Download Cyclophosphamide Modulates Rat Hepatic Cytochrome P450 2C11

[CANCER RESEARCH 53. 2490-2497. June I. 19931
Cyclophosphamide Modulates Rat Hepatic Cytochrome P450 2C11 and Steroid
5tt-Reductase Activity and Messenger RNA Levels through the Combined
Action of Acrolein and Phosphoramide Mustard1
Thomas K. H. Chang and David J. Waxman2
Department ¡ifBioluaical Chemistry and Molecular Phunnaciilogy
ana Dana-Furher Cancer Institute, Hanard
ABSTRACT
Cyclophosphamide treatment of adult male rats leads to sustained de
creases 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 enzyme
expression (G. A. LeBlanc and D. J. Waxman, Cancer Res., 50: 5720-5726,
1990). The present study compares the effects of Cyclophosphamide and its
isomerie analogue ifosphamide on the gender-dependent expression of
hepatic CYP 2C11 and steroid Sa-reductase in adult male rats and also
examines the role of the Cyclophosphamide metabolites acrolein and phosphoramide mustard in feminizing the expression of these liver enzymes.
Ifosphamide (a) suppressed the male-specific CYP 2C11 mRNA and CYP
2C11-catalyzed liver microsomal testosterone 2a-hydroxylation and Cyclo
phosphamide and ifosphamide 4-hydroxylation and (b) elevated the fe
male-dominant liver enzyme steroid 5a-reducta.se and its mRNA 7-9 days
after drug treatment, both occurring in a manner similar to that of cyclophosphamide, but requiring a 50% higher dose (180 mg/kg, single i.p.
injection) to achieve these effects. This pattern of response could not be
achieved by treatment of rats with acrolein or with Cyclophosphamide
analogues that decompose to acrolein without formation of phosphoramiilf 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 mustard
or with the acrolein precursor 4-hydroperoxydechlorocyclophosphamide
(200 mg/kg) alone did not affect liver enzyme expression, whereas the
combination of these agents produced an overall pattern of response that
was similar to that conferred by Cyclophosphamide. These studies estab
lish that ifosphamide is less potent than Cyclophosphamide in modulating
the pattern of cytochrome P450 and steroid 5<x-reductase expression and
that phosphoramide mustard is responsible for the modulation of liver
enzyme expression by Cyclophosphamide, with acrolein potentiating the
modulating activity of the mustard.
Medical Schuol. Bustini. Massachusetts
02115
mustard possesses DNA-alkylating activity and is generally consid
ered to be the therapeutically significant, cytotoxic metabolite of
Cyclophosphamide (1,3). Acrolein. which is an electrophilic aldehyde,
lacks antitumor activity (4. 5) but is highly reactive and binds covalently to proteins, including cytochrome P450 (6-9) and NADPHcytochrome P450 reducÃ-ase(10), leading to enzyme inactivation. The
Cyclophosphamide metabolites 4-hydroxycyclophosphamide
and al
dophosphamide can also be metabolized by aldehyde dehydrogenases
to yield inactive species (11, 12).
Ifosphamide is an isomer of Cyclophosphamide (Fig. 1) that exhib
its important quantitative differences in pharmacokinetics and metab
olism, compared to Cyclophosphamide (13). Although Cyclophospha
mide and ifosphamide are both activated by hepatic cytochrome P450
enzymes to form a 4-hydroxy metabolite that subsequently decom
poses to yield acrolein plus a mustard derivative (isophosphoramide
mustard in the case of ifosphamide), ¡fosphamide is activated at a
lower rate than Cyclophosphamide (14). This appears to reflect both
the lower catalytic efficiency for ifosphamide activation exhibited by
individual cytochromes P450 and changes in the spectrum of cyto
chrome P450 enzymes that can contribute to drug activation (15). In
addition, a quantitatively important pathway of ifosphamide metabo
lism is side-chain /V-dechloroethylation (16), which leads to the for
mation of the therapeutically inactive but neurotoxic metabolite chloroacetaldehyde (17). In contrast. Cyclophosphamide is not subject to
substantial A'-dechloroethylation (1, 18). Whereas Cyclophosphamide
is known to interact with rat hepatic cytochromes P450 via a multi
plicity of mechanisms (19). the potential effects of ifosphamide on
hepatic cytochrome P450 enzyme profiles are not known.
