effective. Although the mechanism for this protection is thought to involve an effective decrease in the reductive metabolism of the nitro-containing a comprehensive model for this metabolism drt&.‘,” has not been presented. However, one phenomenon associated with the metabolism of misonidazole by mammalian cells has been studied in a detailed kinetic manner. That is the binding of products of the metabolism of radioactively-labelled misonidazole (BPMM) to hypoxic cells incubated at 37°C.3.‘5-‘7 Chapman et al. showed that the binding rate was proportional to the square root of the concentration of misonidazole over a considerable range of concentrations.3 We have studied the effect of oxygen concentration on the rate of BPMM to mammalian cells. Our expectation was that information obtained on the oxygen dependency of various metabolic effects of misonidazole would lead to a greater understanding of its overall metabolism.
The culturing and experimental
experiments
Also, we wish to thank Dr. Jim Raleigh for many helpful discussions on radical and other chemical reactions and for preliminary information on the stoichiometry of electron consumption during misonidazole reduction. Accepted for publication 22 March 1984.
Reprint requests to: Dr. Cameron Koch. .~ckno~ledgemenrs-Work supported by the National Cancer Institute of Canada. The authors would like to thank Dr. Don Chapman for generating much enthusiasm in the development of this project and for sharing his radioactive misonidazole.
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thick which equilibrated very rapidly with the gas phase of the chamber.6 At a cell density of lo5 cells/cm’, and an oxygen consumption rate of 3 X IO-” M/cell/set the maximum oxygen gradient across the thin medium layer would be 1.5 PM.* 6 However, at these low oxygen concentrations. cellular oxygen consumption is reduced.’ Since the precise gradient across the medium layer is unknown, all oxygen values quoted in this paper are for gas-phase equilibrium. Radioactive misonidazole (14CZ)was obtained by Dr. J. D. Chapman through the generous auspices of Dr. W. E. Scott at Hoffman-La Roche Inc. (Nutley, NJ). The specific activity was 230 $Zi/mg. Radioactive purity was greater than 90% (determined by Dr. J. Raleigh using thin layer chromatography) and contaminants were not responsible for the binding reported herein, since one could directly compete against binding by purified unlabelled misonidazole. Combinations of radioactive and non-radioactive misonidazole were mixed in the experimental medium. After the gas phase of the chambers had reached the desired oxygen level (30 minutes at room temperature), the chambers containing the dishes with cells were incubated at 37°C in a forced air incubator. Temperature equilibrium was established after 30 min. At various times the chambers were removed from the incubator, the oxygen content of the chamber was tested6 and the chambers were opened and dishes removed. The radioactive medium was removed, the dishes were rinsed twice with 3 ml of medium and than an additional 3 ml (chase medium) was added. The dishes were incubated at 37°C for 15 min in air to allow all non-metabolized radioactive misonidazole to leave the cells. It was established that this rinsing procedure left only background amounts of radioactivity on dishes which contained cells and radioactive medium but without any incubation at 37°C. The dishes were cooled on ice and the chase medium was removed. The dishes were rinsed with ice cold PBS and the cells were scraped into 1.5 ml of 5% TCA. This suspension was added to a centrifuge tube and the dish was rinsed with 1.O ml of TCA which was also added to the centrifuge tube. The tubes were spun at 2500 RPM at 0°C for 20 min. and the supematant was added to a liquid scintillation vial containing 10 ml of Scintiverse (Fisher). This sample was considered to contain acid soluble bound products of the metabolism of misonidazole (ASBPMM). The TCA insoluble pellet was solubilized by incubating at room temperature in 0.3 ml of 1N NaOH for 60 min with vortexing at 30 min. The base was neutralized and the sample centrifuged as above and added to another scintillation vial containing Scintiverse. This
* At the altitude of Edmonton (atmospheric pressure = 700 mm/Hg = 94 kPa) air equilibrated medium at 37T contains approximately 2 IO PM O2 so a direct correspondence exists between the concentration of oxygen in the medium (PM) and
August
1984.
