metabolism and binding of cyclophosphamide and its ... · ports from this laboratory have...

[CANCER RESEARCH 44, 4615-4621, October 1984]

Metabolism and Binding of Cyclophosphamide and Its Metabolite Acroleinto Rat Hepatic Microsomal Cytochrome P-4501

A. J. Mannello,2 S. K. Bansal, B. Paul, P. L. Koser, J. Love, R. F. Struck, and H. L. Gurtoo3

Grace Cancer Drug Center, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263 [A. J. M., S. K. B., B. P., P. L K., J. L, H.L. G.], and Southern Research Institute, Birmingham, Alabama 30305 [R. F. S.]

ABSTRACT

The hepatic cytochrome P-450-mediated metabolism and metabolic activation of [cWoroef/7y/-3H]cyclophosphamide ([cWoro-etfjy/-3H]CP) and [4-14C]cyclophosphamide ([4-14C]CP) were in

vestigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of [14C]acrolein, a metabolite of [4-14C]CP,

were also investigated.The metabolism of [c/J/oroef/7y/-3H]CP and [4-14C]CP to polar

metabolites was found to depend on the presence of NADPHand showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reducÃase.Kâ€žand

Vmaxvalues were essentially similar (Km,0.44 and 0.42 mW; Vâ„¢Â»,4.8 and 7.0 nmol of polar metabolites formed/min/nmol of cytochrome P-450 for [4-14C]CP and [cWoroef/iy/-3H]CP, respec

tively). The patterns of inhibition by microsomal mixed-functionoxidase inhibitors, anti-cytochrome P-450 antibody, and heatdenaturation of the cytochrome P-450 were essentially similar,with subtle differences between [4-14C]CP and [c/7/oroef/)y/-3H]-

CP metabolism. The order of inhibition by various mixed-functionoxidase inhibitors was SKF > a- and 0-naphthoflavones > me-

tyrapone.The in vitro metabolic activation of CP in the reconstituted

system demonstrated predominant binding of [cWoroef/7y/-3H]-CP to nucleic acids and almost exclusive binding of [4-14C]CP to

proteins. Gel electrophoresis-fluorography of the proteins in thereconstituted system treated with [4-14C]CP demonstrated localization of the 14C label in the cytochrome P-450 region. To

examine this association further, hepatic microsomes were modified with [14C]acrolein in the presence and the absence of

NADPH. The results confirmed covalent association between[14C]acrolein and cytochrome P-450 in the microsomes and alsodemonstrated further metabolism of [14C]acrolein, apparently to

an epoxide, which is capable of binding covalently to proteins.The results of these investigations not only confirm the signif

icance of primary metabolism but also emphasize the potentialrole of the secondary metabolism of cyclophosphamide in someof its toxic manifestations.

INTRODUCTION

CP4, an oxazaphosphorine derivative of the classical alkylating

Received November 3,1983; accepted June 25,1984.1This work was supported by USPHS Grants CA-23634 and CA-24538 from

the National Cancer Institute.2A portion of this work was submitted to State University of New York, Buffalo,

in partial fulfillment of the requirement for the Ph.D. degree in Pharmacology.Present address: Albany Medical College, Albany, NY 11209.

3To whom requests for reprints should be addressed.4The abbreviations used are: CP, cyclophosphamide; SKF-525A,2-diethylamino-

ethyl-2,2-diphenylvalerate; [c/7/oroefAiy/-3H]CP, [c/itoroef/>x/-3H]cyclophospharnide;

agent nitrogen mustard, is used commonly in the treatment of avariety of neoplastic diseases (8). In addition, because of itsimmunsuppressive properties, CP has also found use in thetreatment of diseases thought to be of autoimmune etiology (7).CP, inactive per se, requires metabolic activation by the hepaticmicrosomal cytochrome P-450-dependent mixed-function oxidases (Chart 1). In addition to the microsome-mediated biotrans

formation, cytosolic reductases/oxidases also participate in thedetoxification of activated metabolites of CP (4,17).

Since the biological effects of CP are believed to result fromthe binding of activated metabolites to tissue macromolecules,during the past several years, we have investigated the microsomal metabolism-mediated binding of CP to macromolecules,

especially to proteins and nucleic acids. Such microsomal preparations are known to contain several different forms of cytochrome P-450 (10, 11, 23). These different cytochromes P-450,

while possessing some overlapping substrate specificities, areknown to have widely differing specificities in the metabolism ofvarious chemicals (10,11, 23). To define more clearly the role ofCP metabolism in the toxicity of CP, we have now used a purifiedcytochrome P-450 reconstituted system to study the metabolism

and metabolic activation of CP labeled in the chloroethyl sidechain with 3H and in the ring with 14C.These investigations have

helped to delineate the specificity of the macromolecular binding,especially to cytochrome P-450, of various metabolites of CP.