The precise mechanisms by which Cyclophosphamide alters rat
hepatic cytochrome P450 protein levels and enzyme activities remain
to be clarified. Early studies concluded that the effects were due to
INTRODUCTION
denaturation of cytochrome P450 by the Cyclophosphamide metabo
lite acrolein (6. 7). In support of this proposal, sulfhydryl-containing
Cyclophosphamide is a widely used anticancer alkylating agent
prodrug that is bioactivated by the liver CYP3 monooxygenase system
compounds prevent the decrease in cytochrome P450 enzyme activ
( 1). Three specific cytochrome P450 enzymes, forms CYP 2BI (phé ities observed 4 days after treatment of rats with cyclophosphamide
nobarbital inducible). CYP 2C6 (constitutively expressed and gender
(20). However, sulfhydryl compounds do not block the decreases
independent), and CYP 2C11 (constitutively expressed and male spe
observed 7 days after cyclophosphamide treatment (21 ). Rather, these
cific),4 have been identified as major catalysts of Cyclophosphamide
chronic cyclophosphamide-dependent
decreases in liver cytochrome
activation in rat liver (2). The primary metabolite formed by these
P450 enzyme activities are associated with a feminization of liver
enzymes. 4-hydroxycyclophosphamide.
equilibrates with the ringenzyme profiles. Thus, cyclophosphamide suppresses the male-spe
opened aldophosphamide. which undergoes spontaneous decomposi
cific CYP 2A2, 2C11, and 3A2, while it induces the female-predom
tion to yield phosphoramide mustard and acrolein ( 1). Phosphoramide
inant enzymes CYP 2AI and steroid 5a-reductase (9). These effects of
cyclophosphamide are similar to those produced by cisplatin, which
Received 12/21/92: accepted 3/24/93.
feminizes the pattern of liver cytochrome P450 (22. 23) as well as
The costs of publication of this article were defrayed in part by the payment of page
glutathione 5-transferase enzyme expression (24). Thus, the effects of
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
cyclophosphamide are complex and are not simply the result of a
1 Supported in part by Grant CA-49248 from the NIH (D. J. W.). T. K. H. C. was
direct inactivation of cytochromes P450 by acrolein. In principle,
supported by a Canadian Association of Gastroenterology/Janssen
Research Fellowship
(1991-1992) and a Canadian Liver Foundation Research Fellowship (1992-1993).
these major effects of cyclophosphamide on liver cytochrome P450
- To whom requests for reprints should he addressed, at Dana-Farber Cancer Institute.
profiles could be mediated by either acrolein or phosphoramide mus
Room JF-525. 44 Binney Street. Boston. MA 02115.
1The abbreviations used are: CYP. cytochrome P450: HPD-cyclophosphamide.
tard, both of which are reactive electrophilic molecules. Acrolein has
4-hydroperoxydechlorocyclophosphamide;
deCl-cyclophosphamide.
bisfethyllaminocybeen identified as the primary mediator of the urotoxicity that accom
clophosphamide.
panies the clinical use of cyclophosphamide and ifosphamide (25, 26),
4 Individual liver CYP forms are designated according to the systematic nomenclature
(48).
whereas phosphoramide mustard appears to be responsible for the
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ACROI.KIN. PHOSPHOR AMIDI
CICH2CH2x
V
Ml STARI).
vr-C
P
CH,CH
P
XN
acHfttf
O
CICH2CH2
P
u
V
OH
CH,CH2CI
chromatographed on silica gel thin layer chromatography plates developed
with dichloromethane/acetone
(4/1) followed by chloroform/ethyl acetate/
absolute ethanol (4/1/0.7). Metabolites were localized by autoradiography and
quantitated by liquid scintillation counting. Microsomal steroid 5«-reductase
activity was determined by the reduction of |4-l4C|testoslerone to 5a-[4-'4C]dihydrolestosterone in the same assay.
Microsomal cyclophosphamide 4-hydroxylase
v
H
CH,CH,^
IFA
deCI-CPA
ACROLEIN
(CM), il'osphamide (//vl). phosphoramide
mustard (PM ), acrolein. and the acrolein precursors HPD-cyclophosphamide
and deCTcyclophosphamide (di'Cl-CPA).
and ifosphamide 4-hydroxy-
lase activities were determined by a fluorescence assay (31 ). Each incubation
mixture contained 100 nisi potassium phosphate. pH 7.4. 0.1 HIMEDTA. 5 msi
semicarba/ide HC1. 0.5 msi cyclophosphamide or ifosphamide. 100 ug mi
crosomal protein, and I nisi NADPH. in a total volume of 2(X)ul. The reaction
was incubated for 60 min at 37°Cand then stopped by the addition of 80 ul
XP.
