Free radical activation of monomethyl and dimethyl hydrazines in isolated hepatocytes and liver microsomes

Free Radical Biology & Medicine, Vol. 6, pp. 3-8, 1989 Printed in the USA. All rights reserved.

0891-5849/89 $3.00+ .00 © 1989 PergamonPress plc

Original Contribution FREE RADICAL ACTIVATION OF MONOMETHYL AND DIMETHYL HYDRAZINES IN ISOLATED HEPATOCYTES AND LIVER MICROSOMES

EMANUELE ALBANO, ALDO TOMASI*, LUCIA GORIA-GATTI, and ANNA IANNONE* Dipartimento di Medicina ed Oncologia Sperimentale, Universit~ di Torino, Corso Raffaello 30, 10125 Torino, Italy; *Istituto di Patologia Generale Universit~ di Modena, Via G. Campi 287, 41100 Modena, Italy (Received 7 July 1987; Revised 27 October 1987; Accepted 12 November 1987)

Abstract--Isolated hepatocytes and liver microsomes incubated with monomethyl-1,1 dimethyl- and 1,2 dimethylhydrazines produced free radical intermediates which were detected by ESR spectroscopy by using 4-pyridyl-1oxide-t-butyl nitrone (4-POBN) as spin trapping agent. The spectral features of the spin adducts derived from all three hydrazine derivatives corresponded to the values reported for the methyl free radical adduct of 4-POBN. In the microsomal preparations inhibitors of the mixed function oxidase system and the destruction of cytochrome P450 by pretreating the rats with COC12 all decreased the free radical formation. Methimazole, an inhibitor of FAD-containing monoxygenase system, similarly decreased the activation of 1,1 dimethyl-hydrazine, but not that of monomethyl- and 1,2 dimethyl-hydrazines. The addition to liver microsomes of physiological concentrations of glutathione (GSH) lowered by approx. 80% the intensities of the ESR signals. Consistently, incubation of isolated hepatocytes with methyl-hydrazines decreased the intracellular GSH content, suggesting that GSH can effectively scavenge the methyl free radicals. The results obtained suggest that methyl free radicals could be the alkylating species responsible for the toxic and/or carcinogenic effect of methyl-hydrazines. Keywards--Spin trapping, Free radicals, ESR spectroscopy, Methyl-hydrazines, Isolated hepatocytes, Liver microsomes

INTRODUCTION

Monomethyl-hydrazine and 1,1 dimethyl-hydrazine are extensively used in aerospace industry as high energy fuels for rockets,~ but can be also found as natural constituents in tobacco and in some edible mushrooms of the genus Agaricus and Geromitra. 2 The exposure to methyl-substituted hydrazines affects kidney functions, causes methaemoglobinemia and disturbances in the central nervous system leading to convulsions and neuritis. L3 Moreover, some evidences indicated that monomethyl and 1,1 dimethyl-hydrazine can have hepatotoxic effects. 4 1,2 Dimethyl-hydrazine is a well known carcinogen and induces a high incidence of tumors in the colon and rectum of rodents, as well as histiocitomas in many organs. 2,4-6 Recently, it has also been shown that 1,2 dimethyl-hydrazine can initiate liver carcinogenesis in rats. 7 Monomethyl and 1,1 dimethyl-hydrazines are Correspondence should be addressed to: Dr. Emanuele Albano.

similarly carcinogenic in the colon and rectum and also induce lung tumors in mice. 2'4-6 Early investigations have evidenced that both monoalkyl- and dialkyl-substituted hydrazines can be metabolicaly activated, 3,8 but only in recent years the formation of free radical intermediates has been demonstrated. 9 In particular, spin trapping experiments have shown the formation of ethyl and isopropyl free radicals by rat liver microsomes or isolated hepatocytes incubated with, respectively, ethyl and isopropyl-hydrazine, L°,H while methyl free radical has been indentified as the product of horse-radish peroxidasecatalyzed oxidation of 1,2 dimethyl-hydrazine and procarbazine (N-isopropyl- 1-(2-methyl-hydrazino)-ptolbutamide). 12,13 In the present work we have investigated whether free radical species are produced from methyl-substituted hydrazines in a whole cell system using isolated hepatocytes. We also attempted to characterize the metabolic pathway responsible for the free radical activation of these compounds.

