Dec 3, 1992 - Antiandrogen. Flutamide by Rat and Human Cytochromes. P-450, Including. Forms Belonging to the 3A and 1A Subfamilies1. ALAIN. BERSON ...
0022-3565/93/2661-0366$03.00/0 THE
JOURNAL
Copyright
OF PHARMACOLOGY
AND
0 1993 by The American
EXPERIMENTAL
Society
VoL 265, No. 1 in U.S.A.
THERAPEUTICS
for Pharmacology
and Experimental
Therapeutics
Printed
Metabolic Activation of the Nitroaromatic Antiandrogen Flutamide by Rat and Human Cytochromes P-450, Including Forms Belonging to the 3A and 1A Subfamilies1 ALAIN BERSON, CLAUDE WOLF, CLAUDE CHACHATY, CECILE FISCH, JACQUELINE LOEPER, JEAN-CHARLES GAUTHIER, PHILIPPE BEAUNE, PATRICK MAUREL and DOMINIQUE PESSAYRE
DANIEL FAU, DOMINIQUE DENIS POMPON,
EUGENE,
Accepted
for publication
December
3, 1992
ABSTRACT
The in vitro metabolic activation of flutamide, a nitroaromatic antiandrogen which produces hepatitis in a few recipients, was first studied with male rat liver microsomes. There was no electron spin resonance evidence for the reduction of flutamide by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase into a nitro anion free radical. In contrast, flutamide was oxidatively transformed by cytochrome P.450 into reactive metabolite(s) that covalently bound to microsomal proteins. Covalent binding required oxygen and NADPH, and was decreased by the nucleophile glutathione and by the cytochrome P-450 inhibitors SKF 525-A, piperonyl butoxide and troleandomycin (an inhibitor of the cytochrome P450 3A subfamily). Covalent binding was increased markedly by pretreatment with dexamethasone (an inducer of the cytochrome
with /3binding was immunoinhibited markedly by anticytochrome P-450 3A immunoglobulin G and moderately by anticytochrome P450 1 A immunoglobulin G. Covalent binding was much lower with liver microsomes from female rats (not expressing P-450 3A2). Covalent binding of fiutamide also occurred with human liver microsomes (where it was inhibited by troleandomycin), and with yeast microsomes expressing human liver cytochromes P450 1Al, 1A2 or 3A4. We concluded that flutamide was oxidatively transformed into chemically reactive metabolite(s) by rat and human cytochromes P450, including forms belonging to the 3A and 1 A subfamilies.
Flutamide is a nonsteroidal antiandrogen behaving as a competitive antagonist of the androgen receptor. This nitroaromatic compound is proposed in the treatment of metastatic prostatic carcinoma in association with castration. Several
occurrence toxicity
studies
patients cholestatic
have
concluded
to the
beneficial
effects
tion
of this
as judged on clinical and biological 1988; Crawford et at., 1989). After oral administration, flutamide
is rapidly
is extensively
undergoing
several
metabolized
sites
inant,
mainly
1991). Therapeutic Received 1
tories
for
in the
publication
work was supported (Contrat de Prestation
ultraviolet.
ESR,
liver,
et at. , 1988). Urinary form of metabolites
effects
This
ABBREVIATIONS:
366
(Schulz
in the
criteria
of flutamide September
in part
23,
electron
at
is predom-
and
Chrisp,
compounded
by
the
from
Sehering
INSERM/Schering
spin resonance;
Plough
Labors-
Plough NADPH,
89126).
reduced
(as
of hepatitis defined
at 4-fold
be
0.36%
by
in a few subjects. The incidence of liver an increase in serum transaminase
or more in
1091
above
upper
normal
consecutively
limits)
treated
was
prostate
found cancer
(Gometz et at., 1992). Flutamide-induced hepatitis is and/or hepatocellular in type, and fulminant hepatitis is possible (Hart and Stricker, 1989; Moller et at., 1990; Copp#{233}r#{233} et at., 1990; Alperine et at., 1991; Corkery et at., 1991). The chemical structure of flutamide partially resembles that of another antiandrogen, nilutamide (fig. 1). We have shown previously that the toxicity of nilutamide could be attributed to the one-electron reduction of the nitro group (Berson et at., 1991; lung
1992.
by a grant
de Recherche
and
oxidation
(Brodgen
are
et at.,
absorbed
excretion
activity to
associa-
(Beland
P450 3A subfamily) and moderately by pretreatment naphthoflavone (an inducer of the 1A family). Covalent
Berger microsomal
et at.,
1992;
lyzed the one-electron sponding nitro anion nicotinamide
adenine
Fau
et at.,
1992).
