cyclophosphamide modulates rat hepatic cytochrome p450 ... · ifosphamide (a) suppressed the...

[CANCER RESEARCH 53. 2490-2497. June I. 19931

Cyclophosphamide Modulates Rat Hepatic Cytochrome P450 2C11 and Steroid5tt-Reductase Activity and Messenger RNA Levels through the CombinedAction of Acrolein and Phosphoramide Mustard1

Thomas K. H. Chang and David J. Waxman2

Department Â¡ifBioluaical Chemistry and Molecular Phunnaciilogy ana Dana-Furher Cancer Institute, Hanard Medical Schuol. Bustini. Massachusetts 02115

ABSTRACT

Cyclophosphamide treatment of adult male rats leads to sustained decreases in several liver microsomal cytochrome P450 (CYP) activities,including CYP2C11-catalyzed Cyclophosphamide activation, via a process

that is associated with a feminization of the overall pattern of liver enzymeexpression (G. A. LeBlanc and D. J. Waxman, Cancer Res., 50: 5720-5726,1990). The present study compares the effects of Cyclophosphamide and itsisomerie analogue ifosphamide on the gender-dependent expression ofhepatic CYP 2C11 and steroid Sa-reductase in adult male rats and alsoexamines the role of the Cyclophosphamide metabolites acrolein and phos-phoramide mustard in feminizing the expression of these liver enzymes.Ifosphamide (a) suppressed the male-specific CYP 2C11 mRNA and CYP2C11-catalyzed liver microsomal testosterone 2a-hydroxylation and Cyclophosphamide and ifosphamide 4-hydroxylation and (b) elevated the female-dominant liver enzyme steroid 5a-reducta.se and its mRNA 7-9 daysafter drug treatment, both occurring in a manner similar to that of cy-

clophosphamide, but requiring a 50% higher dose (180 mg/kg, single i.p.injection) to achieve these effects. This pattern of response could not beachieved by treatment of rats with acrolein or with Cyclophosphamideanalogues that decompose to acrolein without formation of phosphora-miilf mustard. In contrast, phosphoramide mustard treatment (100 mg/kg) did modulate microsomal CYP 2C11 and steroid 5a-reductase activ

ities. Treatment with a lower dose (50 mg/kg) of phosphoramide mustardor with the acrolein precursor 4-hydroperoxydechlorocyclophosphamide

(200 mg/kg) alone did not affect liver enzyme expression, whereas thecombination of these agents produced an overall pattern of response thatwas similar to that conferred by Cyclophosphamide. These studies establish that ifosphamide is less potent than Cyclophosphamide in modulatingthe pattern of cytochrome P450 and steroid 5<x-reductase expression and

that phosphoramide mustard is responsible for the modulation of liverenzyme expression by Cyclophosphamide, with acrolein potentiating themodulating activity of the mustard.

INTRODUCTION

Cyclophosphamide is a widely used anticancer alkylating agentprodrug that is bioactivated by the liver CYP3 monooxygenase system

( 1). Three specific cytochrome P450 enzymes, forms CYP 2BI (phÃ©nobarbital inducible). CYP 2C6 (constitutively expressed and genderindependent), and CYP 2C11 (constitutively expressed and male specific),4 have been identified as major catalysts of Cyclophosphamide

activation in rat liver (2). The primary metabolite formed by theseenzymes. 4-hydroxycyclophosphamide. equilibrates with the ring-

opened aldophosphamide. which undergoes spontaneous decomposition to yield phosphoramide mustard and acrolein ( 1). Phosphoramide

Received 12/21/92: accepted 3/24/93.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by Grant CA-49248 from the NIH (D. J. W.). T. K. H. C. was

supported by a Canadian Association of Gastroenterology/Janssen Research Fellowship(1991-1992) and a Canadian Liver Foundation Research Fellowship (1992-1993).

- To whom requests for reprints should he addressed, at Dana-Farber Cancer Institute.Room JF-525. 44 Binney Street. Boston. MA 02115.

1The abbreviations used are: CYP. cytochrome P450: HPD-cyclophosphamide.

4-hydroperoxydechlorocyclophosphamide; deCl-cyclophosphamide. bisfethyllaminocy-clophosphamide.

4 Individual liver CYP forms are designated according to the systematic nomenclature

(48).

mustard possesses DNA-alkylating activity and is generally consid

ered to be the therapeutically significant, cytotoxic metabolite ofCyclophosphamide (1,3). Acrolein. which is an electrophilic aldehyde,lacks antitumor activity (4. 5) but is highly reactive and binds co-valently to proteins, including cytochrome P450 (6-9) and NADPH-

cytochrome P450 reducÃase(10), leading to enzyme inactivation. TheCyclophosphamide metabolites 4-hydroxycyclophosphamide and al

dophosphamide can also be metabolized by aldehyde dehydrogenasesto yield inactive species (11, 12).

