oxidative metabolism of n-lsopropyl-Â«-(2 - cancer research

[CANCER RESEARCH 39, 4555-4563. November 1979]0008-5472/79/0039-OOOOS02.00

Oxidative Metabolism of N-lsopropyl-Â«-(2-methylhydrazino)-p-

toluamide Hydrochloride (Procarbazine) by Rat LiverMicrosomes1

Danny L. Dunn,2 Ronald A. Lubet,3 and Russell A. Prough4

The Department of Biochemistry. University of Texas Health Science Center at Dallas, Dallas, Texas 75235

ABSTRACT

The oxidative metabolism of procarbazine (A/-isopropyl-a-(2-methylhydrazino)-p-toluamide hydrochloride) by rat liver micro-

somes has been studied utilizing three different biochemicalassays. Using high-pressure liquid chromatography, it was

observed that the major, stable microsomal metabolite produced was the azo derivative, A/-isopropyl-a-(2-methylazo)-p-

toluamide, which can undergo a rapid chemical conversion toan aldehyde, p-formyl-/V-isopropylbenzamide, and methylhy-

drazine upon the addition of acid. The rate of metabolism couldmost conveniently be determined spectrophotometrically byreacting the resultant methylhydrazine with p-dimethylamino-benzaldehyde. Alternatively, p-formyl-N-isopropyl benzamidecould be extracted directly from the acid-treated mixture withan organic solvent, and its concentration could be determinedspectrophotometrically. Rates of metabolism using all threeassays ranged from 13.4 to 14.1 nmol/min/mg of microsomalprotein. Inhibitor and induction studies indicated that the cy-tochrome P-450-dependent monooxygenase system is respon

sible for this oxidation reaction. Several possible mechanismswere considered: either a direct dehydrogenation or a preliminary /V-oxidation by cytochrome P-450 followed by a rapid

dehydration step.The azo derivative of procarbazine was further oxidized to

two isomerie azoxy derivatives, A/-isopropyl-a-(2-methyl-NNO-azoxy)-p-toluamide and /V-isopropyl-a-(2-methyl-ONN-azoxy)-p-toluamide, at a metabolic rate which is 10 to 15% as large as

the rate of procarbazine oxidation. In addition, the affinity ofthe azo derivative for the monooxygenase is high relative tothat of the parent hydrazine. The reaction was inhibited bycytochrome P-450 inhibitors and was sensitive to the in vitroaddition of several thiono-sulfur-containing compounds. The

azoxy compounds were also metabolized by microsomal fractions from liver and yielded p-formyl-/V-isopropylbenzamide as

a product. This complex in vitro metabolic scheme is analogousto the in vivo metabolism of 1,2-dimethylhydrazine and sug

gests that procarbazine may be metabolically activated to yieldalkylating agents capable of expressing carcinogenic and/ortoxic effects by a mechanism common for at least these two/V.A/'-disubstituted hydrazines.

' This work was supported by Robert A. Welch Foundation Grant 1-616 and

by American Cancer Society Grant BC-153. A preliminary portion was presentedat a Meeting of the American Society of Biological Chemists in San Francisco.Calif., June 1976, and the Third International Symposium on Microsomes andDrug Oxidations, Berlin, Germany, July 21 to 24, 1976 (30).

2 Supported in part by fellowships from the Robert A. Welch Foundation andby USPHS Training Grant T32-CA09082.

3 Recipient of U.S.P.H.S. Research Fellowship 1 F32 CA06019.4 Recipient of USPHS Career Development Award HLCA 00255. To whom

requests for reprints should be addressed.Received February 12, 1979; accepted August 2, 1979.

INTRODUCTION

PCZ5 has been shown to act as an effective antitumor agent

against a number of transplanted tumors in rodents (3, 20, 27).Subsequently, it was shown to be effective in the treatment ofmalignant lymphoma in humans (8). Since its approval by theFood and Drug Administration in 1969, PCZ has been firmlyestablished as a beneficial single agent and as a component ofcombination therapy for Hodgkin's disease and several other

neoplastic disorders (38).PCZ, like other antitumor agents, appears to elicit a number

of carcinogenic and toxic responses. This methylhydrazinederivative induces pulmonary tumors and leukemia in mice(22), mammary adenocarcinoma in rats (23), and myelogenousleukemia and malignant neoplasms in nonhuman primates (36).A number of toxic responses, including nausea, vomiting, andcentral nervous system depression, have been noted when thecompound is administered to humans (7). Studies with animalshave documented PCZ-induced suppression of the immune

system (16), teratogenesis (6), and sterility (17).The relationship between PCZ metabolism and its mode of

action as either an antitumor, carcinogenic, or toxic agent isnot known. In vivo studies in rats, dogs, and humans haveindicated that PCZ is rapidly converted to its azo derivative(34); some controversy exists related to the chemical or enzymatic nature of this reaction in vivo (2, 40). The isopropylamideof terephthalic acid has been found to be the major urinarymetabolite and is not cytostatically active (26, 34). Both carbondioxide and methane are detected as expired products derivedfrom the A/-methyl group of PCZ (1, 10). On the basis of this

metabolite pattern, several laboratories proposed that the following metabolic sequence exists to account for the products(2, 26, 34):

PCZ -Â»AZO â€”HYD -. ALD -. TAC (A)

where TAC is terephthalic acid isopropylamide. In addition, themethylhydrazine formed by hydrolysis of the hydrazone wassuggested to be metabolized to carbon dioxide and methane.

However, Baggiolini et al. (2) showed that, with isolated,perfused rat liver, oxidation of PCZ to AZO proceeded rapidlybut that the azo derivative is a relatively stable chemical species. They could find no evidence to support the assumptionthat AZO undergoes extensive isomerization to its hydrazonetautomer in vitro. In addition, the rate of metabolism of meth-

5 The abbreviations used are: PGZ, W-isopropyl-Â«-(2-methylhydrazino)-p-to-luamide hydrochloride (procarbazine; NSC 77213); AZO, W-isopropyl-Â«-(2-meth-ylazo)-p-toluamide; HYD, fV-isopropyl-o-(2-methylhydrazo)-p-toluamide; ALD, p-formyl-W-isopropylbenzamide; HPLC. high-pressure liquid chromatography;AZOXY. N-isopropyl-Â«-(2-methylazoxy)-p-toluamide; s, singlet; Ehrlich's reagent,

0.05 M p-dimethylaminobenzaldehyde in 1.0 N H2SOÂ«;AM. azomethane; AOM,azoxymethane.

NOVEMBER 1979 4555

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O. L Dunn et al.

ylhydrazine was too slow to account for the formation of carbondioxide from the N-methyl group of PCZ (9). An alternate

metabolic sequence was therefore proposed in which AZO ismetabolized to ALD and methyl diazene. Recently, Weinkamand Shiba (40) have substantiated the slow chemical conversion of AZO to HYD. In addition, they observed the furthermetabolism of the azo derivative to 2 azoxy metabolites andALD. Analysis of extracts of blood from rats treated with PCZand of in vitro reaction mixtures using direct probe chemicalionization mass spectroscopy suggests that several oxidativemetabolic steps occur for this compound (40).