NADPH-cytochrome
reduction free
dinucleotide
radical
Indeed,
P-450
of nilutamide (Berson
phosphate;
rat
liver
reductase
to
et at., 1991;
lgG, immunoglobulin
or
cata-
its
corre-
Berger
et
G; UV,
Downloaded from jpet.aspetjournals.org at ASPET Journals on December 6, 2014
lnstitut National de Ia SantO et de Ia Recherche Medicale (INSEAM) U-24, H#{243}pital Beaujon, Clichy, France (A.B., C.F., D.F., D.E., J.L., D.P.); Laboratoire de Biochimie, Unite de Recherche Associe#{233}(URA) Centre National de Ia Recherche Scientifique (CNRS) 1283, Centre Hospitaller et Universitaire (CHU) SaintAntoine, Paris, France (C.W.); D#{233}partementde Physique GdnOrale, CEN Saclay, Gif-sur-Yvette, France (C.C.); INSEAM U75, CHU Necker, Paris, France (J.C.G., Ph.B.); Centre de GdnOtique MolOculaire du CNRS, Gif-sur-Yvette, France (D.Po.); and INSEAM U-128, Montpellier, France (P.M.)
1993
Metabolic
f
02N
reduction.
/CH3
liquid
Specimens
nitrogen
Animals
\CH3
Charles
FLUTAMIDE
a normal
02N
Some
kg; i.e., 0.4 mg/kg; i.e., phosphate for 3 days,
mmol/kg, 0.37
Fig. 1. Chemical
at.,
Under
1992).
radical
structures
anaerobic
of nilutamide
and nilutamide.
of flutamide
conditions,
was
further
the
reduced
nitro to
anion
reactive
free species
(i.e., the nitroso and the hydroxylamine derivatives) which bound covalently to microsomal proteins (Berson et at., 1991; Berger et aL, 1992). Under aerobic conditions, in contrast, the nitro anion free radical of nilutamide mainly reacted with oxygen. This reaction prevented further reduction of the nitro anion free radical and decreased covalent binding. However, reaction
of the
nitro
anion
radical
with
oxygen
regenerated
the
with concomitant formation of superoxide anion, dismuted to hydrogen peroxide (Berson et at., 1991; Berger et at., 1992). Redox cycling of nilutamide with formation of reactive oxygen species resulted in an oxidative stress in rat isolated hepatocytes (Fau et at., 1992). In the present study, we have investigated the metabolism of
parent
nitro
flutamide
compound
in rat
liver
microsomes.
We
found
that,
contrary
to
nilutamide, flutamide was not reduced detectably to a nitro anion free radical by rat liver microsomes. Instead, flutamide was oxidatively metabolized by microsomal cytochromes P-450, particularly cytochromes P-450 of the 3A and 1A subfamilies, into chemically reactive metabolite(s) responsible for covalent binding to microsomal proteins.
Chemicals.
Flutamide,
activity,
oratories
(Paris,
2-hydroxy-flutamide
and
engineered
mmol/kg,
NADPH was purchased from Boehringer (MannAll other biochemical agents were purchased from Sigma
Chemical
(St.
(Paris,
Human surgery
France). Co.
liver
under
M#{233}dicale from
Specimens reduction cardiac Wisconsin
(1988).
Louis,
specimens.
were
obtained were
Human
from to be
liver
specimens
adult
organ
transplanted
donors in
were
was
continued
during
the
whose
children.
the livers were perfused with a (U.W.) solution at 4’C, as described
arrest,
Perfusion
were
phenobarbital
in 0.5
were
113, UAR, sacrificed
fed
sodium
(100
mgI
9-naphthoflavone
ml of corn
oil)
ad
Villemoisby cervical (100
or dexamethasone
microsomes.
