Inl. J Radmion Oncology Biol. Phys, Vol. 22, pp. 661-663 Pnnted in the U.S.A. All n&s reserved.
0360-3016/92 $5.00 + .I0 Copyright 0 1992 Pergamon Press plc
0 Session D: Bioreductive Mechanisms THE INTERACTION OF REDUCED METRONIDAZOLE DNA BASES AND NUCLEOSIDES JOANNE Chemotherapy
H. TOCHER,
PH.D.
AND DAVID
I. EDWARDS,
WITH
PH.D.
Research Unit, Polytechnic of East London, Romford Road, London, El 5 4LZ, U.K.
The electrochemical behavior of the l-electron couple for the bioreductive drug metronidazole has been examined in the presence and absence of the biological target molecules, DNA bases, and nucleosides, including uracil and uridine. Using cyclic voltammetry as the investigation technique, the change in return-to-forward peak current ratio, ip,/ip, from the control, recorded in the absence of target, was measured as a function of scan rate and biological target concentration. All target molecules, except adenosine and guanine, resulted in interaction with RN02’-, as measured by the decrease in the ip,/ipr ratio in the following order of increasing reactivity : adenine, guanosine, thymine, uracil, uridine, and thymidine (at a metronidazole:target ratio of 1:l). No decrease in ip,/ip, was observed with cytosine or cytidine until ratios of 1:20 and 1:30, respectively, were attained. An approximately linear relationship was found between the percentage change in the CV response and log[target] allowing us to determine the sensitivity of RN02’- to the concentration of the target species. The implication for the biological action of metronidazole and other nitro-heterocyclic drugs is discussed. Metronidazole,
Nitro radical anion, DNA bases, DNA nucleosides.
In addition, bulk reduction of the drugs in the presence of DNA has permitted some evaluation of both the type and extent of DNA damage resulting (4, 14). In this study we have undertaken to investigate the specific reduced drug-DNA interaction. We have used the 5 nitroimidazole metronidazole (1 -P-hydroxyethyl-2methyl-5-nitro-imidazole) as a typical example of monofunctional bioreductive drug action. Our attention was focused on the reversible l-electron couple, RNOz/ RN02’ -, since the nitro radical anion has been proposed as the reduction intermediate responsible for causing the observed damage to DNA (8, 9). A mixed dimethylformamide/Hz0 medium has been used to alter the redox mechanism from the 4-electron RNO,/RNHOH couple in aqueous media to a two-stage process involving the successive addition of 1 and 3 electrons (10, 12). The RN02/RN02 ’- couple can, therefore, be examined without interference from following redox steps.
INTRODUCTION The nitro-aromatics have been extensively studied for their ability to act as radiosensitizers and cytotoxic agents against hypoxic tumor cells. Reduction of the drugs is required for cytotoxic action, which can only be accomplished under conditions of low oxygen tension. The biological target has been identified as DNA, leading to strand breaks and helix destabilization, but the precise nature of the drug-DNA interaction is unknown. To allow the design of more effective bioreductive drugs, it is important that the reduction mechanism of this important range of drugs is fully understood. We have chosen to approach this problem using electrolytic reduction procedures rather than the more usual manner employing pulse radiolysis. By careful choice of the reaction conditions and by using a range of electrochemical techniques, we have undertaken an extensive investigation of the redox characteristics of nitro-aromatic compounds. To date, we have focused our attention on the analysis of the voltammetric response for the compounds, that is, assignment of the reduction steps (lo), identification of coupled chemical reactions (1 I), and measurement of lifetimes of reduction intermediates ( 13).
METHODS
AND
MATERIALS
Metronidazole,* DNA bases and nucleosidest and dimethylformamide$ (spectroscopic grade) were used as received without further purification. Accepted for publication 26 July 199 1. * Rhone-Poulenc, Rorer. t Sigma Chemical Co., Dorset UK. $ Aldrich Chemical Co., Dorset, UK.