Our previous reports have suggested that the predominantbinding of [4-14C]CP to microsomal proteins is most likely due tothe binding of [14C]acrolein produced during the metabolism of

CP (14, 16). Acrolein, a highly reactive olefinic aldehyde, mayitself undergo microsome-mediated metabolic activation, possi

bly to acrolein oxide (27). Acrolein, a toxic chemical (6, 19, 20,25), has been identified recently as the causative agent of CP-

associated hemorrhagic cystitis (2, 3, 5). Moreover, earlier reports from this laboratory have implicated acrolein as the metabolite of CP responsible for the inhibition and denaturation ofhepatic microsomal cytochrome P-450 (16, 24). In the presentreport, using [14C]acrolein and [4-14C]CP, we have also: (a) made

comparative evaluation to define the role of acrolein in the bindingof 14C label from [4-14C]CP to hepatic microsomal cytochrome

P-450; and (b) investigated further metabolism of acrolein toprotein-binding metabolites.

MATERIALS AND METHODS

Chemicals. CP (4-14C, ring-labeled) with a specific activity of 11.25

mCi/mmol was purchased from New England Nuclear (Boston, MA).[Chloroethyl-3H]CP with a specific activity of 440 mCi/mmol was obtainedfrom Amersham/Searle Corp. (Arlington Heights, IL). Both [4-14C]CP and

[c/7/oroef/jy/-3H]CP were repurified prior to use as described previously

[4-uC]CP, [4-14C]cydophosphamide; SDS-PAGE, sodium dodecyl sulfate-polyac-rylamide gel electrophoresis; [1,3-14C]acro)ein, [14C]acrolein.

OCTOBER 1984 4615

on March 15, 2020. © 1984 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

A. J. Mannello et al.

CHIy' CICH.Ci

CP=Cyclophosphamide

P--Phosphomide

M'N(CH2CH2CI)2

Acrolein

Chart 1. Metabolism of CP by hepatic microsomes and cytosdic enzymes.

(14,16). Sources of other chemicalshave been described previously (2,14,16,17,24).

Animal Treatment Male Sprague-Oawleyrats (200 to 250 g), obtained from Marian Sprague-Dawley Industries (Indianapolis, IN) wereused for the isolation of hepatic microsomes. Animals, housed 4 in acage, receivedcommerciallyavailablerat chow and water ad libitum untilthe day of sacrifice.

Animals were given sodium phÃ©nobarbitalas a 0.1% solution (w/v) intheir drinking water for 7 days prior to sacrifice. Hepatic microsomeswere isolated as described previously (16, 24), and protein was determined by the method of Lowry ef al. (22) with bovine serum albumin asthe referencestandard.

Preparation of Purified Microsomal Enzymes. CytochromeP-450and NADPH-cytochromeP-450 reducÃasewere purified from the hepaticmicrosomes of phenobarbital-treated rats according to the method ofGuengerichand Martin (13). Only "Peak Bj" (see Ref. 13) was found tohave significant activity toward CP, either [cÂ«oroef/jy/-3H]CPor [4-14C]-CP, and this peak, which appeared as a single band on SOS-PAGE,was, unless otherwise stated, utilized in the metabolismstudies.

The purified cytochrome P-450 had a specific activity of 14 to 17.5nmol/mg of protein. The protein migrated as a single protein speciesonSDS-PAGE and had an apparent M, of 52,000. The purified NADPH-cytochrome P-450 reducÃasehad a specific activity of 57 /*mol ofcytochrome c reduced per min per mg of protein and had an apparentM, of 75,000.

Metabolismof CP bythe ReconstitutedCytochromeP-450 System.The metabolismof [4-14C]CPand [cWoroef/iy/-3H]CPto aqueous metab

olites was studied in the reconstituted system consisting of purifiedcytochrome P-450, purified NADPH-cytochrome P-450 reducÃase,andsynthetic lipid, dilauroylphosphatidylcholine(Serdary Research Laboratory, Hamilton, Ontario, Canada). Unless otherwise stated in the chartlegends, a typical incubation mixture in phosphate buffer (0.09 M, pH7.4) contained the following: the NADPH-generatingsystem (14,16), 300nmol of labeled cyclophosphamide, 0.25 nmol of cytochrome P-450, 1unit NADPH:cytochromeP-450 reducÃase(1 unit = 1 Â¿tmolof cytochromec reduced/min) and 10 ng of dilauroylphosphatidylcholine in a totalvolume of 1 ml. The reconstituted enzyme system (cytochrome P-450,the reducÃase,and dilauroylphosphatidycholine)was preincubatedfor 5min al 37Â°in ihe presence of Ihe subslrate. The reaction was initialedby Ihe addilion of the NADPH-generatingsystem (incubated previouslyfor 15 min al 37Â°),and Ihe incubation was continued for 30 min al 37Â°.

