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Production of 4-hydroxypyrazole from the interaction of the alcohol dehydrogenase inhibitor pyrazole with hydroxyl radical
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 255, No. 2, June, pp. 217225,1987
Production of 4-Hydroxypyrazole from the Interaction of the Alcohol Dehydrogenase Inhibitor Pyrazole with Hydroxyl Radical’ SUSANA Department
PUNTARULO
of Biochemistl-y,
AND
ARTHUR
I. CEDERBAUM’
Mount Sinai Schnol of Medicine, New York, New York 10029
Received October 10, 1986, and in revised form January
27, 1987
Pyrazole, an effective inhibitor of alcohol dehydrogenase, was previously shown to be a scavenger of the hydroxyl radical. 4-Hydroxypyrazole is a major metabolite in the urine of animals administered pyrazole in vivo. Experiments were conducted to show that 4-hydroxypyrazole was a product of the interaction of pyrazole with hydroxyl radical generated from three different systems. The systems utilized were (a) the iron-catalyzed oxidation of ascorbate, (b) the coupled oxidation of hypoxanthine by xanthine oxidase, and (c) NADPH-dependent microsomal electron transfer. Ferric-EDTA was added to all the systems to catalyze the production of hydroxyl radicals. A HPLC procedure employing either uv detection or electrochemical detection was utilized to assay for the production of 4-hydroxypyrazole. The three systems all supported the oxidation of pyrazole to 4-hydroxypyrazole by a reaction which was sensitive to inhibition by competitive hydroxyl radical scavengers such as ethanol, mannitol, or dimethyl sulfoxide and to catalase. The sensitivity to catalase implicates HzOz as the precursor of the hydroxyl radical by all three systems. Superoxide dismutase inhibited production of 4-hydroxypyrazole only in the xanthine oxidase reaction system. In the absence of ferric-EDTA (and azide), microsomes catalyzed the oxidation of pyrazole to 4-hydroxypyrazole by a cytochrome P-450-dependent reaction which was independent of hydroxyl radicals. This latter pathway may be primarily responsible for the in vivo metabolism of pyrazole to 4-hydroxypyrazole. The production of 4-hydroxypyrazole from the interaction of pyrazole with hydroxyl radicals may be a sensitive, rapid technique for the detection of these radicals in certain tissues or under certain conditions, e.g., increasing oxidative stress. 0 1987 Academic Press, Inc.
by microsomes (6). In vivo administration of pyrazole was shown to induce an isozyme of cytochrome P-450 which appears similar to the isozyme induced by chronic ethanol feeding (7-10). Associated with this induction was an enhancement of the microsomal oxidation of a variety of drugs, including aniline (ll), dimethylnitrosamine (8,12,13), and ethanol (7,lO). Another property of pyrazole was its ability to act as a potent scavenger of the hydroxyl radical (‘OH).3 Pyrazole was shown to inhibit the oxidation of ethanol
Pyrazole is a potent inhibitor of alcohol dehydrogenase and has been widely used to inhibit the oxidation of ethanol (l-4). However, this compound is not specific for alcohol dehydrogenase and can affect other metabolic processes. In vitro, pyrazole was shown to bind to cytochrome P-450 and produce type II spectral changes (5) and block the oxidation of a variety of drugs 1 These studies were supported by USPHS Grants AA-06610 and AA-03312 from The National Institute on Alcohol Abuse and Alcoholism. We thank Ms. Roslyn C. King for typing the manuscript and Dr. Dennis E. Feierman for helpful discussions. ’ To whom correspondence should be addressed.
3 Abbreviations used: ‘OH, hydroxyl dimethyl sulfoxide. 217
radical; DMSO,
0003-9861/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved
218
PUNTARULO
AND
or dimethyl sulfoxide by a variety of ‘OHgenerating systems (14). An approximate rate constant of about 8 X 10’ M-* s-l for the interaction of pyrazole with ‘OH was calculated from the kinetics of the inhibition curves (14), indicating that pyrazole is a powerful scavenger of ‘OH. The product of the interaction of pyrazole with ‘OH was not identified in these earlier studies. Experiments were conducted in the current report to determine if the interaction of pyrazole with ‘OH produces 4-hydroxypyrazole as the primary product. This metabolite has been identified in the urine of mice and rats after administration of 14C-labeled pyrazole (15, 16), and was also produced by isolated microsomal preparations by a NADPH-dependent, carbon monoxidesensitive reaction (17).4A HPLC procedure which allows the detection of small amounts of 4-hydroxypyrazole was developed and utilized for these experiments. MATERIALS
AND
METHODS
Three model ‘OH-generating systems were utilized to study the interaction of pyrazole with ‘OH. All systems were carried out in the presence of ferric-EDTA which catalyzes the production of ‘OH. The xanthine oxidase system (18-20) consisted of 50 mM potassium phosphate, pH 7.4, 1 mM hypoxanthine, 10 mM pyrazole, and 50 pM ferrous-100 ELMEDTA in a final volume of 2 ml. Reactions were initiated by the addition of 0.1 unit of xanthine oxidase and terminated by the addition of perchloric acid to a final concentration of 0.75 N. The iron-catalyzed oxidation of ascorbate system (21, 22) was carried out in a reaction mixture containing 50 mM phosphate buffer, pH 7.4, 10 mM pyrazole, and 167 pM ferrous-333 /.IM EDTA in a final volume of 2 ml. Reactions were initiated by the addition of ascorbate to a final concentration of 2 mM and quenched by perchloric acid (final concentration of 0.75 N). Isolated rat liver microsomes from male Sprague-Dawley rats weighing about 150 g were prepared by differential centrifugation. The microsomes were washed and suspended in 125 mM KC1 and stored at -70°C. The basic reaction system consisted of 100 mM phosphate buffer, pH 7.4, 10 mM MgClz, 0.4 mM NADP+, 10 mM pyrazole, and about 4 mg microsomal protein in a final volume of 2 ml. Reactions were initiated by the addition of a mixture of glucose B-phosphate (final concentration of 10 mM) and 1.4 units of