Reprint requests to: Professor D. 1. Edwards, Chemotherapy Research Unit, Polytechnic of East London, Romford Road, London, El5 4LZ, U.K. Acknowledgements-This work was funded by the Cancer Research Campaign, UK. JHT is a Cancer Research Campaign Senior Research Fellow. 661
662
I. J. Radiation Oncology 0 Biology 0 Physics
Voltammetric investigations employed a PAR 264A polarographic analyzer interfaced with a PAR 303A 3-electrode cell stand and a Bausch and Lomb RE0088 x-y recorder. A hanging mercury drop was used as the working electrode, with a Pt wire as the counter electrode. All potentials were measured against a Ag/AgCl aqueous reference electrode. Cell solutions were purged with H20-saturated N2 prior to all measurements, with a positive pressure of N2 being maintained throughout. The solvent was 33.3% (v:v) dimethylformamide in H20 containing 1.5 X 10-l mol/ dm3 NaCl and 1.5 X 10m2 mol/dm3 trisodium citrate (1 .O SSC buffer) as the supporting electrolyte. The concentration of metronidazole was 2 X 10m4 mol/dm3. The DNA target species was added directly to the electrochemical cell as a pre-weighed solid and ranged in concentration from 4 X 10m5 to 6 X lo-’ mol/dm3 depending on solubility. RESULTS The effect of DNA base and nucleoside addition, including uracil and uridine for completeness, on the reversible RN02/RN02’couple was examined as a function of the DNA target concentration. We were primarily interested in the influence the addition of the DNA target molecules had on the lifetime of the radical anion. The most effective way of monitoring this electrochemically is the return-to-forward peak current ratio, ip,/ip, in the cyclic voltammetric, CV, mode. Choosing a switching potential 100 mV negative of the forward wave peak potential, metronidazole itself had an ip,/ip,- ratio that increased with changing scan rate from v = 10 to 500 mVs_‘, approaching a value of unity. This was used throughout as a control. All the target molecules tested, except adenosine and guanine, resulted in a decrease in ip,/ipf, with the difference from the control value increasing as the concentration of target was increased. By CV, the El,* did not alter significantly with addition of base or nucleoside. For a given base, the decrease in ip,/ip, was found to vary with v with a maximum between 100 and 200 mVs-‘. This behavior was observed throughout the complete target concentration range examined. To allow comparison between the effect produced by the target molecules, the ip,/ip, was best expressed as the percentage change, %A, from the control value. The variation found between the various target molecules was in some instances considerable as illustrated in Table 1. Although the influence of adenosine was examined up to a metronidazole:target ratio of 1:90, no change in ip,/ipr was found. Further increases in the adenosine concentration resulted in interference of the electrode response. A plot of %A vs log[target] gave an approximately straight line relationship, except for cytosine and cytidine, which were curved. Figure 1 illustrates the behavior found for thymine and thymidine, chosen as being representative and to show the difference between base and nucleoside, and cytosine to demonstrate the large contrast found in
Volume
22. Number
4. I992
%A A
30 28 26 24. 22. 20. 16
16 14. 12 10 . 8. 64. 2r -5.4
,,,,,,,,I, -5.0
-4.6
-4.2
-3.8
-3.4
IIIIII -3.0
blog~target) -2.6
-2.2
Fig. I. Plot of %A vs log[target] for thymine (O), thymidine and cytosine (Cl) at a scan rate of 100 mVs_‘.
(X)
some instances. The slope of the straight line can be taken as a measure of the sensitivity of the nitro radical anion to the concentration of the target (Table 1).
DISCUSSION The effect of DNA base addition on the electrolytic response for a number of nitroimidazoles has been examined previously by Declerck and De Ranter (2, 3). Using dc polarography and an aqueous system, they measured the resulting shift in the half-wave potential. A positive shift was found with adenine, cytosine, and guanine, which was assigned to reaction between electrochemically generated intermediates and the added substance, whereas the negative shift observed with thymine was caused by adsorption on to the electrode surface. Such an approach, however, must be treated with caution. The irreversible nature of the 4-electron couple, RN02/RNHOH, observed under these experimental conditions makes it extremely difficult to interpret any potential shift on a theoretical basis (1); and there is no information on which of the reduction products is responsible for the observed effect. For the reasons expressed above, we have chosen to
Table 1. The effect of DNA bases and nucleosides on the ipJip, of the RNO,/RNO, .- couple for metronidazole measured by cyclic voltammetry Target Thymidine Uridine Uracil Thymine Guanosine Adenine Cytosine Cytidine
%A at l:l* 10.2 10.0 6.4 5.0 4.4 3.0
10% change? 9.55 x 1.00x 1.66x 2.14x 2.51 x 3.55 x 1.38 x 3.31 x
lo-6 10-T lop5 1O-5 10-5 10-s lop3 10-3
Gradient$ 10.00 10.60 16.27 14.85 13.55 17.71
*The %A change in ip,/ip, at a drug:target ratio of 1: 1 tThe concentration of target (mol/dm3) required to give a 10% decrease in ip&p,. *Gradient from the %A vs log[target] plot.
