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Mechanism of interaction of in situ produced nitroimidazole reduction derivatives with DNA using electrochemical DNA biosensor
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[32] M e c h a n i s m o f I n t e r a c t i o n o f in Situ P r o d u c e d Nitroimidazole Reduction Derivatives with DNA using Electrochemical DNA Biosensor
By
H. P. S E R R A N O , MAURO A. LA-SCALZA,IVANO G. R. GUTZ, and MARIA L. CRuz
ANA MARIA OLIVEIRA BRETT, SILVIA
Introduction A DNA biosensor has been developed for the evaluation of mechanisms of interaction of drugs with DNA. Damage to DNA can occur by intercalating, alkylating, or strand-breaking agents, but most drugs bind covalently to DNA by cross-linking between two bases on the same strand. The mechanism of the biological action of nitroimidazoles, commonly used to treat infection by anaerobic bacteria, depends on the reduction of the nitro group producing intermediate species which interact with DNA, oxidizing it and resulting in strand breaking and double-helix destabilization. 1,2 The most important nitroimidazole derivatives have substituents in the N-1 position of the heterocyclic ring of 5-nitroimidazole.
O 2 N ~
k/~R2 R1
METRONIDAZOLE : RI =--CH2CH2OH ; R2 = - - C H 3 TINIDAZOLE : RI = - - CH2CH2SO2C2H5 ; R2 = INCH3 SECNIDAZOLE : R1 = --CH2CH2(OH)CH3 ; R2 = --CH3
The electrochemical reduction of nitroimidazoles follows a complex mechanism and in theory the nitro group is able to receive up to six electrons to form the corresponding amine. 3 Under anaerobic and low oxygen prest D. I. Edwards, in " D N A Binding and Nicking A g e n t s , " Vol. 2, C o m p r e h e n s i v e Medicinal Chemistry (C. Hansch, Ed.). P e r g a m o n Press, 1990. 2 D. I. Edwards, J. Antimicrob. Chemother. 31, 9 (1993). 3 p. Z u m a n and Z. Fijalek, J. Electroanal. Chem. 296, 538 (1990).
METHODS IN ENZYMOLOGY, VOL. 300
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 0076-6879199 $30.00
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sure conditions metronidazole (2-methyl-5-nitroimidazole-l-ethanol) follows a reduction mechanism similar to that of nitrobenzene. 3 Electrochemical reduction of nitroimidazole derivatives 4-7 shows two reduction waves in aqueous acid media, the first involving 4 electrons and corresponding to the reduction of the nitro group to form the intermediate hydroxylamine ( - N H O H ) and the second involving 2 electrons and corresponding to the reduction of the hydroxylamine to amine (-NH2). Using three different electrode materials, bare glassy carbon electrode, mercury thin film electrode, and DNA biosensor, we have verified 8-1° that hydroxylamine formation involves 4 electrons and is pH dependent. The results are in good agreement with previous work using the dropping mercury electrode.ll, 12 As will be shown, the DNA biosensor developed by us 8-m,13 provides a new perspective to the research and study of the mechanism of action of nitroimidazoles with DNA. Materials and Instrumentation Metronidazole (MTZ) and secnidazole (SCZ) are supplied by Rhodia Farma Lda Brasil, and tinidazole (TNZ) by Laborat6rios Pfizer Lda Brasil. Calf thymus D N A (sodium salt, type I), is obtained from Sigma Chemical Co. and is used without further purification. Acetate buffer solutions of ionic strength 0.2 at pH 4.5 are used in all experiments and are prepared using analytical grade reagents and purified water from a Millipore (Bedford, MA) Milli-Q system (resistivity -> 18 MI~ cm). All experiments are done in deoxygenated solutions and at room temperature. Electrochemical techniques TM of cyclic voltammetry and differential pulse voltammetry are employed using a tzAutolab potentiostat/galvanostat 4 j. H. Tocher and D. I. Edwards, Biochem. Pharmac. 48, 1089 (1994). 5 p. j. Declerck and C. J. De Ranter, J. Chem. Soc. Faraday Trans 1 83, 257 (1987). 6 D. Dumanovic, J. Volke, and V. Vajgand, J. Pharma. Pharmac. 18, 507 (1966). 7 D. Dumanovic and J. Ciric, Talanta 20, 525 (1973). 8 A. M. Oliveira Bren, S. H. P. Serrano, I. Gutz, and M. A. La-Scalea, Bioelectrochem. Bioenerg. 42, 175 (1997). 9 A° M. Oliveira Brett, S. H. P. Serrano, I. Gutz, and M. A. La-Scalea, Electroanaylsis 9, 110 (1997). 10 A. M. Oliveira Brett, S. H. P. Serrano, I. Gutz, and M. A. La-Scalea, Electroanalysis, 9, 1132 (1997). 11 p. Zuman, Z. Fijalek, D. Dumanovic, and D. Suznjevic, Electroanalysis 4, 783 (1992). 12 C. Karakis and P. Zuman, J. Electroanal. Chem. 396, 499 (1995). 13 A. M. Oliveira Brett, S. H. P. Serrano, T. A. Macedo, D. Raimundo, M. H. Marques, and M. A. La-Scalea, Electroanalysis 8, 992 (1996). 14 C. M. A. Brett and A. M. Oliveira Brett, "Electrochemistry, Principles, Methods and Applications." Oxford University Press, Oxford, 1993.