4 Feierman, D. E., and Cederbaum, A. I., manuscript submitted for publication.
CEDERBAUM glucose-6-phosphate dehydrogenase, and terminated by perchloric acid. In most experiments, 25 pM ferrous-50 pM EDTA and 1 mM sodium azide were also present. The azide was added to inhibit the activity of catalase, which is present as a contaminant in isolated microsomes. After terminating the reactions with 0.1 ml perchloric acid (70% w/v), samples were centrifuged for 5 min in a clinical centrifuge. The supernatant was neutralized with I1 N KOH and the potassium perchlorate was removed by centrifugation. One milliliter of the clear, neutral supernatant was loaded onto a SEP-PAK Cis cartridge (Waters Associates, Milford, MA). The SEP-PAK was previously activated by washing with 6 ml of methanol, followed by 10 ml of water. One milliliter of water (HPLC grade) was added to elute the metabolite, 4-hydroxypyrazole, off the SEP-PAK and two 0.5-ml fractions were collected. The second fraction was utilized for the HPLC analysis of 4-hydroxypyrazole. A lo-p1 aliquot of the sample prepared as described above was injected into a Microsorb Cl8 column (4.6 mm X 25 cm, Rainin Instrument, Woburn, MA) using a Waters universal injector. The 4-hydroxypyrazole was separated from the remaining components of the reaction mixture isocratically, using a mobile phase of 10% acetonitrile plus 1% glacial acetic acid plus 5 mM octanesulfonic acid in HzO. In view of the positive charge on 4-hydroxypyrazole at pH values less than 3, negatively charged paired-ion chromatography was used to aid in the separation of the metabolite from other components of the reaction mixture. Under these conditions, the 4-hydroxypyrazole was detected at 254 nm using a Waters Model 400 detector or a BAS electrochemical detector with a retention time of about 6.8 min. Raising or lowering the concentrations of acetonitrile decreased or increased the retention time for 4-hydroxypyrazole, respectively. A 10% acetonitrile concentration appeared to produce the best separation with minimum peak broadening. Peak height was proportional to the concentration of standard 4hydroxypyrazole and linear standard curves could be generated. The standard 4-hydroxypyrazole was a generous gift from Eli Lilly Co. (Indianapolis, IN). All values represent the mean of two or three experiments, each carried out in duplicate. Variability between experiments did not exceed 10%. All values were corrected for “zero-time” controls in which the perchloric acid was added to the reaction system prior to the addition of the initiating agent for the reaction.
RESULTS
Asmrbate-dependent oxidation of pyraxole. A HPLC procedure was developed for the detection of 4-hydroxypyrazole, an anticipated product of the interaction of pyr-
OXIDATION
OF PYRAZOLE
azole with ‘OH. The iron-catalyzed oxidation of ascorbate was used as a chemical model’OH-generating system (21,22). Figure 1A shows a HPLC profile of a zero-time reaction mixture in which perchloric acid was added to quench the reaction prior to the addition of ascorbate. No prominent peak with retention times around 6.8 min was detected. When standard 4-hydroxypyrazole was coinjected with an aliquot of the zero-time sample, a peak with a retention time of about 6.8 min was observed (Fig. 1B). The height of this peak was directly proportional to the amount of standard 4-hydroxypyrazole that was injected.
A
0
C
D
3’
222 FIG. 1. HPLC profile for the detection of 4-hydroxypyrazole produced from the interaction of pyrazole with hydroxyl radicals generated by the ascorbate system. The oxidation of pyrazole by the ascorbate system was assayed as described under Materials and Methods. (A) HPLC profile for a “zero-time” control in which perchloric acid was added to quench the reaction prior to the addition of ascorbate. No peak with a retention time of about 6.8 min can be observed. (B) HPLC profile for standard 4-hydroxypyrazole (30 pM stock concentration) coinjected with a zero-time control. A peak at 6.8 min retention time is produced under these conditions. (C) HPLC profile for a sample obtained after a lo-min incubation period in the ascorbate system. A metabolite with a retention time of 6.8 min is produced. (D) HPLC profile of the sample from C coinjected with standard I-hydroxypyrazole (30 pM stock concentration, as in B). The increase in height of the peak with a retention time of 6.8 min is equal to the sums found for B plus C. No peak splitting is observed.
219
BY ‘OH
0
20
40
60
TIME hn)
FIG. 2. Time pyrazole when icals generated tem (A) or the (B). Reactions Materials and
course for the production of I-hydroxypyrazole is oxidized by hydroxyl radfrom the ferric-EDTA-ascorbate sysferric-EDTA-xanthine oxidase system were carried out as described under Methods.
When pyrazole was oxidized by the standard ascorbate system, the 6%min peak was produced (Fig. 1C). Production of this peak was linear with time for 10 min (Fig. 2A) and then quickly levelled off. The production of 4-hydroxypyrazole required the presence of ascorbate, pyrazole, and ferricEDTA. Figure 1D depicts the profile obtained when a coinjection of the prepared supernatant from a typical sample (as in Fig. 1C) was made along with the standard 4-hydroxypyrazole. The metabolite produced from the ascorbate-ferric-EDTAdependent oxidation of pyrazole coeluted with the standard 4-hydroxypyrazole (Fig. 1D). The peak height was equal to the sum of the peak heights produced from the metabolite alone (Fig. 1C) plus the standard alone (Fig. 1B). The absence of peak splitting when the metabolite plus standard are mixed together indicates identical coelution. An additional peak with a retention time of about 7.4 min was detected from the interaction of pyrazole with *OH (Fig. 1C or 1D). No attempt was made to identify this peak in the current studies (see Discussion). The amount of 4-hydroxypyrazole produced was dependent on the concentration of ascorbate utilized to initiate the reaction; rates of 4-hydroxypyrazole production were (nmol/min/ml) 2.4,5.2, and 7.8 in the presence of 0.5,1, and 2 mM ascorbate, respectively. A pyrazole concentration of about 5 mM was found to saturate the system. To implicate oxygen radicals in the
220
PUNTARULO
AND TABLE
EFFECTOFOXYGENRADICAL SCAVENGERS ON THE
CEDERBAUM I
OXIDATION
OF PYRAZOLE
TO4-HYDROXYPYRAZOLE
BY THE IRON-ASCORBATE SYSTEM
Addition
Concentration -
Catalase
Superoxide dismutase Ethanol Dimethyl sulfoxide Mannitol Urea
Rate of 4-hydroxypyrazole production (nmol/min/ml)
-
mU mU mU mU mM
8.7 5.1 2.4 0.8 8.0 0.75
-41 -72 -91 -9 -91
30 mM 50 mM 50 IIIM
0.10
-98
0.90 7.8
-90 -10
97 162 325 300 100
Note. The oxidation of pyrazole to 4-hydroxypyrazole by the iron-ascorbate under Materials and Methods, in the presence of the indicated addition.
production of 4-hydroxypyrazole from pyrazole, the effect of various oxyradical scavengers was evaluated. Catalase produced a concentration-dependent inhibition of 4-hydroxypyrazole production (Table I). Boiled catalase was not inhibitory. Superoxide dismutase had no effect on the oxidation of pyrazole by the ascorbate system (Table I). These results indicate that HzOz, but not superoxide is required for the production of the oxidant responsible for the oxidation of pyrazole. However, H202 alone did not oxidize pyrazole to 4hydroxypyrazole. Ethanol, dimethyl sulfoxide, and mannitol, which are effective ‘OH-scavenging agents (23), all produced inhibition of the production of 4-hydroxypyrazole (Table I). By contrast, urea, which is a weak ‘OH scavenger, had no effect. Electrochemical
Effect of addition (%)
system was assayed as described
the metabolite produced from the ascorbate-dependent oxidation of pyrazole as detected by uv absorbance at 254 nm (A)
detection of Q-hydroxy-
pyraxole. To further validate that the metabolite produced from the interaction of pyrazole with ‘OH generated by the ascorbate system was 4-hydroxypyrazole, an electrochemical detection procedure was utilized. An advantage of this system is that one can vary the applied voltage and thus evaluate whether a metabolite and a standard display the same electrochemical response and characteristics (24). Figure 3 shows a comparison of HPLC profiles of
FIG. 3. HPLC profile for the determination of 4hydroxypyrazole produced during pyrazole oxidation by the ascorbate system and detected either by absorbance at 254 nm (A) or electrochemically with an applied voltage of 0.85 V (B). A lo-p1 aliquot of sample was injected and detection was monitored simultaneously with a uv detector (A) or an electrochemical detector (B).