Interaction
of metronidazole
with DNA bases and nucleosides
concentrate on the mixed solvent medium where we can specifically examine the effect of biological target addition on the l-electron reduction product. The RNOz’- species undergoes further reaction via a disproportionation reaction 2RN02’-
- >RNO*
+ RNO.
The corresponding 2nd order rate constant can be calculated from the variation in ip,/ipr with v (11, 12). The disproportionation is pH-dependent (7), with RN02’becoming increasingly stabilized as the pH is increased. Addition of the target species resulted in a decrease in the ip,/ipr ratio, except for adenosine and guanine. In the case of the latter, this might be attributed to the sparing solubility of the base in the medium. As the ip,/ipr value is a direct measure of the concentration of the reduction product, RN02’-, available for re-oxidation on the return potential sweep, any decrease observed upon the addition of the DNA target represents removal of the radical, that is, interaction. From an examination of Table 1, the lifetime of the metronidazole nitro radical anion was quite clearly curtailed by the addition of the target substances, as indicated by a decrease in the ip,/ipr ratio, by an amount that was dependent on the identity of the target. Comparing the data as either the %A at a metronidazole:target ratio of one, or the quantity of target required to give a 10% change in the ip,/ip, the same order of reactivity was observed, with thymine followed by adenine being the most effective DNA bases. The reactivity of RNOz’- with cytosine was markedly less. The differences observed between DNA base and nucleoside, particularly adenine and adenosine, are under further investigation. We also examined the gradient of the linear %A vs log[target] relationship (Fig.
0 J. H. TOCHER AND D. 1. EDWARDS
663
1, Table 1) as an indication of the sensitivity of RNOz.to the concentration of the target species. Although the ordering was different to that found above, we again observed a high susceptibilty to adenine and thymine content with slopes of 17.7 and 14.8, respectively. The high degree of reactivity found for thymine and adenine was in line with previous studies upon the bulk electrolytic reduction of nitroimidazole drugs in an aqueous media, where the greatest damage was found to occur in DNA with the highest AT ratio (5, 6). From pulse radiolysis studies, however, the misonidazole nitro radical
anion
showed
no
reactivity
towards
guanine
P., personal communication). Our studies also failed to detect reaction of metronidazole with guanine. In addition, we have preliminary results which indicate that RNOz’- in a mixed solvent, but stabilized by high pH, shows a decreased, or a complete absence, of reaction with thymine. As the exact conditions used for the radiolysis study are unknown it is difficult to make direct comparisons between the radiolytic and electrolytic reduction procedures. Our studies into the electrochemically induced interaction between nitroheterocyclic compounds and biological target molecules are continuing. From the variation found in %A between the target sites it is apparent that the radical anion does not react indiscriminately. The extension of this work to include a range of nitroheterocyclic compounds, the effect of RN02’- lifetime and the interaction of other reduction products (eg the 2-electron addition product the nitroso) with target substances will result in a more comprehensive understanding of the mode of action of these important bioreductive agents. At present, however, the assignment of RN02’as the DNA damage-causing species remains a possibility. (Wardman,
REFERENCES 1. Bard, A. J.; Faulkner,
2. 3.
4. 5.
6.
7.
L. R. In: Electrochemical methods. Fundamentals and applications. New York: J.Wiley &Sons Inc.; 1980. Declerck, P. J.; De Ranter, C. J. In vitro reductive activation of nitroimidazoles. Biochem. Pharmacol. 35:59-6 1; 1986. Declerck, P. J.; De Ranter, C. J. Polarographic evidence for the interaction of reduced nitroimidazole derivatives with DNA bases. J. Chem. Sot. Faraday Trans. I 35:257-265; 1987. Edwards, D. I. Reduction of nitroimidazoles in vitro and DNA damage. Biochem. Pharmacol. 35:53-58; 1986. Edwards, D. I.; Knox, R. J.; Rowley, D. A.; Skolimowski, I. M.; Knight, R. C. The biochemistry of nitroimidazole drug action. In: Van den Bossche, H., ed. The host-invader interplay. Amsterdam: Elsevier; 1980:673-676. Edwards, D. I.; Rowley, D. A.; Knox, R. J.; Skolimowski, I. M.; Knight, R. C. Nature of DNA damage induced by electrolytically reduced nitroimidazole drugs. In: Nelson, J. D., ed. Current chemotherapy and infectious disease. Washington: American Society for Microbiology; 1980:56 l563. Henry, Y.; Guissani, A.; Hickel, B. Radicals of nitroimidazole derivatives: pH dependence of rates of formation and decay related to acid-base equilibria. Int. J. Radiat. Biol. 5 1:797-809; 1987.