Since the pathway for the oxidative metabolism of PCZ hasnot been characterized, studies utilizing HPLC were initiated tofollow the production of the various metabolites involved. Twomicrosomal monooxygenases have been described which metabolize a variety of nitrogenous compounds, including hydra-

zine derivatives. These 2 enzymes are the liver microsomalcytochrome P-450 (18) and amine oxidase (29, 30, 43). Sincemethyl-substituted hydrazines have been observed to undergometabolism by both enzyme systems (29-32, 42), PCZ may

also be a potential substrate for either monooxygenase. Afterthe development and documentation of a spectrophotometricassay, the effects of various inhibitors and inducers have beenstudied in an attempt to identify the enzyme system(s) responsible for the oxidation of PCZ. Analysis of in vitro incubationmixtures demonstrates the conversion of the azo derivative ofPCZ to azoxy compounds, which may be further metabolizedto reactive intermediates in a manner similar to that suggestedfor 1,2-dimethylhydrazine by Fiala (11 ); Chart 1 shows a generalized scheme for metabolism of at least 1,2-dimethylhydra

zine and PCZ.

MATERIALS AND METHODS

Materials

PCZ was a gift from Hoffmann-La Roche, Inc. (Nutley, N. J.).Radioactive PCZ labeled with 14C in the A/-methyl and ring

positions was provided by the Drug Research and DevelopmentProgram, Division of Cancer Treatment, National Cancer Institute (Bethesda, Md.). Glass-distilled solvents for HPLC were

obtained from Burdick & Jackson Laboratories (Muskegon,Mich.). The authentic sample of ALD used for mixed meltingpoint determinations was a gift from Dr. Donald J. Reed,Department of Biochemistry and Biophysics, Oregon StateUniversity. All other chemicals were commercially availablereagent grade, unless otherwise stated. Benzene was redistilled and used within 1 week of distillation. Catalase (30,000units/mg), isocitrate dehydrogenase (type IV), sodium isocit-rate, NADP+, and NADPH were obtained from the Sigma Chem

ical Co. (St. Louis, Mo.).

Structural Analysis of Metabolites

UV spectra were recorded on a Gary Model 14 (VarianAssociates, Palo Alto, Calif.) or Beckman Model 25 (BeckmanInstruments, Inc., Irvine, Calif.) spectrophotometer, and massspectra were obtained using a Varian Model CH7 mass spectrometer. Nuclear magnetic resonance spectra were recordedon a Perkin-Elmer Model R24A spectrometer (Norwalk, Conn.),

using tetramethylsilane as an internal standard. Melting pointswere measured in a Fisher Scientific Co. Eimer and Amend

RCH2NHNHCH2R'

I

RCH2NZNCH2R

[ ?RCHjNr

.NCHR

I-R'CHO

Chart 1. Schematic representation of the metabolism of A/.W-disubstitutedhydrazines. For 1,2-dimethylhydrazine. R=R'â€” H; for procarbazine. either R=Hand R'=â€” C6H4CONHCH(CH3)2 or R'=H and R=â€” C6H4CONHCH(CH3)2. Thestable in vitro metabolites for PCZ are N-isopropyl-a-(2-methylazo>-p-toluamide(AZO), the benzyl- or methylazoxy isomers (AZOXY; see Footnote 5). and ALD.

melting point apparatus (Pittsburg, Pa.), and are uncorrected.

Synthesis of Metabolites

AZO. A mixture of PCZ (0.5 g), ether (2.5 ml), ethyl alcohol(2.5 ml), and mercuric oxide (1.5 g) was shaken at roomtemperature for 2 hr. The resulting suspension was filtered andevaporated in a vacuum. The residue was taken up in ether(30 ml), filtered, and evaporated in a vacuum; yield, 47%. Thecrude AZO was purified by HPLC (see below); m.p. 109-112Â°;[Bollag ef al. (4), m.p. 111-115Â°]; UV (ethanol) maximum 231

nm (log e = 4.20), 316 nm (log e = 4.10); mass spectrum (90eV) m/e 219 (M*). [14C]AZO was synthesized from ring-labeled

PCZ as described above; the radiolabeled AZO was purifiedby HPLC. The chemical identity and purity of this material wasascertained by its Chromatographie properties prior to andafter acid treatment (acid-catalyzed conversion to ALD).

AZOXY.6 AZO (5.0 mg), 85% m-chloroperbenzoic acid (4.7

mg), and mÃ©thylÃ¨nechloride (10 ml) were shaken at roomtemperature for 3 hr. The mixture was then washed with 10%sodium bisulfite (10 ml), 10% sodium bicarbonate (10 ml), andwater (10 ml). After drying over sodium sulfate, the mÃ©thylÃ¨nechloride was evaporated in a vacuum, and the residue wasrecrystallized from ethyl alcohol in a yield of 38%. The materialwas further purified by HPLC, as described below. Analysis ofthe purified material by HPLC shows 2 closely spaced peaks,

6 The nomenclature used for the azoxy isomers is based on IUPAC TentativeRules which uses the infixes â€”NNOâ€”or â€”ONNâ€”to specify the position of theoxygen For procarbazine. the 2 isomers would be W-isopropyl-Â«-(2-methyl-NNO-azoxy)-p-toluamide for the isomer with the oxygen closest to the benzyl group(I), and W-isopropyl-Â«-(2-methyl-ONN-azoxy)-p-toluamide for the isomer with theoxygen closest to the methyl group (II). For convenience, isomers I and II will bedesignated as the benzylazoxy isomer (I) and methyl-azoxy isomer (II). respectively. The nitrogen numbering system. N-1 or N-2. is based on the nomenclatureof the parent compound, a 2-methyl-1-benzylhydrazine derivative.

O

ICH3â€”N=Nâ€”CH2â€”R

(I)

O

ICH3â€”N=Nâ€”CrV-R

(II)

where

R = â€”C6H4CNHCH(CH3)2

4556 CANCER RESEARCH VOL. 39



Oxidative Metabolism of Procarbazine

indicating that the product is probably a mixture of 2 isomerieazoxy compounds (I and II); m.p. 96-109Â° [Weinkam and Shiba(40), m.p. 95-103Â° for a similar mixture of isomers]; massspectrum (90 eV) m/e 235 (M+); UV (ethanol) maximum 228

nm (log e = 4.47).ALD. 4-Carboxybenzaldehyde (1 7.6 g), thionyl chloride (30

ml), and benzene (20 ml) were refluxed until a clear solutionwas obtained (approximately 20 hr). Benzene and excessthionyl chloride were removed in a vacuum, and the residuewas distilled under reduced pressure to yield 4-formylbenzoylchloride, b.p. 146-152Â°/6 mm; [Slotta and Kethur (37); b.p.

