In vitro metabolism and interactions of pyridostigmine bromide, N,N-diethyl-m-toluamide, and permethrin in human plasma and liver microsomal enzymes

Article abstract of DOI:10.1080/00498250701813230

1. The in vitro human plasma activity and liver microsomal metabolism of pyridostigmine bromide (PB), a prophylactic treatment against organophosphate nerve agent attack, N,N-diethyl-m-toluamide (DEET), an insect repellent, and permethrin, a pyrethroid insecticide, either alone or in combination were investigated. 2. The three chemicals disappeared from plasma in the following order: permethrin > PB > DEET. The combined incubation of DEET with either permethrin or PB had no effect on permethrin or PB. Binary incubation with permethrin decreased the metabolism of PB and its disappearance from plasma and binary incubation with PB decreased the metabolism of permethrin and its clearance from plasma. Incubation with PB and/or permethrin shortened the DEET terminal half-life in plasma. These agents behaved similarly when studied in liver microsomal assays. The combined incubation of DEET with PB or permethrin (alone or in combination) diminished DEET metabolism in microsomal systems. 3. The present study evidences that PB and permethrin are metabolized by both human plasma and liver microsomal enzymes and that DEET is mainly metabolized by liver oxidase enzymes. Combined exposure to test chemicals increases their neurotoxicity by impeding the body's ability to eliminate them because of the competition for detoxifying enzymes.

Full text of DOI:10.1080/00498250701813230

Xenobiotica, March 2008; 38(3): 294–313
In vitro metabolism and interactions of pyridostigmine
bromide, N,N-diethyl-m-toluamide, and permethrin in
human plasma and liver microsomal enzymes
A. W. ABU-QARE* & M. B. ABOU-DONIA
Department of Pharmacology and Cancer Biology, Duke University Medical Center,
Durham, NC, USA
(
Received 8 November 2007; accepted 17 November 2007)
Abstract
. The in vitro human plasma activity and liver microsomal metabolism of pyridostigmine bromide
1
(PB), a prophylactic treatment against organophosphate nerve agent attack, N,N-diethyl-m-toluamide
(DEET), an insect repellent, and permethrin, a pyrethroid insecticide, either alone or in combination
were investigated.
. The three chemicals disappeared from plasma in the following order: permethrin > PB > DEET.
2
The combined incubation of DEET with either permethrin or PB had no effect on permethrin or PB.
Binary incubation with permethrin decreased the metabolism of PB and its disappearance from plasma
and binary incubation with PB decreased the metabolism of permethrin and its clearance from plasma.
Incubation with PB and/or permethrin shortened the DEET terminal half-life in plasma. These agents
behaved similarly when studied in liver microsomal assays. The combined incubation of DEET with
PB or permethrin (alone or in combination) diminished DEET metabolism in microsomal systems.
3
. The present study evidences that PB and permethrin are metabolized by both human plasma and
liver microsomal enzymes and that DEET is mainly metabolized by liver oxidase enzymes. Combined
exposure to test chemicals increases their neurotoxicity by impeding the body’s ability to eliminate
them because of the competition for detoxifying enzymes.
Keywords: Pyridostgimine bromide, permethrin, N,N-diethyl-m-toluamide (DEET), human plasma,
human liver, microsomal enzymes, Gulf War
Correspondence: M. B. Abou-Donia, Department of Pharmacology and Cancer Biology, Duke University Medical Center,
Durham, NC 27710, USA. Tel: þ1-919-684-2221. Fax: þ1-919-681-8224. E-mail: donia@acpub.duke.edu
*
Present address: Bioanalytical R&D, Drug Safety and Metabolism, Wyeth Research, Pearl River, NY 10965, USA.
ISSN 0049-8254 print/ISSN 1366-5928 online ß 2008 Informa UK Ltd.
DOI: 10.1080/00498250701813230