Conlrol incubations contained all of Ihe above except NADPH. Thereaction was slopped by Ihe addition of 6.0 ml of chloroform:isoamylalcohol (95:5), followed by 1 ml of waler. The lubes were capped, Ihecontents were mixed vigorously on a vortex mixer, and Ihe lubes werecentrifugea for 5 min al 2000 rpm. One ml of Ihe aqueous layer wascounted in a Beckman scintillation counter lo determine Ihe formation of

NADPH-medialedaqueous metabolites of [4-14C]CPor [cÂ«oroef/7y/-3H]-

CP. After subtraction of Ihe background, Ihe enzymatic activity is expressed as nmol of [c/)/oroefAiy/-3H]CPor [4-14C]CPrelated aqueousmetabolites formed per nmol of cyfochrome P-450 per min.

Whilephosphoramidemuslard and nor-nilrogen muslard (degradationproduct of phosphoramide mustard) would be among the major metabolites in chloroform-extracted aqueous phase of Ihe incubation mixture,other polar metabolites remaining in the aqueous phase could includesome acrolein and dechlorinated metabolites of CP reported lo beproduced by sheep (1). However, as reported previously (17), Ihe easewilh which Ihe polar metabolitesof CP can be measuredas radioactivityin Ihe aqueous phase has provided a rapid assay for detailed investigations of Ihe enzymology of CP melabolism catalyzed by Ihe purifiedcylochrome P-450 in ihe reconsliluled system.

Bindingof [c/itoroeÂ«iy/-3H]CP,[4-14C]CP,and [14C]Acroleinto Proteins. The bindingof [14C]acroleinper se and/or Ihe metabolism-mediated binding of [c/?/oroei/)y/-3H]CP,[4-14C]CP,and [14C]acroleinto

proteins were sludied wilh Ihe incubation mixlures containing eitherhepatic microsomes or Ihe reconstituted system.

Unless otherwise staled, Ihe incubation mixlures for studies on thebindingof [14C]acroleinand [4-14C]CPto hepatic microsomescontained:

0.09 Mpotassium phosphate buffer, pH 7.4; 5 HIMMgCI2;1.4 ITIMMnCI:-;0.54 mg of hepatic microsomal protein, NADPH generating system (14,16); 65 nmol of acrolein or 150 nmol of CP (final volume of incubationmixture, 0.5 ml). The metabolismof [cWoroeÂ«7y/-3H]CPand [4-14C]CPlo

protein binding metabolites was also sludied in Ihe reconstituted cylochrome P-450 system. The incubation mixlure was similar lo lhal described for sludies on the metabolism of CP to polar metabolites. Thereaction mixture for either type of incubation was incubated for 30 minat 37Â°.At the end of Ihe incubation, proteins were precipitated from the

incubationmixlure by Ihe addilion of 4 ml of cold 5% trichloroaceticacid.The resulting protein pellet was washed with cold 5% perchloric acidfollowed by elhanol and air dried, digesled, and counted as describedbefore (16,17).

Bindingof [cÂ«oroef/iy/-3H]CPand [4-14C]CPto NucleicAcids Catalyzed by the CytochromeP-450 ReconstitutedSystem. The bindingof [cÂ«oroef/7y/-3H]CPand [4-14C]CPto Escherichia coli IRNA was sludied wilh Ihe reconsliluled cylochrome P-450 activation system. A typicalincubation mixture described earliercontained in addition 2 mg of Â£coliIRNA. This mixlure (wilhoul Ihe nucleic acid)was preincubatedfor 5 minal 37Â°,ihe nucleic acid was added, and ihe reaction was initialed by ihe

addilion of NADPH (0.12 ITIM).Conlrol incubation mixlures were exactlysimilar excepl lhal NADPH was omitted. The reaction was allowed toproceed for 2 hr al 37Â°.At various time points during Ihe incubation (20,

40, 60, and 80 min), Ihe reaction was supplemented by Ihe addilion ofNADPH(0.06 mÂ«)and cytochrome P-450 (0.25 nmol).The reaction wasstopped by Ihe addition of 1.0 ml of 0.1 M potassium phosphate buffer

4616 CANCER RESEARCH VOL. 44



Metabolism and Binding of CP and Acrolein to Cytochrome P-450

(pH 7.4) and 7.5 ml of chloroform:isoamyl alcohol (95:5). The mixturewas extracted, and the aqueous phase was reextracted with chloro-form:isoamyl alcohol, transferred to 25-ml Ehrlenmyer flasks, cappedwith parafilm, and incubated for 16 hr at 37Â°.The samples were subse

quently dialyzed for 24 hr against 8 liters of 25 HIMpotassium phosphatebuffer (pH 7.4). The nucleic acids were then precipitated and washedfree of unbound radioactivity, and aliquota were taken for radioactivityand 260 nm absorbance measurements as described before (16, 17).The data are reported as pmol of metabolite bound per mg of nucleicacid.