8. Knox, R. J.; Edwards, D. I.; Knight, R. C. The mechanism of nitroimidazole damage to DNA: coulometric evidence. Int. J. Radiat. Oncol. Biol. Phys. IO: 13 15- 13 18; 1984. for 9. Olive, P. L. Evidence suggesting that the mechanism aerobic and hypoxic cytotoxicity of nitroheterocycles is the same. Int. J. Radiat. Oncol. Biol. Phys. 8:687-691; 1982. 10. Tocher, J. H.; Edwards, D. I. Electrochemical characteristics of nitroheterocyclic compounds of biological interest. I. The influence of solvent. Free Rad. Res. Commun. 4:269- 276; 1988. 1I. Tocher, J. H.; Edwards, D. I. Electrochemical characteristics of biological interest. III. Nitroso derivative formation. Free Rad. Res. Commun. 5:327-332; 1989. 12. Tocher, J. H.; Edwards, D. I. Electrochemical characteristics of nitroheterocyclic compounds of biological interest. IV. Lifetime of the metronidazole radical anion. Free Rad. Res. Commun. 6:39-45; 1989. 13. Tocher, J. H.; Edwards, D. I. Electrochemical characteristics of nitroheterocyclic compounds of biological interest. V. Measurement and comparison of nitro radical lifetimes. Int. J. Radiat. Biol. 57:45-53; 1990. 14. Zahoor, A.; Lafleur, M. V. M.; Knight, R. C.; Loman, H.; Edwards, D. I. DNA damage induced by reduced nitroimidazole drugs. Biochem. Pharmacol. 36:3299-3304; 1987.
0360-3016/92 $5.00 + .I0 Copyright 0 1992 Pergamon Press plc
0 Session D: Bioreductive Mechanisms THE INTERACTION OF REDUCED METRONIDAZOLE DNA BASES AND NUCLEOSIDES JOANNE Chemotherapy
H. TOCHER,
PH.D.
AND DAVID
I. EDWARDS,
WITH
PH.D.
Research Unit, Polytechnic of East London, Romford Road, London, El 5 4LZ, U.K.
The electrochemical behavior of the l-electron couple for the bioreductive drug metronidazole has been examined in the presence and absence of the biological target molecules, DNA bases, and nucleosides, including uracil and uridine. Using cyclic voltammetry as the investigation technique, the change in return-to-forward peak current ratio, ip,/ip, from the control, recorded in the absence of target, was measured as a function of scan rate and biological target concentration. All target molecules, except adenosine and guanine, resulted in interaction with RN02’-, as measured by the decrease in the ip,/ipr ratio in the following order of increasing reactivity : adenine, guanosine, thymine, uracil, uridine, and thymidine (at a metronidazole:target ratio of 1:l). No decrease in ip,/ip, was observed with cytosine or cytidine until ratios of 1:20 and 1:30, respectively, were attained. An approximately linear relationship was found between the percentage change in the CV response and log[target] allowing us to determine the sensitivity of RN02’- to the concentration of the target species. The implication for the biological action of metronidazole and other nitro-heterocyclic drugs is discussed. Metronidazole,
Nitro radical anion, DNA bases, DNA nucleosides.
In addition, bulk reduction of the drugs in the presence of DNA has permitted some evaluation of both the type and extent of DNA damage resulting (4, 14). In this study we have undertaken to investigate the specific reduced drug-DNA interaction. We have used the 5 nitroimidazole metronidazole (1 -P-hydroxyethyl-2methyl-5-nitro-imidazole) as a typical example of monofunctional bioreductive drug action. Our attention was focused on the reversible l-electron couple, RNOz/ RN02’ -, since the nitro radical anion has been proposed as the reduction intermediate responsible for causing the observed damage to DNA (8, 9). A mixed dimethylformamide/Hz0 medium has been used to alter the redox mechanism from the 4-electron RNO,/RNHOH couple in aqueous media to a two-stage process involving the successive addition of 1 and 3 electrons (10, 12). The RN02/RN02 ’- couple can, therefore, be examined without interference from following redox steps.