Volume IO. Number
8
sample was considered to contain acid precipitable bound products of the metabolism of misonidazole (APBPMM). The samples were countedt with correction for quenching. Each experiment contained a set of dishes which were incubated in extreme hypoxia with 20 1M of ‘“C-misonidazole. The rate of binding (approximately 3.000 cpm/ IO6 cells/hr) for this standard condition was taken to be unity and the rate of binding for all other concentrations and conditions was compared with this standard. It was found that the radioactive misonidazole behaved identically to non-radioactive misonidazole. That is. the absolute counts of BPMM for 20 PM “C-misonidazole plus 180 PM non-radioactive misonidazole was the expected ‘/,. of the counts of BPMM for 200 KM “C-misonidazole. Therefore, the normalized rate for any condition and total concentration of misonidazole was computed as: observed rate/relative R=
fraction of radioactive: non-radioactive misonidazole
rate for extremely hypoxic cells at 20 PM all rates being expressed as cpm/ lo6 cells/hr of incubation. It was found in all of the many experiments performed that one could equally compare rates of binding for the ASBPMM as for the APBPMM. (That is there were no differential effects of oxygen concentration or misonidazole concentration on the acid soluble versus insoluble fraction of bound material). Since the acid soluble compartment contained roughly 2-fold more radioactive bound products, the data shown in this paper were computed from this soluble fraction of bound material.
RESULTS The counts of ASBPMM and APBPMM were found (Fig. 1) to increase linearly with time (Fig. 1) over the course of a typical experiment involving incubation for up to 8 hours at 37°C. To determine a rate of binding, the slopes of curves such as those in Figure 1 containing 4-8 points were fit via a linear regression forced through time zero. When the logarithm of the rate of binding was plotted against the logarithm of misonidazole concentration an extremely interesting observation was made. Whereas the slope of the plot was 4 for extremely hypoxic cells, in precise agreement with the results of Chapman ef al.,’ it changed to 1 in the presence of oxygen. That is, the rate of binding changed from a half order to a first order dependence on misonidazole concentration when small amounts of oxygen were made available to the cell. For the Chinese hamster cells used in this study, the
the oxygen partial pressure of the gas phase (thousands of parts/ million = kppm). t Beckman Scintillation Counter.
Binding of ‘C-misonidazole
to hypoxic cells 0 C.
16
0” “Q 8
et al.
1329
dioactive misonidazole at these low binding rates, we have not yet determined the extent of the second component of oxygen-concentration-dependent change in the rate of BPMM to the cells.
.
m 5 12
J. KOCH
. .
t
DK3CUSSION
I
2
3
TIME
(HOURS)
4
5
6
Fig. 1. Rate of binding of “C2-misonidazole (thousands of counts per minute per million cells) as a function of time of incubation for extreme hypoxia at 20 PM drug concentration (m, 0) and for moderate hypoxia (2.1 PM 02) at 1,000 PM drug concentration (100 PM “C2 + 900 PM non radioactive (A, A). The absolute ratios of counts between the two conditions (not corrected for concentration) is 1.80 using the ASBPMM (B, A) and 1.76 using the APBPMM (Cl, A) and these are not signif-
icantly different. kinetic change in the rate of binding was present at only 2 PM oxygen (Fig. 2). As the oxygen concentration was increased further, the overall rate of binding decreased but with the maintenance of first order kinetics. In the range of 7-20 PM oxygen there was essentially no additional inhibition of binding by the increase in oxygen concentration. However, at even higher oxygen concentrations approaching air saturation levels there was yet another increase in the order of the kinetic dependence of binding on misonidazole concentration (Fig. 2). Because of the requirement for very large amounts of ra-
1
kI
RN02 + e- -
A’- c* A’. H’
(1)
The radical may be negative or neutral depending on the pH. The fate of the radical then follows 2 paths, a first order reaction leading to binding, and a second order loss via dismutation or dimerization. k?
i.e. A + Substrate -
Product (covalently bound)
A’ + A’ 2 A + Arcduced(or A - A dimer)
(2) (3)
Since A’ is the agent directly responsible for binding we determine its net rate of change and under steady state conditions let this equal zero:
I
1
The observation that the rate of binding of misonidazole to extremely hypoxic cells varies as the square root of misonidazole concentration over a very broad concentration range suggested a relatively simple chemical kinetic mechanism was operating. Although we were unable to find a description of this type of kinetics in standard chemical texts, there appeared to be a precise analogue of the present results with those of the radiochemical peroxidation of lipids. I3 In the case of lipid peroxidation, a lipid peroxy radical is produced which either propagates in a slow first order reaction with other lipids or disappe.ares in a fast second order reaction. In the case of misonidazole metabolism, assume that misonidazole is reduced in a one electron step by a first order competition for electrons from other metabolic redox-reactions: ‘.I’
0 . f
??