Ifosphamide is an isomer of Cyclophosphamide (Fig. 1) that exhibits important quantitative differences in pharmacokinetics and metabolism, compared to Cyclophosphamide (13). Although Cyclophosphamide and ifosphamide are both activated by hepatic cytochrome P450enzymes to form a 4-hydroxy metabolite that subsequently decom

poses to yield acrolein plus a mustard derivative (isophosphoramidemustard in the case of ifosphamide), Â¡fosphamide is activated at alower rate than Cyclophosphamide (14). This appears to reflect boththe lower catalytic efficiency for ifosphamide activation exhibited byindividual cytochromes P450 and changes in the spectrum of cytochrome P450 enzymes that can contribute to drug activation (15). Inaddition, a quantitatively important pathway of ifosphamide metabolism is side-chain /V-dechloroethylation (16), which leads to the for

mation of the therapeutically inactive but neurotoxic metabolite chlo-

roacetaldehyde (17). In contrast. Cyclophosphamide is not subject tosubstantial A'-dechloroethylation (1, 18). Whereas Cyclophosphamide

is known to interact with rat hepatic cytochromes P450 via a multiplicity of mechanisms (19). the potential effects of ifosphamide onhepatic cytochrome P450 enzyme profiles are not known.

The precise mechanisms by which Cyclophosphamide alters rathepatic cytochrome P450 protein levels and enzyme activities remainto be clarified. Early studies concluded that the effects were due todenaturation of cytochrome P450 by the Cyclophosphamide metabolite acrolein (6. 7). In support of this proposal, sulfhydryl-containing

compounds prevent the decrease in cytochrome P450 enzyme activities observed 4 days after treatment of rats with cyclophosphamide(20). However, sulfhydryl compounds do not block the decreasesobserved 7 days after cyclophosphamide treatment (21 ). Rather, thesechronic cyclophosphamide-dependent decreases in liver cytochrome

P450 enzyme activities are associated with a feminization of liverenzyme profiles. Thus, cyclophosphamide suppresses the male-specific CYP 2A2, 2C11, and 3A2, while it induces the female-predominant enzymes CYP 2AI and steroid 5a-reductase (9). These effects of

cyclophosphamide are similar to those produced by cisplatin, whichfeminizes the pattern of liver cytochrome P450 (22. 23) as well asglutathione 5-transferase enzyme expression (24). Thus, the effects of

cyclophosphamide are complex and are not simply the result of adirect inactivation of cytochromes P450 by acrolein. In principle,these major effects of cyclophosphamide on liver cytochrome P450profiles could be mediated by either acrolein or phosphoramide mustard, both of which are reactive electrophilic molecules. Acrolein hasbeen identified as the primary mediator of the urotoxicity that accompanies the clinical use of cyclophosphamide and ifosphamide (25, 26),whereas phosphoramide mustard appears to be responsible for the

2490

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ACROI.KIN. PHOSPHOR AMIDI Ml STARI). \M> (il.SI. 1 M'KI-SSION

VCICH2CH2x P

acHfttf

CPA

\

IFA

CH,CH2CI

vr-CCH,CH P J

XN O '

CH,CH,'

HPD-CPA

HvCH,CH,^ XP.

CHjCH/

deCI-CPA

CICH2CH2 Pu V

OHCICH2CH2/

ACROLEINFig. 1. Structures of cyclophosphumide (CM), il'osphamide (//vl). phosphoramide

mustard (PM ), acrolein. and the acrolein precursors HPD-cyclophosphamide IUPD-CPA )and deCTcyclophosphamide (di'Cl-CPA).

ovarian toxicity of cyclophosphamide (27) and has also been implicated as an important cardiotoxic metabolite of cyclophosphamide(28).

The present study compares the effects of/Â« vivo ifosphamide andcyclophosphamide treatment of adult male rats on hepatic CYP 2CIIand steroid 5Â«-reduetase enzyme activities and mRNA levels and

examines the role of the metabolites acrolein and phosphoramidemustard. /'/; vivo, in the femini/.ation of these hepatic drug- and ste

roid-metabolizing enzymes. The results obtained establish that (a)

ifosphamide is less potent than cyclophosphamide in suppressing CYP2CI1 and elevating steroid 5ct-reductase mRNA levels and enzymeactivities, including CYP 2C11-catalyzed cyclophosphamide and ifosphamide activation, (h) modulation of these gender-dependent he

patic mRNA levels and enzyme activities by cyclophosphamide is dueto the action of phosphoramide mustard, and (<â€¢)acrolein potentiates

the modulating activity of the mustard.

MATERIALS AND METHODS

Chemicals. Cyclophosphamide. ifosphamide. and phosphoramide mustardIphosphoramidic acid yV.jV-bis(2-chloroethyl). cyclohexylumine salt] were ob

tained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda. MDl. HPD-cyclophosphamide and 4-hydroperoxyitospha-

mide were kindly provided by Dr. J. Pohl (ASTA Pharma. Bielefeld. Germany).Dr. J. Hilton (Johns Hopkins Oncology Center. Baltimore. MD) provideddeCl-cyclophosphamide. Acrolein and [4-l4C]testosterone were purchased

from Aldrich Chemical Co. (Milwaukee. WI) and Amershum Corp. (ArlingtonHeights. IL), respectively.