INTRODUCTION The nitro-aromatics have been extensively studied for their ability to act as radiosensitizers and cytotoxic agents against hypoxic tumor cells. Reduction of the drugs is required for cytotoxic action, which can only be accomplished under conditions of low oxygen tension. The biological target has been identified as DNA, leading to strand breaks and helix destabilization, but the precise nature of the drug-DNA interaction is unknown. To allow the design of more effective bioreductive drugs, it is important that the reduction mechanism of this important range of drugs is fully understood. We have chosen to approach this problem using electrolytic reduction procedures rather than the more usual manner employing pulse radiolysis. By careful choice of the reaction conditions and by using a range of electrochemical techniques, we have undertaken an extensive investigation of the redox characteristics of nitro-aromatic compounds. To date, we have focused our attention on the analysis of the voltammetric response for the compounds, that is, assignment of the reduction steps (lo), identification of coupled chemical reactions (1 I), and measurement of lifetimes of reduction intermediates ( 13).
METHODS
AND
MATERIALS
Metronidazole,* DNA bases and nucleosidest and dimethylformamide$ (spectroscopic grade) were used as received without further purification. Accepted for publication 26 July 199 1. * Rhone-Poulenc, Rorer. t Sigma Chemical Co., Dorset UK. $ Aldrich Chemical Co., Dorset, UK.
Reprint requests to: Professor D. 1. Edwards, Chemotherapy Research Unit, Polytechnic of East London, Romford Road, London, El5 4LZ, U.K. Acknowledgements-This work was funded by the Cancer Research Campaign, UK. JHT is a Cancer Research Campaign Senior Research Fellow. 661
662
I. J. Radiation Oncology 0 Biology 0 Physics
Voltammetric investigations employed a PAR 264A polarographic analyzer interfaced with a PAR 303A 3-electrode cell stand and a Bausch and Lomb RE0088 x-y recorder. A hanging mercury drop was used as the working electrode, with a Pt wire as the counter electrode. All potentials were measured against a Ag/AgCl aqueous reference electrode. Cell solutions were purged with H20-saturated N2 prior to all measurements, with a positive pressure of N2 being maintained throughout. The solvent was 33.3% (v:v) dimethylformamide in H20 containing 1.5 X 10-l mol/ dm3 NaCl and 1.5 X 10m2 mol/dm3 trisodium citrate (1 .O SSC buffer) as the supporting electrolyte. The concentration of metronidazole was 2 X 10m4 mol/dm3. The DNA target species was added directly to the electrochemical cell as a pre-weighed solid and ranged in concentration from 4 X 10m5 to 6 X lo-’ mol/dm3 depending on solubility. RESULTS The effect of DNA base and nucleoside addition, including uracil and uridine for completeness, on the reversible RN02/RN02’couple was examined as a function of the DNA target concentration. We were primarily interested in the influence the addition of the DNA target molecules had on the lifetime of the radical anion. The most effective way of monitoring this electrochemically is the return-to-forward peak current ratio, ip,/ip, in the cyclic voltammetric, CV, mode. Choosing a switching potential 100 mV negative of the forward wave peak potential, metronidazole itself had an ip,/ip,- ratio that increased with changing scan rate from v = 10 to 500 mVs_‘, approaching a value of unity. This was used throughout as a control. All the target molecules tested, except adenosine and guanine, resulted in a decrease in ip,/ipf, with the difference from the control value increasing as the concentration of target was increased. By CV, the El,* did not alter significantly with addition of base or nucleoside. For a given base, the decrease in ip,/ip, was found to vary with v with a maximum between 100 and 200 mVs-‘. This behavior was observed throughout the complete target concentration range examined. To allow comparison between the effect produced by the target molecules, the ip,/ip, was best expressed as the percentage change, %A, from the control value. The variation found between the various target molecules was in some instances considerable as illustrated in Table 1. Although the influence of adenosine was examined up to a metronidazole:target ratio of 1:90, no change in ip,/ipr was found. Further increases in the adenosine concentration resulted in interference of the electrode response. A plot of %A vs log[target] gave an approximately straight line relationship, except for cytosine and cytidine, which were curved. Figure 1 illustrates the behavior found for thymine and thymidine, chosen as being representative and to show the difference between base and nucleoside, and cytosine to demonstrate the large contrast found in
Volume
22. Number
4. I992
%A A
30 28 26 24. 22. 20. 16
16 14. 12 10 . 8. 64. 2r -5.4
,,,,,,,,I, -5.0
-4.6
-4.2
-3.8
-3.4
IIIIII -3.0
blog~target) -2.6
-2.2
Fig. I. Plot of %A vs log[target] for thymine (O), thymidine and cytosine (Cl) at a scan rate of 100 mVs_‘.