CHjCH/
Fig. 1. Structures of cyclophosphumide
1 M'KI -SSION
laheled testosterone, and 1 m.wNADPH. in a total volume of 2(X)ul. Reactions
were incubated for IO min at 37°C.extracted with ethyl acetate, and then
HPD-CPA
CPA
(il.SI.
activity was determined as described previously (30). Incubation mixtures
contained
100 msi 4-(2-hydroxyethyl)-l-pipera/.ineethanesullbnic
acid
(HEPES). (pH 7.4). O.I msi EDTA. 30 ug microsomal protein. 50 UM I4C-
CICH2CH2/
CH,CH,'
\
J
'
\M>
IUPD-CPA )
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 supernatant
were 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 heated
at 9()°Cfor 20 min and then cooled to room temperature before the fluores
ovarian toxicity of cyclophosphamide (27) and has also been impli
cated as an important cardiotoxic metabolite of cyclophosphamide
(28).
The present study compares the effects of/« vivo ifosphamide and
cyclophosphamide treatment of adult male rats on hepatic CYP 2CII
and steroid 5«-reduetase enzyme activities and mRNA levels and
examines the role of the metabolites acrolein and phosphoramide
mustard. /'/; vivo, in the femini/.ation of these hepatic drug- and ste
cence was measured (350-nm excitation wavelength and 515-nm emission
wavelength). 4-Hydroperoxyifosphamide
was used as a standard for acrolein
(32) and was incubated as described above hut with heat-inactivated
roid-metabolizing enzymes. The results obtained establish that (a)
ifosphamide is less potent than cyclophosphamide in suppressing CYP
2CI1 and elevating steroid 5ct-reductase mRNA levels and enzyme
activities, including CYP 2C11-catalyzed cyclophosphamide and if
osphamide activation, (h) modulation of these gender-dependent he
patic mRNA levels and enzyme activities by cyclophosphamide is due
to the action of phosphoramide mustard, and (<•)
acrolein potentiates
NEN. Boston. MA) (29). The nucleotide sequences for the oligonucleotide
probes ON-5 (CYP 2CII). ON-48 (steroid 5«-reductase). and ON-50
(tt-tubulin) are presented elsewhere (29. 33).
microsomes and in the absence of NADPH.
Oligonudeotide
Probes. Gene-specific oligonucleotide
otide kinase. and subsequently
purified on NENSORB 20 columns (DuPont-
Northern Blot Analysis. Total liver RNA samples were isolated from
frozen 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()°Cor 45°C)and tbrmamide concentration (0-25%, v/v) suitable for each
the modulating activity of the mustard.
MATERIALS
probes were syn-
thesi/.cd on an Applied Biosystems DNA synthesizer, purified by high perfor
mance liquid chromalography. ^'-labeled with [-y-12P]ATP by T4 polynucle-
oligonucleotide probe (29. 33). The nylon fillers were then washed and ex
posed to Kodak XAR-5 film, w ith intensifying screens, al -8(FC for 2-6 days.
Each blot was probed with rat «-tuhulinoligonucleotide probe ON-50 to assess
AND METHODS
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
Chemicals. Cyclophosphamide. ifosphamide. and phosphoramide mustard
Iphosphoramidic acid yV.jV-bis(2-chloroethyl). cyclohexylumine salt] were ob
tained from the Drug Synthesis and Chemistry Branch, National Cancer In
stitute (Bethesda. MDl. HPD-cyclophosphamide
and 4-hydroperoxyitospha-
tosterone kit (Diagnostic Products Corp.. Los Angeles. CA).
mide were kindly provided by Dr. J. Pohl (ASTA Pharma. Bielefeld. Germany).
Dr. J. Hilton (Johns Hopkins Oncology Center. Baltimore. MD) provided
deCl-cyclophosphamide.
Acrolein and [4-l4C]testosterone
were purchased
RESULTS
from Aldrich Chemical Co. (Milwaukee. WI) and Amershum Corp. (Arlington
Heights. 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. injections
of cyclophosphamide.
ifosphamide. phosphoramide mustard, acrolein. or
HPD-cyclophosphamide.
at the doses indicated in the text. deCl-cyclophosphamide was administered i.p. at a dose of 2<X)nig/kg in a previous experiment
conducted 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, drugs
were 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 text
for each experiment, rats were killed by cervical dislocation following brief
asphyxiation under CO2. Livers were quickly excised, washed with ice-cold
1.15% KC1 solution, cut into small pieces, frozen in liquid nitrogen, and then
stored at -8()°Cuntil used for microsomal preparation or RNA isolation. Blood
was collected by cardiac puncture and was allowed to clot at 4°C.Serum was
prepared by centrifugaron and then stored at -20CC until use.