Animal Treatments. Adult male Fischer 344 rats (190-200 g. 8-9 weeks

old: Taconic Farms. Germamown. NY) were treated with single i.p. injectionsof cyclophosphamide. ifosphamide. phosphoramide mustard, acrolein. orHPD-cyclophosphamide. at the doses indicated in the text. deCl-cyclophos-

phamide was administered i.p. at a dose of 2<X)nig/kg in a previous experimentconducted by Dr. G. F. Weher of this laboratory. Control rats were given i.p.injections of the vehicle (0.9% NaCI solution). On the day of treatment, drugswere dissolved in the vehicle and administered to rats (4 ml/kg body weight)immediately thereafter. At 7 or 9 days after treatment, as specified in the textfor each experiment, rats were killed by cervical dislocation following briefasphyxiation under CO2. Livers were quickly excised, washed with ice-cold

1.15% KC1 solution, cut into small pieces, frozen in liquid nitrogen, and thenstored at -8()Â°Cuntil used for microsomal preparation or RNA isolation. Bloodwas collected by cardiac puncture and was allowed to clot at 4Â°C.Serum wasprepared by centrifugaron and then stored at -20CC until use.

Knzyme Assays. Microsomes were prepared from individual rat livers by acalcium precipitation method (29) and were then assayed for testosterone,cyclophosphamide. and Â¡fosphamidemetabolism. Testosterone 2Â«-hydroxylase

activity was determined as described previously (30). Incubation mixturescontained 100 msi 4-(2-hydroxyethyl)-l-pipera/.ineethanesullbnic acid(HEPES). (pH 7.4). O.I msi EDTA. 30 ug microsomal protein. 50 UM I4C-

laheled testosterone, and 1 m.wNADPH. in a total volume of 2(X)ul. Reactionswere incubated for IO min at 37Â°C.extracted with ethyl acetate, and then

chromatographed on silica gel thin layer chromatography plates developedwith dichloromethane/acetone (4/1) followed by chloroform/ethyl acetate/absolute ethanol (4/1/0.7). Metabolites were localized by autoradiography andquantitated by liquid scintillation counting. Microsomal steroid 5Â«-reductaseactivity was determined by the reduction of |4-l4C|testoslerone to 5a-[4-'4C]-

dihydrolestosterone in the same assay.Microsomal cyclophosphamide 4-hydroxylase and ifosphamide 4-hydroxy-

lase activities were determined by a fluorescence assay (31 ). Each incubationmixture contained 100 nisi potassium phosphate. pH 7.4. 0.1 HIMEDTA. 5 msisemicarba/ide HC1. 0.5 msi cyclophosphamide or ifosphamide. 100 ug microsomal protein, and I nisi NADPH. in a total volume of 2(X)ul. The reactionwas incubated for 60 min at 37Â°Cand then stopped by the addition of 80 ul

ice-cold 5.5% zinc sulfate solution, followed by 80 pi saturated barium hy

droxide and 40 ul 0.01 si HCI. After centrifugation. 300 ul of the supernatantwere derivati/ed with a IftO-ul solution containing aminophenol (6 mg/ml) and

hydroxvlamine hvdrochloride (ft mg/ml) in I si HCI. This mixture was heatedat 9()Â°Cfor 20 min and then cooled to room temperature before the fluores

cence was measured (350-nm excitation wavelength and 515-nm emissionwavelength). 4-Hydroperoxyifosphamide was used as a standard for acrolein(32) and was incubated as described above hut with heat-inactivated

microsomes and in the absence of NADPH.Oligonudeotide Probes. Gene-specific oligonucleotide probes were syn-

thesi/.cd on an Applied Biosystems DNA synthesizer, purified by high performance liquid chromalography. ^'-labeled with [-y-12P]ATP by T4 polynucle-

otide kinase. and subsequently purified on NENSORB 20 columns (DuPont-

NEN. Boston. MA) (29). The nucleotide sequences for the oligonucleotideprobes ON-5 (CYP 2CII). ON-48 (steroid 5Â«-reductase). and ON-50(tt-tubulin) are presented elsewhere (29. 33).

Northern Blot Analysis. Total liver RNA samples were isolated fromfrozen liver tissue and then electrophoresed in \'/< agarose/O.ftft si formalde

hyde gels as detailed elsewhere (29). The RNA was transferred to nylon (liters(Genescreen; DuPont-NEN) and then UV cross-linked. Prehybridization and

hybridization for Northern blot analysis were carried out at a temperature(4()Â°Cor 45Â°C)and tbrmamide concentration (0-25%, v/v) suitable for each

oligonucleotide probe (29. 33). The nylon fillers were then washed and exposed to Kodak XAR-5 film, w ith intensifying screens, al -8(FC for 2-6 days.Each blot was probed with rat Â«-tuhulinoligonucleotide probe ON-50 to assess

RNA loading consistency and integrity.Serum Testosterone Assay. Serum testosterone concentration was meas

ured by solid-phase '-*! radioimmunoassay with the Coat-A-Count total tes

tosterone kit (Diagnostic Products Corp.. Los Angeles. CA).