Metabolism and interactions
295
Introduction
Pyridostigmine bromide (PB) (Figure 1) that has been used for the treatment of myasthenia
gravis patients (Anquilloins & Hartvig 1980) has also been used as a prophylactic agent
against potential nerve gas attack during the Persian Gulf War in 1990–91 (Abou-Donia
et al. 1996a, b; Young & Evans 1998). Permethrin is a pyrethroid insecticide effective against
mites and head lice (Fraser 1994) and N,N-diethyl-m-toluamide (DEET) is an insect
repellent applied against mosquitoes and biting insects (Brown & Hebert 1997). These
chemicals were used by military personnel during the Gulf War (Young & Evans 1998;
Riviere et al. 2002). Combined exposure to PB, DEET and permethrin enhanced their
neurotoxicity in hens (Abou-Donia et al. 1996a, b), and rats (Abdel-Rahman et al. 2001,
2
004a, b), increased mortality in rats (McCain et al. 1997), and caused neurobehavioral
alterations in male rats (Hoy et al. 2000; Abou-Donia et al. 2004). In addition, the toxicity of
DEET to German cockroaches was increased following combined exposure with permethrin
or PB (Moss 1996). Furthermore, concurrent administration of PB and DEET caused
seizure in rats (Chaney et al. 1999). In addition, following in vitro incubation of DEET and
PB with Escherichia coli, DEET significantly inhibited P-glycoprotein (P-gp), PB uptake and
H C
H C
3
3
O
C
N
N
CH3
Br
Br
O
N
OH
CH3
Pyridostigmine bromide (PB)
N-Methyl-3-hydroxypyridinium bromide
O
C H
2
5
O
O
C
N
C
NH2
C
OH
C H
2
5
CH3
CH3
CH3
DEET (N,N-diethyl-m-toluamide)
m-Toluamide
m-Toluic acid
H C
3
CH3
COOH C
O
O
Cl
Cl
O
C
HC
HOH C
2
2
HOOC
Permethrin
m-Phenoxybenzyl alcohol
m-Phenoxybenzoic acid
Figure 1. Chemical structures of PB, DEET, and permethrin and their metabolites.

2
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A. W. Abu-Qare and M. B. Abou-Donia
decreased resistance against PB (El-Masry & Abou-Donia 2006). Published reports
implicated combined exposure to PB, DEET and permethrin in the development of Gulf
War Illnesses (Abou-Donia et al. 1996a, b; Haley & Kurt 1997; Shen 1998; Riviere et al.
2
002; Hodgson & Rose 2005). Moreover, other studies reported potential interactions
between organophosphorus insecticides such as chlorpyrifos or malathion and the
pyrethroid insecticide, permethrin and the insect repellent, DEET on the inhibition certain
enzymes including trans-permethrin hydrolysis in human liver (Choi et al. 2004; Hodgson &
Rose 2005), alteration of dermal disposition of permethrin by PB (Baynes et al. 2002), and
development of neurotoxicity following combined exposure (Abdel-Rahman et al. 2004b).
PB is metabolized, mostly via hydrolysis to N-methyl-3-hydroxypyridinium bromide (Taylor
et al. 1991; Abu-Qare & Abou-Donia 2000). Metabolism and disposition of DEET in vivo
were rapid following application in human volunteers (Selim et al. 1995), rats (Schoenig
et al. 1996), and mice (Blomquist & Thorsell 1977). DEET is metabolized in vitro via
cytochrome P450-mediated oxidation of the aromatic methyl group and by N-deethylation
following incubation with rat liver microsomes (Taylor 1986; Constantino & Iley 1999)
Metabolism of permethrin has been examined following oral and intravenous dose in rats
(Anadon et al. 1991). Furthermore, Vulule et al. (1999) reported that oxidases and esterases
catalysed permethrin metabolism.
Alteration of pharmacokinetics and metabolism is considered a possible mechanism of
interaction between chemicals following combined exposure that could change bioavail-
ability and concentration at the toxicity targets leading to enhanced toxicity in chemical
mixtures (Abou-Donia et al. 1996a, b, 2004; Moss 1996; Ito et al. 1998; Hodgson & Rose
2005). In vitro interactions between PB, DEET, and permethrin have been previously
reported (Baynes et al. 2002; Usmani et al. 2002).
Because combined exposure to the three test compounds resulted in neurological deficits
among US military personnel during the Persian Gulf War, the main objective of the present
study was to investigate whether the metabolic interactions between them could explain their
enhanced neurotoxicity following combined exposure. Therefore, the study was designed to
do the following:
.
Examine and compare the disappearance and the metabolic pathways of PB, DEET and
permethrin when tested alone and in combination in human plasma and liver microsomal
incubates.
.
.
Identify specific enzyme(s) involved in the metabolism of PB, DEET and permethrin.
Correlate the findings to an enhanced toxicity following in vivo combined exposure to
these chemicals.
Materials and methods
Chemicals
Pyridostigmine bromide (3-dimethylaminocarbonyloxy-N-methylpyridinium bromide, PB;
99%) (Figure 1), m-phenoxybenzyl alcohol, m-phenoxybenzoic acid, coumarin,
ꢀ
tetra-isopropyl pyrophosphoroamide (iso-OMPA), ꢀ-nicotinamide adenine dinucleotide
phosphate (NADPH), glucose 6-phosphate, and glucose 6-phosphate dehydrogenase were
obtained from Sigma Chemical Co., Inc. (St Louis, MO, USA). Permethrin (93.6%,
ꢁ cis/trans-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxy-
phenyl)methyl ester) was obtained from Roussel Uclaf Corp. (Pasadena, TX, USA).
m-Toluamide and m-toluic acid were purchased from Fisher Scientific (Pittsburgh,

