Activation of hydrazine derivatives to free radicals in the perfused rat liver: a spin-trapping study

Biochimica et Biophysica Acta 924 (1987) 261-269

261

Elsevier BBA 22726

Activation of hydrazine derivatives to free radicals in the perfused rat liver: a spin-trapping study Birandra K. Sinha Clinical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD (U.S.A.)

(Received10 December1986)

Key words: Hydrazinederivative; Free radical; Spin trapping; (Rat liver)

Spin-trapping techniques and electron spin resonance spectroscopy have been used to study bioactivations of hydrazine and its derivatives by isolated perfused rat livers. Using phenylbutylnitrone (PBN) as the stable spin trap, it was found that the liver perfusion of hydrazine, acetylhydrazine and isoniazid resulted in the formation of the same carbon-centered radical which was shown to be the acetyl radical. The identity of the acetyl radical was confirmed after organic extraction of the liver perfusates, by comparing its coupling constants with those of the in vitro metal ion- or horseradish peroxidase-catalyzed oxidation products of the hydrazines in the same solvents. The liver perfusion of iproniazid formed the isopropyl radical which was previously observed to result from peroxidase-catalyzed oxidation.

Introduction Hydrazine and its derivatives are carcinogenic and mutagenic [1,2]. However, hydrazine derivatives are extensively used in industry as rocket fuel and in medicine as chemotherapeutic agents for the treatment of cancer (procarbazine), to control hypertension (hydralazine), as antituberculosis agents (isoniazid) and as antidepressants (iproniazid). Hydralazine has been shown to cause base pair substitution [3] in DNA, to increase the incidence of lung tumors in mice [4], and has been associated with the induction of systemic lupus erythematosus [5]. Isoniazid and iproniazid cause

Abbreviations: PBN, phenyl-tert-butylnitrone; DMPO, 5,5-dimethyl-l-pyrroline N-oxide; ESR, electron spin resonance; DETAPAC, diethylenetriaminepentaaceticacid; FC-43, Fluosol. Correspondence: B.K. Sinha, Clinical Oncology Branch, National Cancer Institute, Building 10, Room 6N-119, National Institutes of Health, Bethesda, MD 20892, U.S.A.

irreversible liver damage which m a y result from their metabolism to reactive intermediates and subsequent binding of these intermediates to critical hepatic proteins [6,7]. Although the biochemical or molecular mechanism of hydrazine toxicities are not clearly defined, it has been suggested that the oxidative activation, catalyzed by cytochrome P-450, may be responsible for the cellular damage induced by hydrazines [6,7]. Recent studies have shown that hydrazines are also metabolized to free radical intermediates [8-14] and the subject matter has been recently reviewed [13,14]. Studies from our laboratory have shown that a number of hydrazine derivatives: e.g., hydralazine, phenylhydrazine, isoniazid, and iproniazid, form both nitrogen- and carbon-centered free radicals [15-18] as the result of in vitro activation (enzymatic or catalyzed by metal ions). It is believed that the free radicals may also be formed metabolically in vivo and that these radicals may be responsible for the toxicity of these compounds. Poyer et al. [19] have used spin-trapping techniques to detect formation of free radical inter-

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mediates during in vivo metabolism of CC14, and recently Connor et al. [20] have applied spin-trapping techniques to study the metabolism of CCI 4 in a rat liver perfusion system, indicating that the stable spin traps could be effectively used to study free radical metabolism of toxic xenobiotics in vivo. In this paper, we have examined the activation of hydrazine derivatives in a rat isolated liver perfusion system, as a model to study the in vivo metabolism of hydrazine drugs to reactive radicals. Using spin-trapping ESR techniques, we have found that free radicals are formed as a result of metabolic activation of hydrazines in vivo. Moreover, we have identified these radical intermediates by comparing their hyperfine splitting constants to those of the PBN adducts, prepared by in vitro enzymatic and metal-catalyzed activation of the hydrazine derivatives. Methods and Materials