RESULTS

Modulation of Liver Enzyme Patterns by Ifosphamide. Treatment of adult male rats with ifosphamide or cyclophosphamide led tosignificant decreases in liver microsomal ifosphamide 4-hydroxylase(Fig. 2A ) and cyclophosphamide 4-hydroxylase activities (see Table 2.

below) (9). Since CYP 2C11 is a major catalyst of both ifosphamide(15) and cyclophosphamide (2) activation in adult male rat livermicrosomes, these results suggest that ifosphamide suppresses theexpression of CYP 2CII, just as cyclophosphumide does (9). Weexamined this possibility by comparing the effects of these oxaza-phosphorines on the levels of CYP 2CII, which is an adult male-

specific liver cytochrome P450 enzyme that is suppressed by cyclophosphamide. and on the cytochrome P450-independent enzymesteroid 5a-reductase. which is a female-predominant enzyme whose

levels in the liverare increased by cyclophosphamide treatment. Whenthe drugs were given at equimolar doses (120 mg/kg. single i.p.injection), ifosphamide decreased CYP 2C11-catalyzed hepatic microsomal testosterone 2a-hydroxylase activity by ~50%, compared toan ~80<7r decrease by cyclophosphamide. 7 days after drug treatment

2491



ACROLEIN. PHOSPHORAMIDE MUSTARD. AND GENE EXPRESSION

CD

E

cE

\oEc

(A) IFOSPHAMIDE 4-HYDROXYLASE

(B) TESTOSTERONE 2Â«-HYDROXYUSE

5O<

oIâ€”<Iâ€”zLJOzoÃœ

O)C

(C)STEROID Sa-REDUCTASE .

(D) SERUM TESTOSTERONE

SALINE IFA IFA CPA120 180 120

mg/kg mg/kg mg/kg

Fig. 2. Effect of in vivo ifosphamide and cyclophosphamide treatment on hepaticmicrosomal en/yme activities and serum testosterone levels. Adult male rats were administered single i.p. injections of ifosphamide (IFA ) ( 120 or 180 mg/kg), cyclophosphamide(CPA I ( 120 mg/kg). or saline (control) and were killed 7 days later. Microsomal enzymeactivities and serum testosterone levels were determined as described in "Materials andMethods." Points, determination for each individual rat; bars, mean values for each

treatment group.

(Fig. 2B). At this dose ifosphamide did not significantly alter hepaticmicrosomal steroid 5a-reductase activity, whereas cyclophosphamideincreased it by ~7-fold (Fig. 2C). In contrast, when ifosphamide was

given at a 50% higher dose (180 mg/kg) it suppressed testosterone2a-hydroxylase activity (Fig. 2B) to the same extent as did cyclophosphamide, whereas it increased steroid 5a-reductase activity (Fig.

2C) but to a lesser extent than did cyclophosphamide. Northern blotanalysis of hepatic CYP 2C11 mRNA and steroid 5a-reductase mRN A

levels (Fig. 3, A and B) substantiated the effects of the drugs on thecorresponding enzyme activities. Thus, ifosphamide elicits the sameeffects as cyclophosphamide on these two hepatic enzymes but requires a higher dose.

The feminization of the gender-dependent hepatic cytochrome P450enzymes and steroid 5a-reductase by cyclophosphamide treatment of

adult male rats is associated with depletion of serum testosterone (9).Therefore, serum testosterone concentrations were compared in ifosphamide- and cyclophosphamide-treated adult male rats. As shown in

Fig. 2D. serum testosterone was largely depleted by both drugs, suggesting that the effects of ifosphamide and cyclophosphamide on CYP2C11 and steroid 5a-reductase occur by the same mechanism.

In Vivo Acrolein Treatment. Cyclophosphamide and ifosphamideare both metabolized to yield two electrophilic metabolites, i.e., ac-

rolein. which has been implicated in protein alkylation, and a mustard,which alkylates DNA. Either of these reactive metabolites could, inprinciple, mediate the major changes in liver enzyme patterns thatfollow in vivo treatment with the parent oxazaphosphorine. To addressthis issue, we first examined the effects of acrolein, since this electrophilic aldehyde has been shown to bind to cytochrome P450 enzymes in vitro by interacting with cysteine sulfhydryl groups, resulting in protein denaturation (6-8). As shown in Table 1, at a dose of 3mg/kg (single i.p. injection) acrolein decreased testosterone 2a-hy-droxylase activity by ~50% and increased steroid 5a-reductase activity by 1.5-fold in isolated liver microsomes. This corresponds to afeminization ratio (defined as the ratio of microsomal steroid 5a-reductase activity to microsomal testosterone 2a-hydroxylase activity) of only 1.7, compared to a ratio of 19 for the cyclophosphamide-

treated group (Table 1). At a higher dose of acrolein (5 mg/kg)testosterone 2a-hydroxylase activity was decreased to a level similar

to that observed following cyclophosphamide treatment but steroid5Â«-reductase activity was not further increased (Table 1). However, in

contrast to cyclophosphamide treatment, severe toxicity (body weightloss) and some lethality occurred with this treatment. Rats treated withacrolein at a higher dose ( 10 mg/kg) died within 24 h after injection.Thus, although acrolein can elicit some of the effects of cyclophosphamide. this response is observed only under conditions of severetoxicity.

