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Introduction
Flutamide,
3¡¯-trifluoromethyl-4¡¯-nitro-2-methylpropinoyl-anilide, is a nonsteroidal
antiandrogen[1,2] devoid of other hormonal activity and is recognized worldwide as the most
beneficial drug for the treatment of patients with advanced
prostate cancer[3-5] when used in combination with various
luteinizing hormone-releasing factor agonists. This
antian-drogen is also used in combination with oral contraceptives
for the treatment of hirsutism[6] and benign prostatic
hyperplasia[7, 8].
However, flutamide therapy is associated with hepatitis
in a few subjects. The incidence of liver toxicity (as defined
by an increase in serum transaminase activity of fourfold
greater than upper normal limits) was found to be 0.36% in
1091 consecutively treated prostate cancer
patients[9-11]. Flutamide-induced hepatitis was decreased using piperonyl
butoxide [cytochrome P450 (CYP450) inhibitor] and increased
using b-naphthoflavone (CYP450 1A
inducer)[12].
Previous results show that CYP1A2 is involved in
fluta-mide bioactivation[12,13]. CYP1A2 is a cytochrome P-450
enzyme constitutively expressed in the liver, which catalyzes
the metabolism of many drugs, such as caffeine, phenacetin,
and propranolol[14]. The enzyme also participates in the
metabolic activation of chemical mutagens in cooked food such
as 2-amino-3-methylimidazo[4,5-f]quinoline and
2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
[15]. Previous studies have reported flutamide bioactivation using the P450 enzyme
system generated a nitro anion free radical after
treatment[16]. These radicals can bind covalently to proteins and lipids, or
remove hydrogen atoms from polyunsaturated fatty acids in
lipids, thereby initiating lipid peroxidation and liver cell
injury[17]. Therefore, CYP1A2 increase might be a risk factor
for flutamide-associated hepatotoxicity or carcinogenicity.
In the present study, we investigate the modulation of
CYP1A2 expression in adult male rats given flutamide for 2
weeks.
Materials and methods
Chemicals Flutamide was provided by the Shanghai
Hongqi Pharmaceutics Factory. Trizol reagent was obtained
from Life Technologies and reverse transcription-polymerase
chain reaction kits were obtained from Sangon Biological
Technique. Caffeine and its metabolite, and
b-hydroxyethyl-theophyline (HT) were obtained from Sigma Chemical
Company. Rabbit anti-rat CYP1A2 monoclonal antibody was
obtained from Santa Cruz Biotechnology (kindly provided
by Dr XC MA).
Animals and treatment Adult male Sprague-Dawley rats
weighing between 200 g and 250 g (Experimental Animal
Center of Fudan University, grade II, certification
No 01212) were housed at a temperature of 20
°C-25 °C under a 12 h light/dark cycle with 50% relative humidity in filtered and
pathogen-free air. The animals were acclimatized for 1 week prior
to use. For CYP1A2 induction studies, rats were divided
into five groups of 10 animals each. The first group was
given 0.5% carboxymethylcellulose sodium (CMC) as a
control, and the second group of rats received
3-methylcholanthrene (3-MC, 30 mg/kg, ip, for 5 d) as a positive
control for CYP1A2 induction. Flutamide and 3-MC were also
suspended in 0.5% CMC. The rats were treated once daily,
ig, for 14 consecutive days. All rats were killed on d 16.
Livers were excised and immediately frozen on ice and stored
at -80 °C for total RNA and liver microsomal preparation.
RNA extraction and cDNA synthesis Total RNA was
obtained from frozen rat livers (0.5 g-1 g) using Trizol
reagent. The RNA concentration was determined
spectroscopically at 260 nm, and 5 µg of total RNA was taken for
reverse transcription. Random primer (300 ng) was added
and primer annealing was performed using incubation for
10 min at 65 °C and 10-min progressive cooling on ice.
Deoxynucleotides (dNTP) (4 mmol/L each), 5 µL of
10×reaction buffer, 50 U of block ribonuclease inhibitor, and
200 U of SuperScript II RNase reverse transcriptase were
added to a final volume of 50 µL, and samples were
incubated at 42 °C for 30 min. The reaction was stopped by
heating to 90 °C for 5 min. Control samples contained
distilled water instead of RNA. The synthesized cDNA was
stored at -20 °C until use.