4

E. ALBANO, A. TOMASI, L. GORIA-GATTI, and A. IANNONE M A T E R I A L S AND M E T H O D S

Male Wistar rats (250-300 g body wt) were supplied by Nossan (Corezzana, Italy) and fed ad libitum with a standard laboratoy diet, devoid of antioxidants, prepared by Piccioni (Brescia, Italy). Phenobarbital, 0.1% solution (w/v), was included in the drinking water for at least one week before the sacrifice. Cobalt chloride (60 mg/kg body wt.) was injected subcutaneously as a solution containing 6 mg/ml in 0.85% saline 72 and 48 h before microsome preparation. Monomethyl, 1,1 dimethyl and 1,2 dimethyl-hydrazines, 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN), 1-nitroisothiocyanate (NITC) were purchased from Aldrich-Europe (Bersee, Belgium). Collagenase Type I, 2-methyl- 1,2-di-3-pyridyl- 1-propane (Metyrapone), p-chloromercuri benzoate (pCMB) and methimazole were supplied by Sigma Chemical Co. (St Louis, MI, U.S.A.). Diethylaminoethyl-diphenylaminoacetate (SKF 525A) was kindly provided by Smith, Kline, & French Ltd (Welwyn Garden City, Hefts, U.K.). Isolated hepatocytes were prepared as described in H and 2 ml aliquots of the cell suspensions (7.5 × 10~ cells/ ml) were incubated at 37°C in the presence of the various compounds under study and 25 mM 4POBN as spin trapping agent. Liver microsomes were prepared as described by Slater and Sawyer, ~5 except that the livers were perfused with ice-cold saline to remove the blood before homogenization. For the experiments 0.4 ml of the microsomal suspension (approx. 2 mg proteins) were added to 1.5 ml of an incubation mixture containing 83.5 mM KCI, 37.2 mM Tris-HC1 buffer pH 7.4, 2 mM MgC12, 5 mM glucose-6-phosphate, 0.25 mM NADP +, 10 I.U. of glucose-6-phosphate dehydrogenase and 25 mM 4-POBN. The microsomes were incubated for 30 min at 37°C in 25 ml bottles closed with screw caps. The various compounds were dissolved in water and added to the incubation mixture to make up the final volume of 2 ml. Carbon monoxide was fluxed for 1 min through the microsomal suspension immediately before adding it to the incubation mixture. Free radical adducts were extracted from isolated hepatocytes and microsomal suspensions by shaking them with 1 ml chloroform/methanol (2:1, v/v) mixture and the chloroform phase, separated by centrifugation, was used for the ESR analysis.14 A Bruker 200 D/SCR spectrometer fitted with a variable temperature cavity was used. The instrument settings were as follows: Microwave power 10 roW; modulation frequency 100 MHz; modulation amplitude 1 G; scanning field 100 G; sample temperature - 50°C. Intracellular glutathione (GSH) content was estimated by using the Ellman's reagent as reported in. J6

Copper-catalyzed oxidation of methyl-substituted hydrazines was performed as described in 1°.]2 using a 50 mM solution of NazCO3 in water (pH 10) containing 0.01 mM C u C I 2 , 50 mM of 4-POBN and 1 mM of either monomethyl-, 1,1 dimethyl and 1,2 dimethylhydrazine. The spin adducts were extracted with I ml chloroform-methanol mixture as reported for the experiments in biological systems. The same reaction performed in the absence of the hydrazine did not result in any detectable ESR signal indicating that, under the conditions used, there was no oxidation of the spin traps to nitroxide derivatives. RESULTS

The incubation of isolated hepatocytes with 2 mM monomethyl-, 1,1 dimethyl- or 1,2 dimethyl-hydrazines resulted in the formation of well defined ESR spectra due to 4-POBN spin adducts (Fig. 1). As reported in Table 1 the hyperfine splitting values obtained with all three methyl-hydrazines were prac-

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Fig. 1. ESR spectra of the 4-POBN free radical adducts produced in isolated bepatocytes incubated with methyl-bydrazines. Isolated liver cells were incubated 30 min at 37 ° with 25 mM 4-POBN and 2 mM of, respectively, monomethyl-hydrazine (trace B), 1,1 dimethyl-hydrazine (trace C) 1,2 dimethyl-hydrazine (trace D). The recorder amplification gain was 2 × 106 for trace A and D, 2 x 105 for trace B and 106 for trace C.

