Metabolic Activation of the Nitroaromatic Antiandrogen Flutamide by ...

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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