Peroxidase Substrates Stimulate the Oxidation of Hydralazine to

for a variety of adverse side effects including cancer and drug-induced lupus. However, hydrazines are regarded as a poor peroxidase substrates becaus...
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Chem. Res. Toxicol. 1997, 10, 328-334

Peroxidase Substrates Stimulate the Oxidation of Hydralazine to Metabolites Which Cause Single-Strand Breaks in DNA Christopher A. Reilly and Steven D. Aust* Biotechnology Center, Utah State University, Logan, Utah 84322-4705 Received November 18, 1996X

Hydrazines are believed to be oxidized by peroxidases to reactive intermediates responsible for a variety of adverse side effects including cancer and drug-induced lupus. However, hydrazines are regarded as a poor peroxidase substrates because inactivation of the peroxidase occurs during oxidation of these compounds. We have investigated the hypothesis that efficient peroxidase substrates, termed mediators, may stimulate peroxidase-catalyzed oxidation of hyrazines to intermediates capable of causing DNA damage. Oxidation of hydralazine by horseradish peroxidase was stimulated, enzyme inactivation was significantly decreased, and DNA strand breakage was enhanced by the addition of chlorpromazine. Similar results were obtained using other peroxidases, mediators, and hydrazine derivatives. DNA damage required the addition of a minimum of 3 equiv of hydrogen peroxide, suggesting the involvement of a three-electron oxidation product of hydralazine in DNA damage. Efficient substrates may therefore play a critical role in peroxidase-dependent oxidative metabolism and subsequent damage to biological macromolecules by certain chemicals.

Introduction Peroxidases are heme-containing enzymes which are known to catalyze one-electron oxidation of a variety of structurally diverse organic compounds using hydrogen peroxide as the oxidant (1-3). The peroxidase catalytic cycle was originally described by Chance for horseradish peroxidase (HRP)1 (4) and later modified by Dunford (5). Ferric peroxidase is oxidized by two electrons by hydrogen peroxide to yield water and compound I, an oxyferryl porphyrin π cation radical (P•+) (reaction 1). Compound I accepts one electron from a reducing substrate, yielding compound II, or oxyferryl heme, and 1 equiv of substrate free radical (reaction 2). Compound II oxidizes an additional equivalent of substrate, generating ferric enzyme and the corresponding substrate free radical (reaction 3) (5).

Fe(III) + H2O2 f Fe(IV)dO[P•+] + H2O

(1)

Fe(IV)dO[P•+] + RH2 f Fe(IV)dO + RH•

(2)

Fe(IV)dO + RH2 f Fe(III) + RH• + H2O

(3)

Inherent to the peroxidase catalytic cycle is the generation of free radicals and other oxidized products capable of participating in a variety of secondary reactions (6, 7). The products of peroxidase catalysis may be involved in a number of nonenzymatic reactions such as disproportionation, polymerization, and electron transfer (oxidative or reductive) (8). Redox mediation, a * To whom correspondence should be addressed: Steven D. Aust, Biotechnology Center, Utah State University, Logan, UT 84322-4705. Phone, (801) 797-2730; FAX, (801) 797-2755; E-mail, [email protected]. X Abstract published in Advance ACS Abstracts, February 15, 1997. 1 Abbreviations: HRP, horseradish peroxidase; CPZ, chlorpromazine; MPO, myeloperoxidase; LPO, lactoperoxidase; DMPO, 5,5dimethyl-1-pyroline N-oxide; ABTS, 2,2′-azinobis(ethylbenzthiazoline6-sulfonic acid); CPZ•+, chlorpromazine cation radical; DETAPAC, diethylenetriaminepentacetic acid.