Hydrazine monohydrate, isoniazid, iproniazid phosphate, acetylhydrazine, PBN and DMPO were obtained from Aldrich Chemical Co., Milwaukee, WI; horseradish peroxidase (RZ = 3.0) and DETAPAC were obtained from Sigma Chemical Co., St. Louis, MO. DMPO used in this study was purified by a vacuum distillation and passing through activated charcoal. Liver perfusion was carried out with male Sprague-Dawley rats (250 g). Rats were anesthetized by intraperitoneal injection of sodium pentobarbital and the livers were surgically isolated from circulation by the method of Hems et al. [21]. The carcass containing the cannulated liver was placed on a MX Ambec 210 perfuser (MX International, Aurora, CO), which was equipped to collect samples of the liver effluent and samples from the perfusion reservoir. For these perfusions studies, we chose to use an artificial oxygen carrier, Fluosol-43 (FC-43, manufactured by Green Cross, Japan and distributed by Gibco, New York, NY). FC-43 is a neutral oxygen carrier and consists of: perfluorotributylamine, 0.86 M; and pluronic F-68 (polyoxyproplene-polyoxyethylene copolymer), 26 m g / ml; NaCI, 100 mM; KC1, 4.6 mM; CaC12, 2.5 mM; MgC12, 2.1 mM; N a H C O > 25 mM; glucose,

1 mM and hydroxyethyl starch, 30 m g / m l . Previous studies by Monk and Cyssyk [22] have shown that FC-43 did not interfere with normal liver function for up to 2 h and that the metabolic activity of the liver remained constant. Furthermore, it was advantageous for our studies to use this neutral perfusion media because other perfusion media such as those containing aged erythrocytes could contain broken cells and, thus, contain metal ion contaminants which may artifactually activate hydrazines. The liver perfusion was carried out in the presence of 50-100 mM PBN and the hydrazine derivatives (5-10 mM) were added to the media after 15 min equilibration. The samples were collected at 15 min (passage 1) and 30 min (passage 2) and analyzed for PBN adducts by ESR. Although the traces of hydrazine-derived PBN adducts could be detected at as early as 5 rain of perfusion, the formation of the adducts was time dependent and maximum free radical adducts were formed within 15-30 min. The ESR spectra were recorded either on a Varian E-104 or an IBMBrucker spectrometer (ER 200 D SRC) equipped with a N M R gaussmeter (ER 035) and a TM cavity. Unless indicated otherwise, all ESR spectra presented in this paper with liver perfusion system were recorded on 30 min samples. The g values were measured with diphenylpicylhydrazyl (g = 2.0036) as the standard. In vitro activations of hydrazine derivatives were carried out with horseradish peroxidase (0.25 m g / m l ) , hydrazines (1-2 raM) and spin-trap (PBN or DMPO, 100 mM) and the reactions were initiated by adding H202 (400 /~M) in phosphatebuffered saline (pH 7.4) containing 100 /~M DETAPAC or in FC-43. For the metal-catalyzed reactions of hydrazines, 50 t~M Cu 2÷ was used. Results

A cetylhydrazine The perfusion of the isolated liver with acetylhydrazine in the presence of PNB resulted in the formation of a PBN adduct consisting of a triplet of a doublet with the following splitting constants: a N = 15.9 G; a n = 3.25 G with a g value of 2.0059 (Fig. 1A). These splitting constants are different from those reported by Augusto and

263

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_

A

't

"''"

!/I

Ii

.Yv'

!/ v

Fig. 1. ESR spectra obtained from acetylhydrazine (10 mM) in the presence of PBN (100 mM) in FC-43 during (A) liver perfusion with a r~ =15.9 G; a n = 3.25 G, (B) oxidation of acetyihydrazine (1 mM) with Cu 2+ (50 /tM) and (C) liver perfusion with PBN alone with a ~ =15.5 G; a H = 3.0 G. The ESR settings were: fidd, 3390; field scan, 100 G; modulation amplitude, 1-2 G; modulation frequency, 100 kHz; nominal microwave power, 20 mW; and the receiver gain was 1.25.105 for A; 5.104 for B and 6.3.104 for C.

