is oxidized by rat liver microsomes in the presence of reducednicotinamide adenine dinucleotide phosphate and oxygen toyield two major metabolites as determined by high-pressureliquid chromatography. The isolated products are the two isomerie azoxy derivatives, /V-isopropyl-a-(2-methyl-NNO-azoxy)-p-toluamide (benzylazoxy) and A/-isopropyl-a-(2-methyl-ONN-azoxy)-p-toluamide (methylazoxy), since the mass spectra ofthe metabolites are distinctly different from each other but areidentical to those of the respective chemically synthesizedazoxy standards. In addition, p-formyl-/V-isopropylbenzamide
methylazoxyprocarbazine by rat liver microsomes is inhibitedby a number of specific cytochrome P-450 inhibitors. Azopro
carbazine elicits a very weak spectral complex (type II) with ratliver microsomal cytochrome P-450. These results stronglysuggest that cytochrome P-450-dependent monooxygenase(s)are involved in the N-oxidation of azoprocarbazine to yield two
azoxy isomers of procarbazine. Incubation of liver microsomalprotein with [14C]azoprocarbazine, oxygen, and reduced nico
tinamide adenine dinucleotide phosphate results in a time-
dependent increase in covalent binding of labeled material tomicrosomal protein. More protein-bound label is obtained with[inef/iy/-14C]azoprocarbazine than with [nng-'4C]azoprocarba-
zine, suggesting that the molecule can be metabolically activated to a moiety which preferentially binds the methyl portionof its structure to microsomal protein in vitro.
INTRODUCTION
In a previous paper (7), we demonstrated the oxidativemetabolism of the antitumor agent, procarbazine [W-isopropyl-
a-(2-methylhydrazino)-p-toluamide hydrochloride], by rat liver
microsomes. Evidence was presented which strongly supported the involvement of the microsomal monooxygenase,cytochrome P-450, in at least 3 oxidative steps of procarbazine
metabolism. Attention was focused, however, on the first ofthese oxidation reactions, the formation of the azo derivative ofprocarbazine, /V-isopropyl-a-(2-methylazo)-p-toluamide, by rat
liver microsomal suspensions.The present paper concentrates on the second of these
oxidative steps, that of the oxidation of the azoprocarbazine to2 isomerie azoxy derivatives, /V-isopropyl-a-(2-methyl-NNO-azoxy)-p-toluamide (benzylazoxyprocarbazine) and N-isopro-pyl-a-(2-methyl-ONN-azoxy)-p-toluamide (methylazoxyprocarbazine),4 by rodent liver microsomes. Using rodent liver micro
bazine, and, in an attempt to assess the involvement of themicrosomal monooxygenase cytochrome P-450, the effect of
certain specific inhibitors on this reaction was also investigated.Evidence for the further metabolism of the 2 azoxy derivativesis also reported. The results of experiments designed to measure the covalent binding of azoprocarbazine to microsomalprotein are also reported here in order to shed light on thebiological activity of the metabolites produced, since the relationship between procarbazine metabolism and its mode ofaction as antitumor, carcinogenic, or toxic agent has not yetbeen elucidated.
MATERIALS AND METHODS
The sources and purity of all chemicals and solvents usedare those described previously (7). Procarbazine hydrochloridewas a gift from Hoffman-La Roche Inc., Nutley, N. J., and
1This work was supported in part by Grant 1-616 from the Robert A. WelchFoundation and by Grant B-336 from the American Cancer Society.
2 Robert A. Welch Postdoctoral Fellow.3 Research Career Development Awardee HL 00255. To whom requests for
reprints should be addressed.Received February 20, 1980; accepted June 25, 1980.