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running with GPES version 3 software (Eco-Chemie, Utrecht, Netherlands). Differential pulse voltammetry conditions are: pulse amplitude 50 mV, pulse width 70 msec, and scan rate 5 mV sec -1. The working electrode is the DNA biosensor, the counter electrode is a Pt wire, and the reference electrode is a standard calomel electrode (SCE), all contained in a onecompartment cell.
DNA
Biosensor
The DNA biosensor consists of a glassy carbon electrode covered by a layer of immobilized double-stranded DNA conditioned in a singlestranded solution. The structure of the DNA layer on the electrode surface after conditioning is important for electrode stability and reproducibility. Single-stranded DNA (ssDNA) is prepared by treating an accurately weighed sample of approximately 3 mg of DNA with 0.5 ml of 60% pure perchloric acid; after dissolution, 0.5 ml of 9 M NaOH is then added to neutralize the solution followed by addition of pH 4.5 acetate buffer until complete 10 ml (I ~ 9 M). The DNA biosensor is prepared by covering a glassy carbon electrode (Tokai, GC20, area 0.07 cm2) with 3 mg of DNA dissolved in 80/xl of pH 4.5 acetate buffer and leaving the electrode to dry. After drying, the electrode is immersed in acetate buffer solution and a constant potential of +1.4 V applied during 5 min. A differential pulse scan is then done from 0 V to +1.4 V vs SCE to check that no electrochemical reaction occurs on the surface of the DNA biosensor in supporting electrolyte. The electrode is then transferred to a solution containing single-stranded DNA and differential pulse voltammograms recorded in the range 0 to + 1.4 V until stabilization of the peak currents that correspond to guanine (+0.776 V) and adenine (+1.065 V) electrooxidation occurs. Is,16 The electrode was then dried at room temperature and used to study the mechanism of action of nitroimidazoles with DNA.
Mechanism of Interaction of Nitroimidazoles with DNA The DNA biosensor has been used to study the electrochemistry of metronidazole (MTZ) in detail and also that of two other nitroimidazoles: secnidazole (SCZ), and tinidazole (TNZ) which follow a similar reduction is C. M. A. Brett, A. M. Oliveira Brett, and S. H. P. Serrano, J. Electroanal. Chem. 366~ 255 (1994). 16 m. M. Oliveira Brett and S. H. P. Serrano, J. Braz. Chem. Soc. 6, 97 (1995).
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II
I
"IV
-1'.o'' '-o'.6' :o'.2'o.'2'o15' EIV
vs
o18
SCE
FIG. 1. Cyclic v o l t a m m o g r a m s of 1.0 m M M T Z in p H 4.5 acetate buffer at a D N A biosensor. u, 100 m V sec 1; Edep, --0.6 V; tdep, 2 min with stirring; and Ei, - 0 . 2 V vs SCE.