163-165V15 mm].4-Formylbenzoyl chloride (11.4 g, 0.068 mol), dissolved in

50 ml of benzene, was added dropwise to a mixture of isopro-

pylamine (11.56 g, 0.136 mol) and benzene (50 ml) in an icebath. After being stirred for 18 hr at room temperature, thereaction mixture was washed with 10% hydrochloric acid (v/v), 10% sodium carbonate (w/v), and water before extractionwith 30% sodium bisulfite (w/v). The aqueous layer containingthe sodium bisulfite salt of the aldehyde was separated, andthe pH was adjusted to 14 with 6 N sodium hydroxide, whichcaused the immediate formation of a white precipitate. Theprecipitate was extracted with benzene and evaporated in avacuum to give ALD as a white crystalline solid in 50% yield,m.p. 120-121Â°; an authentic sample of ALD melted at 119-120Â° and a mixed melting point showed no depression; UV

(ethanol) maximum 252 nm (log e = 4.21 ); mass spectrum (90eV) m/e 191 (M +) nuclear magnetic resonance (CCI3D) 1.14(doublet, 6H, â€”CH(CH3)j), 3.91 (multiplet, 1H, â€”CH(CH3)?),6.01 (broad s, 1H, â€”CONHâ€”),7.34 (s, 4H, phenyl), 9.43 (s,1H, â€”CHO).

HYD. p-Formyl-A/-isopropylbenzamide (0.1 g), methylhydra-

zine (0.1 ml), and ethyl alcohol (10 ml) were stirred at roomtemperature for 18 hr (40). Ethyl alcohol was removed in avacuum to yield the hydrazone as a white residue in 74% yield.The structure was confirmed by its conversion to ALD andmethylhydrazine upon addition of acid. A small sample recrys-tallized from methyl alcohol gave m.p. 145-149Â°; UV (ethanol)

maximum 225 nm (log e = 4.07), 312 nm (log Â«= 4.15); massspectrum (90 eV) m/e 219 (M+).

Extraction Efficiency

The extraction efficiency and stability of PCZ and its metabolites were determined by extraction with equal volumes ofredistilled benzene. Solutions of each compound in 0.05 Mpotassium phosphate:0.05 M A/-tris(hydroxymethyl)methyl-

glycine buffer, pH 7.4, were extracted with equal volumes oforganic solvent, and the organic solvent was evaporated withnitrogen. The residue was dissolved in 95% ethanol, and theconcentration of the metabolites was determined by their UVabsorbance; in certain cases the residues were analyzed byHPLC.

PCZ and its azo derivative were noted to be somewhatunstable when extracted from aqueous medium with undistilledether, hexane, and benzene. Considerable concentrations ofAZO can be obtained by extraction of aqueous solutions ofPCZ with commercial solvents, but the amount of AZO extracted can be decreased (50 to 80%) using freshly glass-

distilled benzene or hexane. This result suggests that commercial organic solvents contain materials such as organic perox

ides or peracids which can oxidize PCZ to its azo derivative ordecompose the AZO to its hydrazone or aldehyde analog.Extraction with equal volumes of redistilled benzene allowedthe recovery of the azo compound (89%) without significantdegradation to the aldehyde derivative (ALD); hexane extraction allowed only a 41% recovery of AZO. The extractionefficiencies with equal volumes of benzene for the compoundsare as follows: ALD, 63%; AZO, 89%; AZOXY, 59%; HYD,78%; and PCZ, <2%. Since PCZ and its metabolites havemarked differences in their extraction efficiencies, the metabolites can be quantitatively removed from the aqueous phaseand from PCZ by 4 successive extraction steps with equalvolumes of benzene.

HPLC

Samples were chromatographed using a /uBondapak-CN col

umn (Waters Associates, Milford, Mass.) with a Water ModelALC 202/401 high-pressure liquid Chromatograph. A constant

solvent flow rate of 3.0 ml/min was used. The initial solventcondition for the metabolites of PCZ and its azo derivative(mÃ©thylÃ¨nechloride:hexane, 5:95) was changed by a lineargradient over a 15-min period to a final mÃ©thylÃ¨nechloride:

hexane ratio of 35:65. The initial solvent condition used tostudy AZOXY metabolism (100% hexane) was changed by alinear gradient over a 14-min period to a final mÃ©thylÃ¨ne

chloride:hexane ratio of 15:85.Column effluent was monitored at 254 nm. Fractions were

collected, evaporated, and assayed for radioactivity using ascintillation cocktail of 0.55% PPO (Amersham/Searle Corp.,Arlington Heights, III.) in Triton X-100:to!uene (1:2, v/v) on aBeckman Model LS-230 liquid scintillation counter (Palo Alto,

Calif.). The formation of ALD from AZOXY was calculated from254 nm absorbance using a standard curve obtained for pureALD under the same HPLC conditions. The recovery of radioactivity from the HPLC column ranged from 92 to 102%.

Preparation of Liver Microsomal Fractions

Male Sprague-Dawley CD rats, 150 to 250 g, were pur

chased from the Charles River Breeding Laboratory, Inc. (Wilmington, Mass.), and maintained on water and laboratory chowad libitum. They were given injections in the peritoneal cavityof either corn oil, phÃ©nobarbital in 0.9% NaCI solution (80 mg/kg), or 3-methylcholanthrene in corn oil (20 mg/kg) daily for 4days and starved 18 hr prior to sacrifice by decapitation.Microsomal fractions of liver were prepared using a Teflon-glass Potter-Elvehjem homogenizer (A. H. Thomas Co., Phila

delphia, Pa.), as described by Remmer ef al. (35); the variousfractions were centrifuged at 1,200 x g for 5 min (cell debris,nuclei), at 11,400 x g for 15 min (mitochondria), and at 78,000x g for 60 min (microsomal protein). Protein concentrationwas determined by the method of Lowry ef al. (24).

Metabolism Studies

The metabolism experiments were performed at 37Â°with a

reaction mixture consisting of microsomal protein (2 mg/ml),3 mW sodium DL-isocitrate, isocitrate dehydrogenase (0.8 IU/ml), 0.5 mw NADP+, 5 rriM MgCI4, 3.8 HIM PCZ, and 0.05 M

potassium phosphate:0.05 M A/-tris(hydroxymethyl)methyl-glycine-HCI buffer, pH 7.8. The reaction was initiated by addi-

NOVEMBER 1979 4557



D. L. Dunn et al.

tion of microsomes after a 5-min preincubation period. The

concentrations of AZO and AZOXY used for metabolism studies were 0.5 and 0.07 mw, respectively. For the metabolicstudies, 1.5-ml aliquots were removed at 2-min intervals during

the linear portion of the reaction (15 min). The concentration ofproducts was plotted as a function of time, and specific activities (nmol product formed per min per mg of protein) werecalculated from the slope of the plot. Control experimentsdesigned to measure chemical oxidation were performed; therates of chemical oxidation were less than 10% of the enzymatic rate. Solutions of '"C-labeled PCZ were extracted with

redistilled benzene or hexane before use to remove any non-

polar impurities (AZO). An aliquot was normally removed at 15min for HPLC analysis, and the rate of reaction was determinedby the nmol of product present at this time point. Samples forHPLC analysis either were added to 1.5 ml of 1.0 N H2SOâ€žandextracted with four 5.0-ml portions of benzene, or were directlyextracted with four 5.0-ml portions of benzene. The extracts

were evaporated under nitrogen and the residue was taken upin 0.5 ml of mÃ©thylÃ¨nechloride. Aliquots of this solution (0.1ml) were injected onto the HPLC column for Chromatographieanalysis.

