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YAMAMIYA ET AL.
metabolism, we used 10.5 U/ml of purified TPase. To determine the sponta-
neous degradation of FT, we performed the reaction without TPase. The
reactions were initiated by the addition of cDNA-expressed CYP2A6 or
purified TPase, after preincubation for 5 min at 37°C. The reactions were
performed as described for the assay of FT metabolism in human hepatic
microsomes, cytosol, and S9. To examine the formation of metabolites from
the furan ring of FT, we added a 3.1 mg/ml aliquot of DNPH/HCl solution to
the mixture after the reaction, followed by incubation for 15 min at 37°C. Next,
DNPH derivatives were extracted with ethyl acetate, and the organic layer was
dried under a nitrogen stream. The resultant residue was dissolved in the
mobile phase and injected into the LC-MS/MS system.
Assay to Determine the Formation of GBL/GHB from 4-OH-BTL in
Human Liver Microsomes or Cytosol. The standard mixture contained 62.5
M 4-OH-BTL and 1 mg/ml cytosol in 100 mM phosphate buffer-0.1 mM
EDTA (pH 7.4). We used -NADϩ or an NADPH-generating system as
cofactors in these assays. The spontaneous degradation of 4-OH-BTL to
GBL/GHB was evaluated using inactivated human liver cytosol, which was
prepared by boiling it at 100°C for 5 min. After preincubation for 5 min at
37°C, reactions were initiated by the addition of human liver cytosol, followed
by incubation for 3 min. Reactions were stopped by mixing the samples with
3 volumes of ice-cold acetonitrile. After the centrifugation, the supernatant was
collected and stored at Ϫ80°C until the determination of GBL.
Inhibition Study. We determined the effects of inhibitors of CYP2A6 and
TPase on the formations of the FT metabolites, including 5-FU, GBL/GHB,
SA, and 4-OH-BTL. TCP and TPI were used as inhibitors of CYP2A6 and
TPase, respectively (Fukushima et al., 2000; Zhang et al., 2001). Each inhibitor
was added to the reaction mixture at a concentration of 10 M.
We determined the effects of different oxidase inhibitors, disulfiram (an
aldehyde dehydrogenase inhibitor), 4-methylpyrazole (an alcohol dehydroge-
nase inhibitor), and menadione (an aldehyde oxidase inhibitor), on the forma-
tion of GBL/GHB from 4-OH-BTL in human hepatic cytosol at a concentra-
tion of 100 M (Pietruszko, 1975; Lam et al., 1997; Lake et al., 2002; Obach
et al., 2004). The reactions were performed as described above.
Assay to Determine the Enzyme Activities of Biological Samples (Pos-
itive Control). CYP2A6 and TPase activities of biological samples were
evaluated by measuring coumarin 7-hydroxylase activity and the formation
rate of thymine (from thymidine), respectively. Coumarin 7-hydroxylase ac-
tivity was determined by a fluorometric assay (Bogaards et al., 2000). The
thymine formed from thymidine was measured with high-performance liquid
chromatography. The high-performance liquid chromatography analysis was
performed with a Prominence LC-20 system (Shimadzu, Kyoto, Japan)
equipped with a TSKgel ODS-100V column (4.6 mm i.d. ϫ 150 mm, 3 m;
Tosoh, Tokyo, Japan). The flow rate was 1.0 ml/min, and the column temper-
ature was 25°C. The mobile phases were A (4.5% acetonitrile) and B (aceto-
nitrile). Typical conditions for the elution were as follows: 100% A (0–8 min),
20% A (8.5 min), 20% A (10.5 min), and 100% A (11–25 min). The eluent was
monitored at 256 nm to determine thymine and thymidine. The activities of
aldehyde dehydrogenase and alcohol dehydrogenase were assayed by moni-
toring the formation of -NADH from -NADϩ at a wavelength of 340 nm
during the metabolism of acetaldehyde and ethanol, respectively. The aldehyde
oxidase activity was measured using phenanthridine as the substrate, according
to the published method (Lake et al., 2002).
Measurement of DNPH Derivatives. The LC-MS/MS system consisted of
an HP1100 series liquid chromatograph (Agilent Technologies, Santa Clara,
CA) coupled with an API4000 triple-quadrupole mass spectrometer (Applied
Biosystems, Foster City, CA) equipped with a Turbo V source and ESI
interface. Chromatographic separation of DNPH derivatives was performed on
a XBridge C-18 column (4.6 mm i.d. ϫ 150 mm, 5 m; Waters, Milford, MA)
using 10 mM ammonium acetate and acetonitrile under gradient elution
conditions at a flow rate of 0.2 ml/min. We used the following gradient
programs: 10 mM ammonium acetate-acetonitrile from 70:30 (v/v) to 10:90
(v/v) in 7 min, immediately back to 70:30 (v/v), and then held for 8 min. Oven
temperature was maintained at 40°C. TurboIonSpray was used for ionization
with negative ion detection for the measurements of DNPH derivatives. The
source temperature was set at 600°C, ionization voltage at Ϫ4 kV, and orifice
potential at Ϫ60 V. Multiple reaction monitoring (MRM) was performed in the
negative ionization mode, after the reactions, m/z 247 to m/z 181 (CE 30 eV)
2,4-dinitrophenylhydrazine (DNPH) derivatization procedure,
which is widely used in the analysis of aldehydes and ketones
(Zwiener et al., 2002; Andreoli et al., 2003), to detect unstable
metabolites formed from the furan ring of FT.