mechanism involving a 4-electron transfer to form the corresponding hydroxylamines1,3,11,17 as occurs with metronidazole. The reduction of MTZ has been studied at pH 4.5 using differential pulse voltammetry with a bare glassy carbon electrode as well as with the DNA biosensor. The DNA biosensor shows the advantage of the ability to preconcentrate metronidazole on the electrode surface, and a good linear working range of 1.0 to 54.3/zmol/liter when using 2 min preconcentration at 0.0 V with stirring has been obtained. 9 Preconcentration consists in accumulating the electroactive species onto the electrode surface at a predetermined applied potential during an optimized length of time, which will incorporate either metronidazole or its reduction products on the DNA biosensor. This enables the detection of lower concentration levels of the electroactive analyte in solution when a potential scan is applied in the determination step. In order to investigate the oxidation of the reduced nitro compound, the preconcentration potential is changed from 0.0 V to -0.6 V. Preconcentration at a potential more negative than the reduction potential of MTZ leads to preconcentration of the reduction products rather than of the starting compound. The interaction of MTZ with DNA is shown in the cyclic voltammogram of Fig. 1. Three oxidation peaks can be seen, the first being reversible. 17 p. j. Declerck and C. J. De Ranter, Biochem. Pharmac. 35, 59 (1986).
318
[32]
ASSAYS IN CELLS, BODY FLUIDS, AND TISSUES DNA
DNA (ox)
nitroimidazole RNO 2
hydroxylamine IV
RNHOH
nitroso derivative
I . la
RNO
Condensation of RNHOH t _ _
+ R~O J
R~: NR o2oxy compound
RNH2 ~
R N H . N H R -~
1
RN:NR
SCHEMEI
Identification of the peaks in Fig. 1 is based on the electrochemical behavior of nitrobenzene. I2'ls The reduction product of MTZ at -0.6 V is the corresponding hydroxylamine which is preconcentrated on the DNAbiosensor surface at a constant applied potential for 2 min, consequently permitting its interaction with the DNA. The cyclic voltammetric scan is started in the positive direction at -0.2 V so that oxidation of the nitro compound reduction products can be detected. The first wave, L corresponds to oxidation of the hydroxylamine to a nitroso derivative (Ep = 0.081 V); which can be further oxidized to an azoxycompound 19,2° peak H (Ep = 0.358 V), or, in the cyclic voltammetry reverse scan, reversibly reduced to hydroxylamine 12,18,21peak Ia (Ep = -0.037 V), and the reduction of MTZ in solution corresponds to peak IV (Ep = -0.556 V). The reduction mechanism of the nitroheterocyclic derivatives, responsible for biological activity, and the consequent formation of bimolecular azoxy, azo, and hydrazo derivative compounds can be described by Scheme I. This shows the reversible oxidation of the hydroxylamine derivative (RNHOH) to the corresponding nitroso derivative (RNO) and a condensation reaction between the hydroxylamine and nitroso derivatives to form 18 I. Rubinstein, J. Electroanal. Chem. 183, 379 (1985). 19 N. V. Sidgwick, "Organic Chemistry of Nitrogen." Clarendon Press, Oxford, 1937. 20 Z. Fijalek, M. Pugia, and P. Zuman, Electroanalysis 5, 65 (1993). 21 R. Carlier, E. Raoult, A. Tallec, V. Andre, P. Gauduchon, and J.-C. Lancelot, Electroanalysis 9, 79 (1997).
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the azoxy compound (RNO : NR). The amount of azoxy compound formed depends on the concentration of the nitroso derivative, which rapidly reduced to hydroxylamine)9-21 Concerning peak III (Ep = 0.595 V), it is well known that hydroxylamine displays a large difference in reactivity for single-stranded vs doublestranded DNA, 22-24 which makes it a sensitive detector of H-DNA. H-DNA is the recently discovered nucleic acid triplex structure. = The kinetics of formation of triple-stranded structures is slower than that of double-stranded structures and is dependent on the ionic strength of the medium. This H-DNA conformation, formed by mirror-symmetric homopurine-homopyrimidine sequences, consists of an intramolecular PyPuPy triple-stranded region, i.e., the single strand of an unwound region folds back on the double strand forming Hoogsteen hydrogen bonds. Because the DNA biosensor is prepared by covering a glassy carbon electrode with double-stranded DNA and then transferring it to a solution containing single-stranded DNA for electrochemical conditioning, it is possible that the electrode contains a small amount of H-DNA segments which could be detected by the hydroxylamine. Further evidence for this hypothesis was given by the same experiments using a glassy carbon electrode modified with either only ssDNA or only dsDNA: an irreproducible electrochemical response was obtained showing no peak III. The differential pulse potential of peak III (Fig. 2) is very similar to that of uric acid, Ep = +0.424 V vs SCE in pH 4.5 acetate buffer measured at a newly prepared DNA biosensor) 3 In fact, this comparison is not speculative and is in agreement with the literature that presents the purine bases as the principal target of the oxidative lesion of DNA. s'2s We can therefore assume that peak III corresponds to the formation of deoxypurinic acid derivative26 formed by reaction of the MTZ reduction intermediates with the DNA multilayer immobilized onto the surface of the glassy carbon electrode. Immobilization of DNA on solid substrates by means of electrostatic interactions has been shown27 to have clear advantages compared with chemical bonding.