Gel Electrophoresis and Fluorography. Cytochrome P-450 treatedwith either [14C]acrolein, [4-14C]CP, or [c/)/oroefr/y/-3H]CP in the presence

or absence of NADPH in the reconstituted system was subjected toSDS-PAGE essentially according to the method of Laemmli (21). Prior to

solubilization for gel electrophoresis, the proteins in the incubation mixture were precipitated as follows: the incubation mixture was dialyzed at4Â° for 24 hr against 2 liters of 50 PIM Tris-acÃ©tate buffer (pH 7.4)

containing 20% glycerol and 0.1 mu EDTA and then for an additional 24hr against distilled water. The protein, which precipitated during dialysisagainst distilled water, was collected by centrifugation, solubilized, andsubjected to gel electrophoresis. After electrophoresis, the gels wereprocessed as follows. The gel was immersed in 100 ml of Enhance (NewEngland Nuclear) for 1 hr at room temperature, and it was subsequentlywashed for 45 min with cold tap water prior to drying the gel. Photographic film (Kodak SB-5) was placed over the dried gel to visualize the

radiolabeled proteins.Preparation of Antibody against the Cytochrome P-450. Rabbit

antibody was prepared against the purified phenobarbital-induced cytc-chrome P-450 as described by Thomas ef a/. (31). The IgG fraction from

the immunized sera was isolated as described by Guengerich (9).

Measurement of radioactivity remaining in the chloroform-extracted aqueous phase following the metabolism of [chloro-ethyl-3H]CP or [4-14C]CP indicated that more 3H label than 14C

label was recovered in the aqueous phase. These results aresimilar to those reported previously for the hepatic microsomes(14, 16). The formation of the activated and polar metabolitesdepended upon the presence of NADPH and Cytochrome P-450

since, in the absence of either, very little metabolite formationoccurred.

In order to confirm that protein binding 14Clabel from [4-14C]-

CP was associated with cytochrome P-450 protein, we separated the proteins in the reconstituted system by SDS-PAGEfollowing the metabolism of [4-14C]CP and visualized the radio-

labeled proteins by fluorography. As shown in Fig. 1, the greatestamount of protein-bound radioactivity migrated in one region of

the autoradiogram. This is the area of migration of cytochromeP-450, since comparable experiments in which the proteins werestained have shown that cytochrome P-450 migrates in thisregion of the gel. Essentially no binding was observed when

14C-Cyclophosphomide

-NAOPH NADPH

RESULTS

Metabolism and Binding of CP. As shown in Table 1, themetabolism of [4-14C]CP and [cWoroef/jy/-3H]CP by the recon

stituted cytochrome P-450 system (consisting of purified cytochrome P-450, purified NADPH-cytochrome P-450 reducÃase,

dilauroyl phosphatidycholine, and NADPH) resulted in the formation of protein binding, nucleic acid binding, and polar metabolites. Over 85-fold greater binding of the 14Clabel to cytochromeP-450 protein was observed compared to the binding of the 3H-label. However, the pattern of binding of [c/i/oroef/jy/-3H]CP and[4-14C]CP to nucleic acids was nearly completely reversed. The

metabolism of CP resulted in greater binding (10-fold) to E. colitRNA of the 3H label than that of the 14C label. Similar results

were obtained with calf thymus DNA (data not shown).

CytochromeP-450

Table 1Metabolism of [chloroethyl-aH]CP and [4-"C]CP to polar, protein-binding, and

nucleic acid-binding metabolites catalyzed by a reconstituted purified cytochromep-450 system

Results are shown of 3 different experiments in which different batches of thecytochrome P-450 were used to measure the formation of polar, protein-bound,and nucleic acid-bound metabolites. Each value is a mean of 2 or 3 determinations.

pmol of metabolite bound Polar metabolites(pmol of labeled

Substrate[4-"C]CP[c/itoroef/iy/-3H]CP

To protein (per To nucleic acid metabolites pronmol of cyto- (per mg of Â£. duced/min/nmol of

chrome P-450) coli tRNA) cytochrome P-450"

17,200203

1701,640

3,2608,730

* The incubation mixture was extracted with chlorofomrisoamyl alcohol (95:5),

and the radioactivity remaining in the aqueous phase was used to calculate thequantities of aqueous (polar) metabolites derived from [c/i/oroef/)y/-3H]CP and |4-14C]CP.