When determining the rate of metabolism by assaying for themethylhydrazine present after addition of acid, the aliquotswere mixed with 1.5 ml of Ehrlich's reagent. Precipitated pro

tein was removed by centrifugation at 12,000 x g for 15 min,and the supernatant was decanted. The solutions were equilibrated at 25Â°for 15 min, and the concentration of quinoid ion

hydrazone (39) was calculated from the absorbance at 465 nm(log Ã‰= 3.6; this value was found to vary for different temperatures and pH values). Similarly, if the rate of metabolism wasto be determined by assaying for ALD formation, aliquots wereadded to 1.5 ml of H2SC>4(1.0 N), the mixture was extractedwith four 1.5-ml portions of hexane, the organic phase was

evaporated under nitrogen, and 1.5 ml of ethanol were addedto the residue. The concentration of ALD present was thendetermined by its UV absorbance at 252 nm.

RESULTS

HPLC Analysis. The primary products formed by the oxida-tive metabolism of PCZ can be isolated, using HPLC, andidentified by their chromotographic mobility, UV absorbancespectrum, and mass spectrum. When benzene extracts of a15-min incubation mixture were analyzed by HPLC, the major254-nm-absorbing peak exhibits a retention time and physicalproperties identical to the authentic sample of AZO (Chart 2;Table 1). However, when the incubations were quenched with1 N sulfuric acid instead of benzene, the principal metaboliteisolated was ALD (Chart 2; Table 1). The ALD is most probablyproduced by an acid-catalyzed tautomerization of AZO to HYD,

which undergoes subsequent hydrolysis to ALD and methylhydrazine. A similar conversion of AZO to ALD by aqueousacid solutions has been observed by others (2, 40). Using 14C-

ring-labeled PCZ, this chemical reaction was shown to bequantitative, inasmuch as over 99% of the AZO derivativepresent was converted to ALD.

Exact rates of formation for the various PCZ metaboliteswere obtained by HPLC using '"C-labeled substrate (Table 2).

After extraction with chilled benzene, incubation mixtures containing '"C-ring- and methyl-labeled PCZ showed a total rate of

ALD

AZO

AcidQuench

BenzeneQuench

0 3 6 IZM Â¡Ã±utes

Chart 2. HPLC separation of PCZ metabolites after benzene and acid quenchof the incubation mixture. The initial solvent condition (mÃ©thylÃ¨nechloride:hex-ane, 5:95) was changed by a linear gradient over 15 min to a final mÃ©thylÃ¨nechloride:hexane ratio of 35:65 The flow rate was 30 ml/min.

Table 1

Physical characterization of procarbazine metabolites

CompoundStandardsAZOALDAZOXY-IAZOXY-IIHYDMajor

metabolitesAZOMinor

metabolitesALDAZOXY-IAZOXY-IIHYDRetention

time"(min)5.982879.411.55.78.18.99511.3Absorbancemax

imum [nm (log0!231

(4.20)252(4.21)228(4

47)c228225(4.07)312(4.15)228252228228225,312m/e"219191235235219219

Maior metabolites (acidified)ALD 8.1 252 191

Minor metabolites (acidified)AZOXY-I 8.9 228AZOXY-II 9.5 228HYD 11.2 225,312J These retention times are those obtained using a (iBondapak-CN column

with a 15-min linear gradient elution from 5 to 35% mÃ©thylÃ¨nechloride in hexane.* M '. 90 eV.c The extinction coefficient is for the purified mixture of both isomers obtained

by m-chloroperbenzoic acid oxidation of AZO.

metabolite formation of 13.0 and 12.7 nmol/min/mg protein,respectively, using microsomes from phenobarbital-treatedrats. After addition of acid to the reaction mixture prior to theextraction, metabolism of 14C-ring-labeled PCZ (ALD formation)

proceeded at a rate of 12.0 nmol/min/mg protein. However,





when reaction mixtures containing 14C-methyl-labeled PCZ

were terminated with acid, only trace radioactivity was observed in the ALD peak collected by HPLC (Table 2). UnlikeAZO, the AZOXY metabolites appear to be quite stable totreatment with acid (Chart 2; Table 2). These studies confirmthat the primary stable metabolite of PCZ oxidation is AZO andthat AZO indeed rapidly decomposes into ALD upon the addition of acid.

The 3 most probable mechanisms of AZO formation wouldbe either direct dehydrogenation, a preliminary /V-oxidation bycytochrome P-450 followed by a rapid dehydration step, or C-

hydroxylation at the benzyl carbon followed by dehydrogenation and tautomerization to the azo. Since the hydrazone derivative does not readily tautomerize to yield the azo derivative inaqueous solution (i.e., the equilibrium lies in favor of thehydrazone and hydrazone hydrolysis), the possibility of a C-hydroxylation reaction would appear not to be energeticallyfeasible. Under these metabolic conditions, AZO is probablyfurther /V-oxidized to the methyl- and benzyl-AZOXY derivatives, /V-isopropyl-a-(2-methylazoxy)-p-toluamide. The rates of

AZOXY formation varied between 0.6 and 1.0 nmol/min/mgprotein in all cases, which represents about 5 to 8% of the totalmetabolism. Small amounts of ALD and HYD were also detected above that obtained by incubating PCZ in the absenceof NADPH for 15 min (the blank values for the metabolites wereless than 5 to 10% of the AZO formed and 50 to 60% of theALD and HYD formed). No AZOXY was detected in the 15-minreaction mixtures lacking NADPH.

ALD and Methylhydrazine after Acid Quench. AlthoughHPLC may be used to follow the metabolism of PCZ, thequantitative conversion of AZO and HYD to ALD and methyl-hydrazine by acid allows a less time-consuming measurementof the rates of metabolism. For example, ALD may be extractedfrom the incubation mixture with an organic solvent and itsconcentration determined directly by the absorbance at 252nm. The rate of PCZ metabolism obtained in this manner wasfound to be 12.0 nmol/min/mg protein.