Polymerase chain reaction Reverse transcription sample
of 2.5 mL was added to the corresponding polymerase chain
reaction mixture containing 0.5 µmol/L sense and antisense
primer, 2 mmol/L MgCl2, 10 mmol/L Tris-HCl (pH 9),
50 mmol/L KCl, 0.1% Triton X-100, and 80 µmol/L dNTP. After heating
at 95 °C for 5 min and cooling to the primer-annealing
temperature for 5 min (60 °C), 2 U of
Taq DNA polymerase was added. The following primers were used: CYP1A2 forward
(+127 to +147), 5¡¯-GGACCCTGGGGCTTGCCCTTC-3¡¯; reverse
(+484 to +504), 5¡¯-AGCCTCTTTGCTCACGTGCTC-3¡¯;
b-actin forward (+59 to +69),
5¡¯-CCCAAGTCGCCTCC-GTCCCGC-3¡¯; reverse (+894 to +914),
5´-CCCTCCAGGAGCCCCATGAGC-3¡¯. The specificity of
CYP1A2 primers was confirmed through alignment with the CYP subfamily DNAs. Amplification
conditions for all primer pairs were used as follows: 94 °C 30 s,
60 °C 30 s, and 72 °C 30 s for 30 cycles.
b-actin segment was amplified separately and used as an external standard,
because interaction of primer pairs caused a decrease in signal
intensity. A total 12 µL of each preparation was subjected to
agarose gel electrophoresis in the presence of ethidium
bromide. The gel was photographed using Polaroid 665 film
and the intensity of each band was quantified using the
software provided with the ABI 7700 system. Each assay
was performed in triplicate. Results were expressed as a
ratio of the optical intensity of the
CYP1A2 to that of the
b-actin. This approach allows for the semiquantita-tion of
the expression of the CYP1A2 gene.
Immunoblot analysis Liver microsomes were prepared
as described by Kamataki and
Kitagawa[18]. The protein concentration was determined using Bradford¡¯s
method[19], using a protein assay with bovine serum albumin as a standard.
Three micrograms of microsomal protein were resolved
separately by electrophoresis on 10% SDS-polyacrylamide gel
and then transferred onto a nitrocellulose membrane. The
membrane was incubated with a rabbit anti-rat CYP1A2
monoclonal antibody, and further incubated with goat anti-rabbit
secondary antibody conjugated with horseradish peroxidase,
followed by detection with 3,3¡¯-diaminobenzidine and
hydrogen peroxide. The membrane was scanned with an image
scanner, and the signal intensity was quantified using the
software provided with the ABI 7700 system. In the present
study, the levels of CYP1A2 proteins were in the linear range
for densitometric readings.
CYP1A2 activity Caffeine was used as a probe for
CYP1A2 activity and plasma caffeine (137X) and its
metabolite (17X) were assayed using high performance liquid
chromatography[20]. After the last dose of flutamide and
overnight fast, each rat received 20 mg/kg (ip) of caffeine. Blood
samples (2 mL) were drawn after caffeine administration.
Plasma was separated and frozen at -20°C for later analysis.
The mixtures of plasma containing 17X and 137X was
extracted with chloroform/isopropanol (9:1). The residue
remaining after evaporation was dissolved in 0.1 mL elution
and 20 µL was injected into the chromatographic system.
HT was chosen as an internal standard (IS). All components
were separated isocratically on a reversed-phase column
using 0.05% acetic acid:acetonitrile:methanol (82:8:10) as a
mobile phase at a flow rate of 1 mL/min. Ultraviolet
detection wavelength was 282 nm. The retention times for 17X,
137X, and IS were 4.50 min, 5.03 min, and 8.13 min,
respec-tively. The CYP1A2 activity was expressed as a ratio of the
concentration of 17X to that of 137X observed 6 h after
giving caffeine.
Statistical analysis The statistical significance of the
difference between the means of the two groups was
assessed using the independent t test
(spss software package,
1997). P<0.05 was considered statistically significant.