.
. .
-d[A’] dt
= 2k3[A.]’ + kz[A’] - k,[RNOZ] = 0.
(4)
Solving the quadratic equation: [A’] = $?
+
[ RNO?].
3
-6 LOG
-5
CONCENTRATION
-4 MISONIDAZOLE
(5)
If k1 e k, -3 (MOLAR)
Fig. 2. The rate of binding of “Cz-misonidazole to V79-WNRE Chinese hamster cells as a function of misonidazole and oxygen concentration (all relative lo the rate of extremely hypoxic cells at 20 PM). Kinetic analysis ofthe results gives a l/zorder reaction for extreme hypoxia (0) but a first order reaction at low oxygen levels (0. 2 PM: 0. 7 PM). There is no difference in absolute rate of binding between 7 PM Oz (0) and 20 PM O2 (A) and there is an additional change in the kinetic order of the reaction at 0: levels approaching air (B-180 PM).
r
(6) The simplest interpretation of [A’] from this analysis suggests that the nitro-radical is the important species for binding. However. the reaction mechanism is completely general as long as “some” radical is formed in a reaction which is first order in misonidazole concentration and that radical reacts either in a relatively slow first order
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Radiation Oncology 0 Biology 0 Physics
reaction to produce binding or a fast second order reaction: i.e. RNOz -,-+-+A’-HA’. k, effect’ve
(7) H+
Since there is considerable evidence in the literature that the reduction of agents like misonidazole occurs in three or more electron steps (see refs. 1 & I 1 for review) a more likely candidate for A’ is the nitroso radical (anion?). Indeed our own data suggests that there are more steps in the sequence than reactions l-6 would indicate because oxygen has 2 distinct effects; at very low levels ( 1- 10 PM) and at much higher levels (approximately 100 MM). The absolute number of electrons per reduced misonidazole equivalent would depend on the fate of the second-order loss of A’. If this loss were by dismutation one would expect a 4 electron reduction. However, if this loss were by dimerization one would expect a 3 electron reduction.‘4 The reaction scheme proposed correctly predicts the consequences for the addition of oxygen, since electrons will be transferred rapidly from the radical anion to oxygen leading to the production of superoxide and then hydrogen peroxide.’ kl
i.e. RN02 + e- -
A’- -
A’
(8)
H+ kz
A’- + Subst. -
covalent binding
(9)
kr
A’- + O2 -
A + Oz-
(10)
The steady state concentration of A’- (or A’) will now be first order in RNOz concentration since there will be much less of this radical and the second order loss reaction will become insignificant. We are presently measuring the ratio of binding of metabolic products of “C-misonidazole to another cell line (EMT-6 ed) and preliminary data indicates good agreement with the results found for the V79 cell line
August 1984. Volume 10. Number 8
used in the present experiments. We are also attempting to ascribe kinetic parameters to the rates of killing of moderately hypoxic and extremely hypoxic cells by high concentrations of misonidazole. The dependence of toxicity towards extremely hypoxic cells on misonidazole concentration reported previously’ would be consistent with a square root relationship. However. we have not yet determined whether this relationship changes at low oxygen levels. Previous attempts to measure the oxygen dependence of the metabolism and/or binding of sensitizer molecules’2.‘6 have possibly over-estimated the oxygen requirement for inhibition because of the difficulties involved with maintaining a suspension culture of cells at low oxygen concentrations.4.6,‘8 Similarly, previous measurements of the relative binding rates in N2 vs. air have not found the large differentials reported here because of the low specific activities of “C-Misonidazole used.‘5-‘7.‘9 It is clear from the results shown in Figure 2 that high drug concentrations tend to minimize the differences between aerobic and hypoxic rates of binding. Our results also have some implications for the use of radioactive nitroheterocycles as markers for hypoxic cells and as radiosensitizers,2,3.‘5-‘7.‘9 Since oxygen not only reduces the overall rate of binding of APBPMM and ASBPMM but also changes the kinetics of the reaction(s) it is clear that the differential rate of binding for aerobic versus hypoxic cells increases as the misonidazole concentration decreases (Fig. 2). Thus, if a gamma-emitting isotope were to be used in a nuclear medicine type assay for hypoxic cells one would need a very high specific activity of isotope to achieve the best contrast. Finally, it is crucial to determine if other nitroimidazoles (metronidazole, Sri-2508) have the same type of kinetic behavior of metabolism. The present results demonstrate the feasibility of using the kinetic dependencies of drug metabolism on oxygen concentration to identify the (radical?) species which are important for toxicity and for binding.