Montellano [9] for a PBN adduct formed during the metal ion-catalyzed oxidation of acetylhydrazine in aqueous buffer. This suggested that the

radical intermediate formed and trapped by PBN may not have been the acetyl radical ( C H 3 C O ) . Alternatively, the perfusion medium may have altered the splitting constants of the PBN adduct. In order to investigate these possibilities and to confirm the identity of the radical adduct derived from acetylhydrazine metabolism in the isolated liver, the acetyl radical adduct of PBN was prepared from acetylhydrazine with Cu 2÷ or horseradish peroxidase activation (in FC-43). The resulting spectrum is shown in Fig. 1B, and the coupling constants of a r~ = 16.0 G; a H = 3.25 G are identical to those of the adduct formed during liver perfusion of acetylhydrazine. This would suggest that acetyl radical was formed and subsequently trapped by PBN during the liver perfusion of acetylhydrazine. Furthermore, the PBN adduct from acetylhydrazine formed either chemically or metabolically during liver perfusion and extracted in benzene had the same splitting constants (a N = 14.2 G; a H = 3.2 G), which are identical to those previously reported [21] in benzene. Thus, these observations confirm then that acetyl radicals are formed in vivo in liver from acetylhydrazine. In the absence of acetylhydrazine, perfusing the liver with PBN alone resulted in a PBN adduct which had the following splitting constants: a ~ = 15.2 G; a H = 2.9 G with a g value of 2.0059 (Fig. 1C). The identity of this adduct is not known, however, it may have been formed from the trapping of a lipid or lipid alkoxy radical, since the splitting constants are similar to those reported for a PBN adduct formed during the peroxidation of microsomal lipids in the presence of CC14 [24]. Moreover, the doublet at high field is unsymmetrical and broad as previously noted for a lipid-PBN adduct. lsoniazid Liver perfusion of isoniazid resulted in the formation of a PBN adduct whose ESR spectrum was characterized by the following splitting constants: a N = 15.9 G; a H = 3.2 G with a g value of 2.00595 (Fig. 2A), which are similar to those of acetyl-PBN, obtained previously from acetylhydrazine. This would suggest that acetyl radical was also formed from isoniazid in vivo. In a previous study [16] I showed that isoniazid formed an acyl-type radical (PyCO') during the horsera-

264

a H = 3.2 G, liver perfusion) when extracted in benzene, indicating that the two adducts resulted from the trapping of two different radical species. These observations clearly indicate that acetyl radical was formed from isoniazid and trapped by PBN during the in vivo metabolsim of isoniazid.

/J

I

lproniazid

OQ v

Iproniazid in the presence of PBN during liver perfusion also formed a PBN adduct consisting of a triplicate of a doublet (Fig. 3A) with the follow-

'I j~/V

Fig. 2. ESR spectra obtained from isoniazide (10 mM) in the presence of PBN (100 mM) during (A) liver perfusion with a N =15.9 G; a H = 3.25 G and (B) oxidation of isoniazid (2 mM) with horseradish peroxidase (0.25 mg/ml) and H202 (400 ~M) in FC-43. The ESR settings were similar to those in Fig. 1 except that the receiver gain for A was 1.25.105 with modulation amplitude of 2.0 G and the receiver gain for B was 2.104 with modulation amplitude of 1.0 G.

dish peroxidase-catalyzed reaction. Therefore, we compared the two pathways to identify the free radical formed from isoniazid during the liver perfusion. The PBN adduct formed during the horseradish peroxidase-catalyzed activation of isoniazid had the following splitting constants in FC-43: a N = 15.75 G, a H = 3.4 G (Fig. 2B), which are different from those obtained during liver perfusion. Furthermore, the two PBN-adducts formed from isoniazid by these two pathways, i.e., horseradish peroxidase or the liver perfusion, had different splitting constants (a N = 14.2 G; a H = 2.0 G, horseradish peroxidase and a N = 14.4 G;

'/

j

i /iJ ': l/log I

,

{ i

I

Y

Fig. 3. ESR spectra obtained from iproniazid (10 mM) in the presence of PBN (100 mM) during (A) liver perfusion and with a N =15.8 G; a H = 3.1 G and (B) oxidation with horseradish peroxidase (0.25 m g / m l ) and H202 (400 ~tM) in FC-43 with aN=15.7 G; a l l = 3 . 1 G and aN=15.0 G and a l l = 2 . 8 G. The ESR settings were similar to those in Fig. 1 except the receiver gain for A was 1.105 and 1.25-104 for B with modulation amplitude of 1.0 G for both.