22 V. N. Soyfer and V. N. Potaman, "Triple-Helical Nucleic Acids." Springer-Verlag, New York, 1995. 23 B. Johnson, Methods in Pharmacology 212, 180 (1992). 24 E. Freese and E. B. Freese, Biochemistry 4, 2419 (1965). 25 p. j. Declerck and C. J. De Ranter, Analysis 15, 148 (1987). 26 G. Dryhurst, J. Electrochem. Soc. 116, 1411 (1969). 27 G. B. Sukhorukov, M. M. Montrel, A. I. Petrov, L. I. Shabarchina, and B. I. Sukhorukov, Biosensors and Bioelectronics 11, 913 (1996).
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ASSAYS IN CELLS, BODY FLUIDS, AND TISSUES
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II
-0.2
o .0 E / V
o .2 vs
0.4
0.6
SCE
F[~. 2. Anodic stripping differential pulse voltammograms in pH 4.5 acetate buffer of MTZ: (--) (1) 0.0 mM; (2) 0.2 mM, and (3) 0.5 mM and Edep = -0.5 V vs SCE using the DNA biosensor; (---) 1.0 mM; Edep = --0.7 V vs SCE using a bare glassy carbon, v, 5 mV see-l; AE, 50 mV; td~p,2 min with stirring.
An important parameter is the preconcentration time because of its relation with the formation of hydroxylamine that will interact with D N A and two minutes preconcentration with stirring, was found to be the best. Figure 2 also includes, for comparison, the results at a bare glassy carbon electrode; because it is not possible to preconcentrate when using this electrode, a much higher concentration, 1 raM, of M T Z was u s e d - nevertheless, no peak I I I appeared. However, peak I I I can already be seen for a concentration of 0.2 m M when using the D N A biosensor. This confirms the assumption that peak I I I is due to interactions of the reduction intermediates with the DNA. In order to better understand the biological activity of this type of drug, similar voltammetric experiments using the D N A biosensor were carried out for two other nitroimidazoles, secnidazole and tinidazole (Fig. 3). There is no appreciable difference in the peak potentials; the differences in the peak currents for peaks I and H can be explained, probably, by the difference in the substituent groups in the N1 position. The reduction intermediates' interaction with D N A can be assigned to peak I I I with the same explanation as for MTZ; the nitro radical is always that principally responsible for the biochemical lesion on D N A of anaerobic microorganisms.
[32]
INTERACTION OF NITROIMIDAZOLE DERIVATIVES WITH D N A I
1
- 0 '.2
I1
3
III
C
0 .'0 E
c'
0 .'2 IV
vs
321
0 .'4
0 .'6
SCE
FIG. 3. Anodic stripping differential pulse voltammograms in pH 4.5 acetate buffer using a DNA biosensor of three nitroimidazoles: (a) metronidazole; (b) secnidazole; (c) tinidazole. v, 5 mV sec-1; AE, 50 mV; toep, 2 min with stirring, and Edep = --0.5 V vs SCE; Ei, -0.2 V.
Conclusions The newly developed DNA biosensor is a very promising tool for the investigation and study of the action of drugs specifically designed to interact with DNA. The electrochemical reduction of nitroimidazole drugs was studied in the presence of D N A immobilized onto the surface of a glassy carbon electrode. This enabled preconcentration of the drug onto the electrode surface, which was then electrochemically reduced to the corresponding hydroxylamine followed by reoxidation to give the nitroso compound and subsequently an azoxy compound. Moreover, as the target of the nitroimidazole action was the DNA, the damage caused to DNA on the electrode surface by a reduction product of this drug could be detected in situ.
Acknowledgments We thank CNPq and PAPESP (Brasil) for a fellowship to M. A. La-Scalea and S. H. P. Serano, respectively. Rhodia Farma Lda and Laborat6rios Pfizer Lda very kindly supplied the nitroimidazoles used in this work.