4 The nomenclature used for the azoxy isomers is based on IUPAC Tentative
Rules which uses the infix -NNO- or -ONN- to specify the position of the oxygen.For procarbazine, the 2 azoxy isomers would be /V-isopropyl-«-(2-methyl-NNO-azoxy)-p-toluamide for the isomer with the oxygen closest to the benzyl group(Isomer I) and W-isopropyl-«-(2-methyl-ONN-azoxy)-p-toluamide for the isomerwith the oxygen closest to the methyl group (Isomer II). For convenience, IsomersI and II will be designated as the benzylazoxy Isomer and methylazoxy isomer,respectively. The nitrogen numbering system, N-1 or N-2, is based on thenomenclature of the parent compound, a 2-methyl-1-benzylhydrazine derivative.
radioactive procarbazine labeled with 14Cin either the /v-methyl
or ring positions was provided by the Drug Research andDevelopment Program, Division of Cancer Treatment, NationalCancer Institute, Bethesda, Md. Azoprocarbazine, its 2 isomerie azoxy derivatives, and [14C]azoprocarbazine (50 /¿Ci/
mmol) were prepared as described previously (7, 18) andpurified using the high-pressure liquid chromatography systemdescribed below. [7,10-'4C]benzo(a)pyrene was purchased
from the Amersham/Searle Corp. (Arlington Heights, III.) anddiluted with unlabeled benzo(a)pyrene to obtain a specificradioactivity of approximately 2 fiCi/mmol. Mass spectra wereobtained using a Finnigan 31 ODD mass spectrometer coupledto a 6100 data system. UV and melting point measurementswere carried out as described previously (7).
Animal pretreatment, preparation of liver microsomal fractions, and metabolism studies were essentially as describedpreviously (7). A typical reaction mixture contained microsomalprotein (2 mg/ml), 3 rriM sodium DL-isocitrate, isocitrate dehy-drogenase (0.8 Ill/ml), 0.5 mw NADP+, 5 HIM MgCI2, and 0.05
M potassium phosphate:0.05 M /V-tris(hydroxymethyl)-methylglycine buffer, pH 7.6. The final concentration of azo-procarbazine used in all metabolic studies was 0.25 mM, unlessotherwise stated. The reaction was initiated by addition ofmicrosomes after preincubation of the incubation mixture containing a NADPH-regenerating system for at least 5 min at 37°.After a 10-min incubation period, the reaction was terminated
with 5 ml of benzene and subsequently extracted 3 times with5-ml portions of this solvent. The organic phases were evaporated to dryness and prepared for high-pressure liquid Chro
matographie analysis (7). This extraction system allowed quantitative recovery of the 2 azoxy isomers and the p-formyl-N-
isopropylbenzamide (>97%) as well as of the substrate and itshyd razone tautomer.
Samples were chromatographed using a juBondapak CNcolumn (Waters Associates, Milford, Mass.) with a WatersModel ALC 202/401 high-pressure liquid Chromatograph.Base-line separation of the substrate and its metabolites wasachieved using an isocratic solvent system consisting of hex-
ranged from 95 to 104%.Covalent binding studies were carried out using the same
incubation mixture as that described for the metabolism study.At appropriate time intervals, duplicate 2-ml aliquots were
removed from the reaction mixture, and the reaction was terminated by addition of the aliquots to 2 ml of chilled 1 NTCA.5 Following centrifugation, the precipitate was washed atleast twice with 2 ml of chilled 1 N TCA to remove any nonco-valently bound material. The TCA-insoluble material was ex
tracted with 5 ml of benzene and then digested for 30 min in 1N NaOH at 90°. Aliquots were removed for determination of
radioactivity and protein concentration. Studies were also performed with [7,10-"'C]benzo(a)pyrene as a control. The meth
odology used is essentially as described above, except thatthe benzo(a)pyrene was used at 80 ¿tK/ifinal concentration,microsomal protein was used at 0.5 mg/ml in the incubationmixture, and an ethyl acetate extraction was carried out priorto the digestion of the TCA-insoluble material in 1 N NaOH.
