In vitro metabolism of the new insecticide flupyrazofos by rat liver microsomes

Article abstract of DOI:10.1080/004982597240415

1. The in vitro metabolism of the new insecticide flupyrazofos was studied using rat liver microsomes. Two metabolites were produced and identified as O,O-diethyl O-(1-phenyl-3-trifluoromethyl-5-pyrazoyl) phosphoric acid ester (flupyrazofos oxon) and 1-phenyl-3-trifluoromethyl-5-hydroxypyrazole (PTMHP) based on UV and mass spectral analysis. 2. Cytochrome P450 oxidatively converted flupyrazofos to flupyrazofos oxon, a major metabolite and phenobarbital-induced microsomes increased this desulphuration by 8-fold. 3. Flupyrazofos oxon was converted to PTMHP with a half-life of 47.8 min by chemical hydrolysis and this conversion also proceeded non-enzymatically under our microsomal incubation conditions.

Full text of DOI:10.1080/004982597240415

xenobiotica , 1997, vol. 27, no. 5, 423±429
In vitro metabolism of the new insecticide
upyrazofos by rat liver microsom es
¯
H. S. LEE‹*, S. JEONG‹, K. KIM‹, J. H. KIM‹, S. K. LEE‹,
B. H. KANG‹ and J. K. ROH‹
‹
‹
College of Pharmacy, Wonkwang University, Shinyongdong, Iksan 570-749, Korea
Toxicology Research Center, Korea Research Institute of Chemical Technology,
Yoosung, PO Box 107, Taejeon 305-606, Korea
Received 14 October 1996
1
. The in vitro metabolism of the new insecticide ¯upyrazofos was studied using rat
liver microsomes. Two metabolites were produced and identi®ed as O,O-diethyl O-(1-
phenyl-3-tri¯uoromethyl-5-pyrazoyl) phosphoric acid ester (¯upyrazofos oxon) and 1-
phenyl-3-tri¯uoromethyl-5-hydroxypyrazole (PTMHP) based on UV and mass spectral
analysis.
2
. Cytochrome P450 oxidatively converted ¯upyrazofos to ¯upyrazofos oxon, a major
metabolite and phenobarbital-induced microsomes increased this desulphuration by 8-
fold.
3
. Flupyrazofos oxon was converted to PTMHP with a half-life of 47±8 min by
chemical hydrolysis and this conversion also proceeded non-enzymatically under our
microsomal incubation conditions.
Introduction
Flupyrazofos [O,O-diethyl O-(1-phenyl-3-tri¯uoromethyl-5-pyrazoyl) thio-
phosphoric acid ester] (®gure 1) was developed by the Korea Research Institute of
Chemical Technology (KRICT, Taejeon, Korea) as a new insecticide in 1987. This
organophosphorothionate was found to be very eåective against the diamond-back
moth (Plutella xylostella) acting as a contact and stomach poison inhibiting
acetylcholinesterase as other organophosphorous pesticides do. No phototoxicity
was observed and a low acute toxicity (rat, oral) of 372±605 mg}kg was reported
(
SungBo Chemical’s report, unpublished data). In an aquatic toxicity test, the EC&!
for carp in 48 h was 0±9 ppm, and mutagenicity, teratogenicity and delayed
neurotoxicity were not observed (unpublished data).
Good thermostability (Cho and Han 1992) and photostability (Cho et al. 1993)
were reported with small amounts of degradation products including O,O-diethyl
O-(1-phenyl-3-tri¯uoromethyl-5-pyrazoyl) phosphoric acid ester (¯upyrazofos
oxon), O,S-diethyl O-(1-phenyl-3-tri¯uoromethyl-5-pyrazoyl) phosphorothiolate
and 1-phenyl-3-tri¯uoro-methyl-5-hydroxypyrazole (PTMHP).
The purpose of the present study is to understand in vitro metabolism of
¯
upyrazofos using rat liver microsomes and to characterize the major cytochrome
P450 isozymes involved in ¯upyrazofos metabolism.
M aterials and m ethods
Chemicals
Flupyrazofos (99 %), ¯upyrazofos oxon (99 %) and PTMHP (99 %) were kindly donated from
SungBo Chemicals Co. (Ansan, Korea). "% C-¯upyrazofos (speci®c activity 28 mCi}mmol, " 99 %) was
synthesized by GSF (Germany) in collaboration with KRICT. NADP+, glucose 6-phosphate, glucose 6-
*
Author for correspondence.
049±8254}97 $12±00 ’ 1997 Taylor & Francis Ltd
0