Detection of free radicals from methylhydrazines

5

Table 1. Hyperfine Splitting Constants of the ESR Spectra Due to the 4-POBN Adducts Produced During Biological Activation or Chemical Oxidation of Methyl Substituted Hydrazines Hyperfine Splitting Constants of the ESR Spectra Isolated Hepatocytes aN aH Monomethyl-hydrazine 1,1 dimethyl-hydrazine 1,2 dimethyl-hydrazine

14.97 14.94 14.94

Liver Microsomes aN aH

2.34 2.34 2.33

14.96 14.94 14.90

Chemical Oxidation aN aH

2.32 2.33 2.34

14.97 14.92 14.96

2.34 2.30 2.33

Note. The values are expressed in Gauss and represent the means of 3-5 experiments in the case of biological samples and of two experiments in the case of chemical reactions.

tically identical, and consistent with the trapping of the same carbon-centered free radical species. However, the intensity of ESR signals produced by monomethyl-hydrazine was about ten and five fold higher than those formed by equimolar concentrations of 1,1 dimethyl- and 1,2 dimethyl-hydrazines, respectively. Liver microsomes similarly activated monomethyl-, 1,1 dimethyl- and 1,2 dimethyl-hydrazines to free radical metabolites that were trapped by 4-POBN. The hyperfine splitting constants of these spectra were very similar to each other and almost identical with those detected in isolated hepatocytes (Table 1). In liver microsomes the free radical formation required the presence of a NADPH regenerating system and was inhibited by anaerobic incubation or using heat-denaturated microsomes (Table 2). Monomethyl-hydrazine, however, gave rise to a small but detectable ESR signal even in the presence of denaturated microsomes or when NADP + was omitted from the incubation mixture, most likely as a result of a spontaneous decomposition catalyzed by traces of metal ions present in the microsomal suspensions. The chemical oxidation of monomethyl-, 1,1 dimethyl- and 1,2 dimethyl-hydrazines in the presence of 4-POBN produced ESR spectra having identical hy-

perfine splitting constants (aN -- 14.97, aH = 2.33 G). These values were comparable with those observed in biological systems and were consistent with the spectral features attributed to the methyl free radical adduct of 4-POBN [12]. The role played by monoxygenase enzymes in the generation of the free radical metabolites from methylhydrazines has been investigated in microsomal preparations. As shown in Figure 2, the stimulation of cytochrome P450 system by phenobarbital increased

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Table 2. Intensities of the Methyl-4-POBN ESR Signals Following Various Treatments of Liver Microsomes ESR signal intensity (arbitrary units) Treatments

MMH

1,1 DMH

1,2 DMH

None No NADP ÷ Anaerobic incubation Heat inactivated microsomes + GSH 4 mM

324 46 (86%) 34 (90%)

95 7 (93%) 3 (97%)

64 4 (94%) 2 (97%)

50 (85%) 46 (86%)

5 (96%) 12 (87%)

1 (98%) 12 (82%)

Note. Phenobarbital-induced microsomes were incubated at 37°C with 25 mM 4-POBN and, respectively, 1 mM monomethyl-hydrazine (MMH) or 2 mM 1,1 dimethyl- (1,1 DMH) and 1,2 dimethyl-hydrazine (1,2 DMH). Anaerobic conditions were obtained by flushing 10 min the incubation flasks with nitrogen. Heat inactivated microsomes were boiled 5 min before being added to the incubation mixture. The results are expressed in arbitrary units and are means of two experiments. The values in parentheses represent the percent inhibition with respect to the respective controls.

LU 50

0 MMH

1,1DMH

1.2DMH

Fig. 2. Effects of rat pretreatment with phenobarbital and CoC12 on the intensities of the ESR signals produced by liver microsomes incubated with methyl-hydrazines. Liver microsomes were incubated as described in the Methods section with either 1 mM of monomethyl-hydrazine or 2 mM of, respectively, 1,1 dimethyl- and 1,2 dimethyl-hydrazines. The results are express in arbitrary units and are means of 3-7 experiments -- S.E.M.