(X)
some instances. The slope of the straight line can be taken as a measure of the sensitivity of the nitro radical anion to the concentration of the target (Table 1).
DISCUSSION The effect of DNA base addition on the electrolytic response for a number of nitroimidazoles has been examined previously by Declerck and De Ranter (2, 3). Using dc polarography and an aqueous system, they measured the resulting shift in the half-wave potential. A positive shift was found with adenine, cytosine, and guanine, which was assigned to reaction between electrochemically generated intermediates and the added substance, whereas the negative shift observed with thymine was caused by adsorption on to the electrode surface. Such an approach, however, must be treated with caution. The irreversible nature of the 4-electron couple, RN02/RNHOH, observed under these experimental conditions makes it extremely difficult to interpret any potential shift on a theoretical basis (1); and there is no information on which of the reduction products is responsible for the observed effect. For the reasons expressed above, we have chosen to
Table 1. The effect of DNA bases and nucleosides on the ipJip, of the RNO,/RNO, .- couple for metronidazole measured by cyclic voltammetry Target Thymidine Uridine Uracil Thymine Guanosine Adenine Cytosine Cytidine
%A at l:l* 10.2 10.0 6.4 5.0 4.4 3.0
10% change? 9.55 x 1.00x 1.66x 2.14x 2.51 x 3.55 x 1.38 x 3.31 x
lo-6 10-T lop5 1O-5 10-5 10-s lop3 10-3
Gradient$ 10.00 10.60 16.27 14.85 13.55 17.71
*The %A change in ip,/ip, at a drug:target ratio of 1: 1 tThe concentration of target (mol/dm3) required to give a 10% decrease in ip&p,. *Gradient from the %A vs log[target] plot.
Interaction
of metronidazole
with DNA bases and nucleosides
concentrate on the mixed solvent medium where we can specifically examine the effect of biological target addition on the l-electron reduction product. The RNOz’- species undergoes further reaction via a disproportionation reaction 2RN02’-
- >RNO*
+ RNO.
The corresponding 2nd order rate constant can be calculated from the variation in ip,/ipr with v (11, 12). The disproportionation is pH-dependent (7), with RN02’becoming increasingly stabilized as the pH is increased. Addition of the target species resulted in a decrease in the ip,/ipr ratio, except for adenosine and guanine. In the case of the latter, this might be attributed to the sparing solubility of the base in the medium. As the ip,/ipr value is a direct measure of the concentration of the reduction product, RN02’-, available for re-oxidation on the return potential sweep, any decrease observed upon the addition of the DNA target represents removal of the radical, that is, interaction. From an examination of Table 1, the lifetime of the metronidazole nitro radical anion was quite clearly curtailed by the addition of the target substances, as indicated by a decrease in the ip,/ipr ratio, by an amount that was dependent on the identity of the target. Comparing the data as either the %A at a metronidazole:target ratio of one, or the quantity of target required to give a 10% change in the ip,/ip, the same order of reactivity was observed, with thymine followed by adenine being the most effective DNA bases. The reactivity of RNOz’- with cytosine was markedly less. The differences observed between DNA base and nucleoside, particularly adenine and adenosine, are under further investigation. We also examined the gradient of the linear %A vs log[target] relationship (Fig.