Fig. 1. Gel electrophoresis-fluorography of purified cytochrome P-450 treatedwith [4-"C]CP in the reconstituted system. Purified cytochrome P-450 was incubated in the reconstituted system with (4-"C|CP in the presence and absence of

NADPH. The proteins from the incubation were precipitated and subjected to gelelectrophoresis. Gels with separated radiolabeled proteins were subjected tofluorography. Details are described in the text.

Similar results [reported previously (15)] were obtained when purified cytochromeP-450 in the reconstituted system was treated with [14C]acrolein in the absence ofNADPH. That is, the major band of radioactivity traveled with cytochrome P-450.

OCTOBER 1984 4617




NADPH was omitted from the reaction mixture. In separateexperiments, essentially identical results, i.e., binding of the 14C

label to cytochrome P-450, were obtained when purified cyto-chrome P-450 was treated with [14C]acrolein in the absence of

NADPH. In these experiments, gel electrophoresis-fluorography

of the modified proteins demonstrated that major band of radioactivity is associated with cytochrome P-450. These results,

reported previously (15), are in agreement with our earlier suggestion (14, 16) that the CP metabolite binding to cytochromeP-450 is acrolein.

Kinetic Analysis of [4-"C]CP and [cWoroetfiy/-3H]CP Me

tabolism. Optimum conditions for cytochrome P-450-catalyzedformation of the polar metabolites of [4-14C]CP and [chloroethyl-3H]CP were determined with respect to incubation time, amount

of purified cytochrome P-450, and the amount of NADPH-cyto-chrome P-450 reducÃase. Optimization experiments conductedwith [4-14C]CP and [chloroethyl-3H]CP as the substrate yielded

nearly identical results. Therefore, optimal conditions (e.g., 30min of incubation at 37Â°,0.25 nmol of cytochrome P-450,4 units

of reductase/nmol of cytochrome P-450) defining a linear rate of

reaction were used in subseuqent studies carried out to measurekinetic parameter of [c/?/oroef/7y/-3H]CP and [4-14C]CP metabo

lism.In order to determine whether there are qualitative differences

for the catalysis of [c/7/oroefrty/-3H]CP and [4-14C]CP in the

reconstituted system, Km and Vma,values were measured. Themetabolism data derived for both [4-14C]CP and [chloroethyl-3H]CP could be fitted to linear Lineweaver-Burke plots (Chart 2);[4-14C]CP exhibited a Km of 0.44 HIM and maximum velocity of

4.8 nmol of aqueous metabolites formed/nmol of cytochrome P-450/min, while [c/?/oroef/7y/-3H]CP exhibited similar Kâ„¢and Vm

values (0.42 mw and 7.0 nmol of aqueous metabolites formedper nmol of cytochrome P-450 per min, respectively). In addition,there was an excellent correlation (r2 = 0.99) between the

reciprocals of product formation and substrate concentration inthe Lineweaver-Burk plots for both [4-14C]CP and [chloroethyl-3H]CP metabolism.

16

15

12

VHAX-48nmoi / nmolCytochromeP450/minKy= 044mMr* â€¢0.99

10

CYCLOPHOSPHAMIDE u k

16 20

Effects of Inhibitors on [4-14C]CP and [c/i/oroethy/-3H]CP

Metabolism by the Reconstituted Cytochrome P-450 System.SKF-525A and metyrapone are known inhibitors of mixed-func

tion oxidase enzyme activities induced by phÃ©nobarbital andsimilarly acting chemicals, whereas naphthoflavones (a and ÃŸ)are potent inhibitors of the enzyme activities induced by 3-

methylcholanthrene and similarly acting chemicals (26,30). Studies were conducted to determine the effects of these inhibitorson the metabolism of both [4-14C]CP and [c/)/oroefriy/-3H]CP to

aqueous metabolites. The data are presented in Chart 3. All theinhibitors tested, SKF-525A, metyrapone, a-naphthoflavone and/3-naphthoflavone, produced a concentration-dependent inhibition of the metabolism of both [cWoroeffty/-3H]CP and [4-14C]-

CP, with minor differences between the 2 labels. The order ofpotency was SKF-525A > a- and 0-naphthoflavones > metyra

pone.Effect of Anti-Phenobarbital-induced Cytochrome P-450

Antibody on the Metabolism of [4-14C]CP and [chloroethyl-3H]CP by the Reconstituted Cytochrome P-450 System. TheIgG antibody raised against the cytochrome P-450 was determined to be specific by Ouchterlony double-diffusion analysisagainst the cytochrome P-450 (Peak B2) isolated from pheno-barbital-treated rats. This antibody was tested for its ability toinhibit [cWoroef/7y/-3H]CP and [4-14C]CP metabolism. The anti-

METYRAPONE

-NAPHTHOFLAVONE

[INHIBITOR]M

METYRAPONE

B

Chart 2cytochromemetabolism.