Knzyme Assays. Microsomes were prepared from individual rat livers by a
calcium precipitation method (29) and were then assayed for testosterone,
cyclophosphamide. and ¡fosphamide metabolism. Testosterone 2«-hydroxylase
Modulation of Liver Enzyme Patterns by Ifosphamide. Treat
ment of adult male rats with ifosphamide or cyclophosphamide led to
significant 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 liver
microsomes, these results suggest that ifosphamide suppresses the
expression of CYP 2CII, just as cyclophosphumide does (9). We
examined this possibility by comparing the effects of these oxazaphosphorines on the levels of CYP 2CII, which is an adult malespecific liver cytochrome P450 enzyme that is suppressed by cyclo
phosphamide. and on the cytochrome P450-independent enzyme
steroid 5a-reductase. which is a female-predominant enzyme whose
levels in the liverare increased by cyclophosphamide treatment. When
the drugs were given at equimolar doses (120 mg/kg. single i.p.
injection), ifosphamide decreased CYP 2C11-catalyzed hepatic mi
crosomal testosterone 2a-hydroxylase activity by ~50%, compared to
an ~80<7r decrease by cyclophosphamide. 7 days after drug treatment
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ACROLEIN.
PHOSPHORAMIDE
(A)
IFOSPHAMIDE
4-HYDROXYLASE
(B)
TESTOSTERONE 2«-HYDROXYUSE
CD
E
c
E
\
o
E
c
(C)STEROID
Sa-REDUCTASE
.
5
O
<
(D) SERUM TESTOSTERONE
o
I—
<
I—
z O)
LJ C
O
z
o
Ü
SALINE
IFA
120
IFA
180
CPA
120
mg/kg
mg/kg
mg/kg
MUSTARD.
AND GENE EXPRESSION
levels (Fig. 3, A and B) substantiated the effects of the drugs on the
corresponding enzyme activities. Thus, ifosphamide elicits the same
effects as cyclophosphamide on these two hepatic enzymes but re
quires a higher dose.
The feminization of the gender-dependent hepatic cytochrome P450
enzymes 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 ifos
phamide- and cyclophosphamide-treated adult male rats. As shown in
Fig. 2D. serum testosterone was largely depleted by both drugs, sug
gesting that the effects of ifosphamide and cyclophosphamide on CYP
2C11 and steroid 5a-reductase occur by the same mechanism.
In Vivo Acrolein Treatment. Cyclophosphamide and ifosphamide
are both metabolized to yield two electrophilic metabolites, i.e., acrolein. which has been implicated in protein alkylation, and a mustard,
which alkylates DNA. Either of these reactive metabolites could, in
principle, mediate the major changes in liver enzyme patterns that
follow in vivo treatment with the parent oxazaphosphorine. To address
this issue, we first examined the effects of acrolein, since this elec
trophilic aldehyde has been shown to bind to cytochrome P450 en
zymes in vitro by interacting with cysteine sulfhydryl groups, result
ing in protein denaturation (6-8). As shown in Table 1, at a dose of 3
mg/kg (single i.p. injection) acrolein decreased testosterone 2a-hydroxylase activity by ~50% and increased steroid 5a-reductase ac
tivity by 1.5-fold in isolated liver microsomes. This corresponds to a
feminization ratio (defined as the ratio of microsomal steroid 5areductase activity to microsomal testosterone 2a-hydroxylase activ
ity) of only 1.7, compared to a ratio of 19 for the cyclophosphamidetreated 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 steroid
5«-reductase activity was not further increased (Table 1). However, in
contrast to cyclophosphamide treatment, severe toxicity (body weight
loss) and some lethality occurred with this treatment. Rats treated with
acrolein at a higher dose ( 10 mg/kg) died within 24 h after injection.
Thus, although acrolein can elicit some of the effects of cyclophos
phamide. this response is observed only under conditions of severe
toxicity.
Effects of Acrolein Precursors. In order to better model the liver
metabolism-dependent release of acrolein from 4-hydroxycyclophosphamide that occurs in vivo, we examined the effects of deCl-cyclophosphamide (Fig. 1), which is a cyclophosphamide analogue that
yields acrolein enzymatically but without the formation of phosphoramide mustard. Treatment of rats with deCl-cyclophosphamide
(200
mg/kg. i.p.; the animals were killed 7 days later) did not alter hepatic
CYP 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 de
Fig. 2. Effect of in vivo ifosphamide and cyclophosphamide treatment on hepatic
composition of HPD-cyclophosphamide (Fig. 1). which is another
microsomal en/yme activities and serum testosterone levels. Adult male rats were admin
cyclophosphamide analogue that yields acrolein but not phosphoraistered single i.p. injections of ifosphamide (IFA ) ( 120 or 180 mg/kg), cyclophosphamide
mide mustard. Therefore, we examined the effects of HPD-cyclophos
(CPA I ( 120 mg/kg). or saline (control) and were killed 7 days later. Microsomal enzyme
activities and serum testosterone levels were determined as described in "Materials and
phamide and found that at an i.p. dose of 100. 150, or 200 mg/kg it did
Methods." Points, determination for each individual rat; bars, mean values for each
not affect hepatic microsomal testosterone 2a-hydroxylase or steroid
treatment group.