OXIDATION
OF PYRAZOLE
221
BY ‘OH
from the interaction of pyrazole with ‘OH generated by the ascorbate system (closed circles) as a function of the applied electrode potential is shown in Fig. 4. Both the standard and the ‘OH-dependent metabolite have the same electrochemical characteristics, suggesting that they are identical. ELECTRODE
Xanthine oxidase-dependent oxidation of pyraxole. The coupled oxidation of hypo-
POTENTIAL(vI
FIG. 4. Comparison of the electrochemical characteristics of standard 4-hydroxypyrazole (0) and the metabolite (0) produced from the oxidation of pyrazole by the ascorbate system. A lo-p1 aliquot of a sample prepared as described in Fig. 1B (standard) or 1C (metabolite) was injected and the height of the peak with a retention time of 6.8 min was determined as a function of the applied voltage. The two curves are identical.
or electrochemically (B). Both methods of detection reveal a peak with a retention time of 6.8 min. Peak heights were linearly proportional to the amount of 4-hydroxypyrazole added and standard 4-hydroxypyrazole eluted at the same position as the metabolite without any peak splitting (not shown). A titration curve of the relative peak height (determined electrochemitally) for the standard 4-hydroxypyrazole (open circles) and the metabolite produced TABLE
xanthine by xanthine oxidase, in the presence of ferric-EDTA, was utilized as an enzyme-catalyzed model ‘OH-generating system (M-20). As shown in Fig. 2B, the production of 4-hydroxypyrazole by this reaction system was linear with time over a 60-min period. No 4-hydroxypyrazole was produced if the ferric-EDTA, hypoxanthine, xanthine oxidase, or pyrazole was omitted from the reaction system. The production of 4-hydroxypyrazole was sensitive to inhibition by either catalase or by superoxide dismutase, indicating that both HzOz and the superoxide anion radical are required for the production of ‘OH (Table II). Inhibition of pyrazole oxidation to 4hydroxypyrazole was also observed in the presence of competitive ‘OH scavengers such as ethanol, dimethyl sulfoxide, and mannitol, whereas urea was without any effect (Table II). II
EFFECTOF OXYGEN RADICAL SCAVENGERS• NTHEOXIDATIONOFPYRAZOLETO~-HYDROXYPYRAZOLE BYTHE XANTHINE OXIDASE SYSTEM
Addition
-
Catalase Superoxide Ethanol Dimethyl Mannitol Urea
Concentration
dismutase
sulfoxide
97 162 45 300 100
mU mU mU mU mM 30 mM 50 mM 50 mM
Rate of 4-hydroxypyrazole production (nmol/min/ml)
Effect of additon (S) -
0.40 0.067 0.033 0.142 0.067 0.058 0.06’7 0.067 0.40
Note. The oxidation of pyrazole to 4-hydroxypyrazole by the coupled oxidation oxidase was assayed as described under Materials and Methods.
-83 -92 -64 -83 -86 -83 -83 0 of hypoxanthine
by xanthine
222
PUNTARULO
AND
TME him)
FIG. 5. Time course for the oxidation of pyrazole to 4-hydroxypyrazole by rat liver microsomes in the absence (0) of ferric-EDTA and sodium azide, and in their presence (0). Reactions were carried out as described under Materials and Methods.
Microsmal
oxio?ation of ggraxole.
The
ability of isolated rat liver microsomes to generate ‘OH-like species was shown to require the presence of iron (25). FerricEDTA was a particularly effective stimulant, relative to other iron chelates, of ‘OH production by microsomes (26). Azide also stimulated microsomal ‘OH generation by inhibiting catalase activity found in isolated microsomes (27). Microsomes were incubated in absence and presence of the TABLE
CEDERBAUM
combination of ferric-EDTA plus azide and the production of 4-hydroxypyrazole was determined. In the absence of ferric-EDTA and azide, microsomes oxidized pyrazole to 4-hydroxypyrazole in a time-dependent manner (Fig. 5). The addition of ferricEDTA plus azide produced a fivefold increase in the production of 4-hydroxypyrazole (Fig. 5). There was no production of 4-hydroxypyrazole in the absence of either pyrazole, microsomes, or the NADPHgenerating system. In the absence of ferric-EDTA plus azide, DMSO and ethanol produced some inhibition of the microsomal oxidation of pyrazole (Table III). However, this inhibition was about the same as that found with urea, which is a relatively poor scavenger of ‘OH. Moreover, catalase had no effect on the production of 4-hydroxypyrazole (Table III). These results suggest the possibility that the oxidation of pyrazole is occurring via a ‘OH-independent mechanism. In the presence of ferric-EDTA, ethanol and dimethyl sulfoxide, but not urea, were considerably more effective in blocking microsomal oxidation of pyrazole III
EFFECTOF OXYGEN RADICAL SCAVENGERSONTHE OXIDATIONOF PYRAZOLETOI-HYDROXYPYRAZOLE BY RAT LIVER MICROSOMES Rate of 4-hydroxypyrazole production and effect of addition (nmol/min/mg microsomal protein) and (7%) -Fe EDTA Addition -
Dimethyl
sulfoxide
Concentration -
10 mM
30 mM Ethanol Urea Catalase Superoxide
dismutase
100 mM 50 mM 325 mU 300 mU
+Fe EDTA
A Fe EDTA
Rate
Effect
Rate
Effect
Rate
Effect
0.063 0.060 0.044 0.039 0.043 0.062 0.066
-5 -30 -38 -32 -2 +5
0.276 0.220 0.100 0.066 0.249 0.066 0.338
-20 -64 -76 -10 -76 +22
0.213 0.160 0.056 0.027 0.206 0.004 0.272
-25 -14 -88 -4 -99 +28
Note The oxidation of pyrazole to 4-hydroxypyrazole by microsomes was assayed as described under Materials and Methods in the presence of the indicated additions. Reactions were conducted either in the absence of iron EDTA plus azide or in the presence of 25 FM ferric-50 @i EDTA-1.0 mhl sodium azide. The net increase produced by iron-EDTA is shown in the A Fe EDTA column. For experiments involving catalase, azide was omitted from the reaction system.