Effects of Acrolein Precursors. In order to better model the livermetabolism-dependent release of acrolein from 4-hydroxycyclophos-phamide that occurs in vivo, we examined the effects of deCl-cyclo-

phosphamide (Fig. 1), which is a cyclophosphamide analogue thatyields acrolein enzymatically but without the formation of phosphora-mide mustard. Treatment of rats with deCl-cyclophosphamide (200

mg/kg. i.p.; the animals were killed 7 days later) did not alter hepaticCYP 2C11 or steroid 5a-reductase mRNA levels (Fig. 4, A and B).Preliminary experiments showed that /'/; vitro the formation of acrolein

from deCl-cyclophosphamide catalyzed by uninduced rat liver mi

crosomes occurs with a lower efficiency than does the chemical decomposition of HPD-cyclophosphamide (Fig. 1). which is anothercyclophosphamide analogue that yields acrolein but not phosphora-mide mustard. Therefore, we examined the effects of HPD-cyclophos

phamide and found that at an i.p. dose of 100. 150, or 200 mg/kg it didnot affect hepatic microsomal testosterone 2a-hydroxylase or steroid5a-reductase activity 7 days after treatment (Table 1). Together, these

data indicate that neither acrolein nor the acrolein precursors canmimic the effects of cyclophosphamide with respect to feminization ofthe pattern of hepatic CYP 2CI1 and steroid 5a-reductase enzyme

expression.In Vivo Phosphoramide Mustard Treatment. We next examined

whether phosphoramide mustard might account for the changes inliver enzyme levels observed following oxazaphosphorine treatment.Adult male rats were given single i.p. injections of phosphoramide

2492



ACROLEIN. PHOSPHORAMIDK MUSTARD. AND GENE EXPRESSION

M(120)IFA

(180) (120)IFA CPA M

Fig. 3. Alteration in liver mRNA expression following treatment with ifosphamide. Total liverRNA was prepared from adult male rats (M ) treatedwith saline (Â¡tint's1. 2. and //). ifosphamide (IFA }

( 120 mg/kg. lanes } and 4: 180 mg/kg. lunes 5 and6). or cychiphosphamide iCPA I ( 120 mg/kg, lanes7 and K) and killed 7 days later. RNA samples fromuntreated adult female rat livers (Ft. which do notexpress the male-specific CYP 2CII hut have highlevels of the female-predominant steroid 5o>reduc-tase. are included for comparison (lanes 9 and 10 ).Shown are autoradiograms of a Northern hlot analyzing total liver RN'A (one individual liver RNA

sample per lane), probed sequentially with the CYP2C1I (A) and steroid 5a-reductase (ÃŸlgene-specific oligonucleotides (29). Rat a-iubulin mRNA

levels (C) are indicative of the RNA load and integrity for the samples shown in A and H.

2C11

B. 5ccR

Tubulin

i 8 10 11

mustard at 20-100 mg/kg and then were killed 7 days later. At the

lower doses used in the experiments, phosphoramide mustard did notaffect CYP 2C11-catalyzed testosterone 2a-hydroxylase activity orsteroid 5a-reductase activity (Table I and data not shown), nor did italter CYP 2C11 mRNA or steroid 5a-reductase mRNA levels (Fig. 4.

A and ÃŸ).In contrast, phosphoramide mustard at 100 mg/kg decreasedtestosterone 2a-hydroxylase activity by 90<7cand increased steroid5a-reductase activity by ~4.5-fold, corresponding to a feminization

ratio of 28 (Table 1). Although this dose of phosphoramide mustardaltered these enzyme activities in a manner similar to that effected bycyclophosphamide (120 mg/kg). it also conferred general systemictoxicity, as exemplified by major body weight loss (Table 1). Incyclophosphamide-treated rats, body weight typically decreases by~5-10% during the first 7 days after drug administration but then

stabilizes for the remainder of the experimental period (see Table 3).In contrast, a continual decline in body weight occurred during the7-day observation period in rats treated with the 100 mg/kg dose of

phosphoramide mustard (Table I and data not shown). Thus, phosphoramide mustard can modulate liver enzyme activities in a mannersimilar to that of cyclophosphamide.

Kffects of Phosphoramide Mustard and HPD-cyclophospha-

mide in Combination. To test whether the modulation of the specifichepatic enzymes by cyclophosphamide is achieved through (he combined action of acrolein and phosphoramide mustard, rats were giveni.p. injections of phosphoramide mustard (50 mg/kg). HPD-cyclo-

phosphamide (200 mg/kg). or both agents in combination, and theanimals were killed 9 days later. The doses chosen were shown to haveminimal gross toxicity (body weight loss) in earlier experiments (Ta-

Table I Effect of in VÃ•YOtreatment with cvcloplutsphantitle, acrolein, HPD-c\cltiphcrsphainide. or phosphoratniilt1 tnusuinl un hepatic tnicnisomal c/i-vmc activities anil hntl\

weight

Hepatic microsomes were prepared from adult male rats treated with single i.p. injections of saline (control), cyclophosphamide (CPA). phosphoramide mustard (PM), acrolein. orHPD-cyclophosphamide (HPD-CPA) at the doses indicated and killed 7 days later. Enzyme activities were determined as described in "Materials and Methods." Results are expressed

as mean Â±SD in cases where the number (Â«)of rats was three or four per group or as mean Â±half the range for groups with two rats.