265

reaction of oxygen with the isopropyl radical during the peroxidase reaction of iproniazid.

ing splitting constants: a TM = 15.8 G; a H = 3.1 G with a g value of 2.0095. These splittings are different from those observed for the acetyl-PBN adduct obtained from acetylhydrazine. Since iproniazid has been shown to form isopropyl radical during horseradish peroxidase activation [16], it is possible that this PBN adduct resulted from the trapping of the iproniazid-derived isopropyl radical formed during liver perfusion. The horseradish peroxidase-catalyzed oxidation of iproniazid in FC-43 resulted in the formation of two radical adducts with the following splitting constants: a N = 15.7 G; a H = 3.1 G (adduct 1) a n d a TM = 15.0 G; a n = 2.8 G (adduct 2; Fig. 3B). Furthermore, with time, more of the adduct 1 was formed. When the reaction was carried out under anaerobic conditions, adduct 1 was the major radical adduct and only traces of adduct 2 were detected. Since the splitting constants the PBN adduct obtained during liver perfusion are identical to those of isopropyl-PBN (adduct 1), the liver perfusion of iproniazid formed the isopropyl radical. The radical adduct 2 must be formed from the

Hydrazine Recently Noda et al. [25] have reported that hydrazine radical ( N H 2 N H ) was formed during liver microsomal or Cu2+-catalyzed activation of hydrazine. Liver perfusion was therefore carried out with hydrazine in the presence of PBN, and a radical adduct of PBN was detected which had the following splitting constants: a TM = 16.0 G; a H = 3.25 G (g = 2.0059; Fig. 4A), which are identical to those of the PBN-acetyl adduct formed from acetylhydrazine or isoniazid in the liver perfusion. This suggests that hydrazine is rapidly acetylated in vivo and that acetylhydrazine is subsequently metabolized to the acetyl radical. In order to confirm the possibility that acetyl radical was formed in vivo, we carried out the Cu2+-catalyzed oxidation of hydrazine, as reported by Noda et al. [25]. The radical intermediate trapped by PBN in FC-43 had the following splitting constants: a N = 15.9 G; a H = 3.5 G (Fig. 4B) which are different

TABLE I S U M M A R Y O F SPIN T R A P P I N G OF H Y D R A Z I N E S IN VITRO A N D IN VIVO Com pou nd

Activating system

Splitting constants (G) aN aH Buffer

Radical trapped

FC-43

Benzene

Acetylhydrazine

Ca 2+ HRP liver perfusion

15.5 15.5 -

3.6 3.6

15.9 15.9 15.9

3.3 3.3 3.25

14.2 14.2 14.3

3.2 3.2 3.2

CH3CO CH3CO CH3CO

Isoniazid

HRP liver perfusion

15.8 -

3.6

15.75 15.9

3.4 3.25

14.3 14.2

2.0 3.2

CH3CO"

HRP

16.2

2.5

15.7 15.0 15.8

3.1 2.8 3.1

14.6 14.0 14.6

2.7 1.9 2.7

"C H (C H 3) 2 "O O C H ( C H 3 ) 2 "CH(CH3)2

15.0 15.0

2.8 7.5

14.2

3.2

• OH "H "OH CH3CO" "OH OH

14.6

2.3

OH

14.3

2.7

L

Iproniazid

liver perfusion Hydrazine

-

Cu 2+

16.2

3.2

15.9

3.5

HRP liver perfusion Cu2+/DMPO HRP/DMPO

15.7 14.9 14.9

4.4

15.9 15.9

3.5 3.25

Fe2+//H 202

PBN

16.0

PBN

liver perfusion

HRP, horseradish peroxidase.