OXIDATION
OF PYRAZOLE
BY ‘OH
223
be oxidized to 4-hydroxypyrazole was characterized in three different systems which are known to produce ‘OH, the ironcatalyzed oxidation of ascorbate, the coupled oxidation of hypoxanthine by xanthine oxidase, and NADPH-dependent microsomal electron transfer in the presence of ferric-EDTA. Inhibition by competitive ‘OH scavengers and catalase and the requirement for iron support a role for ‘OH in pyrazole oxidation by these reaction systems. On the basis of the effect of catalase in blocking pyrazole oxidation to 4hydroxypyrazole, it appears that Hz02 serves as the precursor of ‘OH by all three reaction systems. The requirement for iron suggests that a Fenton-type (30) of reaction between ferrous and HzOzis responsible for the generation of ‘OH. The reductant of the ferric iron is different for the three systems. In the xanthine oxidase reaction, reduction of ferric-EDTA is mediated by superoxide radical and ‘OH is generated by an iron-catalyzed Haber-Weiss type of reaction (19,20). This explains the sensitivity of pyrazole oxidation to inhibition by superoxide dismutase. The lack of inhibition by superoxide dismutase in the ascorbate or microsomal reaction system is probably due to reduction of ferric-EDTA by ascorbate (21, 22) and by NADPH-cytochrome P-450 reductase (26, 31). A HPLC procedure using paired ion chromatography was employed to detect 4hydroxypyrazole. The metabolite produced from the interaction of pyrazole with ‘OH exhibits the same retention time and coelutes with the standard 4-hydroxypyrazole under a variety of conditions. The metabolite also displays the same electrochemical properties as the standard 4-hydroxypyrazole and is thus likely to be identical to the standard. Under the conditions described under Materials and Methods, less than 0.02 nmol of 4-hydroxypyrazole can easily be detected. DISCUSSION The interaction of ‘OH with pyrazole may Pyrazole was previously found to be an proceed by hydrogen abstraction or addieffective ‘OH scavenging agent (14). The tion to the ring may occur. Addition of ‘OH present report shows that 4-hydroxypyrto the ring would produce the 4-hydroxyazole is a product of the interaction of pyr- pyrazole radical, which upon dismutation azole with ‘OH. The ability of pyrazole to would generate 4-hydroxypyrazole. Alter-
than in the absence of ferric-EDTA. Moreover, catalase now produced a striking inhibition of 4-hydroxypyrazole production (Table III). Subtraction of rates of production of 4-hydroxypyrazole in the absence of ferric-EDTA from the rates in the presence of ferric-EDTA indicated that the net increase produced by the addition of ferricEDTA was almost completely abolished by catalase and competitive ‘OH scavengers, whereas urea had no effect (Table III, AFe EDTA column). These results suggest that the increase in pyrazole oxidation to 4-hydroxypyrazole produced by the addition of ferric-EDTA to microsomes was due to the increased generation of ‘OH. Superoxide dismutase had no effect on the production of 4-hydroxypyrazole in the absence or presence of ferric-EDTA. In other experiments, the effects of inhibitors of cytochrome P-450-catalyzed reactions or of a competitive drug substrate on production of 4-hydroxypyrazole were determined. Metyrapone (1 mM), carbon monoxide (2.5:1 ratio, CO:02), and aminopyrine (5 mM) produced about 50% inhibition of microsomal pyrazole oxidation when ferric-EDTA and azide were omitted from the reaction system, thus implicating a role for cytochrome P-450 in the oxidation of pyrazole. However, these agents had no effect on the net increase in rates of pyrazole oxidation produced by the addition of ferric-EDTA plus azide (data not shown). This is in agreement with previous studies implicating a role for the NADPHcytochrome P-450 reductase and not cytochrome P-450 in interacting with iron to generate ‘OH (26, 28, 29). Thus, pyrazole can be oxidized by microsomes by a cytochrome P-450-dependent pathway independent of ‘OH, as well as by a ‘OH-dependent reaction which involves the reductase and catalytic amounts of iron chelates.
224
PUNTARULO
AND
natively, the 4-hydroxypyrazole radical can be oxidized, e.g., by ferric-EDTA to produce 4-hydroxypyrazole. Hydrogen abstraction would produce the pyrazole radical, which upon interaction with Ozwould form the peroxy radical. Dismutation of the latter or decomposition via a Russelltype mechanism (32) would yield 4-hydroxypyrazole. It should be emphasized that the HPLC system was maximized only for the detection of 4-hydroxypyrazole and the production of other products from the interaction of pyrazole with ‘OH was not evaluated in these systems. That other products are indeed formed can be seen by the presence of a peak with retention time of 7.4 min. At present, we suspect that the unknown peak may be 3-hydroxypyrazole and experiments to evaluate this possibility and synthesize this agent are underway. 4-Hydroxypyrazole was the product sought in these experiments since this compound was a major metabolite recovered in the urine of mice and rats which had been administered pyrazole in vivo (15, 16). The production of 4-hydroxypyrazole was lowered by the simultaneous in vivo administration of ethanol (15,16) resulting in an increase in the half-life of pyrazole in plasma (33). In this regard, it is of interest that ethanol inhibited the oxidation of pyrazole to 4-hydroxypyrazole by the various ‘OH-generating systems which were utilized. Results shown in Fig. 5 and Table III suggest that pyrazole can also be oxidized to 4-hydroxypyrazole by a microsomal pathway in which ‘OH does not appear to play a role. This microsomal pathway appears to involve cytochrome P-450 and can be induced by prior treatment with certain inducers such as pyrazole, methylpyrazole, or chronic ethanol treatment (17).4 It is likely that cytochrome P-450dependent oxidation of pyrazole to 4-hydroxypyrazole is primarily responsible for the in vivo metabolism of pyrazole although some oxidation of pyrazole by ‘OH may occur under conditions of increased oxidative stress or by certain cell populations, e.g., activated macrophages. In view of the sensitivity, specificity, and simplicity of the assay procedure, the production of
CEDERBAUM
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31. MOREHOUSE, L. A., THOMAS, C. E., AND AUST, S. D. (1984) Arch. Biochem. Biophys. 232, 365377. 32. RUSSELL, G. A. (1957) J. Amer. Chem. Sot 79,38713877. 33. RYDBERG,
U., BUIJTEN,
Pharm. Pharmacol.
J., AND NERI,
24,651-652.
A. (1972)
J.
Production of 4-Hydroxypyrazole from the Interaction of the Alcohol Dehydrogenase Inhibitor Pyrazole with Hydroxyl Radical’ SUSANA Department
PUNTARULO
of Biochemistl-y,
AND
ARTHUR
I. CEDERBAUM’
Mount Sinai Schnol of Medicine, New York, New York 10029
Received October 10, 1986, and in revised form January
27, 1987
Pyrazole, an effective inhibitor of alcohol dehydrogenase, was previously shown to be a scavenger of the hydroxyl radical. 4-Hydroxypyrazole is a major metabolite in the urine of animals administered pyrazole in vivo. Experiments were conducted to show that 4-hydroxypyrazole was a product of the interaction of pyrazole with hydroxyl radical generated from three different systems. The systems utilized were (a) the iron-catalyzed oxidation of ascorbate, (b) the coupled oxidation of hypoxanthine by xanthine oxidase, and (c) NADPH-dependent microsomal electron transfer. Ferric-EDTA was added to all the systems to catalyze the production of hydroxyl radicals. A HPLC procedure employing either uv detection or electrochemical detection was utilized to assay for the production of 4-hydroxypyrazole. The three systems all supported the oxidation of pyrazole to 4-hydroxypyrazole by a reaction which was sensitive to inhibition by competitive hydroxyl radical scavengers such as ethanol, mannitol, or dimethyl sulfoxide and to catalase. The sensitivity to catalase implicates HzOz as the precursor of the hydroxyl radical by all three systems. Superoxide dismutase inhibited production of 4-hydroxypyrazole only in the xanthine oxidase reaction system. In the absence of ferric-EDTA (and azide), microsomes catalyzed the oxidation of pyrazole to 4-hydroxypyrazole by a cytochrome P-450-dependent reaction which was independent of hydroxyl radicals. This latter pathway may be primarily responsible for the in vivo metabolism of pyrazole to 4-hydroxypyrazole. The production of 4-hydroxypyrazole from the interaction of pyrazole with hydroxyl radicals may be a sensitive, rapid technique for the detection of these radicals in certain tissues or under certain conditions, e.g., increasing oxidative stress. 0 1987 Academic Press, Inc.