4
24
H. S. Lee et al.
Figure 1. Structures of ¯upyrazofos and its metabolites. *Site of the "% C label.
phosphate dehydrogenase and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were obtained from Sigma
Chemical Co. (St Louis, MO, USA). Phenobarbital sodium (PB) were obtained from Daehan
Pharmaceutical Co (Seoul, Korea) and the protein assay kit was from Bio-Rad Lab. (Richmond, CA,
USA). Scintillation cocktail (Ready Flow II) for ¯owthrough radioisotope detector was obtained from
Beckman (USA). All other chemicals were the highest grade commercially available.
Instruments
Hplc analysis was conducted with P4000 (Thermo Separation Production, Fremont, CA, USA)
equipped with a Focus scanning detector (TSP) using an Inertsil ODS-2 column (0±46¬15 cm, 5 lm,
GL Science, Tokyo, Japan). A b-RAM radioactivity ¯ow-through detector (IN}US Corp., FL, USA)
was used for the determination of radioactivity. The chromatograms were obtained from an IBM-
compatible computer using PC 1000 software package (TSP), or in part, Win¯ow software package
(
IN}US Corp.).
Mass spectra were recorded on GC}MS (Finnigan MAT GCQ) with electron impact mode using an
MS-Select column (0±25 mm i.d.¬30 m).
Preparation of liver microsomes
Speci®c pathogen-free male Sprague-Dawley rats (160±180 g) were pretreated with either PB
(
80 mg}kg, i.p. for 3 days) in saline, ethanol (5 ml}kg, p.o. for 3 days) or TCDD (10 lg}kg, i.p. 1 day)
in corn oil to enrich speci®c types of cytochrome P450 isozymes. The rats were sacri®ced at 48 h after the
last injection by cervical dislocation before the livers were perfused with saline to remove excess blood
and homogenized with 4 vol ice-cold 1±15 % potassium chloride solution (pH 7±4). The liver homogenates
were centrifuged at 9000g for 10 min at 4 °C and the resulting post-mitochondrial supernatants were
centrifuged again at 105 000g for 60 min at 4 °C. The microsomal pellets were resuspended in 50 mm
potassium phosphate buåer (pH 7±4) containing 0±1 mm EDTA. Aliquots of liver microsomes were
stored at ®70 °C until use. The content of microsomal protein was determined according to the method
of Bradford (1976) using bovine serum albumin as a standard.
Incubations and extraction
Control or induced rat liver microsomes (0±1 mg) were preincubated in 1±0 ml 50 mm potassium
phosphate buåer (pH 7±4) in the presence of 50 lm ¯upyrazofos with or without 0±1 lCi "% C-¯upyrazofos
or 50 lm parathion for 3 min at 37 °C in shaking water bath. The reactions were initiated by the addition
of a NADPH-generating system containing 0±8 mm NADP+, 10 mm glucose 6-phosphate, and 1 unit of
glucose 6-phosphate dehydrogenase into the reaction mixture (®nal volume 1±1 ml). The reactions were
stopped after a 10-min incubation by the addition of 50 ll 43 % phosphoric acid and 2 ml methylene
chloride. After mixing and centrifugation, the organic layer was removed and dried under a stream of
nitrogen gas. The residue samples were stored at ®20 °C until analysis.
Control rat liver microsomes (0±1 mg}ml) were incubated for 10 min with increasing ¯upyrazofos
concentrations ranging between 2 and 100 lm in the presence of the NADPH-generating system and the
formation of ¯upyrazofos oxon and PTMHP were monitored.
The recoveries of ¯upyrazofos, ¯upyrazofos oxon and PTMHP in the concentration range of
±50 nmol}ml were 96±5³4±5, 97±6³3±8 and 94±5³3±9 % respectively.
1
Hydrolysis of ¯upyrazofos oxon
PTMHP could be produced either by enzymatic hydrolysis of ¯upyrazofos and ¯upyrazofos oxon or
by non-enzymatic hydrolysis of ¯upyrazofos oxon. To address the mechanism for the formation of
PTMHP, ¯upyrazofos oxon (10 or 50 lm) was incubated with control rat liver microsomes as described