Similarly, the rate of oxidative metabolism may be obtainedfrom the amount of methylhydrazine present after acid quench.Ehrlich's reagent reacts with methylhydrazine in acid to form a

quinoid ion hydrazine, aldazine, which possesses an absorbance maximum at 465 nm (Chart 3) (39). After testing 30hydrazines covering a wide range of possible structures, it wasestablished that only primary hydrazines and hydrazine itselfexhibit this absorbance when reacted with Ehrlich's reagent.Since PCZ is a A/./V'-disubstituted hydrazine, it cannot form a

quinoid ion hydrazone and does not interfere with the aldazineabsorbance at 465 nm. Further, the absorbance of the aldazinewas shown to be linearly proportional to concentration (i.e.,obeys Beer's relationship), as shown in Chart 3. The rate of

metabolism based on methylhydrazine concentration was 14.1nmol/min/mg protein. Time courses of the various methodsfor determining PCZ metabolism rates are shown in Chart 4.The latter assay method, which uses Ehrlich's reagent to

determine the methylhydrazine concentration, seems to be thesimplest of the various methods to measure metabolism of PCZto AZO; it may be performed rapidly and no extractions arerequired. All of the following enzymology studies on AZOformation were obtained using this method of assay afterverification by HPLC.

Optimization and Localization of Enzymatic Activity (AZO

Table 2

HPLC characterization ol PCZ metabolism

Position of label QuenchingagentRing

BenzeneRing

AcidMethyl

BenzeneMethyl

AcidCompoundAZOALDAZOXYHYDTotalAZOALDAZOXYHYDTotalAZOALDAZOXYHYDTotalAZOALDAZOXYHYDTotalMetabolic

rate"

(nmol/min/mg)10.70.70.61.013.00.010.61.00.412.010.70.10.71.212.70.00.41.00.41.8

These results were corrected for extraction efficiency, percentage of recovery from HPLC. and endogenous rates. The rates were obtained at pH 7.3. usingliver microsomes prepared from phenobarbitaMreated animals, and are basedon the nmol of product present after 15 min.

Formation). The enzymatic conversion of PCZ to AZO wasoptimized with regard to protein concentration, optimal pH, Km,and the effect of certain compounds normally included inmicrosomal reaction mixtures was determined. The reactionwas linearly dependent on protein concentration up to 4 mg/ml and on time up to 15 min. The pH optimum for the reactionwas broad and centered at pH 7.7 to 7.8. An apparent Km forthe reaction was determined using a double reciprocal plot ofvelocity versus substrate concentration and was noted to beapproximately 0.6 rriM. For all subsequent reactions, a substrate concentration of 3.8 HIMwas used, which was sufficientto optimize the reaction. Added catalase (1 mg/ml) or sodiumazide (5 mw) had no effect on the reaction. Dithiothreitol (1mw) inhibited the reaction 40%, while butylated hydroxytoluene(1 HIM) or butylated hydroxyanisole (1 rtiM) slightly stimulatedthe initial rates of metabolism (20 to 40%). The lack of effectof added catalase or azide suggests strongly that PCZ oxidation is not simply the result of a direct chemical oxidation byhydrogen peroxide. Low concentrations of hydrogen peroxide[50 ftM, levels of peroxide which can be generated by micro-

somes in the presence of NADPH, sodium azide, and O? (41)]did not convert PCZ to AZO.

The distribution of NADPH-dependent activity in the subcel

lular organelles separated by differential centrifugation isshown in Table 3. The sum of the activities obtained for all thesubcellular components was about 88% of the total activityexhibited by the initial homogenate. A major portion of theactivity was found to reside in the microsomal fraction of ratliver and accounts for approximately 60% of the total recoveredactivity. Rehomogenization of the cell debris and nuclei, followed by differential centrifugation, greatly decreased theNADPH-dependent metabolism of PCZ;7 the activity in the

crude fraction containing cell debris most likely is due to the

7 M. L. Coomes and R. A Prough, unpublished results.

NOVEMBER 1979 4559



D. L. Dunn et al.

Chart 3. Absorbance due to the quinoid ion hydrazoneformed by reacting p-dimethylaminobenzaldehyde with methylhydrazine (MH).

450 550

Wavelength (nm)

1.0 2.0 3.0Methylhydrazme (MxlO4)

150

100

Table 3

Subcellular distribution of activity

3 6 9 12

time (mm)

Chart 4. Comparison of the 3 biochemical assays for determination of PCZmetabolism. D. HPLC; O, ALD; A. methylhydrazine (by Ehrlich s reagent). Theslope of each line is the rate of reaction; HPLC, 13.4 nmol/min/mg protein; ALD.13.8 nmol/min/mg protein; methylhydrazine. 141 nmol/min/mg protein.

presence of whole cells or poorly extracted microsomal protein.The distribution of oxidative activity was consistent with anNADPH-dependent enzyme residing in the endoplasmic retic-

ulum.Effect of Inhibitors and Inducers on AZO Formation. Sev

eral characteristics of the oxidation of PCZ to AZO were noted.PCZ was found to undergo facile metabolism by liver micro-somes from control animals (9.1 nmol/min/mg protein), atnearly twice the rate of benzphetamine A/-demethylation (5.5nmol/min/mg protein), one of the best substrates for livermicrosomal cytochrome P-450 (33). Pretreatment of the animals with phÃ©nobarbital caused a 1.8-fold increase in the rateof metabolism, but pretreatment with 3-methylcholanthrene

showed no effect (Table 4). Additionally, oxygen and NADPHappear to be absolute requirements for metabolism to occur.Inhibitors of cytochrome P-450, such as carbon monoxide,metyrapone, A/-octylamine, or specific antibodies to liver microsomal NADPH:cytochrome P-450 (cytochrome c) reduc

Ãase,were effective in reducing the rate of metabolism (Table5). However, the specific microsomal amine oxidase inhibitor,methimazole, showed little effect (Table 5). The effect of SKF-525A on metabolism by microsomes from control, phÃ©nobarbital, and 3-methylcholanthrene-pretreated rats is shown in

Chart 5. It had little effect on the reaction catalyzed by liver

FractionHomogenate

Cell debris, nucleiMitochondriaMicrosomesCytosolTotal

protein(mg)12,1707,040

8601.1903.150Total

activity(nmol/min)52,3301

7.600516

27.60863Specific

activity"

(nmol/min/mg)4.32.5

0.61740.1

Rates were obtained at pH 7.8. using liver microsomes from phenobarbital-treated animals.

Table 4

Effect of animal pretreatment on PCZ metabolism

Pretreatment Rate (nmol/min/mg)

ControlPhÃ©nobarbitalMethylcholanthrene

9.116.5

9.0

Rates were obtained at pH 7.8 and represent the average of 3 experimentswith a S.D. of 11%.

microsomes from control or 3-methylcholanthrene-treated an

imals but caused a maximal 45% inhibition at 100 fiM withmicrosomes from phenobarbital-pretreated rats. The remaining

rate was equal to that obtained with control microsomes.Oxidative Metabolism of Azo-PCZ to Azoxy Derivatives.

Since azoxy derivatives can be easily formed by peracid oxidation of azo compounds, the possible enzymatic conversionof the azo derivative of PCZ to azoxy metabolites was investigated using 14C-ring-labeled AZO. Benzene extracts of reactionmixtures containing [14C]AZO, liver microsomes from pheno

barbital-pretreated rats, NADPH, and oxygen were analyzedby HPLC techniques; 2 major metabolites (92%) had retentiontimes identical to the 2 synthetic AZOXY PCZ standards, themethyl- and benzyl-azoxy derivatives. These 2 metabolites had

UV absorbance spectra with maxima at 228 nm and werestable to acid treatment in the same manner as the 2 tentativelyidentified azoxy derivatives in Tables 1 and 2.