ASSAYS IN CELLS, BODY FLUIDS, AND TISSUES
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[32] M e c h a n i s m o f I n t e r a c t i o n o f in Situ P r o d u c e d Nitroimidazole Reduction Derivatives with DNA using Electrochemical DNA Biosensor
By
H. P. S E R R A N O , MAURO A. LA-SCALZA,IVANO G. R. GUTZ, and MARIA L. CRuz
ANA MARIA OLIVEIRA BRETT, SILVIA
Introduction A DNA biosensor has been developed for the evaluation of mechanisms of interaction of drugs with DNA. Damage to DNA can occur by intercalating, alkylating, or strand-breaking agents, but most drugs bind covalently to DNA by cross-linking between two bases on the same strand. The mechanism of the biological action of nitroimidazoles, commonly used to treat infection by anaerobic bacteria, depends on the reduction of the nitro group producing intermediate species which interact with DNA, oxidizing it and resulting in strand breaking and double-helix destabilization. 1,2 The most important nitroimidazole derivatives have substituents in the N-1 position of the heterocyclic ring of 5-nitroimidazole.
O 2 N ~
k/~R2 R1
METRONIDAZOLE : RI =--CH2CH2OH ; R2 = - - C H 3 TINIDAZOLE : RI = - - CH2CH2SO2C2H5 ; R2 = INCH3 SECNIDAZOLE : R1 = --CH2CH2(OH)CH3 ; R2 = --CH3
The electrochemical reduction of nitroimidazoles follows a complex mechanism and in theory the nitro group is able to receive up to six electrons to form the corresponding amine. 3 Under anaerobic and low oxygen prest D. I. Edwards, in " D N A Binding and Nicking A g e n t s , " Vol. 2, C o m p r e h e n s i v e Medicinal Chemistry (C. Hansch, Ed.). P e r g a m o n Press, 1990. 2 D. I. Edwards, J. Antimicrob. Chemother. 31, 9 (1993). 3 p. Z u m a n and Z. Fijalek, J. Electroanal. Chem. 296, 538 (1990).
METHODS IN ENZYMOLOGY, VOL. 300
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 0076-6879199 $30.00
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sure conditions metronidazole (2-methyl-5-nitroimidazole-l-ethanol) follows a reduction mechanism similar to that of nitrobenzene. 3 Electrochemical reduction of nitroimidazole derivatives 4-7 shows two reduction waves in aqueous acid media, the first involving 4 electrons and corresponding to the reduction of the nitro group to form the intermediate hydroxylamine ( - N H O H ) and the second involving 2 electrons and corresponding to the reduction of the hydroxylamine to amine (-NH2). Using three different electrode materials, bare glassy carbon electrode, mercury thin film electrode, and DNA biosensor, we have verified 8-1° that hydroxylamine formation involves 4 electrons and is pH dependent. The results are in good agreement with previous work using the dropping mercury electrode.ll, 12 As will be shown, the DNA biosensor developed by us 8-m,13 provides a new perspective to the research and study of the mechanism of action of nitroimidazoles with DNA. Materials and Instrumentation Metronidazole (MTZ) and secnidazole (SCZ) are supplied by Rhodia Farma Lda Brasil, and tinidazole (TNZ) by Laborat6rios Pfizer Lda Brasil. Calf thymus D N A (sodium salt, type I), is obtained from Sigma Chemical Co. and is used without further purification. Acetate buffer solutions of ionic strength 0.2 at pH 4.5 are used in all experiments and are prepared using analytical grade reagents and purified water from a Millipore (Bedford, MA) Milli-Q system (resistivity -> 18 MI~ cm). All experiments are done in deoxygenated solutions and at room temperature. Electrochemical techniques TM of cyclic voltammetry and differential pulse voltammetry are employed using a tzAutolab potentiostat/galvanostat 4 j. H. Tocher and D. I. Edwards, Biochem. Pharmac. 48, 1089 (1994). 5 p. j. Declerck and C. J. De Ranter, J. Chem. Soc. Faraday Trans 1 83, 257 (1987). 6 D. Dumanovic, J. Volke, and V. Vajgand, J. Pharma. Pharmac. 18, 507 (1966). 7 D. Dumanovic and J. Ciric, Talanta 20, 525 (1973). 8 A. M. Oliveira Bren, S. H. P. Serrano, I. Gutz, and M. A. La-Scalea, Bioelectrochem. Bioenerg. 42, 175 (1997). 9 A° M. Oliveira Brett, S. H. P. Serrano, I. Gutz, and M. A. La-Scalea, Electroanaylsis 9, 110 (1997). 10 A. M. Oliveira Brett, S. H. P. Serrano, I. Gutz, and M. A. La-Scalea, Electroanalysis, 9, 1132 (1997). 11 p. Zuman, Z. Fijalek, D. Dumanovic, and D. Suznjevic, Electroanalysis 4, 783 (1992). 12 C. Karakis and P. Zuman, J. Electroanal. Chem. 396, 499 (1995). 13 A. M. Oliveira Brett, S. H. P. Serrano, T. A. Macedo, D. Raimundo, M. H. Marques, and M. A. La-Scalea, Electroanalysis 8, 992 (1996). 14 C. M. A. Brett and A. M. Oliveira Brett, "Electrochemistry, Principles, Methods and Applications." Oxford University Press, Oxford, 1993.