Desulphuration of ¯upyrazofos
425
Figure 2. Hplc chromatograms of ¯upyrazofos and its metabolites obtained from incubations of
upyrazofos with rat liver microsomes in the presence of an NADPH-generating system. Peaks:
M1, PTMHP; M2, ¯upyrazofos oxon.
¯
for ¯upyrazofos. The hydrolysis of ¯upyrazofos oxon to PTMHP was measured in 50 mm phosphate
buåer (pH 7±4) at 37 °C. Aliquots were removed from the incubations at diåerent time points and the
concentrations of ¯upyrazofos oxon and PTMHP were determined using reversed-phase hplc.
Analysis of metabolites
The samples were dissolved in 100 ll of the mobile phase for the analysis. An aliquot (20 ll) was
analysed by hplc using acetonitrile in 10 mm potassium phosphate (pH 3±0) (60 : 40, v}v) as a mobile phase
with a ¯ow rate of 1±0 ml}min. The eçuent was monitored using a Focus scanning detector at 230 nm and
a b-RAM radioactivity ¯ow-through detector with 2 ml}min scintillation cocktail when "% C-¯upyrazofos
was used.
For the identi®cation of metabolites, the hplc fractions of non-radioactive extraction were evaporated
under reduced pressure and the residues analysed by GC}MS.
Results
From the incubation studies of rat liver microsomes with ¯upyrazofos in the
presence of NADPH-generating system, the formation of its two possible meta-
bolites, ¯upyrazofos oxon and PTMHP (®gure 1) were observed and they were well

4
26
H. S. Lee et al.
Figure 3. Electron impact mass spectrum of the ¯upyrazofos oxon metabolite.
separated by reversed-phase hplc (®gure 2). These metabolites were identi®ed on
the basis of the mass spectral data, UV spectra and hplc elution pro®le comparing
with the authentic compounds (®gures 3 and 4).
The apparent kinetic constants for the formation of ¯upyrazofos oxon (including
PTMHP) were 10±1³3±59 lm and 729³73 pmol}mg protein}min for Km and Vmax
respectively.
In the absence of NADPH-generating system there were no metabolites formed,
thus, inferring, but not absolutely proving, the participation of cytochrome P450
and not microsomal esterases.
To investigate the eåects of diåerent cytochrome P450 isoforms on ¯upyrazofos
and parathion metabolism, the products formed during the incubation of
¯
upyrazofos or parathion with control, PB-, EtOH- and TCDD-induced rat liver
microsomes are shown in table 1. PB-induced microsomes increased the desulphura-
tion of ¯upyrazofos 8-fold when compared with the control microsomes. EtOH and
TCDD-induced microsomes had no signi®cant eåect on the desulphuration of
¯
upyrazofos in comparison with the control microsomes. However, the desulphura-
tion of parathion to paraoxon was increased four times by PB- and TCDD-induced
microsomes in comparison with the control microsomes.
The hydrolysis kinetics of ¯upyrazofos oxon were investigated for the possibility
of PTMHP formation by chemical hydrolysis. The hydrolysis of ¯upyrazofos oxon
to PTMHP was rapid and approximately followed ®rst-order kinetics with a half-
life of 47±8 min at 37 °C and pH 7±4.
To examine the possible enzymatic hydrolysis of ¯upyrazofos oxon to PTMHP
by esterases, ¯upyrazofos oxon was incubated with control rat liver microsomes
in the absence of the presence of an NADPH-generating system. The production
of PTMHP from ¯upyrazofos oxon (10 lm) was the same in both the

