The roles of carboxylesterase and CYP isozymes on the in vitro metabolism of T-2 toxin

Chemicals and reagents

T-2 toxin, HT-2 toxin, neosolaniol (NEO), acetyl T-2, T-2 triol, T-2 tetraol and zearalanone
(ZAN) with a purities greater than 98 %, bis (4-nitrophenyl) phosphate (BNPP), ethyleneglycol-bis
(2-aminoethylether) -tetraacetic acid (EGTA), tetraisopropylpyrophosphamide (iso-OMPA),
phenacetin, dextromethorphan, tolbutamide, ?-naphthoflavone, sulfaphenazole, quinidine
and tranylcypromine of analytical grade were purchased from Sigma Aldrich (St. Louis,
MO, USA). We synthesized 3?-OH T-2 (purity of 99 %) in our laboratory. HPLC grade
methanol and acetonitrile were obtained from DUKSAN (Sungkok, Korea). All other reagents
were of analytical grade or better and purchased from Sino Pharm chemical reagent
Co. Ltd (Beijing, China). Sterilized ultrapure water was generated with a Milli-Qultrapure
water system (Millipore, Billerica, MA, USA). Human liver microsomes and nicotinamide
adenine dinucleotide phosphate (NADPH) were purchased from Beijing Dingguo Changsheng
Biotechnology Co., Ltd. (Beijing, China). CYP450 enzymes and S-mephenytoin were purchased
from BD Gentest (Woburn, MA, USA). Midazolam and ketoconazole were purchased from
the National Institutes for Food and Drug Control (NIFDC, Beijing, China).

All samples were separated in an Agilent 1200 HPLC system equipped with a binary pump,
an Agilent control union, a degasser, and an auto sampler, and then detected by an
Agilent 6430 triple quadrupole mass spectrometry (QqQ MS) equipped with an electrospray
ion source (Agilent Co., California, USA). The introduction of the liquid phase was
accomplished with an Agilent XDB C18 column (50 mm long, 4.6 mm diameter, 1.8 ?m particle
size, Agilent Co., California, USA) coupled with a Waters in-line filter kit (Waters
Co., Milford, USA). All data were collected and analyzed by Agilent ChemStation software
with MassHunter acquisition system (ver. B.04.00).

Inhibitor phenotyping samples 15]

T-2 toxin (50 ?L) at a concentration of 10 ?mol/L was pre-incubated with 3 ?l BNPP
at a concentration of 3 mmol/L (an inhibitor of carboxylesterase), 1 ?L EGTA at a
concentration of 1 mmol/L (an inhibitor of paraoxonase), 5 ?L iso-OMPA at a concentration
50 ?mol/L (an inhibitor of acetylcholine esterase) with human liver microsomes at
37 °C for 5 min, respectively. HLM at a concentration of 0.5 mg/mL was then added
to start the reaction, and 200 ?L solution of 1 mmol/L ZAN in 1:3 methane: acetonitrile
was added to stop the reaction after 30 min.

Chemical inhibition method to determine the contribution of CYP450 enzymes 15]

The sample group was prepared as follows: 100 ?L T-2 toxin at a concentration of 10 ?mol/L
was pre-incubated with 80 ?L of ?-naphthoflavone at a concentration of 10 ?mol/L (an
inhibitor of CYP1A2), sulfaphenazole at a concentration of 40 ?mol/L (an inhibitor
of CYP2C9), tranylcypromine at a concentration of 400 ?mol/L (an inhibitor of CYP2C19),
quinidine at a concentration of 10 ?mol/L (an inhibitor of CYP2D6), or ketoconazole
at a concentration of 10 ?mol/L (an inhibitor of CYP3A4), respectively, in HLMs at
37 °C for 5 min. NADPH (20 ?l) at a concentration of 1 mmol/L was then added to start
the reaction, and a solution of 1 mmol/L ZAN in 1:3 methane: acetonitrile was added
to stop the reaction after 30 min.

The inhibitor group was prepared as follows: all procedures were same as for the sample
group, except that the carboxylesterase inhibitor BNPP was added at the beginning.
For the positive control group, all procedures were same as the sample group, except
that no isozyme inhibitor was added. For the negative control group, all procedures
were same as the sample group, except that no inhibitor was added.

Each sample was prepared in triplicate. After the reactions were terminated, all samples
were vortexed for 2 min and then centrifuged at 14,000 r/min for 10 min. The supernatant
was injected in the HPLC-QqQ MS.

Recombinant CYPs test 15]

All the procedures were same as for the sample group, except that the CYP450 isozyme
was used instead of the liver microsomes. The isozymes included CYP2C19, CYP3A4, CYP2C9,
CYP2A6, CYP2B6, CYP2D6, CYP1A2, CYP2C8 and CYP2E1, at a concentration of 25 nmol/L.

To determine the effects of NADPH, we used the same incubation conditions as for the
sample group. Moreover, we added the stop solution at 0, 1, 2, 3, 4, 5, 10, 15, 20,
30 min to stop the reaction.

Statistical analysis

The data were expressed as the mean?±?SD and analyzed by an ANOVA test for independent
samples using the SPSS 11.5 statistical software. The level of significance was P??0.05.

HPLC-QqQ MS parameters

The analysis was followed our previously established method with slight modifications
16]. The separation was performed on an Agilent XDB C18 column (50 mm long, 4.6 mm diameter,
1.8 ?m particle size) coupled with a Waters in-line filter kit. The mobile phase A
was 5 mmol/L ammonium acetate in water, and the mobile phase B was 5 mmol/L ammonium
acetate in methanol. The gradient profile was 65 % B held from 0 to 0.5 min, followed
by a linear increase to 100 % B over 3 min that was held for 2.5 min, then a decrease
to 65 % B over 0.1 min that was held for 0.9 min, for a total run time of 7.0 min.
The flow rate was 0.6 mL/min, the injection volume was 5 ?l, and the column temperature
was set to 50 °C.

The HPLC eluent was transferred by the XDB-C18 chromatographic column after the guard
column to an Agilent triple-quadrupole mass spectrometer (QqQ MS) in the positive
electrospray ionization mode. The capillary voltage was set to 3500 V, the source
temperature was set at 350 °C, and the drying gas was set at 9 L/min. All of the analytes
were measured in the multiple reaction monitoring (MRM) mode. All other parameters
listed in Table 1.

Table 1. Parameters for the analytes and internal standard in HPLC-QqQ MS