Free radical-mediated activation of hydrazine derivatives.

Hydrazines are known to undergo oxidative activation in several enzymatic systems in vitro. Free radicals or carbonium ions have been proposed as active intermediates during such activation. The toxic effects elicited by hydrazines have also been linked to free radical-mediated activation. In this report, we have reviewed the identification of organic free radicals from hydrazines by direct ESR and ESR-spin trapping.


Introduction
Hydrazines are found naturally in plants (1). In the environment their occurrence is widespread, as they are used as herbicides, rocket fuels, and chemical intermediates (1). They find use in medicine as antitumor drugs, antidepressants, and as antihypertensive agents (l). However, the therapeutic effects are often complicated by adverse side effects such as hepatic necrosis and rheumatoid arthritis (2,3). Some hydrazines have been shown to be tumorigenic and mutagenic (3). Most metabolism studies link these effects to the production of reactive intermediates (i.e., free radicals and carbonium ions) (4)(5)(6). Hydrazines also exhibit organospecificity suggesting the involvement of several activating enzymes (1). A complete review of the biochemical toxicology of hydrazines has been given by Moloney and Prough (3). The present review deals specifically with the production and identification of organic free radicals in biochemical systems and their implications in the biochemical toxicology of hydrazines.

Radical Generation and Identification
Free radicals from hydrazines can be generated in vitro by both one-and two-electron oxidations (7). The stability of the radical derived depends on the substituents: whereas the primary radical cation of tetrasubstituted hydrazines can be observed by direct ESR (electron spin resonance) under static conditions, the radical cation from the trisubstituted hydrazine undergoes dehydrogenation, forming the less stable hydrazyl  (Fig. 1). Similarly, with the mono-and disubstituted hydrazines, the cation radicals are too unstable to be observed under static conditions, although they have been detected under flow conditions in chemical systems (8). However, one can detect the secondary radicals formed in these systems under static conditions. The secondary radicals can be formed from decomposition of primary radical cation or directly by twoelectron oxidation of the parent hydrazine (3,7). Direct ESR is the method of choice for detecting the stable radical cations (7) and the less stable hydrazyl and (secondary) carbon-centered radicals are detected by ESRspin trapping (9,10).
Free radicals from hydrazines can occur in a variety of oxidizing systems containing metal ions, oxyhemoglobins, horseradish peroxidase/hydrogen peroxide, prostaglandin synthase/arachidonic acid, cytochrome P-450/NADPH, and neutrophil-derived oxidants.

Metal-Catalyzed Autoxidation
It was shown in the past that hydrazine enhanced the rate of decomposition of cumene hydroperoxide while also quantitatively converting it to acetophenone in the presence of Fe(III)EDTA complex (10). Several reports on metal-catalyzed one-electron oxidation of hydrazines have now appeared (11)(12)(13)(14)(15)(16). Free radicals (i.e., the hydrazyl radicals) formed during such reactions have now been spin trapped (

Oxidation of Hydrazines by Hemoglobins in Erythrocytes
Phenylhydrazine is the prototype of drugs known to induce hemolysis in red blood cells. The phenyl radical formed during oxidation of phenylhydrazine by hemoglobin was detected by spin trapping (26) and the structure of the spin adduct verified by mass spectrometry (Table 1) (26). Of interest also is the trapping of a lipid radical formed as a result of hydrogen abstraction by phenyl radicals (27). Phenyl radicals were also shown to covalently bind to the heme (23). A complete summary of research in this area has been given by Mason (10. Horseradish Peroxidase/Hydrogen Peroxide It has been reported that iproniazid and other hydrazines react covalently and stochiometrically with the non-heme portion of HRP (horseradish peroxidase) causing time-dependent inactivation of the enzyme (29,30). Nevertheless, this system has been shown to oxidize a number of hydrazines via radical intermediates (17,20,21). Tetramethylhydrazine (TMH) was oxidized via the radical cation intermediate to formaldehyde (17). An interesting aspect of this peroxidatic oxidation is the fact that TMH underwent sequential one-electron oxidation catalyzed by HRP/H202. Perhaps the most interesting aspect of HRP/H202-induced activation of hydrazines is oxygen activation caused by secondary carbon-centered radicals (20). In the presence of oxygen, alkyl peroxyl radicals have been spin-trapped (20); purging the system with N2 caused the disappearance of the ESR spectrum of peroxyl adduct which was replaced by that of DMPO-alkyl adduct (Fig. 2).
Prostaglandin Synthase/Arachidonic Acid The PG-hydroperoxidase component present in PGsynthase has been shown to catalyze the reduction of PGG2 to PGH2 while also oxidizing a number of xenobiotics (31). This activity also was responsible for free radical-mediated metabolism of tetramethylhydrazine (18,19). The production of radical cation (Fig. 3)  responded closely to that of formaldehyde. ESR-spin trapping has allowed the detection of the hydralazyl radical derived from activation of hydralazine by PGsynthase/AA (20) (Fig. 4). The significance of this metabolizing system can be linked to the fact that the target organs (e.g., blood vessels, kidney, and lung) of hydrazine-induced tumorigenicity are all rich in PG-synthase activity (18).
Cytochrome P-450/NADPH Free radicals have been detected (by spin trapping) during microsomal and subcellular metabolism of several monosubstituted hydrazines (20)(21)(22)24). The spin adducts were characterized either by mass spectrometry (24) or by comparison with those spin adducts formed in a purely chemical system (14). Sensitivity to carbon monoxide indicated the involvement of cytochrome P-450 (22). It is not clear at present whether these radicals are produced by N-hydroxylation followed by dehydration or by an hydroxyl radical-dependent oxidation (32). In either case, the corresponding diazene intermediate is most likely to be formed. In most cases, the activity of cytochrome P-450 decreased during the course of hydrazine metabolism (33).