0 J. H. TOCHER AND D. 1. EDWARDS
663
1, Table 1) as an indication of the sensitivity of RNOz.to the concentration of the target species. Although the ordering was different to that found above, we again observed a high susceptibilty to adenine and thymine content with slopes of 17.7 and 14.8, respectively. The high degree of reactivity found for thymine and adenine was in line with previous studies upon the bulk electrolytic reduction of nitroimidazole drugs in an aqueous media, where the greatest damage was found to occur in DNA with the highest AT ratio (5, 6). From pulse radiolysis studies, however, the misonidazole nitro radical
anion
showed
no
reactivity
towards
guanine
P., personal communication). Our studies also failed to detect reaction of metronidazole with guanine. In addition, we have preliminary results which indicate that RNOz’- in a mixed solvent, but stabilized by high pH, shows a decreased, or a complete absence, of reaction with thymine. As the exact conditions used for the radiolysis study are unknown it is difficult to make direct comparisons between the radiolytic and electrolytic reduction procedures. Our studies into the electrochemically induced interaction between nitroheterocyclic compounds and biological target molecules are continuing. From the variation found in %A between the target sites it is apparent that the radical anion does not react indiscriminately. The extension of this work to include a range of nitroheterocyclic compounds, the effect of RN02’- lifetime and the interaction of other reduction products (eg the 2-electron addition product the nitroso) with target substances will result in a more comprehensive understanding of the mode of action of these important bioreductive agents. At present, however, the assignment of RN02’as the DNA damage-causing species remains a possibility. (Wardman,
REFERENCES 1. Bard, A. J.; Faulkner,
2. 3.
4. 5.
6.
7.
L. R. In: Electrochemical methods. Fundamentals and applications. New York: J.Wiley &Sons Inc.; 1980. Declerck, P. J.; De Ranter, C. J. In vitro reductive activation of nitroimidazoles. Biochem. Pharmacol. 35:59-6 1; 1986. Declerck, P. J.; De Ranter, C. J. Polarographic evidence for the interaction of reduced nitroimidazole derivatives with DNA bases. J. Chem. Sot. Faraday Trans. I 35:257-265; 1987. Edwards, D. I. Reduction of nitroimidazoles in vitro and DNA damage. Biochem. Pharmacol. 35:53-58; 1986. Edwards, D. I.; Knox, R. J.; Rowley, D. A.; Skolimowski, I. M.; Knight, R. C. The biochemistry of nitroimidazole drug action. In: Van den Bossche, H., ed. The host-invader interplay. Amsterdam: Elsevier; 1980:673-676. Edwards, D. I.; Rowley, D. A.; Knox, R. J.; Skolimowski, I. M.; Knight, R. C. Nature of DNA damage induced by electrolytically reduced nitroimidazole drugs. In: Nelson, J. D., ed. Current chemotherapy and infectious disease. Washington: American Society for Microbiology; 1980:56 l563. Henry, Y.; Guissani, A.; Hickel, B. Radicals of nitroimidazole derivatives: pH dependence of rates of formation and decay related to acid-base equilibria. Int. J. Radiat. Biol. 5 1:797-809; 1987.
8. Knox, R. J.; Edwards, D. I.; Knight, R. C. The mechanism of nitroimidazole damage to DNA: coulometric evidence. Int. J. Radiat. Oncol. Biol. Phys. IO: 13 15- 13 18; 1984. for 9. Olive, P. L. Evidence suggesting that the mechanism aerobic and hypoxic cytotoxicity of nitroheterocycles is the same. Int. J. Radiat. Oncol. Biol. Phys. 8:687-691; 1982. 10. Tocher, J. H.; Edwards, D. I. Electrochemical characteristics of nitroheterocyclic compounds of biological interest. I. The influence of solvent. Free Rad. Res. Commun. 4:269- 276; 1988. 1I. Tocher, J. H.; Edwards, D. I. Electrochemical characteristics of biological interest. III. Nitroso derivative formation. Free Rad. Res. Commun. 5:327-332; 1989. 12. Tocher, J. H.; Edwards, D. I. Electrochemical characteristics of nitroheterocyclic compounds of biological interest. IV. Lifetime of the metronidazole radical anion. Free Rad. Res. Commun. 6:39-45; 1989. 13. Tocher, J. H.; Edwards, D. I. Electrochemical characteristics of nitroheterocyclic compounds of biological interest. V. Measurement and comparison of nitro radical lifetimes. Int. J. Radiat. Biol. 57:45-53; 1990. 14. Zahoor, A.; Lafleur, M. V. M.; Knight, R. C.; Loman, H.; Edwards, D. I. DNA damage induced by reduced nitroimidazole drugs. Biochem. Pharmacol. 36:3299-3304; 1987.