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running with GPES version 3 software (Eco-Chemie, Utrecht, Netherlands). Differential pulse voltammetry conditions are: pulse amplitude 50 mV, pulse width 70 msec, and scan rate 5 mV sec -1. The working electrode is the DNA biosensor, the counter electrode is a Pt wire, and the reference electrode is a standard calomel electrode (SCE), all contained in a onecompartment cell.
DNA
Biosensor
The DNA biosensor consists of a glassy carbon electrode covered by a layer of immobilized double-stranded DNA conditioned in a singlestranded solution. The structure of the DNA layer on the electrode surface after conditioning is important for electrode stability and reproducibility. Single-stranded DNA (ssDNA) is prepared by treating an accurately weighed sample of approximately 3 mg of DNA with 0.5 ml of 60% pure perchloric acid; after dissolution, 0.5 ml of 9 M NaOH is then added to neutralize the solution followed by addition of pH 4.5 acetate buffer until complete 10 ml (I ~ 9 M). The DNA biosensor is prepared by covering a glassy carbon electrode (Tokai, GC20, area 0.07 cm2) with 3 mg of DNA dissolved in 80/xl of pH 4.5 acetate buffer and leaving the electrode to dry. After drying, the electrode is immersed in acetate buffer solution and a constant potential of +1.4 V applied during 5 min. A differential pulse scan is then done from 0 V to +1.4 V vs SCE to check that no electrochemical reaction occurs on the surface of the DNA biosensor in supporting electrolyte. The electrode is then transferred to a solution containing single-stranded DNA and differential pulse voltammograms recorded in the range 0 to + 1.4 V until stabilization of the peak currents that correspond to guanine (+0.776 V) and adenine (+1.065 V) electrooxidation occurs. Is,16 The electrode was then dried at room temperature and used to study the mechanism of action of nitroimidazoles with DNA.
Mechanism of Interaction of Nitroimidazoles with DNA The DNA biosensor has been used to study the electrochemistry of metronidazole (MTZ) in detail and also that of two other nitroimidazoles: secnidazole (SCZ), and tinidazole (TNZ) which follow a similar reduction is C. M. A. Brett, A. M. Oliveira Brett, and S. H. P. Serrano, J. Electroanal. Chem. 366~ 255 (1994). 16 m. M. Oliveira Brett and S. H. P. Serrano, J. Braz. Chem. Soc. 6, 97 (1995).
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INTERACTION OF NITROIMIDAZOLE DERIVATIVES WITH D N A
317
II
I
"IV
-1'.o'' '-o'.6' :o'.2'o.'2'o15' EIV
vs
o18
SCE
FIG. 1. Cyclic v o l t a m m o g r a m s of 1.0 m M M T Z in p H 4.5 acetate buffer at a D N A biosensor. u, 100 m V sec 1; Edep, --0.6 V; tdep, 2 min with stirring; and Ei, - 0 . 2 V vs SCE.