6

E. ALBANO, A. TOMASI, L. GORIA-GATTI, and A. IANNONE

Table 3. Effects Produced by Inhibitors of the Microsomal Monoxygenase Systems on the Free Radical Activation of Monomethylhydrazine (MMH), 1,1 Dimethyl-hydrazine (1,1 DMH) and 1,2 Dimethyl-hydrazine (1,2 DMH) by Rat Liver Microsomes Intensities of the ESR signals (% of the control values) Additions SKF 525A l m M Metyrapone 0.5 mM Carbon monoxide l-nitro-isothiocyanate 0.1 mM p-chloromercuribenzoate 0.1 mM Methimazole 1 mM

MMH 72 60 74 27 52 96

-+ -+ -+ -+ ÷ -+

8.0 8.7 6.6 7.5 2.2 5.2

1,1 DMH

1,2 DMH

68 61 40 32 49 14

70 56 54 35 47 92

-+ 4.8 -+ 6.4 +- 5.3 + 4.1 ÷ 3.5 +_ 4.3

-+ 4.2 -+ 8.9 -+ 6.2 +- 3.0 ÷ 4.2 _+ 3.2

Note. Phenobarbital-induced liver microsomes were incubated 30 min at 37°C with, respectively, 1 mM monomethyl-hydrazine or 2 mM of either 1,1 dimethy]- and 1.2 dimethyl-hydrazines and 25 mM 4-POBN. The values are expressed as percent of the respective controls incubated without inhibitors. The results were calculated as the means of 3 4 differents experiments -+ SE.M.

by 2 - 4 fold the free radical production from all the hydrazines tested. Conversely, cobalt chloride administration, which lowered by approx. 60% the cytochrome P450 content in the microsomes, caused a decrease in the ESR signal intensities ranging from 35% up to 78% (Table 3). These results indicated the cytochrome P450 as the probable activation site of alkyl hydrazines. Table 3 shows that the addition of inhibitors of cytochrome P450 such as SKF 525A and metyrapone or the interference in the electron supply to cytochrome P450 caused by p-chloromercuribenzoate (pCMB) lowered by 30-50% the ESR signal intensities of the spin adducts derived from all three methyl hydrazines. The cyanate derivative 1-nitro-isothiocyanate which has been shown previously to prevent both the toxicity ~7 and the free radical activation ~t of acetyland isopropyl-hydrazine inhibited by about 70% the formation of free radical also in the case of methylderivatives. On the contrary, carbon monoxide which is regarded as a typical blocker of cytochrome P450 displayed variable activity: it decreased by 5 6 - 6 0 % the activation of the two dimethyl-hydrazines, but had a minimal effect on that of monomethylhydrazine (Table 3). It has been reported that 1,1 dimethyl-hydrazine is a better substrate for FAD-containing monoxygenase rather than for the cytochrome P450 system. ~8Indeed, the addition of 1 mM methimazole, a competitive inhibitor of the former enzyme, reduced by 86% the free radical formation in microsomes incubated with 1,1 dimethyl-hydrazine, whereas it did not affect the activation of monomethyl- and 1,2 dimethyl-derivatives (Table 3). The free radical metabolites of methylhydrazines were found to interact with glutathione and the addition to microsomal suspensions of physiological concentrations (4 mM) of GSH decreased by 86%, 87%, and 82% the trapping of methyl radicals from, respectively, monomethyl-, 1,1 dimethyl- and 1,2 dimethyl-hydra-

zine (Table 2). On the contrary, glutathione did not interfere with the free radical formation when added at the end of the incubation period (not shown), indicating that the lowering of the ESR signals was not due to an interaction with the nitroxide adducts. When added to isolated hepatocytes methyl substituted hydrazines caused a time-dependent depletion of intracellular glutathione. Following 4 hour incubation with 1 mM monomethyl-hydrazine GSH loss accounted for approx. 62% of the total content (Fig. 3). Both 1,1 dimethyl and 1,2 dimethyl-hydrazines were less effective and at 2raM concentration decreased hepatocyte glutathione by only 20% and 35%, respectively (Fig. 3), consistent with the lower free radical production observed with these latter compounds.