-

14.9 14.9 3.12 15.5

2.9

PyCO

266

#

/ f

than those o b s e r v e d for the P B N a d d u c t o b t a i n e d d u r i n g liver perfusion. This P B N a d d u c t was also f o r m e d f r o m the h o r ser ad i sh p e r o x i d a s e a c t i v a t i o n o f h y d r a z i n e ( a N = 15.9 G ; a H = 3.5 G). Extraction of the Cu2+-catalyzed radical a d d u c t ( a N = 16.2 G; a H = 3.2 G ) in b e n z e n e showed a very c o m p l e x E S R s p e c t r u m (Fig. 4C) which m a y be i n t e r p r e t e d as resulting f r o m the t r a p p i n g of two radical species: P B N - O H ( a N = 15.0 G ; a n = 2.8 G) an d P B N - H ( a N = 15.0 G; a 2H = 7.5 G). T h e Cu2+-catalyzed o x i d a t i o n of h y d r a z i n e in b u f f e r did n o t show this c o m p l e x E S R s p e c t r u m suggesting that Cu 2 + m a y interact with the n i t r o x ide radical. In o r d e r to c o n f i r m this, we reextracted the P B N a d d u c t s f r o m b e n z e n e into w at er an d the s p e c t r u m sh o w n in Fig. 4 D is a c o m p o s i t e of P B N - O H ( a N = 16.0 G; a H = 3.1 G) an d P B N H ( a N = 16.5 G ; a 2H = 10.5 G). This s p e c t r u m is similar to that o b s e r v e d in b e n z e n e an d suggests that the m e t a l ions i n t e r a c t e d with the P B N add u c t in buffer such that only P B N - O H was detected. This was further c o n f i r m e d by p r e p a r i n g the P B N - H by N a B H 4 r e d u c t i o n of P B N in w at er an d a d d i n g Cu 2+ which i m m e d i a t e l y b r o a d e n e d the P B N - H s p e c t r u m so that it could be d e t e c t e d (Fig. 4E and F). Th ese o b s e r v a t i o n s clearly show that the nitrog e n - c e n t e r e d h y d r a z i n e radical was n o t f o r m e d f r o m h y d r a z i n e d u r i n g Cu Z+ - m ed i at ed o x i d a t i o n as r e p o r t e d previously. H o w e v e r , o x y g e n - c e n t e r e d radicals were f o r m e d f r o m h y d r a z i n e by either Cu 2+ or horseradish p e r o x i d a s e activation. This

Fig. 4. ESR spectra obtained from hydrazine (5 mM) in the presence of PBN (100 mM) during (A) liver perfusion with aN=15.9 G; a H= 3.25 G. (B) Cu2+-catalyzed oxidation of hydrazine with aN=15.9 G; a H= 3.5 G. (C) ESR spectrum obtained when the PBN adduct obtained in aqueous buffer (a N =16.2 G; all= 3.2 G) from hydrazine and Cu2+-catalyzed reaction in B was extracted into benzene with a N= 15.0 G; all=2.8 G for PBN-OH and aN=15.0 G; aZH=7.5 G for PBN-H. The spectrum (D) was obtained when the adduct from spectrum C was re-extracted in water with a N= 16.0 G; a H 3.1 G for PBN-OH and arq=16.5 G; a TM =10.5 G for PBN-H. PBN-H spectrum (E) was obtained when PBN was reduced with NaBH 4 in aqueous buffer and F was obtained when Cu 2+ was added to the PBN-H adduct. The ESR settings were identical to those in Fig. 1 except the receiver gain was 1.105 for A, 6.3.104 for B and 2.104 for C-F. The modulation amplitude was 10 G for A, B, D, E and F and 0.1 G forC.

267 was confirmed by using DMPO as a spin-trap since the DMPO-OH/OOH adducts are more characteristic than the PBN adducts. In vitro activation of hydrazine in the presence of DMPO resulted in the characteristic 1,2,2,1 DMPO-OH adduct spectrum with a TM = a H = 14.9 G (data not shown). Some DMPO-H adducts were also present. The formation of the DMPO-OH adducts was significantly decreased under anaerobic conditions, suggesting that oxygen was required for the formation/propagation of the O H from hydrazine. When the reaction was carried out in 2H20, very little isotopic effect was observed. This is unexpected because the hydrazine radical adduct of DMPO should show considerable splitting from the nitrogens. Discussion

The formation of free radical intermediates of hydrazine derivatives has been postulated and confirmed in vitro during the metabolism of hydrazines [8-14]. The formation of acetyl and isopropyl radicals from isoniazid and iproniazid, respectively, have been implicated in the induction of liver damage. However, the formation of radical intermediates of these drugs has never been demonstrated in vivo. In the present study, ESR spin-trapping evidence that reactive free radical intermediates are produced during the metabolism of hydrazines in vivo is presented. Using spintrapping techniques with PBN, we have also identified these radicals. The formation of acetyl radical was confirmed in vivo by its hyperfine splitting constants. It is interesting to note that in a previous study [9] the acetyl radical could not be trapped by PBN during the Cu2+-catalyzed reaction of acetylhydrazine. However, in our study acetyl radical was readily formed and trapped during Cu 2÷ or horseradish peroxidase-catalyzed oxidation of acetylhydrazine. The splitting constants were found to be different from those reported by previous workers [9] but are identical to those reported by Jenzen et al. [23] for acetyl-PBN adduct. Isoniazid also formed a free radical intermediate in vivo. The splitting constants for the trapped radical formed in vivo by PBN were identical to those of the acetyl adduct of PBN formed