Neutrophil-Derived Oxidants
Stimulated neutrophils generate HOCI/-OCI as an integral part of their microbial metabolism (34). HOCl is converted into a stable taurine chloramine in the presence of taurine (neutrophils contain relatively large amounts of taurine). We found that the taurine chloramine oxidizes phenylhydrazine to phenyl radicals which were spin-trapped (35). Azide (a myeloperoxidase inhibitor) inhibited formation of the DMPO-phenyl spin adduct while superoxide dismutase did not have any effect. Oxidation of phenylhydrazine by stimulated neutrophils may thus represent a new pathway for metabolism of hydrazines.
The hepatic necrosis elicited during therapeutic administration of isoniazid and iproniazid has been attributed to covalent binding of acetyl and isopropyl radicals (to proteins) formed from hydrazines that were liberated metabolically (3)(4)(5). Procarbazine and hydralazine, when fortified with microsomes and NADPH were found to covalently bind to proteins, quite possibly via free radical intermediates (36,37).
Hydrocarbon evolution was detected during metab- olism of several hydrazines: propane from iproniazid (4,5), ethyl benzene from phenelzine (24), benzene from phenylhydrazine, and methane from methylhydrazine and procarbazine (37). The precursor alkyl radicals could conceivably abstract an hydrogen atom, forming the various hydrocarbons (Table 1) (3). With some hydrazines, incubation with thiols increased the production of hydrocarbons (by hydrogen abstraction), while reducing the covalent binding.
Peroxidation of red cell membrane lipids has been induced in different amounts in vivo during the administration of the following hydrazines: phenylhydrazine > acetylphenylhydrazine > hydrazine (38). The organic free radicals probably contribute to the lipid peroxidation to a certain extent. The lipid radical produced during the metabolism of phenylhydrazine by erythrocytes has been detected; spin traps also inhibit lipid peroxidation presumably via trapping of the lipid or the phenyl radical (27).
Reactive oxygen species have been implicated in hydrazine-induced inactivation of transforming DNA and also in the unscheduled DNA synthesis found in repairdeficient xeroderma pigmentosum cells following treatment with isoniazid and metals (40). However, the possible involvement of organic free radicals was not discussed. Recently, it was shown that the carbon- centered free radical (e.g., 2-phenylethyl radical) specifically interacted with the DNA apparatus, causing single-strand breaks (nicks) in DNA, and that this action was inhibited by the spin trap (25). During the microsomal metabolism of hydrazines inactivation of cytochrome P-450 occurs; carbon-centered IOG I B FIGURE 4. ESR spectrum obtained from 2 mM hydralazine with (A) incubation was with 1 mg/mL RSV microsomes, 400 ,uM AA, 100 mM DMPO, in the presence of 1.0 mM diethylenetriaminepentaacetic acid (DETAPAC), at pH 7.6; (B) identical with (A) except that the RSV microsomes were preincubated with 400 ,uM indomethacin.
radical derived from the hydrazines presumably add to the heme frame-work resulting in the formation of Nalkylheme adducts. Isolation and identification of N(2phenylethyl) protoporphyrin IX and N-phenylprotoporphyrin IX isolated during metabolism of phelezine and phenylhydrazine support the interraction of 2-phenylethyl and phenyl radicals, respectively, with the heme.
This work was supported by National Institutes of Health Grant RR-01008.