Human
liver Specimens
or
cerevisiae
W(R),
strain
which overexpresses
8 ml
of
20
mM
2-(N-morpholino)ethanesulfonic
acid
6, containing 1.5 M sorbitol, 12 mg of cytohelicase and 2 mg of zymoliase 60000 (Seikagabu Kogyo) until most cells were converted to spheroblasts. After washing, spheroblasts were lysed in a buffer,
pH
20 mM 3-(N-morpholino)propane 7 with
Tris
sulfonide
base
sulfonic
containing
fluoride
by
acid buffer
0.6 M sorbitol
short
sonication
and
adjusted
1 mM
(3 x 30 sec,
to pH
phenylethane-
60 W).
The
lysate
1,100 X g and then 20 mm at 14,000 X g. CaCl2 at a concentration of 18 mM was added to the supernatant, and the mixture was incubated for 15 mm at O’C. Microsomes were harvested by centrifugation (20 mm, 14,000 x g), washed with 50 mM Tris was
centrifuged
7.4,
for
5 mm
containing
as a suspension
1 mM
or with
EDTA
in the same a Bruker
and
25%
glycerol,
kept at
and
buffer.
Free radical
measurements.
E-9
at
ESR spectra
ER-200,
X-band
were recorded spectrometer
with
at room
22’C.
The UV-generated spectrum was achieved by UV irradiation of the ESR cell containing flutamide (72 mM) diluted in the electron donor solvent, methyltetrahydrofuran. UV were generated with a 500-W highpressure mercury arc (SP-500 Philips lamp) emitting at wavelengths superior to 2500 A. In experiments using rat liver microsomes as reductants, a 1-ml solution
of
0.1
mM
sodium-potassium
phosphate
buffer
containing
hepatic
MO).
obtained
at
a protocol approved by the Comit#{233} Consultatif d’Ethique the Centre Hospitalier et Universitaire Bichat-Beaujon.
in size,
i.p.,
of hepatic
in
Tris-HC1
ESR
heim, F.R.G.).
tories
Rats
given
i.p., in 0.5 ml of water),
Saccharomyces
suspended
a Varian
LabLabora-
Animals
(Autoclave
yeast NADPH-cytochrome P-450 reductase when grown on galactose as a carbon source. Transformed cells were cultured into S5 galactose medium at 28’C, and were harvested during the late exponential phase. Samples of 1 g of wet yeast cells were incubated for 10 mm at 30’C in 5 ml of 0.1 M Tris-HC1 buffer, pH 8, containing 5 mM EDTA, 100 mM KC1 and 5 mM dithiothreitol. After centrifugation, the cells were
-80’C
[14C]flutamide
15 mCi/mmol) were given by Schering-Plough France). Nilutamide was given by Cassenne
female Sprague-Dawley, g, were purchased from
300 France).
were
in
(50 mg/kg, i.e., 0.13 mmol/kg, i.p., in 0.5 ml of water) daily and were sacrificed 24 hr after the last dose of the inducer.
temperature,
(specific
rats
frozen
rat livers were weighed, minced and homogenized in 3 volumes of icecold 0.154 M KC1 and 0.01 M sodium-potaSSium phosphate buffer, pH 7.4. The homogenate was centrifuged at 10,000 x g for 10 mm. The supernatant was centrifuged at 100,000 X g for 60 mm. The microsomal fraction was washed with the same buffer and centrifuged at 100,000 x g for 60 mm. Microsomes were stored at -80’C until they were used (within 8 days after preparation). Protein concentration was determined by the method of Lowry et at. (1951). Transformation, cell culture and preparation of microsomes from yeast Saccharomyces cerevisiae. Human cytochromes P-450 1A1, 1A2, 2D6 and 3A4 were expressed separately in yeast using previously described procedures (Cullin and Pompon, 1988; Pompon, 1990; Renaud et aL, 1990; Urban et at., 1990). Briefly, the coding sequence from each isoenzyme was inserted into a yeast expression vector. Each expression vector was separately introduced into the
HC1, pH
Methods
until
male
were
of microsomes.
modified
process
livers, At
the
University
by Jamieson of graft
the
after time
microsomes (10 mg/ml) and 1 mM flutamide, 1 mM 2-hydroxyflutamide or 1 mM nilutamide was gassed with nitrogen for 10 mm in 10-ml stoppered flasks. The reaction was then initiated by adding NADPH (5 mM), and the sample was transferred anaerobically into ESR
cell.