Materials and Methods
Chemicals. FT, 5-chloro-2,4-dihydroxypyridine (CDHP), 5-chloro-6-(2-
iminopyrrolidin-1-yl) methyl-2,4(1H,3H)-pyrimidinedione hydrochloride
(TPI), and 4-OH-BTL were synthesized at Taiho Pharmaceutical Co.
(Saitama, Japan). Succinaldehyde disodium bisulfite was purchased from
Tokyo Chemical Industry Co. (Tokyo, Japan). 5-FU, -NADϩ, glucose
6-phosphate, GBL-d6, DNPH, tranylcypromine hydrochloride (TCP),
phenanthridine, menadione sodium bisulfite, and 4-methylpyrazole were
purchased from Sigma-Aldrich (St. Louis, MO). Magnesium chloride hexa-
hydrate, disulfiram, coumarin, thymidine, thymine, acetaldehyde, ethanol,
and GBL were purchased from Wako Pure Chemical Industries (Osaka,
Japan). -NADPϩ and glucose-6-phosphate dehydrogenase were purchased
from Oriental Yeast Co. (Tokyo, Japan). Other chemicals used were of the
highest grade commercially available.
Enzymes. Human liver samples were purchased from XenoTech, LLC
(Kansas City, KS). Membranes prepared from Escherichia coli expressing
CYP2A6 (Bactosomes) were obtained from Cypex Ltd (Dundee, UK). Control
membranes expressed only the vector. Purified TPase from E. coli was pur-
chased from Sigma-Aldrich.
Preparation of Standard of DNPH Derivatives. The standard DNPH
derivative of SA was prepared using the following procedures. Succinaldehyde
disodium bisulfite was dissolved in an HCl aqueous solution. The reaction was
initiated by the addition of a 3.1 mg/ml aliquot of DNPH/HCl solution to the
SA/HCl aqueous solution, and the mixture was incubated at 37°C for 15 min.
Next, the DNPH derivative was extracted with ethyl acetate. The organic layer
was dried, and the resultant residue was purified by silica gel column chro-
matography (Wakogel-C200; Wako Pure Chemical Industries). The molecular
structure of the derivative was confirmed by use of a hybrid quadrupole
time-of-flight (QqTOF) mass spectrometer and 1H NMR analysis. The stan-
dard DNPH-derivative of 4-OH-BTL was also prepared using the same pro-
cedure as that used for the preparation of SA-DNPH. 4-OH-BTL was incu-
bated in DNPH/HCl solution at 37°C. The DNPH derivative was extracted
with ethyl acetate, and the collected organic layer was evaporated. The deriv-
ative of 4-OH-BTL was characterized using its parent ion, and the subsequent
fragmentation pattern was characterized using LC-MS/MS.
Assay to Determine the Formations of 5-FU and GBL/GHB from FT in
Human Liver Microsomes, Cytosol, or S9. The decrease in the amount of
5-FU formed from FT may be attributed to extensive metabolism of 5-FU by
contaminated DPD. Therefore, a potent DPD inhibitor, CDHP, was added to
all incubations to inhibit the degradation of 5-FU. The standard reaction
mixture contained FT, 1 mM CDHP, and an NADPH-generating system
consisting of 1.3 mM -NADPϩ, 3.3 mM glucose 6-phosphate, 3.3 mM
magnesium chloride, and 0.4 U/ml glucose-6-phosphate dehydrogenase, in 100
mM phosphate buffer-0.1 mM EDTA (pH 7.4). The reactions were initiated by
the addition of human hepatic microsomes, cytosol, or S9, after preincubation
for 5 min at 37°C. All incubations were performed for 30 min at 37°C.
Reactions were stopped by the addition of 3 volumes of ice-cold acetonitrile to
the mixtures. After the centrifugation, the supernatant was collected and stored
at Ϫ80°C until the determinations of 5-FU and GBL. Both microsomes and
cytosol were used at a concentration of 1 mg/ml, and S9 was used at a
concentration of 2 mg/ml. The spontaneous degradation of FT was evaluated
using inactivated S9, which was prepared by boiling it at 100°C for 5 min.
Assay to Determine the Formations of 5-FU, GBL/GHB, SA, and
4-OH-BTL from FT in cDNA-Expressed CYP2A6 or Purified TPase. The
standard reaction mixture contained FT and 1 mM CDHP in 100 mM phos-
phate buffer-0.1 mM EDTA (pH 7.4). To examine CYP2A6-mediated metab-
olism of FT, cDNA-expressed CYP2A6 and an NADPH-generating system
were added to the reaction mixtures. The spontaneous degradation of FT was
evaluated using control membranes. To investigate the effect of human liver
cytosol on CYP2A6-mediated formations of 5-FU and GBL/GHB, cytosol was
added to bacterial membranes containing expressed CYP2A6 or control mem-
branes, and the final concentration was 1 mg/ml. In the case of TPase-mediated