mechanism involving a 4-electron transfer to form the corresponding hydroxylamines1,3,11,17 as occurs with metronidazole. The reduction of MTZ has been studied at pH 4.5 using differential pulse voltammetry with a bare glassy carbon electrode as well as with the DNA biosensor. The DNA biosensor shows the advantage of the ability to preconcentrate metronidazole on the electrode surface, and a good linear working range of 1.0 to 54.3/zmol/liter when using 2 min preconcentration at 0.0 V with stirring has been obtained. 9 Preconcentration consists in accumulating the electroactive species onto the electrode surface at a predetermined applied potential during an optimized length of time, which will incorporate either metronidazole or its reduction products on the DNA biosensor. This enables the detection of lower concentration levels of the electroactive analyte in solution when a potential scan is applied in the determination step. In order to investigate the oxidation of the reduced nitro compound, the preconcentration potential is changed from 0.0 V to -0.6 V. Preconcentration at a potential more negative than the reduction potential of MTZ leads to preconcentration of the reduction products rather than of the starting compound. The interaction of MTZ with DNA is shown in the cyclic voltammogram of Fig. 1. Three oxidation peaks can be seen, the first being reversible. 17 p. j. Declerck and C. J. De Ranter, Biochem. Pharmac. 35, 59 (1986).
318
[32]
ASSAYS IN CELLS, BODY FLUIDS, AND TISSUES DNA
DNA (ox)
nitroimidazole RNO 2
hydroxylamine IV
RNHOH
nitroso derivative
I . la
RNO
Condensation of RNHOH t _ _
+ R~O J
R~: NR o2oxy compound
RNH2 ~
R N H . N H R -~
1
RN:NR
SCHEMEI
Identification of the peaks in Fig. 1 is based on the electrochemical behavior of nitrobenzene. I2'ls The reduction product of MTZ at -0.6 V is the corresponding hydroxylamine which is preconcentrated on the DNAbiosensor surface at a constant applied potential for 2 min, consequently permitting its interaction with the DNA. The cyclic voltammetric scan is started in the positive direction at -0.2 V so that oxidation of the nitro compound reduction products can be detected. The first wave, L corresponds to oxidation of the hydroxylamine to a nitroso derivative (Ep = 0.081 V); which can be further oxidized to an azoxycompound 19,2° peak H (Ep = 0.358 V), or, in the cyclic voltammetry reverse scan, reversibly reduced to hydroxylamine 12,18,21peak Ia (Ep = -0.037 V), and the reduction of MTZ in solution corresponds to peak IV (Ep = -0.556 V). The reduction mechanism of the nitroheterocyclic derivatives, responsible for biological activity, and the consequent formation of bimolecular azoxy, azo, and hydrazo derivative compounds can be described by Scheme I. This shows the reversible oxidation of the hydroxylamine derivative (RNHOH) to the corresponding nitroso derivative (RNO) and a condensation reaction between the hydroxylamine and nitroso derivatives to form 18 I. Rubinstein, J. Electroanal. Chem. 183, 379 (1985). 19 N. V. Sidgwick, "Organic Chemistry of Nitrogen." Clarendon Press, Oxford, 1937. 20 Z. Fijalek, M. Pugia, and P. Zuman, Electroanalysis 5, 65 (1993). 21 R. Carlier, E. Raoult, A. Tallec, V. Andre, P. Gauduchon, and J.-C. Lancelot, Electroanalysis 9, 79 (1997).
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the azoxy compound (RNO : NR). The amount of azoxy compound formed depends on the concentration of the nitroso derivative, which rapidly reduced to hydroxylamine)9-21 Concerning peak III (Ep = 0.595 V), it is well known that hydroxylamine displays a large difference in reactivity for single-stranded vs doublestranded DNA, 22-24 which makes it a sensitive detector of H-DNA. H-DNA is the recently discovered nucleic acid triplex structure. = The kinetics of formation of triple-stranded structures is slower than that of double-stranded structures and is dependent on the ionic strength of the medium. This H-DNA conformation, formed by mirror-symmetric homopurine-homopyrimidine sequences, consists of an intramolecular PyPuPy triple-stranded region, i.e., the single strand of an unwound region folds back on the double strand forming Hoogsteen hydrogen bonds. Because the DNA biosensor is prepared by covering a glassy carbon electrode with double-stranded DNA and then transferring it to a solution containing single-stranded DNA for electrochemical conditioning, it is possible that the electrode contains a small amount of H-DNA segments which could be detected by the hydroxylamine. Further evidence for this hypothesis was given by the same experiments using a glassy carbon electrode modified with either only ssDNA or only dsDNA: an irreproducible electrochemical response was obtained showing no peak III. The differential pulse potential of peak III (Fig. 2) is very similar to that of uric acid, Ep = +0.424 V vs SCE in pH 4.5 acetate buffer measured at a newly prepared DNA biosensor) 3 In fact, this comparison is not speculative and is in agreement with the literature that presents the purine bases as the principal target of the oxidative lesion of DNA. s'2s We can therefore assume that peak III corresponds to the formation of deoxypurinic acid derivative26 formed by reaction of the MTZ reduction intermediates with the DNA multilayer immobilized onto the surface of the glassy carbon electrode. Immobilization of DNA on solid substrates by means of electrostatic interactions has been shown27 to have clear advantages compared with chemical bonding.