The
spectra
were
recorded
in
the
microwave
of
frequency
of
The particular instrument settings for ESR measurements in the figure legends. Computer simulation of the experimental was carried out by the multiple nuclei Varian program.
et at. volume
band
X
at 20 mW.
are given spectra
Downloaded from jpet.aspetjournals.org at ASPET Journals on December 6, 2014
NILUTAMINE
and to
diet sacrifice.
standard
France)
dislocation.
Preparation H3
250
moiety
preparation
Male
weighing
367
of Flutamide
liver
until
(Saint-Aubin-les-Elbeuf,
libitum with son-sur-Orge,
CF
unused
at -80’C
treatments. rats,
River
the
stored
and
Crl:CD(SD)BR
NHCOCH
from
and
Activation
368
Bersonetal.
Vol. 265
Oxygen consumption, superoxide anion oxide production. Polarographic measurement tion
was
carried
out
water-jacketed
with
chamber
a Gilson
oxygraph
maintained
The microsomal
hydrogen perof oxygen consump-
and
at
suspension
equipped
37’C
and
contained
with
using
a 1.4-ml
a Clark
dee-
rat liver microsomes
(1.75 mg protein/ml) suspended in 1.5 mM EDTA, 0.15 mM KC1 and 0.1 M sodium-potassium phosphate buffer, pH 7.4. In some experiments, either flutamide (1 mM) or nilutamide (1 mM) was added. Oxygen consumption was measured after initiating the reaction with 1 mM NADPH. trode.
Production
of superoxide
(Misra
assay
and
anion
Fridovich,
was
1972)
determined
by the
by measuring
nm minus 575 nm, and using an absorption cm1. The microsomal suspension was the
adrenochrome
the absorption
coefficient
of
at 480
mM’.
2.96
same as that described for of oxygen consumption, except that incubations were carned out in the presence of 1 mM epinephrine. The microsomal suspension was divided between two cuvettes warmed at 37’C. NADPH (0.05 assessment
mM) was added
in the sample
rome
was
formation
of superoxide
depending
on
difference
dismutase
superoxide
between
Formation
anion
the
of
two
and the initial
rate of adrenoch-
30 sec in the absence (1 mg/mi). Adrenochrome
or in the formation
during
production
was
calculated
from
the
total
volume
of 500
ice and centrifuged remaining
l.
A 250-zl
incubation
similarly
processed.
microsomai
zero-time
at 100,000 mixture
x g for
was
Supernatants
proteins
was
first were
measured
sample
was
60
at
mm
taken, 4C,
incubated
at 37’C
discarded.
Covalent
described
above.
as
placed
presence of preimmune IgG (serving as alternative binding) decreased covalent binding to microsomal lent binding observed in the presence of anti-P-450 as a percentage of that in the presence of a similar mune IgG.
on
whereas
and binding
the
then to
Because
the
for covalent proteins, the covaIgG was expressed amount of preim-
targets
values.