TreatmentSalineCPAAcroleinHPD-CPAPMn4322"2'32222Dose(mg/kg)1203510100ISO20050100Testosterone2a-hydroxylaseactivity

(nmol/min/mg)"1

.47 Â±0.400.33

Â±0.130.75

Â±0.170.23NA'1.31

Â±0.371.18Â±0.190.83

Â±0.141.79

Â±0.090.14Â±0.02Steroid

5Â«-reduclasc

activity(nmol/min/mg)"0.88

Â±0.306.38

Â±2.581.30

Â±0.121.47NA1.12*0.120.55

Â±0.530.97Â±0.430.82

Â±0.083.94Â±0.49Feminization

ratio''0.6191.7fi.4NA0.90.51.20.528Changein

body weight(g)'+32

Â±4-21

Â±10-3

Â±3-59NA+

I6Â±5+I0Â±1+5

Â±3+

15Â±3-56Â±4

" Activity expressed as nmol product formed/min/mg protein.'' Ratio of steroid 5a-reductase to testosterone 2a-hydroxylase activity. Typical feminization ratio for untreated adult female rats is >30.1 Difference between body weights on the day of sacrifice and the day of drug treatment.

Of the two rats treated with this dose, one died within 24 h after injection.'' Of the two rats treated with this dose, both died within 24 h after injection.

' NA, not applicable.

2493



ACROLEIN. PHOSPHORAMIDI: MUSTARD. AND GENE EXPRESSION

<jF M u g. -a

A.

2C11

B.

t 5aR

c.

Tubulin

1 23456Fig. 4. Phosphoramide mustard (20 mg/kg) and deCI-cyclophosphamide (200 mg/kg)

do not alter hepatic CYP 2CII and steroid 5a-reductase mRNA levels. Adult male rats(M) were given i.p, injections of single doses of saline (lime* 3 and 4), cyclophosphamide(CPA ) ( 120 mg/kg. lane 5 ), phosphoramide mustard (PM ) (20 mg/kg. lane f>). or deCl-cyclophosphamide ttleCI-CPA} (2(X) mg/kg. lane 7) and were killed 7 days later. Totalliver RNA was prepared and Northern blot analysis was performed as in Fig. 3, RNAsamples from untreated adult female rat livers (Fi are included for comparison (lanes Iand 2). A. CYP 2C11 mRNA: B, steroid 5a-reductase mRNA; C. a-tubulin mRNA(control).

ble 1). Neither agent, when administered alone, had major effects onhepatic microsomal enzyme activities, serum testosterone levels (Table 2). or body weight profiles (Table 3). CYP 2C1I mRNA andsteroid 5Â«-reductase mRNA levels were also unaffected (Fig. 5. A and

B). Of the 12 rats treated with both phosphoramide mustard and

HPD-cyclophosphamide. half responded to the drug combination. Inthe responsive rats. CYP 2C11-catalyzed microsomal cyclophospha-mide hydroxylation and testosterone 2a-hydroxylation were decreased by -45% and -90%, respectively, hepatic CYP 2C11 mRNA

became undetectable. and serum testosterone was depleted. This pattern of response is similar to that observed following cyclophosphamide administration (Table 2 and Fig. 5A). Steroid 5a-reductase ac

tivity and mRNA were both increased in the responsive rats but to alesser extent than in the cyclophosphamide-treated rats (Table 2 and

Fig. 5B}. Body weights of the responsive rats declined for 5 days aftertreatment and then stabilized for the remainder of the experimentalperiod but were still lower than those of the cyclophosphamide-treated

rats (Table 3). Overall, these findings demonstrate that, while phosphoramide mustard is responsible for modulation of liver enzymeexpression by cyclophosphamide. acrolein potentiates the modulatingactivity of the mustard.

DISCUSSION

Previous studies have shown that the decreases in total liver cyto-chrome P450 content and cytochrome P45()-dependent enzyme activ

ities following acute cyclophosphamide treatment of adult male rats(reviewed in Ref. 19) are the result of changes in the levels of specificliver cytochrome P450 enzymes (9). While some cytochromes P450were found to decrease, others increased substantially following cyclophosphamide administration. The overall effect is to feminize thepattern of expression of liver enzymes in a manner that is similar to.but mechanistically distinct from, that observed with the alkylatingagent cisplatin (22, 23). The present study establishes that ifospha-

mide is also capable of feminizing the expression of these liver enzymes and that the effects occur at a pretranslational step, involvingsuppression of the male-specific CYP 2C11 mRNA and elevation ofthe female-dominant steroid 5a-reductase mRNA. The present studyalso provides insight into the mechanism by which these oxazaphos-

phorines alter the expression of these mRNAs. Phosphoramide mustard is shown to be responsible for the modulation of liver enzymeexpression by cyclophosphamide. while acrolein potentiates theseeffects of the mustard.