The N-oxidation reaction was nearly maximal at AZO con

centrations of 250 fiM, suggesting that, relative to PCZ, the azoderivative has a high affinity for the monooxygenase catalyzingthe reaction. The reaction required oxygen and NADPH (Table6). Metyrapone was shown to inhibit this reaction, suggesting





Table 5

Effects of inhibitors on PCZ metabolism

Addition ordeletionControl-02-

NADPH+Anti-reductaseglobulin+NonimmuneglobulinCO:O2

(4:1 gasmixture)+Methimazole (1mM)+W-Octylamine (5mM)+

Metyrapone (5 mM)Rate3

(nmol/min/mg)9.40.40.52.39.24.09.61.53.8

These rates were obtained at pH 7.8 using liver microsomes from untreatedrats and are the average of 3 experiments with a S.D. of 12%. Similar resultswere obtained with liver microsomes from phÃ©nobarbital- and 3-methylcholan-threne-pretreated rats.

IOO

oo

60

20

OMC

D ControlA PB

concentration (mM)Chart 5. Effect of SKF-525A on the rate of metabolism by rat liver micro

somes. Animal pretreatment regimen: PB, phÃ©nobarbital; MC, 3-methylcholan-threne.

imazole, and the result suggests that experiments utilizing thecompound as a specific amine oxidase inhibitor must be performed in conjunction with other cytochrome P-450 inhibitors.

Oxidative Metabolism of Azoxy Derivatives. A mixture ofthe 2 azoxy isomers (70 /*M) was incubated in the presence ofliver microsomes from phenobarbital-pretreated rats, NADPH,

and oxygen, and the benzene extracts were analyzed by HPLCtechniques. Chart 6 shows that the azoxy derivatives apparently were enzymatically converted to ALD and that NADPHwas required for metabolism. The azoxy derivative with theshortest HPLC retention time (benzyl-azoxy-PCZ) appeared to

be preferentially metabolized and may yield the aldehyde derivative as its product. The rate of ALD production was calculated to be approximately 0.04 nmol ALD formed per min permg microsomal protein. Campbell ef al. (5) have shown thatazoxymethane is also metabolized at a rate which is significantly lower than expected for a number of N-methyl com

pounds. Studies are in progress to characterize the microsomalmetabolism of the azoxy derivatives.

DISCUSSION

Most hydrazine derivatives are easily air oxidized, and considerable interest exists regarding the enzymatic oxidation ofthese compounds. As noted in "Materials and Methods,"

measurable quantities of AZO can be formed by extraction ofaqueous solutions of PCZ with commercial organic solvents. Inaddition, the azo derivative is not completely stable in commercial solvents. However, if one uses freshly distilled benzeneto extract the azo derivative from aqueous solutions of PCZ,

Table 6Metabolism of AZO-PCZ by liver microsomes

Metabolic rate9

ConditionControl-

NADPH-02MetyraponeMethimazoleDisulfiramTotal1.980.280300

560.721.13ALD0.180.050.050200.280.14Benzylazoxy0.250020030.040.070.13Methylazoxy1.260

020020.130.110.66H

YD0

290.190.200.29021030

Obtained with liver microsomes from phenobarbital-pretreated rats at pH7.5 and expressed as nmol product formed per min per mg microsomal protein.The values are an average of 3 experiments with a S.D of 14%.

that cytochrome P-450 may be involved in the /V-oxidation

reaction. Methimazole and disulfiram also inhibited this reaction. The effect of these thiono-sulfur compounds on PCZ N-

oxidation is consistent with the studies of Fiala et al. (12) onthe effects of disulfiram and other sulfur compounds on themetabolism of another A/./V'-disubstituted hydrazine, 1,2-di-

methylhydrazine. Studies with purified pig liver microsomalamine oxidase have shown that AZO is not a substrate for thisenzyme and that disulfiram is not an effective inhibitor of thedimethylaniline W-oxidase or methimazole S-oxidase activity ofthe enzyme.8 Prough and Ziegler (33) have demonstrated that,

at 1 mM final concentration, a large number of cytochrome P-450-dependent reactions are not affected by addition of methimazole, 1-methyl-2-thioimidazole. It is of interest that certaincytochrome P-450-dependent reactions are sensitive to meth-

0.06

004

.2 0.02

0.00

IO II 9

t ime (min)

IO II

1R. A. Prough. unpublished results

Chart 6. Metabolism of AZOXY-PCZ by microsomes from phenobarbital-pretreated rats The initial solvent conditions (100% hexane) were changed by alinear gradient over 14 min to a final mÃ©thylÃ¨nechloride:hexane ratio of 15:85.The flow rate was 3.0 ml/min. A. no NADPH; B. 0.5 mM NADPH QuantitÃ¤tenwas performed using the peak height or area of the 254 nm trace relative to astandard curve for ALD. The eluted materials have retention times equal to ALD,N-1 (the benzylazoxy isomer), and N-2 (the methylazoxy isomer).

NOVEMBER 1979 4561



D. L. Dunn et al.

there is a decreased amount of AZO extracted, and the AZOderivative formed is more stable (;'.e., less formation of HYD

and ALD). The observations may, in part, explain the pastuncertainty regarding the enzymatic nature of azo compoundformation from hydrazines.

The analysis of organic-soluble metabolites of PCZ by HPLC

techniques demonstrates that the azo derivative is the majorstable metabolite. Since AZO can be further oxidized to yieldAZOXY and ALD, the azo derivative most probably is theprimary stable, organic-soluble product of the oxidative metab

olism of PCZ. The effect of animal pretreatment with phÃ©nobarbital and the effect of various inhibitors indicate that cyto-chrome P-450 is most probably the enzyme responsible for theNADPH-dependent oxidation of PCZ to AZO. This conclusion

is not in complete agreement with the interpretation of experiments using isolated perfused liver (2). Baggiolini ef al. (2)noted that the inclusion of SKF-525A in perfusion media de

creased PCZ oxidation by only 17%, whereas animal pretreatment with 3-methylcholanthrene caused only a 28% increase

in PCZ oxidation in the perfused rat liver. They suggested that,based on these results, the major portion of PCZ oxidation maynot be accounted for by an enzyme resembling a microsomalhydroxylase. Our induction and inhibition experiments clearlyindicate that a microsomal hydroxylase (cytochrome P-450) isinvolved in this reaction and that the results of previouslyreported liver perfusion experiments (2) can, in part, be explained based on the observations that SKF-525A is not a goodinhibitor of this cytochrome P-450-dependent reaction. Exper

iments in progress indicate that phÃ©nobarbital pretreatment ofrats affects the blood levels of the azo derivative upon administration of PCZ, and that SKF-525A only causes small de

creases in the amount of the azo derivative formed by rathepatocytes.6 However, the existence of other enzymes in

volved in the oxidation of PCZ to its azo derivative cannot beexcluded, and the relative contribution of the cytochrome P-450-dependent enzyme system cannot be defined accuratelyto date.