hydrogen
peroxide
was
assayed
by
the
horseradish
method (Green and Hill, 1984). Rat liver microsomes (3 mg ofprotein in 1.5 mM EDTA, 0.15 mM KC1 and 0.1 M sodium-potassium buffer, pH 7.4) were incubated in the presence of 0.5 mM sodium azide for 10 mm. The reaction was initiated by adding 1 mM NADPH in the presence of either 1 mM flutamide or 1 mM nilutamide. The mixture (1 ml) was incubated with shaking under air at 37’C for 15 mm. The reaction was stopped by adding 0.5 ml of 5% trichloroacetic acid. After centrifugation (22,000 x g for 10 mm), the supernatant was brought back to pH 7. Supernatant samples (0.5 ml) were then mixed with 1ml aliquots of a solution containing 24.8 mM phenol, 4.3 mM 4-aminoantipyrine and horseradish peroxidase (19 enzyme units/ml) in 0.1 M sodium-potassium phosphate buffer, pH 6.9. The coupled oxidation of
Results
peroxidase
phenol
and 4-aminoantipyrine by H2O2 in the presence of horseradish peroxidase gave rise to a quinone imine adduct exhibiting a maximal 505
A standard curve with known amounts of for calculations. In vitro covalent binding. The incubation mixture consisted of 0.5 ml of 1 mM EDTA, 0.25 M sodium-potassium phosphate buffer, pH 7.4, [14C]flutamide (0.5 Ci/mi; 0.5 mM), 1 mM NADPH and either 5 mg of human or rat liver microsomal protein/mi or 1 mg of yeast absorbance
at
hydrogen
peroxide
microsomal
somes was
nm.
was
used
The reaction was initiated by mixing plus flutamide solution. A zero-time
protein/mi.
with the NADPH removed
the
and
remaining
mixture
was
incubated
microsample
with
shaking
under air at 37’C for either 10 (in experiments using human or rat liver microsomes) or 20 mm (in experiments using yeast microsomes). Anaerobic incubations microsomal suspension rately under nitrogen initiated mal
by
proteins
Proteins with
were carried and NADPH
or CO at 37’C for 10 mm before
mixing
the
two
was
measured
in the zero-time
components.
Proteins
Covalent
as described
previously
and the incubated
1 ml of 10% perchioric
ethylacetate.
out in the same way except plus flutamide were gassed
acid and
were
then
samples
extracted
washed
the
reaction
was
binding
to
(Berson
microso-
et at., 1991).
were precipitated
four times
with,
that sepa-
successively,
with
1 ml of 10%
per-
chioric acid, acetone, ethanol and 0.1 M sodium-potassium phosphate buffer, pH 7.4. After drying, the pellets were dissolved in 1 ml of 1 N NaOH. Aliquots were acidified with 12 N HC1 and counted for ‘4C radioactivity. Another aliquot was used to determine protein concentration. Nonspecific binding (in the zero-time sample) was subtracted from
that
in the incubated
sample.
Immunoinhibition experiments. Antibodies to highly purified rabbit liver cytochrome P.450 1A2 and rabbit liver cytochrome P-450 3c (P-450 3A6) were raised in sheeps and in goats, respectively. The IgG fraction
We verified hibited
was
isolated
as previously
that the anti-rabbit
the rat liver
1A2 ortholog
described
cytochrome by showing
(Daujat
P.450 that
et at., 1985).
1A2 IgG immunointhese
IgG inhibited
UV irradiation of a solution containing fludiluted in the hydrogen donor solvent methyltetrahydrofuran, generated a multiline ESR spectrum (fig. 2). No ESR signal was detected when the experiment was carried out in the absence of flutamide. Computer simulation of the flutamide experimental spectrum allowed the derivation of the hyperfine splitting constants and the assignment of the spectrum to the flutamide nitro anion free radical in its protonated form (fig. studies.
ESR
tamide,
2).
In contrast, anaerobic incubation of flutamide in the presence of NADPH and male rat liver microsomes did not form the flutamide nitro anion free radical in amounts detectable by ESR, even in the presence of high concentrations of either flutamide or liver microsomes (fig. 3). Likewise, no ESR signal was
detected
when
of 2-hydroxy-flutamide flutamide (Schulz
the
incubations
cubation
of
multiline
ESR spectrum to the nilutamide
attributed at., 1991). Oxygen
were
made
in the
(fig. 3), the main plasma et at., 1988). For comparison,
nilutamide
the
in
same
presence
metabolite anaerobic
of in-
generated
a
conditions
(fig. 3) which has been nitro anion free radical
previously (Berson et
consumption and formation of reactive oxyFlutamide (1 mM) did not increase oxygen consuperoxide anion formation or hydrogen peroxide by male rat liver microsomes (table 1). For compar-
gen species. sumption,
formation ison,
the
in
oxygen
(table
same
conditions, and
consumption
nilutamide reactive
(1
oxygen
mM)
species
increased formation
1).