While ifosphamide and cyclophosphamide were both effective inmodulating liver enzyme levels, ifosphamide was found to be lesspotent than cyclophosphamide. Higher doses of ifosphamide are alsorequired to achieve an equivalent plasma alkylating activity in cancerpatients (3.8 g/m2 versus 1.1 g/m2 for cyclophosphamide) (34). The

Table 2 Effecl of etnnbineil pliosplturuinitle mustard ami HPD-cvclttphosphantitle treatment on hepatic inicnistirtwl eii'vme adivines antl serum testosterone levels

Adult male rats were treated with single i.p. injections of saline (control), cyclophosphamide (CPA) ( 120 mg/kg). phosphoramide mustard (PM) (50 mg/kg). HPD-cyclophosphamide(HPD-CPA) (200 mg/kg). or both phosphoramide mustard and HPD-cyclophosphamide. and the animals were killed 9 days later. Hepatic microsomes were isolated and enzymeactivities and serum testosterone concentrations were determined as described in "Materials and Methods." Results are expressed as mean Â±SD for the indicated number (in of rats

per treatment group.

TreatmentSalineCPAPMHPD-CPAPM

+HPD-CPAResponders

Nonrespondersn13g6666Cyclophosphamide

4-hydroxylase

activity(nmol/min/mg)"4.

12Â±0.48''2.01

Â±0.86''4.32

Â±0.663.70

Â±0.732.33

Â±0.494.03 Â±0.59Testosterone

2tt-hydroxy]aseactivity

(nmol/min/mg)"1.53

Â±0.300.23

Â±0.041.31

Â±0.271

. 11 Â±0.250.15Â±O.I5

I.I7Â±O.I8Steroid

5a-reductase

activity(nmol/min/mg)"0.98

Â±0.488.47

Â±2.081

.02 Â±0.31

.49 Â±0.652.79

Â±1.021.02 Â±0.49Femini/alion

ratio'10.6370.81.3190.9Serumtestosterone

(ng/ml)'2.6

Â±1.9I.I

Â±0.53.4

Â±2.12.8

Â±1.9<0.042.5

Â±1.0" Activity expressed us nmol product formed/min/mg microsomal protein.h Fernini/ution ratio defined us in Tuble I.' Inlerindividuul variations in serum testosterone are commonly observed in adult male rats duell n = 6 rats used tor these activity measurements.

2494

to the intermittent release of testosterone bv the testis.




Table 3 Effect ofphosphoramide mustard und HPD-c\clopho\plmniide combination treatment un hody weight

Data shown are tor the same groups of adult male rats treated on day 0 that are described in Table 2. Data are expressed as mean Â±SD tor the indicated number in) of rats pertreatment group.

Body weight (g)

TreatmentSalineCPAPMHPD-CPAPM

+HPD-CPAResponders

Nonrespondersn986666Day

(I"199

Â±5204

Â±4205

Â±10205

Â±4205

Â±6207Â±1Day

1201

Â±6192

Â±6201

Â±10I90Â±ll190

Â±6194Â±9Day

5218*8193

Â±3217Â±

12204

Â±16161

Â±10205Â±12Day

7226

Â±8180

Â±5225

Â±13215

Â±13160

Â±13215Â±11Day

9235

Â±8188

Â±7236

Â±15225

Â±13159

Â±17225Â±13"

Day after treatment.

observed differences in the potency of these two drugs can largely beexplained by quantitative differences in their metabolism. Cyclophos-phamide is metaboli/ed predominantly at the C4 position of the ox-a/aphosphorine ring and side-chain /V-dechloroethylation is minor(<IO%) (I, 18. 35), whereas in the case of ifosphamide side-chain

metabolism accounts for an estimated 5()9r of the administered dose(16). Therefore, given the same dose, ifosphamide generates lessmustard and less acrolein than does cyclophosphamide.

The suppression by cyclophosphamide and ifosphamide of hepaticCYP 2CII occurred at a pretranslational step. It is likely that CYP2C1I transcription is the step that is affected in oxazaphosphorine-treated rats, because the male-specific expression of the CYP 2C1I

gene is regulated at the level of transcript initiation in adult rats (36).The underlying causes for these effects on CYP 2C11 and steroid5a-reductase levels could involve effects on one or more of thehypothalamo-pituitary and gonadal factors that regulate expression of

these genes (37). rather than direct effects on the liver. Indeed, thesuppression of CYP 2CI1 mRNA and the elevation of steroid 5a-

reductase mRNA by cyclophosphamide and ifosphamide. or by thecombination of HPD-cyclophosphamide and phosphoramide mustard.

is shown to be associated with a substantial decrease in serum levelsof testosterone, which is required for maintenance of the sexuallydimorphic expression of these enzymes (38â€”40).However, while endogenous androgen secretion in cyclophosphamide-treated rats can bestimulated by the luteinizing hormone analogue chorionic gonadotro-

pin. the resultant increase in serum testosterone does not reverse thesuppression of hepatic CYP 2C11 (9). This observation is analogousto the finding that the suppression of CYP 2CII by 3.4.5,3'.4',5'-

hexachlorobiphenyl is also not causally related to the associated depletion of serum testosterone (41 ). Consequently, modulation of liverenzyme expression by cyclophosphamide and ifosphamide may involve action at the hypothalamo-pituitary axis, which establishes thesex-dependent plasma growth hormone profile that in turn determinesthe expression of hepatic CYP 2C11, steroid 5a-reductase, and otherdrug- and steroid-metaboli/.ing enzymes in adult male rats (37, 42,