5a-reductase activity 7 days after treatment (Table 1). Together, these
(Fig. 2B). At this dose ifosphamide did not significantly alter hepatic
data indicate that neither acrolein nor the acrolein precursors can
microsomal steroid 5a-reductase activity, whereas cyclophosphamide
mimic the effects of cyclophosphamide with respect to feminization of
increased it by ~7-fold (Fig. 2C). In contrast, when ifosphamide was
the pattern of hepatic CYP 2CI1 and steroid 5a-reductase enzyme
given at a 50% higher dose (180 mg/kg) it suppressed testosterone
expression.
2a-hydroxylase activity (Fig. 2B) to the same extent as did cyclo
In Vivo Phosphoramide Mustard Treatment. We next examined
phosphamide, whereas it increased steroid 5a-reductase activity (Fig.
whether phosphoramide mustard might account for the changes in
liver enzyme levels observed following oxazaphosphorine treatment.
2C) but to a lesser extent than did cyclophosphamide. Northern blot
analysis of hepatic CYP 2C11 mRNA and steroid 5a-reductase mRN A Adult male rats were given single i.p. injections of phosphoramide
2492
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ACROLEIN.
PHOSPHORAMIDK
MUSTARD.
M
Fig. 3. Alteration in liver mRNA expression fol
lowing treatment with ifosphamide. Total liver
RNA was prepared from adult male rats (M ) treated
with saline (¡tint's1. 2. and //). ifosphamide (IFA }
( 120 mg/kg. lanes } and 4: 180 mg/kg. lunes 5 and
6). or cychiphosphamide iCPA I ( 120 mg/kg, lanes
7 and K) and killed 7 days later. RNA samples from
untreated adult female rat livers (Ft. which do not
express the male-specific CYP 2CII hut have high
levels of the female-predominant steroid 5o>reductase. are included for comparison (lanes 9 and 10 ).
Shown are autoradiograms of a Northern hlot ana
lyzing total liver RN'A (one individual liver RNA
AND GENE EXPRESSION
(120)
(180)
(120)
IFA
IFA
CPA
M
2C11
B.
5ccR
sample per lane), probed sequentially with the CYP
2C1I (A) and steroid 5a-reductase (ßlgene-spe
cific oligonucleotides (29). Rat a-iubulin mRNA
levels (C) are indicative of the RNA load and in
tegrity for the samples shown in A and H.
Tubulin
i
mustard at 20-100 mg/kg and then were killed 7 days later. At the
lower doses used in the experiments, phosphoramide mustard did not
affect CYP 2C11-catalyzed testosterone 2a-hydroxylase activity or
steroid 5a-reductase activity (Table I and data not shown), nor did it
alter CYP 2C11 mRNA or steroid 5a-reductase mRNA levels (Fig. 4.
A and ß).In contrast, phosphoramide mustard at 100 mg/kg decreased
testosterone 2a-hydroxylase activity by 90<7cand increased steroid
5a-reductase activity by ~4.5-fold, corresponding to a feminization
ratio of 28 (Table 1). Although this dose of phosphoramide mustard
altered these enzyme activities in a manner similar to that effected by
cyclophosphamide (120 mg/kg). it also conferred general systemic
toxicity, as exemplified by major body weight loss (Table 1). In
cyclophosphamide-treated
rats, body weight typically decreases by
~5-10% during the first 7 days after drug administration but then
Table I Effect of in VÕYO
treatment with cvcloplutsphantitle,
10
8
11
stabilizes for the remainder of the experimental period (see Table 3).
In contrast, a continual decline in body weight occurred during the
7-day observation period in rats treated with the 100 mg/kg dose of
phosphoramide mustard (Table I and data not shown). Thus, phos
phoramide mustard can modulate liver enzyme activities in a manner
similar to that of cyclophosphamide.
Kffects of Phosphoramide Mustard and HPD-cyclophosphamide in Combination. To test whether the modulation of the specific
hepatic enzymes by cyclophosphamide is achieved through (he com
bined action of acrolein and phosphoramide mustard, rats were given
i.p. injections of phosphoramide mustard (50 mg/kg). HPD-cyclophosphamide (200 mg/kg). or both agents in combination, and the
animals were killed 9 days later. The doses chosen were shown to have
minimal gross toxicity (body weight loss) in earlier experiments (Ta-
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. or
HPD-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 ±SD in cases where the number («)of rats was three or four per group or as mean ±half the range for groups with two rats.