RESULTS
The oxidative metabolism of azoprocarbazine gives rise to 2major isomerie products, benzylazoxy- and methylazoxypro-carbazine, which can be isolated using high-pressure liquidchromatography and identified by their Chromatographie mobility, UV absorbance, and mass spectra (Table 1; Chart 2).The mass spectra presented in Chart 2 indicate not only thatthe 2 metabolites can be distinguished from each other by thedifferences in their fragmentation patterns (Chart 2, C versusD) but also that the mass spectra of the 2 metabolites producedon incubation of 0.25 mM azoprocarbazine with isolated ratliver microsomes from phenobarbital-induced animals, NADPH,
and oxygen are identical to those obtained from the authenticstandards (Chart 2, A versus C and B versus D).
It is tempting to speculate that upon mass spectral analysischemically synthesized benzylazoxyprocarbazine yields a parent ion at m/e 235 and one major fragmentation peak at M-59which might represent the loss of CH3—N=NO (Chart 2A). It
is known that many azoxy derivatives fragment at the bond a
to the N-oxide (17). The methylazoxyprocarbazine also has a
parent peak at m/e 235 and has mass fragments at m/e 218(M-17), 191 (M-44), and 177 (M-58), which might representsuccessive fragmentation of the CH3—NO—Ngroup (Chart
2C). While the fragmentation patterns do not allow unambiguous identification of structure due to the similarities of theazoxy and isopropylamide groups, the order of retention of our2 metabolites is as expected on /^Bondapak Ci8 columns (40%methanol in water) based on the results of Weinkam and Shiba(18), and chemical synthesis with m-chloroperbenzoic acidresulted in a 1:2 ratio of the 2 putative isomers, benzylazoxy-
rat liver microsomes is shown in Chart 3 and Table 2. Followinga 10-min incubation of liver microsomes from untreated ratswith 0.25 HIM azoprocarbazine at 37°,only the methylazoxy
A qualitatively similar induction profile is noted when rabbitliver microsomal suspensions are incubated with 0.25 mwazoprocarbazine. A small but measurable rate of benzylazox-
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Chart 2. Mass spectra of chemically synthesized and metabolically formed benzylazoxy- and methylazoxyprocarbazine isomers. Spectra were obtained by directprobe analysis using a Finnigan 31 ODD mass spectrometer at 70 eV coupled to a 6100 data system. Right abscissa, percentage of the total ¡onflux represented bythe line spectra. A, chemically synthesized benzylazoxyprocarbazine; B, chemically synthesized methylazoxyprocarbazine; C, benzylazoxyprocarbazine metabolite;D, methylazoxyprocarbazine metabolite.
Table 2Effect of animal pretreatment on rate of formation of benzylazoxy- and methylazoxyprocarbazine and p-formyl-N-isopropylbenzamide by
rodent liver microsomes incubated with azoprocarbazine
nmol metabolite/min/mg microsomal protein with following animal pretreatment regimen
yprocarbazine formation is seen with liver microsomes fromcontrol rabbits, and pretreatment with 5,6-benzoflavone causesa 1.9-fold increase in activity. The increase in the rate ofmethylazoxyprocarbazine production (2.9-fold), however, is
the same with either inducer (Table 2).Further examination of Chart 3 reveals that an aldehyde, p-
formyl-A/-isopropylbenzamide, is also formed by rat liver micro
in the rate seen with untreated rat liver microsomes (Table 2).These results are consistent with the suggestion that thisproduct is a further metabolite of one or both of the azoxyisomers (7), since the overall induction profile of these metabolites is similar to that shown in Chart 3 and Table 2.
Table 3 summarizes the effects of a number of specificcytochrome P-450 inhibitors on the W-oxidation of azoprocar
bazine by phenobarbital-induced rat liver microsomes. SKF
525A at a final concentration of 0.5 mM was found to be themost potent inhibitor, decreasing the rate of total azoxyprocar-
bazine formation to 3% of the control value. This is in markedcontrast to the lesser inhibitory effect of SKF 525A (20 to 40%)on the initial rate of A/-oxidation of the parent hydrazine, pro-carbazine (7). n-Octylamine (1.0 mM) and the inhibitory antibody to liver microsomal NADPH-cytochrome c (P-450) reducÃasewere also very effective inhibitors, reducing the overallmetabolic rate to 13% and 10%, respectively, of the control. Itshould be noted that neither nonimmune globulin nor pyrazole(1 or 10 mM) is effective in inhibiting these reactions (Table 3).Inhibition of azoprocarbazine /V-oxidation is also seen when
the reaction is carried out in the presence of a CO:O? atmosphere (4:1, v/v); the overall rate decreased to 32% of the valueobtained in air.