43).The present study demonstrates that, while phosphoramide mustard

is primarily responsible for the modulation of hepatic CYP 2C11 andsteroid 5a-reductase by cyclophosphamide. acrolein potentiates the

modulating activity of the mustard. When administered alone, neither

M F CPA PM HPD-Cm PM+HPD-CPA

Fig. 5. Effect of phosphoramide mustard undHPD-cyclophosphamide combination treatment onhepatic CYP 2C11 and steroid So-rcductase mRNAlevels. Adult male rats (M\ were treated with saline(lanes I and 2}, cyclophosphamide (CPA} (120mg/kg. lanes 5 and 6). phosphoramide mustard(PM] (50 mg/kg. lanes 7-V). HPD-cyclophosphamide (HPD-CPA} (200 mg/kg. lanes 10-12). or acombination ol phosphoramide mustard (50 mg/kg)plus HPD-cyclophosphamide (2(K) mg/kg) (lanes13-16) and were killed 9 days later. Total liverRNA was prepared and Northern blot analysis wasperformed as in Fig. 3. RNA samples from untreated adult female rat livers (/â€¢")are included for

comparison (lanes 3 and 4). A. CYP2C1I mRNA;B, steroid 5a-reductase mRNA; C. u-tubulin

mRNA (control). The reduced signal in Â¡tine5 fortubulin mRNA indicates that the RNA analyzed inthis lane was underloaded, compared to the othersamples.

â€¢¿�â€¢

B.

- 2C11

5aR

Tubulin

123456 8 9 10 11 12 13 14 15 16

2495




acrolein nor acrolein precursors (HPD-cyclophosphamide and deCl-

cyclophosphamide) feminized the expression of these enzymes to thesame extent as did cyclophosphamide. In contrast, phosphoramidemustard altered the expression of CYP 2C11 and steroid So-reductase,

similarly to cyclophosphamide. hut this effect was achieved at a doseof mustard ( KM)mg/kg) that produced greater body weight loss thandid cyclophosphamide. This difference in toxicity might be due todifferences in the pharmacokinetics and tissue distribution of phosphoramide mustard given as a bolus i.p. injection, compared to phosphoramide mustard derived from chemical decomposition of the primary 4-hydroxy metabolite in cyclophosphamide-treated rats.

Whereas treatment with a lower dose of phosphoramide mustard (50mg/kg) or with the acrolein precursor HPD-cyclophosphamide (200

mg/kg) alone did not affect liver enzyme profiles, the combination ofthese two agents suppressed CYP 2CI1 and elevated steroid 5a-

reductase enzyme activities and mRNA levels, in addition to depletingserum testosterone, an overall pattern that is similar to that producedby cyclophosphamide. These effects of the combination treatmentwere observed in only half of the animals tested and suggest that aconcentration threshold exists at the site(s) of action of these metabolites to elicit the modulation of liver enzyme expression. Such athreshold has been documented for acetaminophen-induced hepaticnecrosis (44). The glutathione-depleting agent buthionine sulfoximinealso appears to potentiate phosphoramide mustard-mediated car-

diotoxicity in rodents (28). Acrolein or cyclophosphamide treatmentof rats can decrease cellular glutathione content, whereas phosphoramide mustard is only somewhat effective at glutathione depletionwhen given at high doses (28, 45). Thus, acrolein may render targetcells more sensitive to the modulating activity of phosphoramidemustard by reducing intracellular glutathione levels. As noted above,the effects of cyclophosphamide and the combination of phosphoramide mustard and HPD-cyclophosphamide on liver mRNA levels

indicate that these sensitizing effects of acrolein probably occur atendocrine secretory organs, rather than the liver. Glutathione is foundnot only in liver but also in many other tissues, including brain (46).

Cyclophosphamide and ifosphamide are typically administered tocancer patients as part of a combination chemotherapy regimen. Thepresent study indicates that these alkylating agent prodrugs not onlycan alter their own biotransformation but also may affect cytochromeP450-mediated bioactivation or deactivation of concurrently admin

istered drugs. Clinical studies have shown that chronic administrationof oxazaphosphorines to cancer patients can increase drug clearance(14, 18), suggesting that human liver cytochrome P450 enzymes arealso subject to modulation by these oxazaphosphorines, albeit bymechanisms that lead to increased rates of drug metabolism. Since ourstudies implicate phosphoramide mustard as the primary mediator ofthe effects of the parent drug on liver drug metabolism, it may not bepossible to design active oxazaphosphorines that lack these potentialdrug interactions. Finally, the finding that acrolein potentiates theenzyme-modulating effects of phosphoramide mustard has a broader

implication, insofar as environmental exposure to acrolein may triggeror exacerbate drug- and xenobiotic agent-induced systemic toxicity.

Indeed, significant levels of acrolein are found in tobacco smoke (47).

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1993;53:2490-2497. Cancer Res Thomas K. H. Chang and David J. Waxman Phosphoramide MustardLevels through the Combined Action of Acrolein and

-Reductase Activity and Messenger RNAα2C11 and Steroid 5Cyclophosphamide Modulates Rat Hepatic Cytochrome P450

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