S0893-228x(96)00189-0 CCC: $14.00

nonenzymatic reaction, is of particular interest because of the possible toxicological implications (8, 9). In this process, diffusible free radical intermediates generated by peroxidases in turn indiscriminately oxidize secondary chemicals which may not be efficient peroxidase substrates (9, 10). This can result in efficient indirect oxidation of chemicals to potentially toxic reactive intermediates (6). Exposure to hydrazine-containing compounds is almost inevitable. Hydrazines are found naturally in plants and throughout the environment, and are widely used in industry and medicine (11). Pharmaceutical use of hydrazines in the treatment of disorders such as depression, hypertension, and tuberculosis is frequently complicated by the adverse side effects associated with drug use (11). Hydralazine, an aromatic hydrazine derivative, has been used for long-term treatment of hypertensive disorders (12). Associated with hydralazine exposure is the development of symptoms consistent with the autoimmune disorder systemic lupus erythematosus (1214). In addition, hydralazine has been shown to be genotoxic to isolated hepatocytes (15), mutagenic in bacterial test systems (16), and carcinogenic to mice (17). The oxidative metabolism of hydralazine and other hydrazines to specific intermediates, possibly catalyzed by peroxidases, has been suggested to be essential to the development of such situations (18). Both metal-catalyzed oxidation of hydralazine as well as peroxidasecatalyzed oxidation have been shown to result in degradation of 32P-labeled DNA, although peroxidases are rather inefficient (12). Recent work in our laboratory has shown that peroxidase-catalyzed oxidation of hydrazines may be significantly enhanced by the presence of efficient substrates acting as redox mediators (19). This is due in part to protection of the enzyme from inactivation since, in the presence of a mediator, the oxidation of the hydrazine occurs at some distance from the active site. It has been shown that oxidation of hydrazine-containing compounds © 1997 American Chemical Society

Single-Strand Breaks in DNA by Hydralazine

by peroxidases generates free radicals which can covalently modify the heme group and inactivate the enzyme (20). We have investigated the effects of model peroxidase substrates on the oxidation of hydrazine derivatives by peroxidases and DNA strand breaks that occur as a result of hydrazine oxidation. We propose that these findings may serve as a potential model for peroxidase-dependent oxidation of hydralazine in vivo and propose a possible mechanism for DNA damage by hydrazine derivatives in general.

Materials and Methods Caution: Most of the chemicals used in this study are hazardous and should be handled carefully. Proper protection should be used while these chemicals are handled. Chemicals. Hydrogen peroxide (H2O2), hydralazine, isoniazid, iproniazid, φX174 (RF-1) plasmid DNA, ethidium bromide, horseradish peroxidase Type IV (EC 1.11.1.7) (HRP), human leukocyte myeloperoxidase (MPO), bovine milk lactoperoxidase (LPO), diethylenetriaminepentacetic acid (DETAPAC), chlorpromazine, thioridazine, triflupromazine, 2,2′azinobis(ethylbenzthiazoline-6-sulfonic acid) (ABTS), 5,5-dimethyl1-pyroline N-oxide (DMPO), and guaiacol were purchased from Sigma (St. Louis, MO). Seakem LE agarose was purchased from FMC BioProducts (Rockland, ME). Phenol was from Aldrich (Milwaukee, WI). DMPO was repurified using the method described by Thornally and Bannister and the concentration determined using an extinction coefficient of 7.7 × 103 M-1 cm-1 at 234 nm (21). Prior to use, enzymes were desalted using a Sephadex G-25 (Pharmacia Biotech, Uppsala, Sweden) column equilibrated with Chelex 100 (Bio-Rad, Richmond, CA) treated 50 mM NaCl (pH 7.0). Enzyme concentrations were determined spectrophotometrically using the extinction coefficients of 402 ) 1.02 × 105 M-1 cm-1 for HRP (8), 412 ) 2.14 × 105 M-1 cm-1 for LPO (1), and 430 ) 1.78 × 105 M-1 cm-1 for MPO (22). Concentrations of H2O2 were determined spectrophotometrically using the extinction coefficient of 240 ) 39.4 M-1 cm-1 (23). DNA was reprecipitated with 2.5 volumes of cold ethanol plus 10% 3 M NaCl and resuspended in Chelex-treated 50 mM NaCl. Plasmid DNA concentrations were determined by absorbance at 260 nm. All other reagents were treated with Chelex prior to use. ESR Spectra of Radical Intermediates. ESR spectra were recorded at room temperature in a flat cell using an ECS106 spectrometer (Bruker Instruments, Billerica, MA). The instrument was operated at a frequency of 9.60 GHz with a modulation frequency of 50 kHz. Instrument gain was set at 2 × 104 with a modulation amplitude of 0.946 G and microwave power of 20.1 mW. Sweep time was 83.89 s and sweep width was 100 G. UV-Visible Spectra of CPZ•+. All electronic absorption spectra were recorded using a Shimadzu UV2101-PC spectrophotometer. Chlorpromazine cation radical (CPZ•+) concentrations were determined using the extinction coefficient of 525 ) 12 100 M-1 cm-1 (8). Inhibition of CPZ•+ accumulation was determined by incubating chlorpromazine with HRP and H2O2 in 50 mM NaCl with varied hydralazine concentrations. Reduction of enzymatically generated CPZ•+ by hydralazine was monitored by acquiring absorption spectra prior to and after the addition of hydralazine. Oxidation of Hydralazine by HRP. Changes in the UV spectrum of hydralazine (0.3 mM) were monitored at 1 min intervals during oxidation by HRP (25 nM) in the presence or absence of 50 µM CPZ. Blanks contained all reactants except hydralazine. All reactions were performed in 50 mM NaCl, pH 7.0. DNA Single-Strand Break Assay. Damage to φX174 (RF-1) plasmid DNA was assayed using the method described by Spear and Aust (24) with minor modifications. Briefly, 500 ng of φX174 supercoiled DNA was incubated for 15 min in the dark with the appropriate reactants in a 20 µL reaction mixture.