22 V. N. Soyfer and V. N. Potaman, "Triple-Helical Nucleic Acids." Springer-Verlag, New York, 1995. 23 B. Johnson, Methods in Pharmacology 212, 180 (1992). 24 E. Freese and E. B. Freese, Biochemistry 4, 2419 (1965). 25 p. j. Declerck and C. J. De Ranter, Analysis 15, 148 (1987). 26 G. Dryhurst, J. Electrochem. Soc. 116, 1411 (1969). 27 G. B. Sukhorukov, M. M. Montrel, A. I. Petrov, L. I. Shabarchina, and B. I. Sukhorukov, Biosensors and Bioelectronics 11, 913 (1996).
320
ASSAYS IN CELLS, BODY FLUIDS, AND TISSUES
[321
II
-0.2
o .0 E / V
o .2 vs
0.4
0.6
SCE
F[~. 2. Anodic stripping differential pulse voltammograms in pH 4.5 acetate buffer of MTZ: (--) (1) 0.0 mM; (2) 0.2 mM, and (3) 0.5 mM and Edep = -0.5 V vs SCE using the DNA biosensor; (---) 1.0 mM; Edep = --0.7 V vs SCE using a bare glassy carbon, v, 5 mV see-l; AE, 50 mV; td~p,2 min with stirring.
An important parameter is the preconcentration time because of its relation with the formation of hydroxylamine that will interact with D N A and two minutes preconcentration with stirring, was found to be the best. Figure 2 also includes, for comparison, the results at a bare glassy carbon electrode; because it is not possible to preconcentrate when using this electrode, a much higher concentration, 1 raM, of M T Z was u s e d - nevertheless, no peak I I I appeared. However, peak I I I can already be seen for a concentration of 0.2 m M when using the D N A biosensor. This confirms the assumption that peak I I I is due to interactions of the reduction intermediates with the DNA. In order to better understand the biological activity of this type of drug, similar voltammetric experiments using the D N A biosensor were carried out for two other nitroimidazoles, secnidazole and tinidazole (Fig. 3). There is no appreciable difference in the peak potentials; the differences in the peak currents for peaks I and H can be explained, probably, by the difference in the substituent groups in the N1 position. The reduction intermediates' interaction with D N A can be assigned to peak I I I with the same explanation as for MTZ; the nitro radical is always that principally responsible for the biochemical lesion on D N A of anaerobic microorganisms.
[32]
INTERACTION OF NITROIMIDAZOLE DERIVATIVES WITH D N A I
1
- 0 '.2
I1
3
III
C
0 .'0 E
c'
0 .'2 IV
vs
321
0 .'4
0 .'6
SCE
FIG. 3. Anodic stripping differential pulse voltammograms in pH 4.5 acetate buffer using a DNA biosensor of three nitroimidazoles: (a) metronidazole; (b) secnidazole; (c) tinidazole. v, 5 mV sec-1; AE, 50 mV; toep, 2 min with stirring, and Edep = --0.5 V vs SCE; Ei, -0.2 V.
Conclusions The newly developed DNA biosensor is a very promising tool for the investigation and study of the action of drugs specifically designed to interact with DNA. The electrochemical reduction of nitroimidazole drugs was studied in the presence of D N A immobilized onto the surface of a glassy carbon electrode. This enabled preconcentration of the drug onto the electrode surface, which was then electrochemically reduced to the corresponding hydroxylamine followed by reoxidation to give the nitroso compound and subsequently an azoxy compound. Moreover, as the target of the nitroimidazole action was the DNA, the damage caused to DNA on the electrode surface by a reduction product of this drug could be detected in situ.
Acknowledgments We thank CNPq and PAPESP (Brasil) for a fellowship to M. A. La-Scalea and S. H. P. Serano, respectively. Rhodia Farma Lda and Laborat6rios Pfizer Lda very kindly supplied the nitroimidazoles used in this work.