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40

30

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~ 10

TIME

[hr]

Fig. 3. Time-course of the glutathione depletion induced by methylhydrazines in isolated hepatocytes. Hepatocytes (5 × 106 cells/ml) were incubated at 37°C with, respectively, no addition (Q)); 1 mM monomethyl-hydrazine ( • ) ; 2 mM 1,1 dimethyl-hydrazine ( I ) ; 2 mM 1,2 dimethyl-hydrazine ( A ) . The results are means of three different experiments -+ S.D.

Detection of free radicals from methylhydrazines DISCUSSION

Spin trapping experiments have shown that isolated hepatocytes and liver microsomes activate monomethyl and dimethyl-hydrazines with the formation of free radical intermediates. Both biological and chemical oxidation of methyl-substituted hydrazines give rise to the same free radical species ascribed to the trapping of methyl free radical. The spectral features here reported are consistent with those observed by Augusto et al. j2 during horse-radish peroxidase catalyzed oxidation of 1,2 dimethyl-hydrazine and attributed to the methyl-4-POBN adduct. Spectral analysis have demonstrated that mono- and 1,2 disubstituted hydrazines interact with cytochrome P450 giving type II binding spectra and cause a loss of the CO-reactive heamoprotein. ~9 The results obtained using liver microsomes prepared from CoC12treated rats and the effects exerted by inhibitors of cytochrome P450 suggest that microsomal monoxygenase system is the probable site for the free radical activation of methyl-hydrazines, in agreement with the observations performed with other hydrazine derivatives. ~'2° One electron oxidation, as catalyzed by horse radish peroxidase and prostaglandin synthetase, has been shown to produce free radicals from several alkyl-hydrazines. 9'j2,~3 However, the reaction mediated by cytochrome P450 appears to involve a two electron transfer process, leading to a postulated diazene intermediate. 2~ According to Battioni et a1.,22 in fact, the destruction of cytochrome P450 caused by methyl-hydrazine can result from the formation of ferrous cytochrome-diazene complexes. Studies by the group of Prough have demonstrated that the oxidative metabolism of 1,1 disubstituted hydrazines is largely mediated by FAD-containing monoxygenase ~8 since the oxidation products, 1,1 dialkyl-diazenium ions, forms a complex with cytochrome P450 which terminate its function. 23 We have observed that both methimazole, a competitive inhibitor of FAD-monoxygenases and cytochrome P450 blockers decrease the free radical activation of 1,1 dimethyl-hydrazine. FAD-catalyzed oxidation of 1,1 dimethyl-hydrazine it is known to produce stoichiometric amounts of formaldehyde and monomethyl-hydrazine. ~7 It is, therefore, possible that this latter compound might be the precursor of the methyl free radical and thus explain the effect of both FAD-dependent and cytochrome P450-dependent monoxygenase inhibitors. The formation of alkylating metabolites from 1,2 dimethyl-hydrazine is postulated to involve azomethane, azoxymethane and methylazoxy-methanol as intermediates. 24 However, studies using procarbazine

7

(N-isopropyl-2-(2-methylhydrazino)-p-tolbutamide) have recently shown that azoprocarbazine, but not azoxyprocarbazine, is metabolized by liver microsomes to a reactive species, possibly the methyl free radical. 25 During preliminary experiments, the incubation of azoxymethane with liver microsomes in the presence of NADPH and 4-POBN did not result in any detectable ESR spectrum, suggesting that the azoxy derivative is not a precursor of the free radical intermediate. Thus, it is likely that the oxidation of azomethane would result in the formation of methyl free radical which, on its turn, can act as alkylating agent towards DNA bases. 3 In conclusion, the results obtained indicate that free radical activation of mono and dimethyl hydrazines is taking place in liver ceils. The metabolic pathways involved are similar to those observed with other alkyl hydrazines, 9-~,2° although disubstituted derivatives seem to require preliminary oxidation. The detection of methyl free radicals suggest that such reactive species could be responsible for the toxic and/or carcinogenic effects of methyl-hydrazines. Acknowledgements--This work has been supported by the Ministero della Pubblica Istruzione, Project "Patologia da Radicali Liberi" and by the Consiglio Nazionale delle Ricerche, Project "Rischio Tossicologico, Gruppo Gastroenterologia" contract no. 86.00506.04. A.T. and A.I. are grateful to the International Association for Cancer Research, London for financial support.

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