Chem. Res. Toxicol., Vol. 10, No. 3, 1997 329 Following incubation, 4 µL of electrophoresis loading buffer containing 50% glycerol, 0.025% xylene cyanol, and 0.025% bromophenol blue was added, and the DNA separated by electrophoresis on 0.6% agarose gels containing 0.06% ethidium bromide. DNA single-strand breaks were quantitated using scanning densitometry. Results are expressed as percent of DNA migrating as single-stranded DNA relative to control. HRP Inactivation Experiments. Effects of hydralazine oxidation on HRP activity were determined using the method of Goodwin et al. (19). Briefly, 10 µM HRP was incubated with 5 mM hydralazine, 5 mM H2O2, and (1.25 mM CPZ in either 100 mM Chelex-treated formate (pH 3.0), acetate (pH 5.0), carbonate (pH 7.0) buffers, or 50 mM NaCl (pH 7.0). After 5 min of incubation, 10 µL aliquots were removed and added to 990 mL of 50 mM NaCl (pH 7.0). Next, 100 mL of diluted enzyme was then added to 900 µL of assay buffer containing 1 mM H2O2, 1 mM CPZ, and 1 mM guaiacol. The reaction rate was determined by monitoring the increase in absorbance at 465 nm using an extinction coefficient of 6100 M-1 cm-1 for oxidized guaiacol (25). Percent remaining activity was determined by comparison with an identical enzyme aliquot taken at time zero.