by microsomes (6). In vivo administration of pyrazole was shown to induce an isozyme of cytochrome P-450 which appears similar to the isozyme induced by chronic ethanol feeding (7-10). Associated with this induction was an enhancement of the microsomal oxidation of a variety of drugs, including aniline (ll), dimethylnitrosamine (8,12,13), and ethanol (7,lO). Another property of pyrazole was its ability to act as a potent scavenger of the hydroxyl radical (‘OH).3 Pyrazole was shown to inhibit the oxidation of ethanol
Pyrazole is a potent inhibitor of alcohol dehydrogenase and has been widely used to inhibit the oxidation of ethanol (l-4). However, this compound is not specific for alcohol dehydrogenase and can affect other metabolic processes. In vitro, pyrazole was shown to bind to cytochrome P-450 and produce type II spectral changes (5) and block the oxidation of a variety of drugs 1 These studies were supported by USPHS Grants AA-06610 and AA-03312 from The National Institute on Alcohol Abuse and Alcoholism. We thank Ms. Roslyn C. King for typing the manuscript and Dr. Dennis E. Feierman for helpful discussions. ’ To whom correspondence should be addressed.
3 Abbreviations used: ‘OH, hydroxyl dimethyl sulfoxide. 217
radical; DMSO,
0003-9861/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved
218
PUNTARULO
AND
or dimethyl sulfoxide by a variety of ‘OHgenerating systems (14). An approximate rate constant of about 8 X 10’ M-* s-l for the interaction of pyrazole with ‘OH was calculated from the kinetics of the inhibition curves (14), indicating that pyrazole is a powerful scavenger of ‘OH. The product of the interaction of pyrazole with ‘OH was not identified in these earlier studies. Experiments were conducted in the current report to determine if the interaction of pyrazole with ‘OH produces 4-hydroxypyrazole as the primary product. This metabolite has been identified in the urine of mice and rats after administration of 14C-labeled pyrazole (15, 16), and was also produced by isolated microsomal preparations by a NADPH-dependent, carbon monoxidesensitive reaction (17).4A HPLC procedure which allows the detection of small amounts of 4-hydroxypyrazole was developed and utilized for these experiments. MATERIALS
AND
METHODS
Three model ‘OH-generating systems were utilized to study the interaction of pyrazole with ‘OH. All systems were carried out in the presence of ferric-EDTA which catalyzes the production of ‘OH. The xanthine oxidase system (18-20) consisted of 50 mM potassium phosphate, pH 7.4, 1 mM hypoxanthine, 10 mM pyrazole, and 50 pM ferrous-100 ELMEDTA in a final volume of 2 ml. Reactions were initiated by the addition of 0.1 unit of xanthine oxidase and terminated by the addition of perchloric acid to a final concentration of 0.75 N. The iron-catalyzed oxidation of ascorbate system (21, 22) was carried out in a reaction mixture containing 50 mM phosphate buffer, pH 7.4, 10 mM pyrazole, and 167 pM ferrous-333 /.IM EDTA in a final volume of 2 ml. Reactions were initiated by the addition of ascorbate to a final concentration of 2 mM and quenched by perchloric acid (final concentration of 0.75 N). Isolated rat liver microsomes from male Sprague-Dawley rats weighing about 150 g were prepared by differential centrifugation. The microsomes were washed and suspended in 125 mM KC1 and stored at -70°C. The basic reaction system consisted of 100 mM phosphate buffer, pH 7.4, 10 mM MgClz, 0.4 mM NADP+, 10 mM pyrazole, and about 4 mg microsomal protein in a final volume of 2 ml. Reactions were initiated by the addition of a mixture of glucose B-phosphate (final concentration of 10 mM) and 1.4 units of
4 Feierman, D. E., and Cederbaum, A. I., manuscript submitted for publication.
CEDERBAUM glucose-6-phosphate dehydrogenase, and terminated by perchloric acid. In most experiments, 25 pM ferrous-50 pM EDTA and 1 mM sodium azide were also present. The azide was added to inhibit the activity of catalase, which is present as a contaminant in isolated microsomes. After terminating the reactions with 0.1 ml perchloric acid (70% w/v), samples were centrifuged for 5 min in a clinical centrifuge. The supernatant was neutralized with I1 N KOH and the potassium perchlorate was removed by centrifugation. One milliliter of the clear, neutral supernatant was loaded onto a SEP-PAK Cis cartridge (Waters Associates, Milford, MA). The SEP-PAK was previously activated by washing with 6 ml of methanol, followed by 10 ml of water. One milliliter of water (HPLC grade) was added to elute the metabolite, 4-hydroxypyrazole, off the SEP-PAK and two 0.5-ml fractions were collected. The second fraction was utilized for the HPLC analysis of 4-hydroxypyrazole. A lo-p1 aliquot of the sample prepared as described above was injected into a Microsorb Cl8 column (4.6 mm X 25 cm, Rainin Instrument, Woburn, MA) using a Waters universal injector. The 4-hydroxypyrazole was separated from the remaining components of the reaction mixture isocratically, using a mobile phase of 10% acetonitrile plus 1% glacial acetic acid plus 5 mM octanesulfonic acid in HzO. In view of the positive charge on 4-hydroxypyrazole at pH values less than 3, negatively charged paired-ion chromatography was used to aid in the separation of the metabolite from other components of the reaction mixture. Under these conditions, the 4-hydroxypyrazole was detected at 254 nm using a Waters Model 400 detector or a BAS electrochemical detector with a retention time of about 6.8 min. Raising or lowering the concentrations of acetonitrile decreased or increased the retention time for 4-hydroxypyrazole, respectively. A 10% acetonitrile concentration appeared to produce the best separation with minimum peak broadening. Peak height was proportional to the concentration of standard 4hydroxypyrazole and linear standard curves could be generated. The standard 4-hydroxypyrazole was a generous gift from Eli Lilly Co. (Indianapolis, IN). All values represent the mean of two or three experiments, each carried out in duplicate. Variability between experiments did not exceed 10%. All values were corrected for “zero-time” controls in which the perchloric acid was added to the reaction system prior to the addition of the initiating agent for the reaction.