Covalent binding with rat liver microsomes. Incubation of [‘4C]flutamide in the presence of male rat liver microsomes and NADPH resulted in the irreversible binding ofa radioactive material to microsomal proteins. Covalent binding was linear with time for the first 15 mm of incubation (not shown). An incubation time of 10 mm was selected for further experiments (tables
2-4).
Covalent creased
piperonyl nucleophile Covalent
binding in
the
presence
required of
NADPH the
cytochrome
and
02, P-450
and
was
de-
inhibitors
butoxide and SKF 525-A or in the presence of the glutathione (table 2). binding was increased markedly by pretreatment of
Downloaded from jpet.aspetjournals.org at ASPET Journals on December 6, 2014
presence
cuvette,
recorded
the 7-ethoxyresorufin-O-deethylase activity of rat liver microsomes in a dose-dependent manner, inhibition being approximately 50% with an anti-1A2 IgG/microsomai protein concentration ratio of 0.5 (not shown). The anti-rabbit cytochrome P-450 3c (3A6) IgG have been shown previously to cross-react with, and immunoinhibit, the orthologous proteins ofrat (P-450 3A1, 3A2) and humans (P-450 3A4) (Wrighton et at., 1985b; Watkins et aL, 1985; Lett#{233}ron et at., 1989; Larrey et at., 1990). Rat liver microsomes (2.5 mg) were incubated in the presence of [‘4C]flutamide (0.5 jCi/ml; 0.5 mM), NADPH (1 mM) and either preimmune IgG, anti-P-450 1A2 IgG or anti-P-450 3c (3A6) IgG, in a
1993
Metabolic
Activation
369
of Flutamlde
A lOG
lOG
B
C
Fig. 2. ESR spectrum of the flutamide anion freeradical produced by IN photolysis. Tracing A shows the spectrum of the protonated flutamide anion free radical recorded during IN photolysis of 72 mM flutamide diluted in the electron donor solvent methyltetrahydrofuran. The microwave power (band X) was 20 mW and the modulation amplitude 0.25 G. Tracing B shows the computer simulation of the flutamide nitro anion free radical. The hyperfine coupling constants were: aS = 1 6.9 G, F ‘r’ H _norr’ H _nr’ H r a3.,a-L.o,a-L,aracung shows the spectrum of the flutamide anion free radical under the same conditions as tracing A, except that modulation amplitude was 2 G. Tracing D shows the computer simulation of flutamide nitro anion free radical. The hyperfine coupling constants were as in tracing C and the line width was 2 G. #
,‘.
the
animals
moderately by pretreatments with either /3-naphthoflavone or phenobarbital (table 3). Troleandomycin, which selectively forms an inactive cytochrome P-450-Fe(II)-metabolite complex with cytochrome(s) P-450 of the 3A family (Wrighton et at., 1985a), decreased covalent binding by 26% in microsomes from nontreated male rats and by 79% in microsomes from dexamethasone-treated male
with
(table
rats
Covalent much express
dexamethasone
the
with
(mean ± S.E.M. microsomes from
male-specific
isoform
for four experiments) female rats, which P-450
3A2
(Gonzales,
was poorly 1990),
than with microsomes from male rats: 5.5 ± 0.3 pmol/mg protein/mm with female rat liver microsomes as compared to 10.2 ± 1.3 with male rat liver microsomes (P < .01). Immunoinhibition experiments with rat liver microsomes. As compared to that with identical amounts of preimmune
IgG,
covalent
binding
decreased anti-cytochrome P-450 with 5 mg of anti-P-450 proteins
was
TABLE
1
Comparison
of the effects and production rat liver mlcrosomes
to
untreated
rat
liver
microsomal
by 26% by preincubation with 8 mg of 1A2 IgG and by 47% by preincubation 3c IgG (table 4).
of nllutamide of reactive
consumption Oxygen mation ods.
and flutamide
oxygen
Oxygen
SueAn
-
Control Nilutamide (1 mM) Flutamide (1 mM) .P