Results Hydralazine was oxidized by HRP to a nitrogencentered radical and trapped by DMPO in both the absence (Figure 1A) and presence (Figure 1, B and D) of redox mediators. The hyperfine splitting constants of the 18 line spectrum obtained (aN ) 15.1 G; aHβ ) 16.7 G; aNβ ) 2.6 G) were consistent with published values for the DMPO-hydralazyl spin adduct (26). No nitrogencentered DMPO adduct was observed in the absence of peroxidase (Figure 1C). Oxidation of CPZ to CPZ•+ was monitored using UVvisible spectrophotometry. Figure 2A represents the absorption spectra of the CPZ•+ with an absorbance maximum at 525 nm. Upon the addition of equimolar amounts of hydralazine, the visible absorption spectrum of CPZ•+ was diminished, consistent with reduction of the CPZ•+ by hydralazine. In the presence of hydralazine, during the steady state oxidation of chlorpromazine by HRP, CPZ•+ accumulation was inhibited (Figure 2B). Upon complete oxidation of reductant, a sharp increase in absorbance at 525 nm due to CPZ•+ formation was observed. The time required to observe an increase in absorbance was proportional to hydralazine concentration (lines a-d). When sufficient hydralazine was included, hydrogen peroxide became limiting and no CPZ•+ accumulation was observed (line e). The ultimate rate of CPZ•+ accumulation was not affected by hydralazine oxidation, indicating that chlorpromazine was not consumed in this process (Figure 2B). Both the rate and extent of peroxidase-catalyzed oxidation of hydralazine were increased in the presence of chlorpromazine (Figure 3). Similar results were obtained using isoniazid and iproniazid in place of hydralazine (data not shown) (19). The addition of CPZ to a reaction mixture containing hydralazine, HRP, and hydrogen peroxide significantly enhanced DNA strand breakage, as represented by decreased DNA migration upon electrophoresis in an agarose gel (Figure 4). In the presence of peroxidase, hydrogen peroxide, and chlorpromazine, hydralazine was oxidized to intermediates that caused complete (97 ( 4%) relaxation of φX174 supercoiled DNA. Incubation of DNA with hydralazine alone did not result in strand breakage. Addition of hydrogen peroxide and HRP to the incubation mixture also did not cause damage to DNA. Negligible

330 Chem. Res. Toxicol., Vol. 10, No. 3, 1997

Figure 1. Electron spin resonance spin trapping of radicals produced during the oxidation of hydralazine by horseradish peroxidase. Spectrum A was obtained when 500 µM hydralazine was oxidized in the presence of 25 nM HRP, 250 µM H2O2, and 50 mM DMPO. Spectrum B was obtained when 125 µM CPZ was included in the reaction mixture described in (A). Spectrum C was obtained when HRP was omitted from (B). Spectrum D was obtained when 125 µM ABTS replaced CPZ in reaction mixture (B). Reactions were adjusted to pH 7.0 with minimal NaOH prior to initiation by the addition of HRP.

DNA damage occurred upon incubation with CPZ, HRP, and H2O2. The observed DNA damage was inhibited by 100 mM DMPO (data not shown). Under identical conditions as those described in the legend of Figure 4, lactoperoxidase and myeloperoxidase were capable of catalyzing DNA strand breakage (data not shown). Similar to the results obtained for horseradish peroxidase, no DNA damage occurred in the absence of chlorpromazine. When hydralazine was replaced by either isoniazid or iproniazid, 78 ( 3% and 31 ( 4% DNA damage was observed, respectively. DNA damage by these hydrazine derivatives also required the addition of chlorpromazine. Substitution of chlorpromazine with thioridazine (Table 2) also resulted in enhanced DNA strand breakage. When hydralazine served as the sole reductant for HRP, within 2 min the enzyme was inactivated to ∼2% of its original activity. This reaction was pH dependent and was favored under acidic conditions (Table 1). Peroxidase inactivation in the presence of hydrogen peroxide and hydralazine was mitigated by chlorpromazine, maintaining approximately 50% of the original enzyme activity over a 5 min period (Table 1). The DNA single-strand breakage exhibited a similar pH depen-

Reilly and Aust

Figure 2. UV-visible absorption spectra of the CPZ•+ in the absence or presence of hydralazine. Panel A shows the absorption spectrum of CPZ•+ prior to (solid line) and immediately after (dashed line) the addition of equimolar amounts of hydralazine. Panel B represents the inhibition of CPZ•+ accumulation as a function of hydralazine concentration. (a) 0 µM, (b) 25 µM, (c) 50 µM, (d) 75 µM, (e) 100 µM. Reactions included 100 µM CPZ, 25 nM HRP, 100 µM H2O2, and 50 mM NaCl, pH 7.0.