5«-reduclasc
activity
activity
(nmol/min/mg)"1
(nmol/min/mg)"0.88
TreatmentSalineCPAAcroleinHPD-CPAPMn4322"2'32222Dose
(mg/kg)1203510100ISO20050100Testosterone2a-hydroxylase
in
ratio''0.6191.7fi.4NA0.90.51.20.528Change
body(g)'+32
weight
0.400.33
.47 ±
0.306.38
±
±4-21
±0.130.75
2.581.30
±
10-3±
70.23NA'1.31
±0.1
±0.121.47NA1.12*0.120.55
3-59NA+
±
41.79±0.1
0.530.97
±
0.430.82
±
I6±5+
1+5
I0±
±3+
±0.090.14
±0.02Steroid
0.083.94
±
±0.49Feminization
±3-56
15
±4
±0.371.18
±0.190.83
" 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.
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ACROLEIN.
F
M
PHOSPHORAMIDI:
MUSTARD.
HPD-cyclophosphamide. half responded to the drug combination. In
the responsive rats. CYP 2C11-catalyzed microsomal cyclophosphamide hydroxylation and testosterone 2a-hydroxylation
were de
creased by -45% and -90%, respectively, hepatic CYP 2C11 mRNA
<j
u g. -a
2C11
became undetectable. and serum testosterone was depleted. This pat
tern of response is similar to that observed following cyclophospha
mide administration (Table 2 and Fig. 5A). Steroid 5a-reductase ac
tivity and mRNA were both increased in the responsive rats but to a
lesser extent than in the cyclophosphamide-treated
rats (Table 2 and
Fig. 5B}. Body weights of the responsive rats declined for 5 days after
treatment and then stabilized for the remainder of the experimental
period but were still lower than those of the cyclophosphamide-treated
rats (Table 3). Overall, these findings demonstrate that, while phos
phoramide mustard is responsible for modulation of liver enzyme
expression by cyclophosphamide. acrolein potentiates the modulating
activity of the mustard.
5aR
DISCUSSION
A.
B.
t
Previous studies have shown that the decreases in total liver cytochrome 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 specific
liver cytochrome P450 enzymes (9). While some cytochromes P450
were found to decrease, others increased substantially following cy
clophosphamide administration. The overall effect is to feminize the
pattern of expression of liver enzymes in a manner that is similar to.
but mechanistically distinct from, that observed with the alkylating
agent cisplatin (22, 23). The present study establishes that ifosphamide is also capable of feminizing the expression of these liver en
zymes and that the effects occur at a pretranslational step, involving
suppression of the male-specific CYP 2C11 mRNA and elevation of
the female-dominant steroid 5a-reductase mRNA. The present study
also provides insight into the mechanism by which these oxazaphosphorines alter the expression of these mRNAs. Phosphoramide mus
tard is shown to be responsible for the modulation of liver enzyme
expression by cyclophosphamide. while acrolein potentiates these
effects of the mustard.
While ifosphamide and cyclophosphamide were both effective in
modulating liver enzyme levels, ifosphamide was found to be less
potent than cyclophosphamide. Higher doses of ifosphamide are also
required to achieve an equivalent plasma alkylating activity in cancer
patients (3.8 g/m2 versus 1.1 g/m2 for cyclophosphamide) (34). The
c.
Tubulin
1
AND GENE EXPRESSION
23456
Fig. 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 deClcyclophosphamide ttleCI-CPA} (2(X) mg/kg. lane 7) and were killed 7 days later. Total
liver RNA was prepared and Northern blot analysis was performed as in Fig. 3, RNA
samples from untreated adult female rat livers (Fi are included for comparison (lanes I
and 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 on
hepatic microsomal enzyme activities, serum testosterone levels (Ta
ble 2). or body weight profiles (Table 3). CYP 2C1I mRNA and
steroid 5«-reductase mRNA levels were also unaffected (Fig. 5. A and
B). Of the 12 rats treated with both phosphoramide
mustard and
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 enzyme
activities and serum testosterone concentrations were determined as described in "Materials and Methods." Results are expressed as mean ±SD for the indicated number (in of rats
per treatment group.
TreatmentSalineCPAPMHPD-CPAPM
4-hydroxylase
activity
(nmol/min/mg)"4.