Azoprocarbazine exhibits a spectral complex with microsomal cytochrome P-450 similar to those noted for many othernitrogenous compounds (Amax430 to 440 nm; Amin400 to 410nm). The interaction, however, is very weak as indicated bythe binding parameters shown in Table 4. Application of simpleMichaelis-Menten kinetics to the oxidation of azoprocarbazineby rat liver microsomes to the benzylazoxy- and methylazoxy
benzoflavone results in alteration of both the Km and Vma. ofthe monooxygenase(s) involved in the oxidation of the methylend of the molecule. Animal pretreatment with 5,6-benzofla
0 Mean ±S.D. from 3 separate experiments performed in duplicate.
and benzylazoxy isomers are identical (approximately 120/UM)when microsomes from phenobarbital-treated rats are usedas a source of enzyme, but that the Vmax'sare not equal (Chart
4). The Vmaxfor methylazoxyprocarbazine production is over 3times larger than that seen for the benzyl isomer (Chart 4;Table 5).
The time courses for the production of material which iscovalently bound to rat liver microsomal protein upon incubation with either 0.25 HIM [mefhy/-"'C]azoprocarbazme or [ring-14C]azoprocarbazine are shown in Chart 5. It can be seen that,following a 20-min incubation period at 37°,the level of cova-
lent binding of the methyl-labeled material was over 3 timesgreater than that detected for benzyl-labeled material. However, it must be realized that, over the same incubation period,the amount of covalent binding to microsomal protein afterincubation with 80 ¿AMbenzo(a)pyrene, NADPH, and oxygen,were approximately 3.5 times larger than that produced by[mer/iy/-14C]azoprocarbazine. Experiments to measure cova
lent binding to exogenous DMA have not been attempted, sinceradiolabeled substrate with sufficient specific radioactivity tomeasure DNA alkylation is not available.
DISCUSSION
The metabolism, distribution, and excretion of the chemo-
therapeutic agent, procarbazine, has been investigated, bothin vivo (4, 5, 16, 18) and in vitro (1, 4, 7, 14, 18, 19), byseveral research groups. Evidence, mainly from this laboratory(7, 14), has suggested that the first oxidation step to the azoderivative may take place in the liver and is mediated, at leastin part, by the microsomal cytochrome P-450 mixed-function
oxidase system. Further, the results of work by Weinkam andShiba (18) and by our laboratory (Refs. 7 and 14; this report)demonstrate that azoprocarbazine is W-oxidized to 2 isomerieazoxy derivatives, benzylazoxy-and methylazoxyprocarbazine.
The inhibition studies presented here (Table 3) demonstratethat the inclusion of a carbon monoxide atmosphere, a specificantibody to the NADPH-cytochrome P-450 reducÃase, n-octy-
lamine, or SKF 525A to the reaction mixture results in significant inhibition of this oxidative step, suggesting the possibleinvolvement of the cytochrome P-450-dependent monooxy-
genase in the reaction. However, pyrazole, a known inhibitorof alcohol dehydrogenase and of the further oxidation of meth-
ylazoxymethane (14), had no effect.In addition, the results of the induction studies (Table 2)
imply that there may be more than one species of cytochrome
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Chart 4. Lineweaver-Burk plot of azoprocarbazine oxidation by liver microsomal suspensions from phenobarbital-pretreated rats. Microsomes (approxi
mately 20 mg/ml) were incubated with various concentrations of azoprocarbazine for 10 min at 37°.The azoprocarbazine and its metabolites were extractedand chromatographed as described in Materials and Methods. " Values are the
means of at least 3 separate experiments differing by less than 10%. •,methylazoxyprocarbazine; •benzylazoxyprocarbazine.