dence, with maximal damage resulting near pH 5.0 (Table 1). Stimulation of DNA damage by mediators seemed to involve protection against enzyme inactivation (Table 2). Replacement of CPZ with thioridazine protected the enzyme from inactivation and resulted in enhanced DNA single-strand breaks. When triflupromazine was used, the enzyme was protected to a lesser degree, but no DNA damage was observed. When either phenol or ABTS was used as mediators, the enzyme was rapidly inactivated and no DNA damage occurred. Use of chloride as a mediator by MPO was ineffective at promoting enhanced DNA damage. The agarose gel in Figure 5 represents DNA incubated with increasing equivalents of H2O2, hydralazine, HRP, and CPZ. Addition of 0-2 equiv of H2O2 resulted in hydralazyl radical generation, but no DNA damage. Reactions including a minimum of 3 equiv of H2O2 displayed almost complete DNA supercoil relaxation. Incubation with 4 equiv of H2O2 showed slightly less DNA damage than 3 equiv, possibly because some of the

Single-Strand Breaks in DNA by Hydralazine

Chem. Res. Toxicol., Vol. 10, No. 3, 1997 331

Figure 4. Agarose gel electrophoresis of DNA incubated with hydralazine and HRP. Lane 1 represents a standard of 500 ng of φX174 (RF-1) DNA. Lane 2 represents DNA that was incubated with 0.5 mM hydralazine, 0.1 mM DETAPAC, and 1 mM H2O2 in 50 mM NaCl, pH 7.0, for 15 min. Lane 3 was the same as lane 2 with 25 nM HRP included in the incubation mixture. Lane 4 was the same as lane 3 with 0.25 mM chlorpromazine added to the incubation mixture. Lane 5 represents DNA that was incubated with chlorpromazine, H2O2, and HRP. Figure 3. UV-visible absorption spectra of hydralazine during oxidation by HRP in the absence or presence of CPZ. Panel A represents the spectral changes observed during the direct oxidation of 300 µM hydralazine by 25 nM HRP in the presence of 300 µM H2O2 and 50 mM NaCl. The conditions for panel B are identical to those described for panel A, except that 50 µM CPZ was included.

CPZ•+.

reactive metabolite was consumed by These data suggest that a three-electron oxidation product of hydralazine must be generated to cause DNA single-strand breaks.

Discussion The results of this investigation provide a possible mechanism of peroxidase-dependent hydrazine oxidation and subsequent damage to DNA. Essential to this mechanism is the presence of a mediator which promotes rapid enzyme turnover rates and is oxidized to an oxidizing radical. In turn, these radicals must mediate the oxidation of secondary chemicals to reactive intermediates. CPZ and thioridazine promoted both oxidation of hydralazine and DNA damage. Additionally, other hydrazine derivatives could be oxidized to intermediates which caused DNA damage. Similar results were observed with MPO, LPO, and HRP. Consistent with the redox mediation model proposed by Goodwin et al. (8, 9, 19), oxidation of hydralazine by

Table 1. Effects of Reaction pH and Redox Mediation on Enzyme Inactivation and Relationship to DNA Single-Strand Breakage

pH of incubation

remaining activity without CPZa (%)

remaining activity with CPZa (%)

% DNA damaged without CPZa

% DNA damaged with CPZa

3.0 5.0 7.0 unbuffered NaCl

2.0 ( 0.8 78 ( 7 95 ( 18 1.0 ( 0.5

45 ( 12 82 ( 9 97 ( 8 43 ( 3

4.0 ( 1 12 ( 3 13 ( 2 6(1

no migration 83 ( 13 25 ( 4 97 ( 4

a

Measurements represent mean ( SD of three experiments.