Recently, the in vivo metabolism of 1,2-dimethylhydrazine, a

symmetrical dialkylhydrazine which is a potent lower tractcarcinogen, has been partially elucidated. Fiala ef al. (13) havedescribed the HPLC separation of 1,2-dimethylhydrazine andits postulated metabolites: AM, AOM, and methylazoxymetha-

nol. In addition, the appearance of these metabolites as urinaryproducts has been described (13). AM and CO2 were noted asrespired products (14). In vivo administration of disulfiram andrelated compounds inhibited CO2 production and stimulatedthe respiration of AM from 1,2-dimethylhydrazine (12). Basedon the metabolism of cycasin, a glycoside of methylazoxymeth-anol, they have postulated the following metabolic sequence toaccount for the metabolites and suggested that disulfiramblocked at least one of the oxidative steps (probably AM toAOM).

DMH â€”AM -> AOM -Â»MAOM â€”CO? (B)

where DMH is dimethylhydrazine and MAOM is methylazoxy-

methanol.The observations of Weinkam and Shiba (40) and of this

report strongly suggest that PCZ metabolism in vitro leads tothe formation of the azo, azoxy, and aldehyde derivatives andthat this metabolic pattern resembles that proposed by Fiala(11) for 1,2-dimethylhydrazine. Mass spectral analysis of PCZ

metabolites in rat plasma suggests the existence of a C-hy-

droxyazoxy derivative and its postulated degradation product(40). Our results show that disulfiram and other thiono-sulfurcompounds inhibit azoxy-PCZ formation, and that at least one

product of the further metabolism of the azoxy derivatives isthe aldehyde derivative. This result is similar to the observationthat CO2 is the in vivo product of 1,2-dimethylhydrazine metabolism (14) and that formaldehyde (5) and methylazoxymethanol(15) are formed from AOM in the presence of liver microsomalprotein, NADPH, and oxygen. Evidence presented in this reportstrongly indicates that cytochrome P-450 most probably is the

microsomal monooxygenase involved in at least 3 oxidativesteps in PCZ metabolism. The role of cytochrome P-450 incertain W-hydroxylation reactions is not without precedent. 2-Acetylaminofluorene (19), p-chloroacetanilide (21), isoniazid

(25), iproniazid (25), and acetaminophen (28) have been shownto be A/-hydroxylated by cytochrome P-450, and the toxic

reactions of these compounds are possibly due to their activation to reactive intermediates. Considerable effort will berequired to characterize the enzymatic reactions of /V./V'-disub-

stituted hydrazines and to assess the involvement of theirmetabolites in the therapeutic, toxic, and tumorigenic activityof PCZ.

ACKNOWLEDGMENTS

The authors wish to thank Phil Freeman. Bill Parkhill, and Golden Pan for theirtechnical assistance and Dr. Bettie Sue Masters for her constant encouragement.In addition, they wish to acknowledge Cato McDaniel, Department of Chemistry.North Texas State University, for obtaining the mass spectra of several compounds used in this study.

REFERENCES

1. Baggiolini, M., and Bickel. M. H. Demethylation of ibenzmethyzin (Natulan),monomethylhydrazine and methylamine by the rat in vivo and by the isolatedperfused rat liver. Life Sci., 5. 795-802, 1966.

2. Baggiolini, M., Dewald, B., and Aebi, H. Oxidation of p-(/V'-methylhydrazi-nomethylJ-rV-isopropyl benzamide (procarbazine) to the methylazo derivativeand oxidative cleavage of the N2-C bond in the isolated perfused liver.Biochem Pharmacol.. Ã•8: 2187-2196. 1969.

3 Bollag, W. The tumor-inhibitory effects of the methylhydrazine derivative Ro4-6467/1 (NSC-77213). Cancer Chemother. Rep., 33: 1-4, 1963.

4. Bollag, W., Kaiser, A., Langmann, A., and Zeller, P. Methylazo and meth-ylazoxy compounds; new types of antitumor agents Experientia (Basel), 20503, 1964.

5. Campbell, R. L., Suppnick, J. D., Hettrick, J. M., and Migro, N. D. Rat livermicrosome-mediated W-demethylation and mutagenicity of azomethane.Cancer Res., 38 4585-4590. 1978.

6. Chaube, S., and Murphy, M. L. Fetal malformations produced in rats byW-isopropyl-Â«-(2-methylhydrazino)-p-toluamide hydrochloride (Procarbazine). Teratology, 2 23-31, 1969.

7. DeVita. V. T., Hahn, M. A., and Oliverio, V. T. Monoamine oxidase inhibitionby a new carcinostatic agent, /V-isopropyl-Â«-(2-methylhydrazino)-p-toluam-ide(MIH). Proc. Soc. Exp. Biol. Med., Â»20.561-565. 1965.

8 DeVita, V. T., Jr., Young, R. C., and Cannellos, G. P. Combination versussingle-agent chemotherapy: a review of the basis for selection of drugtreatment for cancer. Cancer (Phila.), 35 98-110, 1975.

9 Dewald, B.. Baggiolini, M , and Aebi. H. A/-Demethylation of p-(/V'-methyl-hydrazino methyl)-W-isopropyl benzamide (procarbazine), a cytostaticallyactive methylhydrazine derivative, in the intact rat and in the isolatedperfused rat liver. Biochem. Pharmacol., 18: 2179-2186, 1969.

10. Dost. F. N.. and Reed, D. J. Methane formation in vivo from N-isopropyl-o-(2-methylhydrazino)-p-toluamide hydrochloride, a tumor inhibiting methylhydrazine derivative. Biochem. Pharmacol., J6: 1741-1746. 1967.

11. Fiala, E. S. Investigation into the metabolism and mode of action of the coloncarcinogen, 1,2-dimethylhydrazine Cancer (Phila.), 36: 2407-2412. 1975.

12. Fiala. E. S., Bobotas, G.. Kulakis, C.. Wattenberg, L. W., and Weisburger,J. H. Effects of disulfiram and related compounds on the metabolism in vivoof the colon carcinogen. 1,2-dimethylhydrazine. Biochem. Pharmacol., 26:1763-1768, 1977.

13. Fiala. E. S.. Bobotas. G., Kulakis. C., and Weisburger, J. H. Separation of1,2-dimethylhydrazine metabolism by high pressure liquid chromatography.





J. Chromatogr., 117: 181-185, 1976.14. Fiala. E. S.. Bobotas, G.. Kulakis, C., and Weisburger. J H Inhibition of

1,2-dimethylhydrazine metabolism by disulfiram. Xenobiotica. 7: 5-9. 1977

15. Fiala. E. S.. Kulakis. C., Christiansen, G., and Weisburger, J. H Inhibition ofthe metabolism of the colon carcinogen, azoxymethane, by pyrazole. CancerRes., 38. 4515-4521, 1978

16. Floersheim. G. L. Prolonged survival time of skin homotransplants in micewith a methylhydrazine derivative. Experimentia (Basel). 19: 546-547.1963.