400-,
E-oC13OJD
200-
Chart 5. Time course of covalent binding of radiolabeled azoprocarbazine toliver microsomal protein from phenobarbital-pretreated rats. Microsomes (approximately 2.0 mg/ml) were incubated at 37° with 0.25 mM [methyl-"'C}-azoprocarbazine. 0.25 mM [ring-"C] azoprocarbazine, or 80 fiM ["CJ-
benzo(a)pyrene for the times indicated, and the amount of covalently boundmaterial was assessed radiometrically as described in Materials and Methods.The values are means of at least 2 separate experiments differing by less than10%. •methyl-labeled azoprocarbazine; A, benzyl-labeled azoprocarbazine;•B(a)P.
dation of azoprocarbazine (Table 5) support this contention. Itshould be noted that preliminary studies on the metabolism ofthe azoxy isomers indicate that the compounds have lowerrates of metabolism at 100 ¡IMsubstrate concentrations thandoes azoprocarbazine.
Azoprocarbazine has a far greater affinity for the monooxy-
genase catalyzing its conversion to azoxy metabolites thandoes the parent hydrazine, procarbazine. The Km for the conversion of procarbazine to azoprocarbazine is approximately0.6 HIM(7) while the Kmfor azoxy formation ranges from 25 to125 fiM. Despite the apparent increase in the affinity of themonooxygenase for azoprocarbazine as compared to procarbazine, the azoprocarbazine appears to form poorly a bimolec-
ular complex (measured as a type II binding spectrum) with themicrosomal cytochrome P-450 (Table 4). As might be ex
pected, the apparent Kmfor the reaction may have no relationto the formation of a low-spin complex of the cytochrome upon
substrate binding. It should be noted that preliminary studieson the metabolism of the azoxy isomers indicate that thecompounds have lower rates of metabolism at 100 JUMsubstrateconcentrations than that of azoprocarbazine.6 The low rates of
metabolism of the azoxy metabolites suggest that the kineticstudies reported measured the initial rates of azo metabolism,not a steady-state rate of metabolism.
The further metabolism of the benzylazoxy- and methylazox-
yprocarbazines by rat liver microsomes has been reportedpreviously to possibly yield an aldehyde, p-formyl-A/-isopropyl-
benzamide (7). Data presented in this paper (Table 2) confirmthis finding, but our current understanding related to the formation of this important intermediary metabolite is scant. Further studies will be necessary to fully document this reaction.
The increased binding of the methyl portion of the moleculeas compared to that of the benzyl end lends support to thehypothesis that a reactive intermediate similar to methyldiazon-
ium ion may be an active species in procarbazine metabolismrather than a diazonium ion involving the benzyl portion of themolecule (Chart 5). Such findings are consistent with the proposed metabolic schemes for procarbazine (7, 15, 16) in vivoand in vitro and for 1,2-dimethylhydrazine, the potent lowertract carcinogen (6, 8-11), in vivo. In addition, the apparent
reactivity of the methyl portion of the molecule may possiblycorrelate with the fact that only the methyl derivative of procarbazine is an effective anticancer agent (2). Since the metabolic fate of these two 1,2-disubstituted hydrazines is thought
to proceed by very similar reaction mechanisms, it is notunreasonable to suggest that they may possess a common
6 R. A. Prough and S. W. Cummings. unpublished results.
active species responsible for the carcinogenic and/or toxicresponses engendered in vivo and that information gained withprocarbazine may well assist in identifying one or more activespecies responsible for the biological effects of 1,2-dimethylhydrazine. However, these results do not provide any insightinto an understanding of the unique organ specificity of carci-nogenesis of these two 1,2-disubstituted hydrazines (6).
ACKNOWLEDGMENTS
The authors wish to express their thanks to Drs. John Lomont and JamesGarriot of the Southwestern Institute of Forensic Sciences for performing themass spectral analyses and to Scott Cummings tor his technical assistance.