Metabolism and interactions
297
PA, USA), while DEET (ꢁ97%, N,N-diethyl-m-toluamide) was obtained from Aldrich
Chemical Co., Inc. (Milwaukee, WI, USA). N-methyl-3-hydroxypyridinium bromide was
prepared in the authors’ laboratory following a method by Somani et al. (1972). Human liver
microsomes were obtained from In Vitro Technologies, Inc. (Baltimore, MD, USA).
HPLC system
The analysis was carried out according to the authors’ published method (Abu-Qare &
Abou-Donia 2000).
Calibration procedures, detection limits, and recovery
Five different calibration standards of a mixture of PB, N-methyl-3-hydroxypyridinium
bromide, DEET, m-toluamide, m-toluic acid, permethrin, m-phenoxybenzoic acid, and
m-phenoxybenzyl alcohol were prepared by adding different concentrations of DEET and
ꢂ1
metabolites, permethrin and metabolites ranged from 100–1000 ng ml to 1 ml of human
ꢂ1
plasma, or 50 ml of (20 mg protein ml ) of human liver microsomes (without adding
an NADPH-generating system). After incubation, the incubation tube was removed to an
ice bath. The samples were acidified, extracted and cleaned up as described below before
analysis by HPLC. Linear calibration curves were obtained by plotting peak areas of the
individual chemicals as a function of concentrations. The standard curves were used to
determine recovery of the compounds from plasma and liver microsomal incubation
reactions. Limits of detection were determined at the lowest concentration can be detected,
taking into consideration a 1:3 baseline noise:calibration point ratio.
Plasma incubations
Concentrations of 20–100 mM of PB, DEET and permethrin, alone and in combination,
ꢃ
were incubated with 100 ml of human plasma from 5 to 60 min at 37 C in a shaking water
bath. DEET was further incubated for 8 h. The reaction was initiated by the addition of
substrate and terminated by the addition of 100 ml of 70% perchloric acid. Plasma
butyrylcholinestrase activity was determined according to the method of Ellman et al.
(1961). The protein content was determined using the method of Smith et al. (1985).
Liver microsomal incubations
ꢂ1
Microsomal incubations were performed by adding 100 ml of stock solution of 20 mg ml
protein of human liver microsomes to test tube in ice bath then a volume of 640 ml
of 100 mM Tris buffer (pH 7.4) was added, followed by a 10 ml of solutions of 100–1000 mM
of PB, DEET, and permethrin, both alone and in combination. The total reaction mixture
was adjusted to 1 ml by the addition of 250 ml of an NADPH-regenerating system (NRS)
ꢂ1
ꢂ1
in 2% sodium bicarbonate containing 0.5 mg ml NADP solution, 2.0 mg ml glucose
ꢂ1
6
-phosphate, and 1.5 unit ml
glucose 6-phosphate dehydrogenase. The mixture was
ꢃ
incubated from 5 to 60 min at 37 C using a shaking metabolic incubator. The reaction was
terminated by transferring the incubation tubes into an ice bath and adding 100 ml of 70%
perchloric acid. The mixture was centrifuged and the supernatant removed prior to analysis