2tt-hydroxy]ase
activity
(nmol/min/mg)"1.53
5a-reductase
activity
(nmol/min/mg)"0.98
testosterone
ratio'10.6370.81.3190.9Serum
(ng/ml)'2.6
±0.48''2.01
12
±0.300.23
0.488.47
±
1.9I.I
±
±0.86''4.32
0.041.31
±
2.081±
±0.53.4
0.663.70
±
±0.271
0.31
.02 ±
±2.12.8
0.732.33
±
0.250.15±O.I5
. 11 ±
0.652.79
.49 ±
1.9<0.042.5
±
HPD-CPAResponders
+
±0.49
±1.02
Nonrespondersn13g6666Cyclophosphamide
4.03 ±0.59Testosterone
I.I7±O.I8Steroid
1.02 ±0.49Femini/alion
" 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 due to the intermittent release of testosterone bv the testis.
ll n = 6 rats used tor these activity measurements.
2494
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±1.0
ACROLEIN.
Table 3 Effect ofphosphoramide
PHOSPHORAMIDE
MUSTARD.
AND GENE EXPRESSION
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 ±SD tor the indicated number in) of rats per
treatment group.
Body weight (g)
TreatmentSalineCPAPMHPD-CPAPM
(I"199
1201
±5204
±6192
±4205
±6201
10205
±
10I90±ll190
±
5218*8193
±4205
7226
9235
±8180
±8188
±3217±
±5225
±7236
12204
13215
±
15225
±
16161
±
13160
±
13159
±
10205
±
±12Day
13215
±
±11Day
17225
±
±
HPD-CPAResponders
+
±6
194±9Day
6207±
±1Day
13" Nonrespondersn986666Day
Day after treatment.
observed differences in the potency of these two drugs can largely be
explained by quantitative differences in their metabolism. Cyclophosphamide is metaboli/ed predominantly at the C4 position of the oxa/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 less
mustard and less acrolein than does cyclophosphamide.
The suppression by cyclophosphamide and ifosphamide of hepatic
CYP 2CII occurred at a pretranslational step. It is likely that CYP
2C1I transcription is the step that is affected in oxazaphosphorinetreated 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 steroid
5a-reductase levels could involve effects on one or more of the
hypothalamo-pituitary and gonadal factors that regulate expression of
these genes (37). rather than direct effects on the liver. Indeed, the
suppression of CYP 2CI1 mRNA and the elevation of steroid 5areductase mRNA by cyclophosphamide and ifosphamide. or by the
combination of HPD-cyclophosphamide and phosphoramide mustard.
M
is shown to be associated with a substantial decrease in serum levels
of testosterone, which is required for maintenance of the sexually
dimorphic expression of these enzymes (38—40).However, while en
dogenous androgen secretion in cyclophosphamide-treated rats can be
stimulated by the luteinizing hormone analogue chorionic gonadotropin. the resultant increase in serum testosterone does not reverse the
suppression of hepatic CYP 2C11 (9). This observation is analogous
to the finding that the suppression of CYP 2CII by 3.4.5,3'.4',5'hexachlorobiphenyl is also not causally related to the associated de
pletion of serum testosterone (41 ). Consequently, modulation of liver
enzyme expression by cyclophosphamide and ifosphamide may in
volve action at the hypothalamo-pituitary axis, which establishes the
sex-dependent plasma growth hormone profile that in turn determines
the expression of hepatic CYP 2C11, steroid 5a-reductase, and other
drug- 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 and
steroid 5a-reductase by cyclophosphamide. acrolein potentiates the
modulating activity of the mustard. When administered alone, neither
F
CPA
PM
HPD-Cm
PM+HPD-CPA
•¿â€¢
Fig. 5. Effect of phosphoramide mustard und
HPD-cyclophosphamide combination treatment on
hepatic CYP 2C11 and steroid So-rcductase mRNA
levels. Adult male rats (M\ were treated with saline
(lanes I and 2}, cyclophosphamide (CPA} (120
mg/kg. lanes 5 and 6). phosphoramide mustard
(PM] (50 mg/kg. lanes 7-V). HPD-cyclophospha
mide (HPD-CPA} (200 mg/kg. lanes 10-12). or a
combination ol phosphoramide mustard (50 mg/kg)
plus HPD-cyclophosphamide
(2(K) mg/kg) (lanes
13-16) and were killed 9 days later. Total liver
RNA was prepared and Northern blot analysis was
performed as in Fig. 3. RNA samples from un
treated adult female rat livers (/•")
are included for
- 2C11
B.
5aR
comparison (lanes 3 and 4). A. CYP2C1I mRNA;
B, steroid 5a-reductase mRNA; C. u-tubulin
mRNA (control). The reduced signal in ¡tine5 for
tubulin mRNA indicates that the RNA analyzed in
this lane was underloaded, compared to the other
samples.