17. Fox, B. W., and Fox, M. Biochemical aspects of the actions of drugs onspermatogenesis. Pharmacol. Rev., 19: 21-57, 1967.

18. Gillette, J. R . Davis. D. C.. and Sasame. H. A. Cytochrome P-450 and itsrole on drug metabolism. Annu. Rev. Pharmacol., 12: 57-84. 1972

19. Grantham, P. H., Weisburger. E. K.. and Weisburger. J. H. Dehydroxylationand deacetylation of A/-hydroxy-A/-2-fluorenylacetamide by rat liver andbrain homogenates. Biochim. Biophys. Acta, 107: 414-424. 1965.

20. Grumberg, E., and Prince, H. N. The activity of ibenzmethyzin hydrochlorideagainst the human transplantable tumors, human epithelioma No. 3 andhuman adenoma No. 1. Experientia (Basel), 22. 324-325, 1966.

21. Hinson, J. A.. Mitchell. J. R., and Jollow, D. J. Microsomal N-hydroxylationof p-chloroacetanilide. Mol. Pharmacol., 11: 462-469. 1975.

22. Kelly, M. G., O'Gara. R. W., Gadekar. K.. Yancey, S. T.. and Oliverio. V. T.

Carcinogenic activity of a new antitumor agent, /N/-isopropyl-a-(2-methyl-hydrazino)-p-toluamide hydrochloride (NSC-77213). Cancer Chemother.Rep.. 39: 77-80. 1964.

23. Kelly, M. G., O Gara, R. W. Yancey, S. T.. and Botkin. C. Induction of tumorsin rats with procarbazine hydrochloride. J. Nati. Cancer Inst., 40: 1027-1051. 1968.

24. Lowry, O. H., Rosebrough, N. J.. Farr, A. L., and Randall, R. J. Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem., /93. 265-275,1951.

25. Nelson. S. D., Mitchell, J. R., Timbress. J. A., Snodgrass. W. R., andCorcoran, G. B. Isoniazid and iproniazid: activation of metabolites to toxicintermediates on man and rat. Science, 193. 901-903, 1976.

26. Oliveria. V. T., Denham, C., DeVita. V. T., and Kelly, M G. Some pharmacologie properties of a new antitumor agent. W-isopropyl-u-<2-methylhydra-zino)-p-toluamide, hydrochloride (NSC-77213). Cancer Chemother. Rep.,42: 1-7. 1964.

27. Oliveria, V. T., and Kelly. M. G. Contributions to the biological and clinicaleffect of a methylhydrazine derivative. II. Some preliminary observations onits physiological disposition, antitumor activity and carcinogenicity. In: P. A.Plattner (ed.), Proceedings of the International Symposium on the Chemotherapy of Cancer, Lugano, April 28 to May 1, 1964, pp. 221-227. Amsterdam: Elsevier Publishing Co.. 1964.

28. Potter, W. Z., Davies, D. C., Mitchell, J. R., Jollow, D. J., Gillette, J. R., andBrodie, B. B. Acetaminophen-induced hepatic necrosis III. Cytochrome P-

450-mediated covalent binding in vitro. J. Pharmacol. Exp. Ther., 187: 203-210, 1973.

29. Prough, R. A. The N-oxidation of alkylhydrazines catalyzed by the micro-somal mixed-function amine oxidase. Arch. Biochem. Biophys.. 758 442-444. 1973.

30. Prough, R. A.. Coomes. M. L.. and Dunn, D. L. The microsomal metabolismof carcinogenic and/or therapeutic hydrazines. In: V. Ullrich. I. Roots. A GHildebrandt, R. W. Estabrook, and A. H. Cooney (eds.), Microsomes andDrug Oxidations, Vol. 3. pp. 500-507. London: Pergamon Press. 1977.

31. Prough. R. A , Wittkop, J. A., and Reed. D. J. Evidence for the hepaticmetabolism of some monoalkylhydrazines Arch. Biochem. Biophys.. 131:369-373, 1969.

32. Prough, R. A., Wittkop, J. A., and Reed, D. J. Further evidence on the natureof microsomal metabolism of procarbazine and related alkylhydrazines.Arch. Biochem Biophys.. 140: 450-458. 1970

33. Prough, R. A..and Ziegler, D. M. The relative participation of liver microsomalamino oxidase and Cytochrome P-450 in W-demethylation reactions. Arch.Biochem. Biophys.. 180: 363-373, 1977.

34. Raaflaub, J., and Schwartz, D. E. Ãœberder Metabolismus eines cytostatischwirksamen Methylhydrazine-Derivates (Natulan). Experientia (Basel). 21:44-45, 1965.

35. Remmer, H.. Greim, H.. Schenkman. J. B., and Estabrook. R W. Methodsfor the elevation of hepatic microsomal mixed function oxidase levels andCytochrome P-450 Methods Enzymol.. 10: 703-708. 1967.

36. Sieber. S M.. Correa. P , Dalgard, D W., and Adamson. R H. Carcinogen-esis and other adverse effects of procarbazine in nonhuman primates.Cancer Res., 38. 2125-2134. 1978.

37. Slotta, K. H.. and Kethur, R Synthese der Terephthal- und Isophthalalde-hydsaure-ester. Chem. Ber.. 71: 335-341. 1938.

38. Spivack. S. D. Procarbazine. diagnosis and treatment-drugs five years later.Ann. Intern. Med.. 81: 795-800. 1974.

39. Watt, G. W.. and Chrisp. J. D. A spectrophotometric method for the determination of hydrazine. Anal. Chem.. 24: 2006-2008. 1952.

40. Weinkam. R. J., and Shiba. D. A. Metabolic activation of procarbazine. LifeSci.. 22. 937-946. 1978.

41. Werringloer. J. The formation of hydrogen peroxide during hepatic microsomal electron transport reactions. In: V Ullrich, I. Roots. A. G. Hildebrandt,R. W. Estabrook. and A. H. Conney (eds.), Microsomes and Drug Oxidations,Vol. 3. pp. 261-268. London: Pergamon Press, 1977.

42. Wittkop, J. A.. Prough. R. A., and Reed. D J. Oxidative demethylation of N-methylhydrazines by rat liver microsomes. Arch Biochem. Biophys.. 134:308-315, 1969.

43. Ziegler. D M.. and Mitchell. C. H. Microsomal oxidase IV: Properties of amixed-function amine oxidase isolated from pig liver microsomes. Arch.Biochem. Biophys.. 150: 116-125. 1972.

NOVEMBER 1979 4563



1979;39:4555-4563. Cancer Res Danny L. Dunn, Ronald A. Lubet and Russell A. Prough Microsomes-toluamide Hydrochloride (Procarbazine) by Rat Liver

p-(2-methylhydrazino)-α-Isopropyl-NOxidative Metabolism of

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