Lineweaver-Burk plot of [4-14C]CP metabolism by the reconstitutedP-450 system. A similar plot was obtained for [c/itoroef/7y/-3H]CP

Other details are described in the text.

[INHIBITOR]M

Charts. Effects of inhibitors of microsomal mixed-function oxidases on themetabolism of [4-14C]CP (A) and [c/i/oroe%/-3H]CP (B) by the reconstituted cyto-

chrome P-450 system. Reaction conditions are as described in the text. Controlvalues for [4-"C]CP and [cft/oroef/iy/-3H]CP polar metabolites generated were 70nmol of polar metabolites/nmol of cytochrome P-450 and 97 nmol of polar metab-olites/nmol of cytochrome P-450, respectively.





cytochrome P-450 antibody produced a concentration-dependent inhibition of [4-14C]CP metabolism, reaching a complete

inhibition at an antibody concentration of 1 mg. The anti-cyto-chrome P-450 antibody also produced a concentration-dependent inhibition of [c/j/oroefrÂ»y/-3H]CPmetabolism; however, at an

antibody concentration of 1 mg, the metabolism of [chloroethyl-3H]CP was inhibited by only 70%. These results show subtledifferences in the inhibition of the metabolism of [4-14C]CP and[cWoroef/7y/-3H]CP by the anti-cytochrome P-450 antibody.

Thermolability of the Purified Cytochrome P-450 Protein. Inthis experiment, cytochrome P-450 was preincubated for varioustimes (5,10, or 15 min) at varying temperatures (37, 42, or 50Â°).

After the incubation period, the remaining components of thecomplete reaction mixture were added to the cytochrome P-450,and the cytochrome P-450 was tested at 37Â°for its ability tosupport the metabolism of [cWoroef/7y/-3H]CP and [4-14C]CP to

aqueous metabolites. When cytochrome P-450 was preincubated at 37Â° for 5, 10, and 15 min, there was a 5 to 20%decrease in the metabolism of either [4-14C]CP or [chloroethyl-3H]CP. At 42Â°,the metabolism of [4-14C]CP or [cAi/oroef/jy/-3H]-CP was decreased by 10 to 20%. However, at 50Â°,there was a

time-dependent loss of activity, reaching about 80% at 15 minfor both [4-14C]CP and [c/7/oroef/7//-3H]CP metabolism.

Effect of Thiols on the Binding of Acrolein. We have reportedpreviously that compounds containing free sulfhydryl group(s)block the binding of 14Clabel from [4-14C]CP to microsomes (14,

16). To further support our hypothesis that these chemicalsblocked the binding by interacting with acrolein formed during[4-14C]CP metabolism, we studied the effects of compounds

containing a free sulfhydryl and/or a free amino group on thebinding of [14C]acrolein to the hepatic microsomes. The results

of this study are given in Table 2. For comparison, we haveincluded data on the binding of [4-14C]CP in this table. Of the

chemicals tested, glutathione was the most potent in blockingthe binding of [14C]acrolein and 14C label from [4-14C]CP; thebinding of both [14C]acrolein and [4-14C]CP was inhibited by

90%. Cysteine, which was a very potent inhibitor of the bindingof 14Clabel from [4-14C]CP, was equally effective in preventingthe binding of [14C]acrolein. A/-Acetylcysteine was somewhat

less effective than was cysteine in preventing the binding of theradiolabel from both sources. Lysine was only marginally effective in blocking the binding either of [14C]acrolein or of 14Clabelfrom [4-14C]CP.

