metabolism of diallyl disulfide by human liver microsomal ...

DIALLYL DISULFIDE METABOLISM BY HUMAN LIVER MONOOXYGENASES
NADPH, 50 mM Tris-HCl pH 7 in a total volume of 500 �l. After 30 min at
37°C, the reaction was stopped by adding 320 �l of acetonitrile. After 15 min
of protein precipitation, the mixture was centrifuged at 10,500g for 10 min and
40 �l of the supernatant was analyzed by HPLC. The same protocol was
applied with 25 pmol for each cDNA-expressed human CYP microsome. In
case of cDNA-expressed human FMO3 microsomes, 100 �g of protein was
added in a total volume of 125 �l. Many DADS concentrations were used to
determine the kinetic parameters.
Thermal inactivation of microsomes was performed by a preincubation of
microsomes in Tris-HCl buffer for 10 min at 37°C in the absence of NADPH,
or in the presence of NADPH for control microsomes. Then DADS and
NAPDH were added subsequently to start the incubation for 30 min.
Inhibition of DADS Metabolism. Inhibitors were added to the incubation
mixtures before initiation of the reaction. Only with the mechanism-based
inhibitors such as diethyldithiocarbamate or aminobenzotriazole, microsomes
were preincubated for 10 min at 37°C before the addition of DADS. Nonhydrosoluble
inhibitors were dissolved in 100% ethanol and the following volumes
of ethanol were added to the incubation medium (total volume, 500 �l):
0.2 �l for sulfaphenazole and orphenadrine, 0.3 �l for coumarin, 0.4 �l for
diethyldithiocarbamate, 0.5 �l for aminobenzotriazole and quinidine, 0.8 �l
for �-naphthoflavone, and 2.5 �l for nifedipine. The human samples KS1,
K12, K25, KS28, K33, K40, KS41, and KS63 were used for the inhibitory
experiments.
HPLC Analysis. HPLC analysis was carried out using a Waters (Saint
Quentin-en-Yrelines, France) system equipped with a model 600 pump, a
model 717 auto sampler, a model 996 photodiode array UV detector, and a GL
Sciences Inc. (Tokyo, Japan). Interstil ODS-3 column (4.6 � 150 mm). The
flow rate was 0.6 ml/min and the solvent-isocratic program was 30:70 (v/v,
acetonitrile/water) for 20 min. The spectrum from 190 to 300 nm was used to
detect DADS and its metabolites. The quantification was made at 254 nm. Data
were processed by Waters Millenium software.
Determination of Kinetic Constants. K m and V max were determined with
a range of substrate concentrations of 0 to 7.5 mM with human liver microsomes
and FMO cDNA-expressed microsomes, and 0 to 5 mM with CYP
cDNA-expressed microsomes. The values were estimated by fitting the
Michaelis-Menten equation using a nonlinear regression program of SAS
Software (Cary, NC). The human samples KS1, KS30, K33, and K40 were
used in this experiment. The apparent V max of FMO3 cDNA-expressed microsomes
was determined considering that 100 �g of protein corresponded to
approximately 100 pmol of enzyme. This consideration was based on the
specific activity of recent lots of FMO3 from Gentest.
Enzyme Assays. Detailed information for these assays are given Table 2.
The coumarin 7-hydroxylase (COH) activity was determined according
to Maurice et al. (1991); dextromethorphan O-demethylase (DOD) according
to Bourrié et al. (1996); 7-ethoxycoumarin deethylase (ECOD) according to
Ullrich and Weber (1972); ethoxyresorufin O-deethylase (EROD) according to
Burke et al. (1985); laurate hydroxylase (LAH) according to Parker and Orton
(1980) with few modifications; mephenytoin hydroxylase (MpH) according to
Meier et al. (1985); methimazole oxidase (MMO) according to Dixit and
Roche (1984); nifedipine oxidase (NfO) according to Guengerich et al. (1986);
p-nitrophenol hydroxylase (PNPH) according to Tassaneeyakul et al. (1993);
and tolbutamide hydroxylase (TDH) according to Bourrié et al. (1996).
Correlation Analysis. To investigate the involvement of different CYP
isoenzymes in DADS oxidation, marker activities of several isoenzymes
(CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and
CYP4A) were measured for at least 24 samples of human liver microsomes.
The enzyme assays were performed as described above. Calculations of the
correlation coefficient between the rate of transformation of DADS and the
marker CYP activities were made with the SAS system (Cary, NC) with the
following equation: r � covariance (x,y) / (variance (x) � variance (y)) 1/2 .
Results
Identification of Metabolite. When DADS was incubated with
human liver microsomes and NADPH, only one peak was detected by
HPLC (Fig. 2). This peak was identified as DADSO by comparing its
retention time in different HPLC gradients and its absorption spectrum
with that of synthesized DADSO. No other metabolite was
detected for various incubation times of DADS. When either NADPH
or microsomes were omitted, no DADSO was detected.
Kinetics of Reaction. The formation of DADSO was linear over a
period of 45 min. An incubation time of 30 min was therefore
routinely used. A concentration of 1 mM NADPH was optimal for the
reaction. The kinetic of formation of DADSO by liver microsomes
was consistent with the Michaelis-Menten equation. The apparent Km was 0.61 � 0.2 mM and the apparent Vmax was 18.5 � 4.2 nmol/
min/mg protein. Values are means � S.E.M. for four samples.
Contribution of FMOs to DADS Oxidation. To evaluate the
respective roles of FMOs and CYP in the oxidation of DADS, we
initiated incubations of DADS in the presence of : 1) 1-aminobenzotriazole,
a suicide inhibitor of CYPs (De Montellano and Mathews,
1981; Fig. 3 shows the inhibition of DADS oxidation by this inhibi-
TABLE 2
Incubation conditions for the determination of marker enzyme activities in human liver microsomes
COH DOD ECOD EROD LAH MpH MMO NfO PNPH TDH
Buffer A B C D E B F G H B
NADPH (mM) 1 1 0.05 0.25 1.5 1 0.1 1 1 1
Microsomal proteins
(mg/ml)
0.28 1 0.13 1 0.125 2 0.3 2 1 2
Substrate (mM) 0.1 0.05 1 0.001 0.13 0.2 1 0.2 0.1 2
Total volume (ml) 0.36 0.5 2 0.4 0.5 0.5 1 0.5 0.5 1
Temperature (°C) 37 37 30 30 37 37 37 37 37 37
Incubation time (min) 3 20 4 3 15 30 5 10 20 30
Stop reagent (�l) HCl 1N HCl 1N HCl 1N AcN/TCA
20%, 50/50
Perchloric acid 0.15 M H3PO4 HPLC separation Yes Yes Yes Yes Yes Yes
UV detection (nm) 412 254 340 230
Radiometric detection 14C 14C Fluorometric
detection
Excitation (nm)
380 280 380 522
Emission (nm) 450 312 450 586
A: 50 mM sodium phosphate buffer (pH 7.4)
B: 100 mM potassium phosphate buffer (pH 7.4)
C: 100 mM Tris-HCl buffer (pH 7.7), 1 mM EDTA
D: 100 mM Tris-HCl buffer (pH 7.7), 25 mM MgCl 2
E: 66 mM Tris-HCl buffer (pH 7.4)
F: 0.1 M tricine (pH 8.4), 1 mM EDTA, 0.06 mM 5,5�-dithiobis(2-nitrobenzoate), 6 mM potassium phosphate (pH 8.4), 0.02 mM dithiothreitol
G: 50 mM potassium phosphate buffer (pH 7.4), 3 mM MgCl 2
H: 100 mM sodium phosphate buffer (pH 6.8)
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