RESULTS
Asmrbate-dependent oxidation of pyraxole. A HPLC procedure was developed for the detection of 4-hydroxypyrazole, an anticipated product of the interaction of pyr-
OXIDATION
OF PYRAZOLE
azole with ‘OH. The iron-catalyzed oxidation of ascorbate was used as a chemical model’OH-generating system (21,22). Figure 1A shows a HPLC profile of a zero-time reaction mixture in which perchloric acid was added to quench the reaction prior to the addition of ascorbate. No prominent peak with retention times around 6.8 min was detected. When standard 4-hydroxypyrazole was coinjected with an aliquot of the zero-time sample, a peak with a retention time of about 6.8 min was observed (Fig. 1B). The height of this peak was directly proportional to the amount of standard 4-hydroxypyrazole that was injected.
A
0
C
D
3’
222 FIG. 1. HPLC profile for the detection of 4-hydroxypyrazole produced from the interaction of pyrazole with hydroxyl radicals generated by the ascorbate system. The oxidation of pyrazole by the ascorbate system was assayed as described under Materials and Methods. (A) HPLC profile for a “zero-time” control in which perchloric acid was added to quench the reaction prior to the addition of ascorbate. No peak with a retention time of about 6.8 min can be observed. (B) HPLC profile for standard 4-hydroxypyrazole (30 pM stock concentration) coinjected with a zero-time control. A peak at 6.8 min retention time is produced under these conditions. (C) HPLC profile for a sample obtained after a lo-min incubation period in the ascorbate system. A metabolite with a retention time of 6.8 min is produced. (D) HPLC profile of the sample from C coinjected with standard I-hydroxypyrazole (30 pM stock concentration, as in B). The increase in height of the peak with a retention time of 6.8 min is equal to the sums found for B plus C. No peak splitting is observed.
219
BY ‘OH
0
20
40
60
TIME hn)
FIG. 2. Time pyrazole when icals generated tem (A) or the (B). Reactions Materials and
course for the production of I-hydroxypyrazole is oxidized by hydroxyl radfrom the ferric-EDTA-ascorbate sysferric-EDTA-xanthine oxidase system were carried out as described under Methods.
When pyrazole was oxidized by the standard ascorbate system, the 6%min peak was produced (Fig. 1C). Production of this peak was linear with time for 10 min (Fig. 2A) and then quickly levelled off. The production of 4-hydroxypyrazole required the presence of ascorbate, pyrazole, and ferricEDTA. Figure 1D depicts the profile obtained when a coinjection of the prepared supernatant from a typical sample (as in Fig. 1C) was made along with the standard 4-hydroxypyrazole. The metabolite produced from the ascorbate-ferric-EDTAdependent oxidation of pyrazole coeluted with the standard 4-hydroxypyrazole (Fig. 1D). The peak height was equal to the sum of the peak heights produced from the metabolite alone (Fig. 1C) plus the standard alone (Fig. 1B). The absence of peak splitting when the metabolite plus standard are mixed together indicates identical coelution. An additional peak with a retention time of about 7.4 min was detected from the interaction of pyrazole with *OH (Fig. 1C or 1D). No attempt was made to identify this peak in the current studies (see Discussion). The amount of 4-hydroxypyrazole produced was dependent on the concentration of ascorbate utilized to initiate the reaction; rates of 4-hydroxypyrazole production were (nmol/min/ml) 2.4,5.2, and 7.8 in the presence of 0.5,1, and 2 mM ascorbate, respectively. A pyrazole concentration of about 5 mM was found to saturate the system. To implicate oxygen radicals in the
220
PUNTARULO
AND TABLE
EFFECTOFOXYGENRADICAL SCAVENGERS ON THE
CEDERBAUM I
OXIDATION
OF PYRAZOLE
TO4-HYDROXYPYRAZOLE
BY THE IRON-ASCORBATE SYSTEM
Addition
Concentration -
Catalase
Superoxide dismutase Ethanol Dimethyl sulfoxide Mannitol Urea
Rate of 4-hydroxypyrazole production (nmol/min/ml)
-
mU mU mU mU mM
8.7 5.1 2.4 0.8 8.0 0.75
-41 -72 -91 -9 -91
30 mM 50 mM 50 IIIM
0.10
-98
0.90 7.8
-90 -10
97 162 325 300 100
Note. The oxidation of pyrazole to 4-hydroxypyrazole by the iron-ascorbate under Materials and Methods, in the presence of the indicated addition.
production of 4-hydroxypyrazole from pyrazole, the effect of various oxyradical scavengers was evaluated. Catalase produced a concentration-dependent inhibition of 4-hydroxypyrazole production (Table I). Boiled catalase was not inhibitory. Superoxide dismutase had no effect on the oxidation of pyrazole by the ascorbate system (Table I). These results indicate that HzOz, but not superoxide is required for the production of the oxidant responsible for the oxidation of pyrazole. However, H202 alone did not oxidize pyrazole to 4hydroxypyrazole. Ethanol, dimethyl sulfoxide, and mannitol, which are effective ‘OH-scavenging agents (23), all produced inhibition of the production of 4-hydroxypyrazole (Table I). By contrast, urea, which is a weak ‘OH scavenger, had no effect. Electrochemical
Effect of addition (%)
system was assayed as described
the metabolite produced from the ascorbate-dependent oxidation of pyrazole as detected by uv absorbance at 254 nm (A)
detection of Q-hydroxy-
pyraxole. To further validate that the metabolite produced from the interaction of pyrazole with ‘OH generated by the ascorbate system was 4-hydroxypyrazole, an electrochemical detection procedure was utilized. An advantage of this system is that one can vary the applied voltage and thus evaluate whether a metabolite and a standard display the same electrochemical response and characteristics (24). Figure 3 shows a comparison of HPLC profiles of
FIG. 3. HPLC profile for the determination of 4hydroxypyrazole produced during pyrazole oxidation by the ascorbate system and detected either by absorbance at 254 nm (A) or electrochemically with an applied voltage of 0.85 V (B). A lo-p1 aliquot of sample was injected and detection was monitored simultaneously with a uv detector (A) or an electrochemical detector (B).