Desulphuration of ¯upyrazofos
427
Figure 4. UV spectra of rat liver microsomal ¯upyrazofos metabolites. (A) PTMHP ; (B) M2,
upyrazofos oxon ; (C) ¯upyrazofos.
¯
Table 1. Metabolism of ¯upyrazofos and parathion by rat liver microsomes. Flupyrazofos (50 lm) or
parathion (50 lm) and rat liver microsomes (0±1 mg}ml) were incubated in the presence of
NADPH-generating system for 10 min at 37 °C as described in the Materials and methods.
Results are expressed as mean³SD from separate microsomal preparations (n ¯ 4).
Metabolite production (pmol}mg protein}min)
Metabolites
Control
EtOH-induced
TCDD-induced
PB-induced
Flupyrazofos
Flupyrazofos
PTMHP
417³178
197³109
390³76
206³84
503³145
159³58
3198³417**
288³106
Parathion
Paraoxon
p-Nitrophenol
1080³295
355³104
1303³219
404³142
3811³415**
763³236**
3750³554**
1203³402**
!
±
* p 0 01 (compared with control).
*
absence and the presence of the NADPH-generating system (1±18³0±08 and
±24³0±10 lmol}incubation) and was not signi®cantly diåerent from that
1±34³0±03 lmol}incubation) in buåer-only incubations. As shown in ®gure 5,
upyrazofos oxon decreased but PTMHP increased with duration of incubation
1
(
¯
time. These results suggest that PTMHP formation could be formed non-
enzymatically from ¯upyrazofos oxon and not by microsomal esterase and}or
dearylase activity.

4
28
H. S. Lee et al.
Figure 5. Time-course for the metabolism of ¯upyrazofos by control liver microsomes. Incubations
contained "% C-¯upyrazofos (50 lm), control liver microsome (0±1 mg}ml), and an NADPH-
generating system. Flupyrazofos (^), ¯upyrazofos oxon (* ), PTMHP (D ). Each point represents
the mean and SEM of four experiments.
Discussion
In the present study, we investigated the metabolic pro®le of ¯upyrazofos by rat
liver microsomes and the contribution of cytochrome P450 to the metabolism of
¯
upyrazofos. Two metabolites were produced from ¯upyrazofos by rat liver
microsomes and this reaction was NADPH-dependent, suggesting that cytochrome
P450 is involved in the desulphuration of ¯upyrazofos. Those metabolites were
identi®ed as ¯upyrazofos oxon and PTMHP on the basis of UV and EI mass spectra.
Similar results were reported from microsomal oxidation of parathion and
fenitrothion which were metabolized to the corresponding oxon and phenol (Neal
and Halpert 1982, Neal 1985, Levi et al. 1988). This result demonstrated that the
initial metabolic reaction was oxidative desulphuration which is common for
organophosphorothionates (Kulkarni and Hodgson 1980).
The biotransformation of ¯upyrazofos or ¯upyrazofos oxon was particularly
enhanced by the pretreatment of PB, which is an inducer of cytochrome P4502B1}2
isozymes amongst others (Okey 1990). The biotransformation of parathion and
fenitrothion (Levi et al. 1988) to their oxon forms was particularly enhanced by the
pretreatment of phenobarbital and TCDD. These results indicate that cytochrome
P4502B1}2 isozymes are responsible for the desulphuration process of ¯upyrazofos,
but does not exclude the participation of additional, PB-inducible cytochrome
P450s. The diåerent eåect of TCDD pretreatment on parathion (or fenitrothion)
and ¯upyrazofos could be rationalized by their chemical properties and reactivities.
During the incubation of ¯upyrazofos with rat liver microsomes, the amount of
¯
upyrazofos oxon decreased with a corresponding increase of PTMHP. From these
studies, PTMHP was produced from ¯upyrazofos oxon to a similar extent,

Desulphuration of ¯upyrazofos
429
regardless of incubation conditions, suggesting that PTMHP can be formed from
chemical hydrolysis rather than from an enzyme-mediated process. This observation
was interesting because p-nitrophenol and m-cresol were formed from parathion and
fenitrothion by a monoxygenase-catalyzed reaction (Kamataki et al. 1976, Levi et al.
1
988).
In conclusion, the new organophosphorous insecticide ¯upyrazofos was de-
sulphurated in vitro to ¯upyrazofos oxon by cytochrome P450, and the hydrolytic
transformation of ¯upyrazofos oxon to another metabolite PTMHP was catalysed
non-enzymatically.
Acknow ledgem ent
The hplc system provided by Insung Hitech Co. is gratefully appreciated. This
study was supported by Wonkwang University in 1996.
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