Tubulin
123456
8
9
10
11
12
13
14
15
16
2495
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ACROLEIN.
PHOSPHORAMIDE
MUSTARD.
acrolein nor acrolein precursors (HPD-cyclophosphamide and deClcyclophosphamide) feminized the expression of these enzymes to the
same extent as did cyclophosphamide. In contrast, phosphoramide
mustard altered the expression of CYP 2C11 and steroid So-reductase,
similarly to cyclophosphamide. hut this effect was achieved at a dose
of mustard ( KM)mg/kg) that produced greater body weight loss than
did cyclophosphamide. This difference in toxicity might be due to
differences in the pharmacokinetics and tissue distribution of phos
phoramide mustard given as a bolus i.p. injection, compared to phos
phoramide mustard derived from chemical decomposition of the pri
mary 4-hydroxy metabolite in cyclophosphamide-treated
rats.
Whereas treatment with a lower dose of phosphoramide mustard (50
mg/kg) or with the acrolein precursor HPD-cyclophosphamide (200
mg/kg) alone did not affect liver enzyme profiles, the combination of
these two agents suppressed CYP 2CI1 and elevated steroid 5areductase enzyme activities and mRNA levels, in addition to depleting
serum testosterone, an overall pattern that is similar to that produced
by cyclophosphamide. These effects of the combination treatment
were observed in only half of the animals tested and suggest that a
concentration threshold exists at the site(s) of action of these metab
olites to elicit the modulation of liver enzyme expression. Such a
threshold has been documented for acetaminophen-induced hepatic
necrosis (44). The glutathione-depleting agent buthionine sulfoximine
also appears to potentiate phosphoramide mustard-mediated cardiotoxicity in rodents (28). Acrolein or cyclophosphamide treatment
of rats can decrease cellular glutathione content, whereas phosphora
mide mustard is only somewhat effective at glutathione depletion
when given at high doses (28, 45). Thus, acrolein may render target
cells more sensitive to the modulating activity of phosphoramide
mustard by reducing intracellular glutathione levels. As noted above,
the effects of cyclophosphamide and the combination of phosphora
mide mustard and HPD-cyclophosphamide
on liver mRNA levels
indicate that these sensitizing effects of acrolein probably occur at
endocrine secretory organs, rather than the liver. Glutathione is found
not only in liver but also in many other tissues, including brain (46).
Cyclophosphamide and ifosphamide are typically administered to
cancer patients as part of a combination chemotherapy regimen. The
present study indicates that these alkylating agent prodrugs not only
can alter their own biotransformation but also may affect cytochrome
P450-mediated bioactivation or deactivation of concurrently admin
istered drugs. Clinical studies have shown that chronic administration
of oxazaphosphorines to cancer patients can increase drug clearance
(14, 18), suggesting that human liver cytochrome P450 enzymes are
also subject to modulation by these oxazaphosphorines, albeit by
mechanisms that lead to increased rates of drug metabolism. Since our
studies implicate phosphoramide mustard as the primary mediator of
the effects of the parent drug on liver drug metabolism, it may not be
possible to design active oxazaphosphorines that lack these potential
drug interactions. Finally, the finding that acrolein potentiates the
enzyme-modulating effects of phosphoramide mustard has a broader
implication, insofar as environmental exposure to acrolein may trigger
or exacerbate drug- and xenobiotic agent-induced systemic toxicity.
Indeed, significant levels of acrolein are found in tobacco smoke (47).
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2344-2350. 1989.
Colvin. M., and Hilton. J. Pharmacology of cyclophosphamide and metabolites.
Cancer Treat. Rep.. 65 (Suppl. 3): 89-95. 1981.
Brock. N. Comparative pharmacologie study in vitro and m vivo with cyclophospha
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Wrabetz, E.. Peter. G., und Hohorst. H. J. Does acrolein contribute to the cytotoxicity
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23.
24.
26.
27.
28.
29.
ACKNOWLEDGMENTS
The authors wish to thank Dr. J. Pohl (ASTA Pharma. Bielefeld. Germany)
and Dr. J. Hilton (Johns Hopkins Oncology Centre. Baltimore. MD) for kindly
providing the acrolein precursors used in this study.
30.
31.
32.
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2497
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Cyclophosphamide Modulates Rat Hepatic Cytochrome P450
2C11 and Steroid 5 α-Reductase Activity and Messenger RNA
Levels through the Combined Action of Acrolein and
Phosphoramide Mustard
Thomas K. H. Chang and David J. Waxman
Cancer Res 1993;53:2490-2497.
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