Results
I>CYP1A2 mRNA levels The levels of
CYP1A2 mRNA were normalized using a comparison with
b-actin, constitutively expressed in the rat liver. The increase in
CYP1A2 mRNA was expressed as a percentage of the control. After
flutamide treatment (50 mg/kg) of rats for 14 d, liver
CYP1A2 mRNA levels were similar to control levels, but
CYP1A2 mRNA levels were increased 1.86 and 3.11-fold relative to
controls in rats in flutamide 100 mg/kg and 200 mg/kg group,
respectively. CYP1A2 mRNA levels were increased
15.6-fold by 3-MC treatment (Figure 1).
CYP1A2 protein Similar results were obtained for the
analysis of the P450 protein. After 50 mg/kg flutamide
treatment, CYP1A2 protein in rat liver microsomes was
similar to that in the control group. However, CYP1A2 protein
levels increased 1.78 and 2.89-fold respectively, after 100
mg/kg and 200 mg/kg flutamide treatment as compared to
those in controls. In contrast, 3-MC treatment increased the
CYP1A2 protein content 13.8-fold (Figure 2).
CYP1A2 enzyme activity Caffeine is hydroxylated to
form paraxanthine (17X) using CYP1A2. Concentration ratio
of caffeine metabolite paraxanthine to caffeine (17X/137X)
was used to express the activity of CYP1A2. After giving
caffeine for 6 h, CYP1A2 activity in rats treated with flutamide
(50 mg/kg) was similar to that in the control group. In rats
given 100 mg/kg and 200 mg/kg of flutamide, CYP1A2
enzyme activity increased 1.65 and 2.83-fold, respectively, as
compared to that in the control group. 3-MC increased
CYP1A2 activity 8.50-fold more than those in the control
group (Figure 3).
Discussion
In the present study, CYP1A2 mRNA, protein, and
enzyme activity were induced by flutamide treatment in rats.
It is reasonable to speculate that flutamide is a CYP1A2
inducer in rats. This P450 induction is dose-dependent as it
increases with increasing amounts of flutamide in the liver.
The magnitude of CYP1A2 induction assessed using three
different parameters, that is, mRNA, protein, and enzyme
activity, was found to be comparable. The differences
between the mRNA and protein induction and between protein
and enzyme activity in rats were less than 2-fold; therefore, it
is highly probable that the CYP1A2 induction occurs mainly
at a pretranslational level, and we speculate that flutamide-
mediated CYP1A2 induction is a result of increased rate of
transcription.
CYP1A2 is one of the major constitutively expressed
P-450 in the liver tissue. It catalyzes steroid hydroxylations,
such as 17b-estradiol
2-hydroxylation[21,22]. Of considerable
interest is the ability of P4501A2 to catalyze the
N-hydroxylation of carcinogenic aryl amines and heterocyclic
amines[23-25]. The oxidized products can modify DNA, either directly or
following conjugation with acryl or sulfate
groups[26,27]. The heterocyclic amines are of considerable interest in that they
are found in charbroiled food and cigarette smoke. There is
some epidemiological evidence that elevated levels of
CYP1A2 can be a predisposing factor to colon cancer,
although the risk is marginal unless high levels of
N-acetyl-transferase are present along with high charbroiled meat
consumption.
Flutamide can be metabolized by CYP1A2 to form the
reactive metabolites. Nitro radical and one-electron
reduction products have toxic effects on the liver
cells[16]. Induction of CYP1A2 can increase the formation of toxic
metabo-lites, and increase the incidence of liver toxicity. This might
explain why flutamide had no significant liver toxicity after
low doses (50 mg/kg), although it caused liver toxicity at
higher doses (100 mg/kg and 200 mg/kg) in previous
studies[28]. Therefore, the induction of CYP1A2 might have a large
influence on the therapeutic efficacy and liver toxicity associated
with flutamide treatment. Induction of CYP1A2 using
flutamide treatment and its possible effects on the
metabolism of other drugs and the carcinogenesis of heterocyclic
amines in human beings would be useful topics of further
study.
Acknowledgments
We thank Yi WANG for kindly supplying b-actin
primer. The supports from PhD Xin WU and Hong-Xin ZHU from
our university are also acknowledged.
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