from acetylhydrazine in vitro or in vivo. The formation of acetyl radical has been suggested during in vivo metabolism of isoniazid as a reslt of the hydrolysis and subsequent metabolism of acetylhydrazine. Our results confirm this observation. The metabolic activation of hydrazine catalyzed either by Cu 2÷ or horseradish peroxidase is complicated and, in our study, no hydrazine radical could be detected. In both activating systems, the O H and the "H formed from the reaction of hydrazine radical with oxygen and the reduction of PBN by hydrazine, respectively, were detected. We have confirmed these observations by using a more characteristic spin-trap (DMPO) for 'OH and by comparing the ESR parameters by trapping "OH radical with PBN formed from a hydroxyl radical-generating system (Fenton reaction). The liver perfusion of hydrazine in the presence of PBN, however, gave an adduct whose splitting constants were identical to the previously trapped radical adduct (acetyl-PBN) formed in vivo from either acetylhydrazine or isoniazid. This indicates that acetyl radical was also formed from hydrazine, and thus must have resulted from the acetylation of hydrazine in vivo followed by its metabolism to the acetyl radical. Iproniazid was also metabolized in vivo to the free radical intermediate, isopropyl radical, as the splitting constants were identical to those obtained from the horseradish peroxidase-catalyzed oxidation of iproniazid. In a previous study I showed that iproniazid formed isopropyl radical during horseradish peroxidase-catalyzed oxidation. However, no peroxy radical, formed from the reaction of the isopropyl radical with oxygen was detected during the liver perfusion. Thus, it would appear that in vivo the metabolism of iproniazid is similar to that of peroxidase-catalyzed one-electron oxidation. In conclusion, it is clear that hydrazine derivatives (acetylhydrazine, isoniazid, iproniazid and hydrazine) form carbon-centered radicals (a summary of spin-trapping study is presented in Table I) during rat liver perfusion. The formation of acetyl radical from hydrazine and isoniazid indicates that these compounds are acetylated to acetylhydrazine and acetylisoniazide, respectively. Nelson et al. [7] have proposed that acetylisonia-

268

zid is then hydrolyzed by acylamidase to acetylhydrazine which then is metabolized by cytochrome P-450 to the acetyl radical. Iproniazid has been reported to form isopropyl radical during in vivo metabolism [7] (identified as the alkylating species derived from iproniazid) and during peroxidase oxidation [16]. Recently, Moloney et al. [26] have shown that microsomal oxidation of iproniazid results in the formation of propane and propylene, presumably via an isopropyl radical which was formed and detected during liver perfusion in this study. The primary drug radicals or reactive secondary radicals such as O H formed during the reaction of the drug radicals with oxygen may induce cellular damage by a lipid peroxidation or DNA damage mechanism. Recently, Kubow et al. [27] reported that 3-methyhndole-derived free radical was formed in vitro and in vivo in lung, which initiated lipid peroxidation. It is also likely, that hydrazine-derived free radicals formed in vivo may bind to critical macromolecules. Covalent binding of hydrazine-derived alkylating species to proteins and its relationship to liver necrosis is well studied [6,7]. Procarbazine and hydralazine have been shown to bind to microsomal proteins, and free radicals are implicated in this bioalkylation [28,29]. Recently, carboncentered free radicals derived from the metabolic activation of phenelzine have been shown to alkylate the prosthetic heine of cytochrome P-450 which leads to its inactivation [11]. Hydrazine-derived free radicals may also bind to DNA which could result in irreversible damage to DNA synthetic and replicative machinery. The phenylethyl radical, derived from phenylethyl hydrazine, has been shown to cause DNA nicking which was inhibited by spin traps [30]. Thus, covalent binding of hydrazine-derivied free radicals, formed as a result of metabolic activation in vivo, to critical cellular macromolecules may ultimately cause cell death. The relationship between metabolic activation of hydrazines to free radicals and their therapeutic and toxic effects are currently under further investigation.

Acknowledgement The author is grateful to Dr. B. Kalyanaraman of National Biomedical ESR Center, Milwaukee,

for his helpful criticism of the study and valuable suggestions on spin-trapping studies.

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