OXIDATION
OF PYRAZOLE
221
BY ‘OH
from the interaction of pyrazole with ‘OH generated by the ascorbate system (closed circles) as a function of the applied electrode potential is shown in Fig. 4. Both the standard and the ‘OH-dependent metabolite have the same electrochemical characteristics, suggesting that they are identical. ELECTRODE
Xanthine oxidase-dependent oxidation of pyraxole. The coupled oxidation of hypo-
POTENTIAL(vI
FIG. 4. Comparison of the electrochemical characteristics of standard 4-hydroxypyrazole (0) and the metabolite (0) produced from the oxidation of pyrazole by the ascorbate system. A lo-p1 aliquot of a sample prepared as described in Fig. 1B (standard) or 1C (metabolite) was injected and the height of the peak with a retention time of 6.8 min was determined as a function of the applied voltage. The two curves are identical.
or electrochemically (B). Both methods of detection reveal a peak with a retention time of 6.8 min. Peak heights were linearly proportional to the amount of 4-hydroxypyrazole added and standard 4-hydroxypyrazole eluted at the same position as the metabolite without any peak splitting (not shown). A titration curve of the relative peak height (determined electrochemitally) for the standard 4-hydroxypyrazole (open circles) and the metabolite produced TABLE
xanthine by xanthine oxidase, in the presence of ferric-EDTA, was utilized as an enzyme-catalyzed model ‘OH-generating system (M-20). As shown in Fig. 2B, the production of 4-hydroxypyrazole by this reaction system was linear with time over a 60-min period. No 4-hydroxypyrazole was produced if the ferric-EDTA, hypoxanthine, xanthine oxidase, or pyrazole was omitted from the reaction system. The production of 4-hydroxypyrazole was sensitive to inhibition by either catalase or by superoxide dismutase, indicating that both HzOz and the superoxide anion radical are required for the production of ‘OH (Table II). Inhibition of pyrazole oxidation to 4hydroxypyrazole was also observed in the presence of competitive ‘OH scavengers such as ethanol, dimethyl sulfoxide, and mannitol, whereas urea was without any effect (Table II). II
EFFECTOF OXYGEN RADICAL SCAVENGERS• NTHEOXIDATIONOFPYRAZOLETO~-HYDROXYPYRAZOLE BYTHE XANTHINE OXIDASE SYSTEM
Addition
-
Catalase Superoxide Ethanol Dimethyl Mannitol Urea
Concentration
dismutase
sulfoxide
97 162 45 300 100
mU mU mU mU mM 30 mM 50 mM 50 mM
Rate of 4-hydroxypyrazole production (nmol/min/ml)
Effect of additon (S) -
0.40 0.067 0.033 0.142 0.067 0.058 0.06’7 0.067 0.40
Note. The oxidation of pyrazole to 4-hydroxypyrazole by the coupled oxidation oxidase was assayed as described under Materials and Methods.
-83 -92 -64 -83 -86 -83 -83 0 of hypoxanthine
by xanthine
222
PUNTARULO
AND
TME him)
FIG. 5. Time course for the oxidation of pyrazole to 4-hydroxypyrazole by rat liver microsomes in the absence (0) of ferric-EDTA and sodium azide, and in their presence (0). Reactions were carried out as described under Materials and Methods.
Microsmal
oxio?ation of ggraxole.
The
ability of isolated rat liver microsomes to generate ‘OH-like species was shown to require the presence of iron (25). FerricEDTA was a particularly effective stimulant, relative to other iron chelates, of ‘OH production by microsomes (26). Azide also stimulated microsomal ‘OH generation by inhibiting catalase activity found in isolated microsomes (27). Microsomes were incubated in absence and presence of the TABLE
CEDERBAUM
combination of ferric-EDTA plus azide and the production of 4-hydroxypyrazole was determined. In the absence of ferric-EDTA and azide, microsomes oxidized pyrazole to 4-hydroxypyrazole in a time-dependent manner (Fig. 5). The addition of ferricEDTA plus azide produced a fivefold increase in the production of 4-hydroxypyrazole (Fig. 5). There was no production of 4-hydroxypyrazole in the absence of either pyrazole, microsomes, or the NADPHgenerating system. In the absence of ferric-EDTA plus azide, DMSO and ethanol produced some inhibition of the microsomal oxidation of pyrazole (Table III). However, this inhibition was about the same as that found with urea, which is a relatively poor scavenger of ‘OH. Moreover, catalase had no effect on the production of 4-hydroxypyrazole (Table III). These results suggest the possibility that the oxidation of pyrazole is occurring via a ‘OH-independent mechanism. In the presence of ferric-EDTA, ethanol and dimethyl sulfoxide, but not urea, were considerably more effective in blocking microsomal oxidation of pyrazole III
EFFECTOF OXYGEN RADICAL SCAVENGERSONTHE OXIDATIONOF PYRAZOLETOI-HYDROXYPYRAZOLE BY RAT LIVER MICROSOMES Rate of 4-hydroxypyrazole production and effect of addition (nmol/min/mg microsomal protein) and (7%) -Fe EDTA Addition -
Dimethyl
sulfoxide
Concentration -
10 mM
30 mM Ethanol Urea Catalase Superoxide
dismutase
100 mM 50 mM 325 mU 300 mU
+Fe EDTA
A Fe EDTA
Rate
Effect
Rate
Effect
Rate
Effect
0.063 0.060 0.044 0.039 0.043 0.062 0.066
-5 -30 -38 -32 -2 +5
0.276 0.220 0.100 0.066 0.249 0.066 0.338
-20 -64 -76 -10 -76 +22
0.213 0.160 0.056 0.027 0.206 0.004 0.272
-25 -14 -88 -4 -99 +28
Note The oxidation of pyrazole to 4-hydroxypyrazole by microsomes was assayed as described under Materials and Methods in the presence of the indicated additions. Reactions were conducted either in the absence of iron EDTA plus azide or in the presence of 25 FM ferric-50 @i EDTA-1.0 mhl sodium azide. The net increase produced by iron-EDTA is shown in the A Fe EDTA column. For experiments involving catalase, azide was omitted from the reaction system.