Mixed Function Oxidase-mediated Activation of Acrolein.The above studies on the binding of [14C]acrolein to microsomes

were done in the absence of NADPH. Therefore, it was of interestto determine whether metabolism-mediated binding of [14C]ac-

Tabte2Binding of ["C]acrolein and [4-"C]CP to hepatic microsomes isolated from

phenobarbital-treated rats

Treatment (4mm)None

GlutathioneCysteineW-AcetylcysteineLysine[14C]Acrolein

bound

as % ofcontrol(-NADPH)"100

10182888[4-14C]CP

bound as

% of control(+NADPH)6100

101230

105

rolein to microsomal proteins occurred. Metabolism of [14C]-

acrolein was assayed with microsomes isolated from phenobarbital-treated rats, and the results of this study are given in Table3. While [14C]acrolein per se in the absence of metabolism

showed significant binding to the microsomes, this binding wassubstantially enhanced in the presence of NADPH, suggestingthat a metabolite of acrolein was also binding to the microsomes.The enhancement in the binding of [14C]acrolein is clearly metab

olism-dependent, since the presence of 1 mw SKF-525A com

pletely abolished the difference in the binding observed betweenplus and minus NADPH. In addition, compared to phenobarbital-

treated microsomes, control microsomes did not generate asubstantial amount of the NADPH-mediated metabolite (e.g.,2707 pmol of metabolite/mg of protein versus 23 pmol of metab-

olite/mg of protein).

DISCUSSION

CP Metabolism by Cytochrome P-450: Kinetics and Inhibi

tion. Since it was of interest to study whether the differentmoieties of the CP molecule are metabolized similarly by cytochrome P-450, we studied the metabolism of [c/7/oroef/7y/-3H]CPand [4-14C]CP in the reconstituted cytochrome P-450 system.Any observable differences between the metabolism of [4-14C]-CP and [c/7/oroefA)y/-3H]CP could possibly be detected by meas

uring the following: kinetic constants (Kâ€žand Vâ„¢Â»),sensitivity toknown mixed function oxidase inhibitors, effects of heat dena-turation of cytochrome P-450, and/or sensitivity to anti-cytochrome P-450 antibody.

Lineweaver-Burke analysis of the metabolism of [4-14C]CPand [c/)/oroef/7y/-3H]CP revealed similar Km values for the for

mation of the labeled polar metabolites. The Michaelis constant(Kn,) was 0.42 mw and 0.44 rriM for [c/7/oroefrjy/-3H]CP and [4-14C]CP, respectively. These values obtained for the metabolism

of the differentially labeled CP by the reconstituted system arevery similar to the Km values reported for CP metabolism bymicrosomes from phenobarbital-treated rats (28). Sladek (28)reported that hepatic microsomes from phenobarbital-treated

Table 3Metabolism-mediated binding of [i4CÂ¡acrolein to hepatic microsomes isolated

from phenobarbital-treated rats

As shown in Table 2, acrolein per se binds to proteins. The amount of themetabolite bound Â¡scalculated as the difference between incubations carried outin the presence and absence of NADPH. Values represent radioactivity bound overand above the binding of acrolein per se. Each value is a mean of duplicate values.The concentration of acrolein was as follows: Experiment 1, 0.130 HIM; Experiment2, 0.1 mm.

NADPH-mediated ["Cjacrolein me

tabolite bound to microsomes(pmol of metabolite bound/mg of

protein)

Treatment8 Experiment 1 Experiment 2

* Control value for acrolein alone averaged 37 nmol of [14C]acrolein bound/mg

of protein.6 Control value for CP alone averaged 45 to 55 nmol of [4-14C]CP metabolite

bound/mg of protein.

(PBMCS6 + NADPH) - (PBMCS - 2707 (100)c 5700 (100)

NADPH)(PBMCS + NADPH + 1 muÃSKF-525A) 6 (0.2) <6 (<0.1)

- (PBMCS - NADPH + 1 mm SKF-

525A)(MCS + NADPH) - (MCS - NADPH) 23 (4.5) <10 (<0.1)

''in Experiment 1, rats received phÃ©nobarbitalin drinking water (0.1%) for 1

week prior to sacrifice. In Experiment 2, rats received 7 i.p. injections of PB (40mg/injection/kg) over a period of 4 days, and the rats were killed on Day 5.

PBMCS, hepatic microsomes from phenobarbital-treated rats; MCS, hepaticmicrosomes from saline-treatedcontrols.

c Numbers in parentheses, percentageof control value.

OCTOBER 1984 4619




rats had a Kâ„¢value for CP metabolism of 0.57 mw and that theK,,, value for untreated microsomes was 1.39 HIM, suggesting,therefore, that phÃ©nobarbitalpretreatment results in the formation of cytochrome(s) P-450 which exhibit greater affinity for CP

substrate.We studied the effects of inhibitors of mixed function oxidase-

mediated reactions on the metabolism of [4-"C]CP and [chlo-roethyl-3H\CP. Inhibitors with preference toward certain types of

induced forms of cytochrome P-450 have become importanttools in differentiating between variants of the hemoprotein. SKF-525A and metyrapone preferentially inhibit mixed-function oxidase activities induced by phÃ©nobarbital and similarly-actingchemicals, whereas 7,8-benzoflavone (a-naphthoflavone) and5,6-benzoflavone (/3-napthoflavone) are potent inhibitors of 3-methylcholanthrene-inducible mixed function oxidase activities

(26, 30).While all 4 inhibitors tested produced inhibition of the metab

olism of [c/7/oroef/jy/-3H]CP and [4-14C]CP in a concentration-

dependent manner, SKF-525A was by far the most potent inhibitor. This observation further strengthens the report that pheno-barbital-inducible cytochrome P-450 preferentially metabolizes

CP (14), and these results are in accord with the results of Sladek(29) on the microsomal metabolism of CP.