REFERENCES Biaglow.J.E., Varnes. M.E.. Koch. C.J.. Sridhar, R.: Metabolic activation of carcinogenic nitro-compounds to oxygen reactive intermediates. In Free Radicals and Cancer, R.E. Floyd (Ed.). New York. Marcel Dekker. 1983, pp. 441502. 2. Chapman, J.D.: Hypoxic sensitizers-Implications for radiation therapy. New Eng. J. Med. 301: 1429-1432, 1979. 3. Chapman, J.D.. Baer. K.. Lee, J.: Characteristics of the metabolism-induced binding of misonidazole to hypoxic mammalian cells. Cuncer Res. 43: 1523-1528. 1983. 4. Franko, A.J., Koch. C.J.: The oxygen dependence ofbinding of ‘%Z-misonidazole to multi-cell spheroids and rodent tumors. Inf. J. Radial. Oncol. Biol. Phys. 10: (In press) 1984. 5. Holtzman, J.L.. Crankshaw. D.L.. Peterson. F.J.. Palnaszek, C.F.: The kinetics of the aerobic reduction of nitrofurantoin I.
by NADPH Cytochrome P-450 (c) reductase. Molec. Phurm. 20: 669-673. 1981. 6. Koch, C.J.: A “thin-film” culturing technique allowing rapid gas-liquid equilibration (6 seconds) with no toxicity to mammalian cells. Rudiut. Res. 97: 434-442, 1984. Koch. C.J., Biaglow, J.E.: Respiration of mammalian cells at low concentrations of oxygen: I. Effect of hypoxic cell radiosensitizing drugs. Br. J. Cancer 37(Suppl. III): l63167. 1978. Koch, C.J., Howell, R.L.: Combined radiation-protective and radiation-sensitizing agents: 11. Radiosensitivity of hypoxic or aerobic Chinese hamster fibroblasts in the presence of cysteamine and misonidazole: Implications for the “oxygen effect.” Rudiur. Res. 87: 265-283. 1981. Koch. C.J., Howell, R.L.: Misonidazole: Inter-related factors
Binding of “C-misontdazole affecting 693-696.
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C.J.. Howell. R.L.. Biaglow, J.E.: Ascorbate anion potentiates cytotoxicity of nitro-aromatic compounds under hypoxic and anoxic conditions. Br. J. Cancer 39: 32 l-329. 1979.
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R.P.: Free radical intermediates of foreign comand their toxicological significance. In Review in Biochemical Toxicologic. Vol. I. E. Hodgson. J.R. Bend and R.M. Philpot (Eds.). Amsterdam. Elsevier North Holland. 1973. pp. 151-200. 12. Olive. P.L.. Durand. R.E.: Fluorescent nitroheterocycles for identifying hypoxic cells. Cancer Res 43: 3276-3280. pounds
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J.A., Kremers. W.: Promotion of radiation perin models of lipid membranes by caesium and counter-ions: micellar linoleic and linolenic acids.
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Reductive
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15. Taylor. Y.C.. Rauth. A.M.: Differences in the toxicity and metabolism of the 2-nitroimidazole misonidazole (Ro-070582) in HeLa and Chinese hamster ovary cells. C’anwr Res. 38: 2745-2752. 1978. 16. Taylor. Y.C.. Rauth. A.M.: Sulphydryls. ascorbate and oxygen as modifiers of the toxicity and metabolism of misonidazole in vitro. Br. J. Cancer 41: 892-900. 1980. 17. Varghese. A.J., Whitmore. G.F.: Binding to cellular macromolecules as a possible mechanism for the cytotoxicity of misonidazole. Cancer Rex 40: 2 165-2 169. 1980. 18. Whillans. D.W., Rauth. A.M.: An experimental and analytical study ofoxygen depletion in stirred cell suspensions. Radial. Rex 84: 97-l 14. 1980. 19. Wong. T.W., Whitmore. G.F.. Gulyas. S.: Studies on the toxicity and radiosensitizing ability of Ro-07-0582 under Radtat. Rev. 75: 54 I conditions of prolonged incubation. 555, 1978.