Table 2. Ability of Other Mediators To Protect HRP from Inactivation and To Enhance DNA Damage peroxidase mediator

protection of HRP activity

enhanced DNA damage

chlorpromazine thioridazine ABTS triflupromazine phenol

yesa yesa no yesa no

yes yes no no no

a

A minimum of 25% original activity remained.

peroxidases was stimulated in the presence of CPZ. This was due in part to a kinetic difference in substrate oxidation (8), as well as protection of the enzyme from modification by radical attack and subsequent inactivation (19, 20). It has been proposed that peroxidasecatalyzed oxidation of hydralazine is inefficient at causing DNA damage because of the inability of peroxidases to

332 Chem. Res. Toxicol., Vol. 10, No. 3, 1997

Figure 5. Agarose gel electrophoresis of DNA incubated with hydralazine, HRP, and various equivalents of hydrogen peroxide. Lane 1 represents a 500 ng standard of φX174 (RF-1) DNA. Lane 2 represents DNA incubated with 50 nM HRP, 1 mM chlorpromazine, and 0.5 mM hydralazine in 50 mM NaCl, pH 7.0. Conditions for lane 3 were identical to lane 2 including 1 equiv (0.25 mM) of H2O2. Lanes 4-6 contained 2, 3, and 4 equiv of H2O2, respectively.

further oxidize the hydralazyl radical (12). We have found that the hydralazyl radical, or a related oxidation product, was capable of inactivating the peroxidase, particularly at low pH. A similar phenomena was observed by Goodwin et al. using isoniazid (19). Inactivation of the enzyme appears to prevent generation of the necessary species to cause DNA damage. However, we have demonstrated that the presence of a redox mediator protected the enzyme from inactivation, promoted extensive oxidation of hydralazine, and enhanced DNA damage over a range of pH values. At pH 7.0 and 5.0, the unprotonated and protonated DMPO-hydralazyl spin adducts were observed using ESR (data not shown). These data are consistent with results obtained by Sinha, who showed that at pH 6.0 both protonated and unprotonated hydralazyl radicals exist (26). These data may have significance to this model. Previous work by Hofstra and Uetrecht (18) has shown that there is a pH-dependent formation of specific intermediates. It was proposed that, at low pH, formation of both the diazene and diazonium ion was favored, and that the diazonium ion may be the damaging oxidized metabolite of hydralazine (12, 18). Our results suggest a necessity for formation of a three-electron oxidation product favored by acidic conditions in order to cause DNA damage. Consistent with other hypotheses (18), a lower pH appears to stabilize an intermediate, permitting further oxidation of that species to one capable of causing DNA strand breakage. The increase in DNA damage as the pH decreased from 7.0 to 5.0 suggests this

Reilly and Aust

may be the case. These data also suggest that the protonation state of particular intermediates may be critical in the generation of a damaging intermediate. The inability of DNA damage to occur at pH 3.0 could be due to decreased peroxidase turnover rate, or decreased CPZ•+-mediated oxidation of hydralazine (19). Although strong evidence exists for the interaction of hydralazyl radical with DNA, specifically at adenine and guanine residues (12), our results do not support this hypothesis. The data presented in Figure 5 clearly indicate that little or no DNA damage occurred under conditions in which only the hydralazyl radical was generated (lane 3). Addition of 3.0 equiv of H2O2 was essential to cause DNA single-strand breakage. This suggests the involvement of a multiple oxidation product, possibly a diazene radical intermediate of hydralazine, in DNA strand breakage. An explanation for the observed discrepancies among our data and those presented by Yamamoto and Kawanishi (12) could be that hydralazyl radical may directly interact with adenine and guanine, but does not cause strand breakage in supercoiled φX174 DNA. Based on kinetic data presented in Figure 2B, it would appear that CPZ was not consumed in the reaction since the initial rate of CPZ•+ accumulation was not affected (9). Possible explanations for the inability of certain chemicals to serve as effective mediators could be that an essential part of this process involves the type of interaction between the mediator and the enzyme, or simply the reaction rate for the particular substrate mediator (8). This may be the case with intermediates of hydralazine that are unstable and do not exist for significant time periods (18); however, DNA damage requires that these intermediates be oxidized. Both thioridazine and chlorpromazine, which enhance DNA damage, serve as excellent reductants of HRP compound I and compound II, far better than triflupromazine, ABTS, or phenol (data not shown) (8). Another possible explanation could be that radical mediators must have a high enough oxidation potential to generate the necessary intermediates. This was probably not the case. CPZ•+ (E° ) 0.78 V) (27) was effective at enhancing DNA damage, whereas phenol (E° > 0.8 V) (27) was not. The inability of phenol to function as a mediator could best be explained by the fact that phenol itself will promote inactivation of peroxidases (10, 28). This may be reversed by redox mediators (10). The ability of various chemicals to serve as redox mediators in peroxidase catalysis appears to be dependent upon a variety of factors. In the present study, many radicals produced by peroxidase were capable of oxidizing hydralazine (data not shown). Of those tested, only CPZ, thioridazine, and triflupromazine could protect the enzyme from being inactivated. Of those capable of protecting the enzyme, only those with high enough oxidation potential or low enough Km could promote DNA damage. The explanation of these findings is obviously far more complex than the scope of this investigation and will require further research. The toxicological significance to these findings would be primarily dependent upon physiological constraints such as location and pH. Using an electron microscopy technique, it has been shown that endogenous peroxidase activity exists in hepatic macrophage rough endoplasmic reticulum, nuclear envelope, and cytoplasm (29). Prostaglandin H synthase, another type of peroxidase, also resides within the endoplasmic reticulum which is contiguous with the nuclear membrane (30). These studies