OXIDATION
OF PYRAZOLE
BY ‘OH
223
be oxidized to 4-hydroxypyrazole was characterized in three different systems which are known to produce ‘OH, the ironcatalyzed oxidation of ascorbate, the coupled oxidation of hypoxanthine by xanthine oxidase, and NADPH-dependent microsomal electron transfer in the presence of ferric-EDTA. Inhibition by competitive ‘OH scavengers and catalase and the requirement for iron support a role for ‘OH in pyrazole oxidation by these reaction systems. On the basis of the effect of catalase in blocking pyrazole oxidation to 4hydroxypyrazole, it appears that Hz02 serves as the precursor of ‘OH by all three reaction systems. The requirement for iron suggests that a Fenton-type (30) of reaction between ferrous and HzOzis responsible for the generation of ‘OH. The reductant of the ferric iron is different for the three systems. In the xanthine oxidase reaction, reduction of ferric-EDTA is mediated by superoxide radical and ‘OH is generated by an iron-catalyzed Haber-Weiss type of reaction (19,20). This explains the sensitivity of pyrazole oxidation to inhibition by superoxide dismutase. The lack of inhibition by superoxide dismutase in the ascorbate or microsomal reaction system is probably due to reduction of ferric-EDTA by ascorbate (21, 22) and by NADPH-cytochrome P-450 reductase (26, 31). A HPLC procedure using paired ion chromatography was employed to detect 4hydroxypyrazole. The metabolite produced from the interaction of pyrazole with ‘OH exhibits the same retention time and coelutes with the standard 4-hydroxypyrazole under a variety of conditions. The metabolite also displays the same electrochemical properties as the standard 4-hydroxypyrazole and is thus likely to be identical to the standard. Under the conditions described under Materials and Methods, less than 0.02 nmol of 4-hydroxypyrazole can easily be detected. DISCUSSION The interaction of ‘OH with pyrazole may Pyrazole was previously found to be an proceed by hydrogen abstraction or addieffective ‘OH scavenging agent (14). The tion to the ring may occur. Addition of ‘OH present report shows that 4-hydroxypyrto the ring would produce the 4-hydroxyazole is a product of the interaction of pyr- pyrazole radical, which upon dismutation azole with ‘OH. The ability of pyrazole to would generate 4-hydroxypyrazole. Alter-
than in the absence of ferric-EDTA. Moreover, catalase now produced a striking inhibition of 4-hydroxypyrazole production (Table III). Subtraction of rates of production of 4-hydroxypyrazole in the absence of ferric-EDTA from the rates in the presence of ferric-EDTA indicated that the net increase produced by the addition of ferricEDTA was almost completely abolished by catalase and competitive ‘OH scavengers, whereas urea had no effect (Table III, AFe EDTA column). These results suggest that the increase in pyrazole oxidation to 4-hydroxypyrazole produced by the addition of ferric-EDTA to microsomes was due to the increased generation of ‘OH. Superoxide dismutase had no effect on the production of 4-hydroxypyrazole in the absence or presence of ferric-EDTA. In other experiments, the effects of inhibitors of cytochrome P-450-catalyzed reactions or of a competitive drug substrate on production of 4-hydroxypyrazole were determined. Metyrapone (1 mM), carbon monoxide (2.5:1 ratio, CO:02), and aminopyrine (5 mM) produced about 50% inhibition of microsomal pyrazole oxidation when ferric-EDTA and azide were omitted from the reaction system, thus implicating a role for cytochrome P-450 in the oxidation of pyrazole. However, these agents had no effect on the net increase in rates of pyrazole oxidation produced by the addition of ferric-EDTA plus azide (data not shown). This is in agreement with previous studies implicating a role for the NADPHcytochrome P-450 reductase and not cytochrome P-450 in interacting with iron to generate ‘OH (26, 28, 29). Thus, pyrazole can be oxidized by microsomes by a cytochrome P-450-dependent pathway independent of ‘OH, as well as by a ‘OH-dependent reaction which involves the reductase and catalytic amounts of iron chelates.
224
PUNTARULO
AND
natively, the 4-hydroxypyrazole radical can be oxidized, e.g., by ferric-EDTA to produce 4-hydroxypyrazole. Hydrogen abstraction would produce the pyrazole radical, which upon interaction with Ozwould form the peroxy radical. Dismutation of the latter or decomposition via a Russelltype mechanism (32) would yield 4-hydroxypyrazole. It should be emphasized that the HPLC system was maximized only for the detection of 4-hydroxypyrazole and the production of other products from the interaction of pyrazole with ‘OH was not evaluated in these systems. That other products are indeed formed can be seen by the presence of a peak with retention time of 7.4 min. At present, we suspect that the unknown peak may be 3-hydroxypyrazole and experiments to evaluate this possibility and synthesize this agent are underway. 4-Hydroxypyrazole was the product sought in these experiments since this compound was a major metabolite recovered in the urine of mice and rats which had been administered pyrazole in vivo (15, 16). The production of 4-hydroxypyrazole was lowered by the simultaneous in vivo administration of ethanol (15,16) resulting in an increase in the half-life of pyrazole in plasma (33). In this regard, it is of interest that ethanol inhibited the oxidation of pyrazole to 4-hydroxypyrazole by the various ‘OH-generating systems which were utilized. Results shown in Fig. 5 and Table III suggest that pyrazole can also be oxidized to 4-hydroxypyrazole by a microsomal pathway in which ‘OH does not appear to play a role. This microsomal pathway appears to involve cytochrome P-450 and can be induced by prior treatment with certain inducers such as pyrazole, methylpyrazole, or chronic ethanol treatment (17).4 It is likely that cytochrome P-450dependent oxidation of pyrazole to 4-hydroxypyrazole is primarily responsible for the in vivo metabolism of pyrazole although some oxidation of pyrazole by ‘OH may occur under conditions of increased oxidative stress or by certain cell populations, e.g., activated macrophages. In view of the sensitivity, specificity, and simplicity of the assay procedure, the production of
CEDERBAUM
4-hydroxypyrazole from the interaction of pyrazole with ‘OH may have some practical use for the detection of ‘OH in tissues with very low levels of cytochrome P-450 or after addition of agents which promote the production of oxygen radicals. REFERENCES 1. LI, T. K., AND THEORELL, H. (1969) Actu Chem. Stand 23,892-902. 2. THEORELL, H., AND YONETANI, Y. (1963) B&hem. 2. 338,537-553. 3. GOLDBERG, L., AND RYDBERG, U. (1969) B&hem. PharmucoL l&1749-1762. 4. LESTER, D., AND BENSON, G. D. (1970) Science 169, 282-283. 5. RUBIN, E., GANG, H., AND LIEBER, C. S. (1971) B&hem. Biophys. Res. Commun 42, l-8. 6. LIEBER, C. S., RUBIN, E., DECARLI, M., MISRA, P., AND GANG, H. (1970) Lab. Invest. 22,615-621. 7. KRIKIJN, G., AND CEDERBAUM, A. I. (1984) Biochim. Biophys. Acta 801, 131-137. 8. YANG, C. S., TV, Y. Y., KOOP, D. R., AND COON, M. J. (1985) Cancer Res. 45,1140-1145. 9. KOOP, D. R., CRUMP, B. L., NORDBLOM, G. D., AND COON, M. J. (1985) Proc. NatL Acad. Sci. USA 82,4066-4069. 10. KRIKUN, G., FEIERMAN, D. E., AND CEDERBAUM, A. I. (1986) J. PharmacoL Exp. Ther. 237,10121019. 11. POWIS, G., AND GRANT, L. (1976) B&hem. PharmacoL 25,2197-2201. 12. Tu, Y. Y., SONNENBERG,J., LEWIS, K. F., AND YANG, C. S. (1981) Biochem. Biophys. Res. Commun. 103,905-912. 13. EVARTS, R. P., HALIDAY, E., NEGESHI, M., AND HJELMELAND, L. M. (1982) Biochem PharwwxoL 31,1245-1249. 14. CEDERBAUM, A. I., AND BERL, L. M. (1982) Arch Biochem. Biophgs. 216,530~543. 15. CLAY, K. L., WATKINS, D. W., AND MURPHY, R. C. (197’7) Drug Metub. Disp. 5,149-156. 16. DEIS, F. H., LIN, G. W. J., AND LESTER, D. (1977) in Alcohol and Aldehyde Metabolizing Systems (Thurman, R. G., Williamson, J. R., Drott, H. R., and Chance, B., Eds.), Vol. III, pp. 399405, Academic Press, New York. 17. FEIERMAN, D. E., AND CEDERBAUM, A. I. (1986) Fed Proc. 45, p. 1663 (Abstract). 18. BEAUCHAMP, C., AND FRIDOVICH, I. (1970) J. Biol. Chem 245,464-4646. 19. MCCORD, J. M., AND DAY, E. D. (1978) FEBSLett. 86,139-142. 20. HALLIWELL, B. (1978) FEBS Z&t. 96,238-242.
OXIDATION
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