T. Fukami et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
3
2.5. Other hydrolase activities
2.9. Kinetic analyses
Flutamide [21], indiplon [22], phenacetin [15], rifampicin [23],
fenofibrate [13], and irinotecan [24] hydrolase activities were
determined according to our previous reports. Individual HLM
from 24 donors were used as the enzyme sources. The protein con-
centrations were as follows: fenofibrate and irinotecan hydrolase
activities, 0.2 mg/ml; flutamide, phenacetin, and indiplon
hydrolase activities, 0.4 mg/ml; rifampicin hydrolase activity,
The kinetic analyses of KC hydrolysis and DAK N-hydroxylation
were performed with 2–150 M and 2–500 M of reaction mix-
tures, respectively. The kinetic parameters were estimated from a
curve fitted using a computer program designed for nonlinear
regression analysis (KaleidaGraph; Synergy Software, Reading,
PA). The following equations were used:
l
l
Michaelis—Menten equation : V ¼ Vmax ꢁ ½Sꢂ=ðKm þ ½SꢂÞ
0.5 mg/ml. The substrate concentrations were as follows: 500
flutamide, 50 M indiplon, 1 M phenacetin, 50 M rifampicin,
M fenofibrate and 2 M irinotecan.
lM
l
l
l
2
Substrate inhibition equation : V ¼ Vmax ꢁ ½Sꢂ=ðKm þ ½Sꢂ þ ½Sꢂ =KiÞ
5
l
l
where V is the velocity of the reaction, S is the substrate concentra-
tion, Km is the Michaelis–Menten constant, Vmax is the maximum
velocity, and Ki is the substrate inhibition constant.
2.6. DAK N-hydroxylase activity
The DAK N-hydroxylase activity was determined as follows: a
typical incubation mixture (final volume of 0.2 ml) containing
100 mM potassium phosphate buffer (pH 7.4 or pH 8.8), various
enzyme sources (HLM: 0.5 mg/ml; recombinant FMO1, FMO3:
0.02 mg/ml, recombinant FMO5: 0.075 mg/ml) and DAK (final con-
2.10. HepaRG cell culture
Human hepatoma-derived HepaRG cells obtained from Bio-
predic International (Rennes, France) were grown according to
the manufacturer’s instructions as follows: the cells were seeded
in culture dishes and maintained in growth medium (William’s E
Medium with no glutamine (Life Technologies, Gaithersburg,
MD), supplemented with 10% fetal bovine serum (FBS) (Life
centration of 300 lM). DAK was dissolved in methanol such that
the final concentration of methanol in the incubation mixture
was 1%. In a preliminary study, we confirmed that the DAK
N-hydroxylase activity was linear with respect to protein concen-
tration (HLM: <0.5 mg/ml; recombinant FMO1 and FMO3:
<0.08 mg/ml; recombinant FMO5: <0.1 mg/ml) and incubation
time (HLM: <10 min; recombinant FMO1 and FMO3: <5 min;
recombinant FMO5: <60 min). The reaction was initiated by the
addition of an NADPH-GS after a 2-min pre-incubation at 37 °C.
After a 10-min (HLM), 3-min (recombinant FMO1 and FMO3), or
45-min (recombinant FMO5) incubation at 37 °C, the reaction
Technologies), 100 units/ml penicillin, 100 lg/ml streptomycin,
2 mM glutamine, 5
at 37 °C under an atmosphere of 5% CO2 and 95% air. The medium
l
g/ml insulin, and 5 ꢀ 10ꢃ5 M hydrocortisone)
was renewed every 3 days. After 14 days, the cells were detached
by gentle trypsinization and seeded in
a 24-well plate at
0.55 ꢀ 105 cells/well with the growth medium. After 14 days, the
culture medium was changed to a differentiation medium (growth
medium containing 2% DMSO) and the cell culture was maintained
for 14 days to differentiate the cells.
was terminated by the addition of 100
After removing the protein by centrifugation at 20,380g for
5 min, 5- aliquot of the supernatant was subjected to
ll of ice-cold acetonitrile.
a
ll
LC–MS/MS. The LC–MS/MS apparatus and conditions were the
same as described above except that the conditions for elusion
were as follows: 20–90% B (0–3 min), 90% B (3–11 min), 90 to
20% B (11–11.01 min), and 20% B (11.01–18 min). The collision
energy was 40 V. Two m/z ion transitions were monitored in
MRM mode: m/z 505.2 and 81.0 for N-hydroxy DAK.
2.11. Overexpression of human AADAC in HepaRG cells
Differentiated HepaRG cells were infected with recombinant
adenoviruses expressing AADAC (AdAADAC) or green fluorescent
protein (AdGFP) at MOI 2.5. The AdAADAC and AdGFP were
constructed in previous studies [23,25]. After 24 h, the medium
was changed to an adenovirus-free medium, and the cells were
incubated for 72 h. To confirm overexpression of AADAC in HepaRG
cells, flutamide hydrolase activity in cellulo was measured as an
AADAC marker activity as follows: The cells were incubated with
2.7. Identification of N-hydroxy DAK
Because an authentic standard of N-hydroxy DAK was not com-
mercially available, LC–MS/MS analysis was performed to examine
whether the formed metabolite was N-hydroxy DAK. The samples
were prepared as described above except that 100 mM potassium
phosphate buffer (pH 8.8), recombinant FMO3 (0.2 mg/ml), and
500 lM flutamide for 1 h, and the medium was collected to
measure the FLU-1 concentration with high-performance liquid
chromatography (HPLC) as previously described [21]. The protein
concentrations of total cell homogenates were determined accord-
ing to the method of Bradford [26] using
standard.
c-globulin as the
DAK (250 lM) were used. In the single-quadrupole mode
(Q1-scan) with LC–MS/MS, full-scan spectra were acquired with a
scan range of m/z 50–550. Subsequently, in the product ion scan
mode, Q1 spectra at m/z 505.2 was monitored and fragment ions
were scanned in the range of m/z 50–550.
2.12. Cytotoxicity assay using HepaRG cells
To evaluate the cytotoxicity of KC and DAK, a WST-8 (2-(2-meth
oxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-te
trazolium monosodium salt) assay, which is a modified MTT assay,
was performed as follows: the differentiated HepaRG cells were
2.8. Quantification of N-hydroxy DAK
The N-hydroxy DAK formation was quantified based on the
treated with 0, 10, 25, and 50 lM KC or DAK, which were dissolved
decreased amount of DAK. DAK (50
l
M) was incubated with
in methanol such that the final concentration of methanol in the
medium was 2%. After 24 h, CCK-8 reagent was added and the
absorbance of the medium at 450 nm was measured. The effect
of human AADAC on KC-induced cytotoxicity was evaluated as
follows: The differentiated HepaRG cells infected with AdAADAC
or AdGFP at MOI 2.5 for 24 h were treated with 10, 25, and
recombinant FMO3 (0.2 mg/ml) for 0, 1, 2, and 3 min. The
decreased amounts of DAK were quantified and applied to
the peaks of formed N-hydroxy DAK. After determining the peak
area per known content of N-hydroxy DAK, the value was used
to calculate the amount of N-hydroxy DAK formed in the
incubation mixture.
50 lM KC for 24, 48, or 72 h. CCK-8 reagent was added, and the
Please cite this article in press as: T. Fukami et al., Human arylacetamide deacetylase hydrolyzes ketoconazole to trigger hepatocellular toxicity, Biochem.