REFERENCES
1. Baggiolini, M.. Dewald. B., and Aebi. H. Oxidation of p-(/V'-methylhydrazino
methyl)-N-isopropyl benzamide (procarbazine) to the methylazo derivativeand oxidative cleavage of the N2—C bond in the isolated perfused liverBiochem. Pharmacol., 18: 2187-2196. 1969.
2. Bollag, W., and Grunberg, E. Tumor inhibitory effects of a new class ofcytotoxic agents: methylhydrazine derivatives Experientia (Basel). 19:130-131.1963
3. Dean, W. L, and Coon, M. J. tmmunochemical studies on two electropho-retically homogeneous forms of rabbit liver microsomal cytochrome P-450:P-45ÛLMÎand P-450LM«.J Biol. Chem., 252 3255-3261. 1977.
4. Dewald, B.. Baggiolini, M.. and Aebi, H. N-Demethylation of p-(W-methyl-hydrazino methyl)-rV-isopropyl benzamide (procarbazine). a cytostaticallyactive methylhydrazine derivative, in the intact rat and in the isolatedperfused rat liver. Biochem. Pharmacol., 18: 2179-2186, 1969.
5. Dost. F. N.. and Reed. D. J. Methane formation in vivo from A/-isopropyl-<i-(2-methylhydrazino)-p-toluamide hydrochloride. a tumor inhibiting methylhydrazine derivative. Biochem Pharmacol.. 16: 1741-1746. 1967.
6 Druckrey. H. Production of colonie carcinomas by 1,2-dialkyihydrazines andazoxyalkanes In: W. J. Burdette (ed.). Carcinoma of the Colon and Antecedent Epithelium, pp. 267-279 Springfield. Ill : Charles C Thomas. Publisher, 1970
7 Dunn. D. L., Lubet. R. A., and Prough. R A Oxidative metabolism of N-isopropyl-<r-(2-methylhydrazino)-p-toluamide hydrochloride (procarbazine)by rat liver microsomes Cancer Res., 39 4555-4563, 1979
8. Fiala. E. S. Investigation into the metabolism and mode of action of the coloncarcinogen, 1,2-dimethylhydrazine Cancer (Phila.). 36 2407-2412, 1975.
9. Fiala. E. S., Bobotas. G.. Kulakis. C., Wattenburg. L. W., and Weisburger.J. H. Effects of ili',iilfir.im and related compounds on the metabolism in vivoof the colon carcinogen, 1,2-dimethylhydrazine. Biochem Pharmacol.. 261763-1768, 1977.
10. Fiala, E. S., Bobotas, G., Kulakis, C., and Weisburger. J H. Inhibition of1,2-dimethylhydrazine metabolism by disulfiram Xenobiotica. 75-9. 1977
12. 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.
13. Haugen, D. A., and Coon, M. J. Properties of electrophoretically homogeneous phenobarbital-induced and ß-naphthoflavone-induced forms of livermicrosomal cytochrome P-450 J. Biol Chem.. 25Õ 7929-7939, 1976.
14. Prough. R. A.. Coomes. M. L.. and Dunn, D L The microsomal metabolismof carcinogenic and/or therapeutic hydrazine In: V Ullrich. I. Roots, A. G.Hildebrandt. R W Estabrook. and A H. Conney (eds ). Microsomes andDrug Oxidations. Vol. 3. pp. 500-507. New York: Pergamon Press. 1977.
15. Reed. D. J , and May. H. E Cytochrome P-450 interactions with the 2-chloroethylnitrosoureas and procarbazine. Biochimie (Paris) 60 989-995.1978
16. Schwartz, D. E.. Bollag. W.. and Obrecht. P. Distribution and excretionstudies of procarbazine in animals and man Arzneim Forsch.. ; 7. 1389-1393, 1967.
17. Tarn. S. W. Mass spectra of hydrazo. azo and azoxy compounds. In. S. Palai(ed.). The Chemistry of the Hydrazo. Azo and Azoxy groups, pp. 109-127.New York: John Wiley & Sons. Inc.. 1975.
18. Weinkam, R. J., and Shiba. D. A. Metabolic activation of procarbazine LifeSci.. 22. 937-946. 1978.
19. 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.
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