Activation of CP by the Cytochrome P450 ReconstitutedSystem. The data presented in Table 1 clearly demonstrate thatbinding of the 14C label from [4-14C]CP to cytochrome P-450protein exceeds that of the 3H label from [cWoroef/jy/-3H]CP, andthat the binding of the 3H label to E. coli tRNA exceeds thebinding of the "C label. A similar pattern of binding of the 14Clabel was observed when |14C]acrolein in the absence of metab

olism was used to modify nucleic acids and cytochrome P-450

protein (data not shown). These results suggest that differentmoieties of the CP molecule bind to proteins and nucleic acidsby different mechanisms. As shown in Chart 1, during themetabolism of [c/?/oroef/jy/-3H]CP, the radioactivity would be

retained in phosphoramide mustard which, being a very reactivealkylating agent, would avidly bind to nucleophiles located innucleic acids (7). On the other hand, the metabolism of [4-14C]-

CP would be expected to yield radiolabeled acrolein, a veryreactive unsaturated aldehyde which denatures cytochrome P-

450 by possibly alkylating critical sulfhydryl groups (16, 24). Ourdata discussed in context of the metabolic scheme presented inChart 1 suggest that predominant binding of the 3H label tonucleic acids and selective binding of the "C label to cytochrome

P-450 proteins are presumably due to the binding, respectively,

of phosphoramide mustard and acrolein. These suggestions arein accord with our earlier observations with hepatic microsomes(24) and our previous suggestion that the denaturation of cytochrome P-450 by CP is due to the binding of acrolein to criticalsites located at or near the active site of cytochrome P-450 (20).

Binding of Acrolein to Cytochrome P-450 and the Effect of

Thiols. The data presented here are in agreement with our earliersuggestion that acrolein, a metabolite of the anticancer agentCP, binds to cytochrome P-450 via alkylation of sulfhydryl groups

located in the apoprotein (16). Acrolein contains a reactive,aldehyde-conjugated double bond with a strong reactivity toward

free sulfhydryl groups (18). The treatment of hepatic microsomesand/or cytochrome P-450 in the reconstituted system (15) with[14C|acrolein resulted in the covalent binding of the radiolabeled

acrolein to proteins (Table 2). In particular, most of the protein-

bound radioactivity migrated in the region of the autoradiogram

where cytochrome P-450 migrates. The effects of various chemicals (Table 2) on the binding of (14C]acrolein to microsomes are

essentially identical to the effects of these chemicals on thebinding of NADPH-mediated [4-14C]CP metabolite; these resultssuggest that the binding of the "C label from [4-14C]CP is due

to the binding of acrolein generated during the metabolism of [4-14C]CP.

It has been reported that acrolein can undergo mixed-functionoxidase-mediated metabolism, which could possibly result in the

production of acrolein oxide (glycidaldehyde) (27). In our studies,we observed a metabolism-dependent binding of [14CJacroleinto

microsomes over and above the binding observed in the absenceof NADPH (Table 3). Our results suggest that the increasedbinding of [14C]acrolein to hepatic microsomal proteins in the

presence of NADPH could result from the binding of the radio-

labeled oxide metabolite reported to be formed during the metabolism of acrolein and acrylonitrile (12, 27).

In conclusion, diverse lines of evidence suggest that: (a) ac-rolein-induced destruction of cytochrome P-450 results from thealkylation of critical sulfhydryl groups located in cytochrome P-

450; (b) acrolein in the presence of NADPH and mixed functionoxidase is capable of further metabolism to a reactive metabolite,presumably an alkene oxide, which can alkylate proteins inmicrosomes; and (c) acrolein is the metabolite of CP responsiblefor some of the toxic effects associated with the parent compound.

ACKNOWLEDGMENTS

The authors would like to thank Karen Mane Schrader for her assistance in thepreparation of this manuscript.

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1984;44:4615-4621. Cancer Res A. J. Marinello, S. K. Bansal, B. Paul, et al. P-450Metabolite Acrolein to Rat Hepatic Microsomal Cytochrome Metabolism and Binding of Cyclophosphamide and Its

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