Single-Strand Breaks in DNA by Hydralazine

provide sufficient evidence to assume that peroxidases exist in close enough proximity to DNA that they may be a concern. Although DNA damage occurs at pH 7.0 in this model system, acidic conditions significantly enhance the effect. Additional studies have found that hydralazine itself was capable of acidifying intracellular and extracellular environments, possibly contributing to the observed cytotoxicity in vitro (31, 32). Additionally, cytotoxicity of hydralazine was enhanced in cells that were acidified before exposure (31, 32). During hydralazine oxidation by the CPZ/HRP/H2O2 system in 50 mM NaCl, the pH was lowered due to proton release. Concomitant to this process was maximal DNA damage. These data suggest that the acidic conditions which favor DNA damage are intrinsic to the oxidation of hydralazine, and possibly other hydrazines, by peroxidases. The extent to which this reaction sequel may occur in vivo may be a point of concern. Endogenous reductants including ascorbate, glutathione, unsaturated lipid, NADH, and a variety of other compounds will serve as reductants for CPZ•+, hence inhibiting CPZ•+-mediated oxidation of hydralazine (27, 33). Additionally, a variety of compounds may react with any number of the intermediates of hydralazine, preventing further oxidation of that particular intermediate. The mechanism proposed in this study was developed using model peroxidase substrates. It has been shown, however, that several physiological reductants of peroxidases may serve as redox mediators including halides, tyrosine, indoles, catechols, and amines (19, 27, 34-36). At any point in time, a number of these compounds may be available for peroxidase-catalyzed generation of radicals which may oxidize hydralazine, eliminating the requirement for CPZ or a related compound. Given the oxidizing nature of most radicals generated by peroxidase catalysis, endogenous antioxidants may be depleted by extensive redox cycling, hence mitigating cellular defenses to oxidative damage. Thus, mediated oxidation of hydralazine, and possibly other xenobiotics including hydrazines, may have significance in toxicity (8, 19). The results of this investigation provide insight as to the role of redox mediators in peroxidase-catalyzed oxidation of hydralazine in vitro. Additionally, insight into the mechanism of peroxidase dependent DNA damage by hydralazine has been obtained. Combined with overwhelming evidence for the toxicity of hydralazine and adverse drug interactions, it seems realistic to propose that redox mediation may serve as a valid model for peroxidase-dependent metabolism of hydralazine and its ability to damage DNA. Given the results obtained using isoniazid and iproniazid, it would appear that a similar mechanism operates during DNA damage by other hydrazine-containing compounds. The findings presented here may serve as a model to help understand the possible requirements for hydralazine, and possibly other hydrazines, to modify DNA in vivo.

Acknowledgment. This work was supported by NIH Grant ES05056. The authors would like to thank Dr. Douglas C. Goodwin for helpful discussions and Terri Maughan for secretarial assistance in the preparation of the manuscript.

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