2
98
A. W. Abu-Qare and M. B. Abou-Donia
by HPLC. Control samples were prepared in which substrate (PB, DEET, permethrin),
NADPH-regenerating system, or microsomal enzymes were omitted.
Plasma butyrylcholinesterase inhibition
Incubation of PB and permethrin with human plasma was undertaken as described above.
Concentrations of 10, 20, 30, 40, 500, 80, and 100 mM of the selective inhibitor
butyrylcholinestrase tetra-isopropyl pyrophosphoramide (iso-OMPA) was included and
ꢃ
the incubation was carried out for 60 min at 37 C. The reaction was terminated by adding
100 ml of 70% perchloric acid, centrifuged, and the supernatant was removed prior to
analysis by HPLC.
Kinetics analysis
IC50 values were also determined by plotting the inhibitor concentration versus concentration
of the metabolite. The kinetic analysis of test compounds in plasma and microsomal incubates
was performed by plotting the log concentration as a function of time, and fitting the curve
using linear regression. The terminal half-life of each test compound was calculated from the
apparent first-order disappearance rate constant, K, that was obtained by linear regression of
the terminal linear decline in test compound concentration using the formula:
0
:693
t1=2 ¼
:
K
Statistical analysis
The mean values for each of five assays were calculated separately for each metabolism
incubation reaction with single compounds or combinations. The mean values between
different reactions were compared separately for each of the test compounds, alone and in
combinations using a one-way analysis of covariance (ANOVA) with a Student–Newman–
Keuls multiple comparison post-hoc test. A p-value < 0.05 was considered as being
statistically significant.
Results
Recovery and detection limits of PB, DEET, permethrin, and metabolites
PB, DEET, permethrin, and their metabolites were analysed in human plasma and
liver microsomal homogenates using HPLC, according to the present authors’ published
method (Abu-Qare & Abou-Donia 2000). Average percentage recoveries of PB, N-methyl-
3
-hydroxypyridinium bromide, DEET, m-toluamide, m-toluic acid, permethrin,
m-phenoxybenzoic acid, and m-phenoxybenzyl alcohol from human plasma were 75, 78,
1, 73, 80, 84, 85 and 77%, respectively. Recoveries from human liver microsomal
incubates were 77, 76, 75, 79, 68, 83, 89, and 90%, respectively. Detection limits
7
ꢂ1
ꢂ1
were 100 ng ml for PB, N-methyl-3-hydroxypyridinium bromide and DEET; 50 ng ml
for m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol, and m-phenoxy benzoic acid;
ꢂ1
and 20 ng ml for permethrin, respectively.

Metabolism and interactions
299
Kinetics of test compounds disappearance
In vitro human plasma incubation. Substrate disappearance data for PB, DEET, and
permethrin following incubation with human plasma, alone or in combination are shown
in Figures 2, 3, and 4, respectively. The apparent first-order disappearance rate constants
ꢂ1
(
K, min ) and the half-lives (min) for each test compound, alone or in combination, are
presented in Table I. Following incubation with human plasma, the three chemicals
disappeared at different rates in the following order: permethrin > PB >> DEET. Permethrin
disappeared at rates of 1.5 and 26 times faster than that of PB and DEET, respectively. The
ꢂ1
apparent first-order disappearance constants (K, min ) and half-lives (min) were: (0.026,
2
6.6), (0.017, 40.2), and (0.001, 665) for permethrin, PB and DEET, respectively.
Following binary incubations, permethrin greatly affected the rate of disappearance of PB.
The addition of permethrin reduced the rate of PB disappearance by 3.3-fold, whereas the
addition of DEET had no effect on the disappearance rate of PB. Thus, binary incubation of
PB with permethrin or tertiary incubation with both permethrin and DEET equally
ꢂ1
diminished the metabolism of PB with K (min ) and half-life (min) of: 0.005, 131; and
0
.005, 127, respectively. Binary combinations of permethrin with PB decreased the
disappearance of permethrin by 5.5-fold, in contrast to DEET that had no effect on the
metabolism of permethrin. In addition, permethrin metabolism was greatly diminished in
the presence of both PB and DEET by 11.2-fold. Binary incubation of PB or permethrin
with DEET greatly enhanced the metabolism of DEET and decreased its half-life to
0
.32- and 0.39-fold of DEET alone, respectively. Furthermore, combined incubation of
DEET with PB and permethrin decreased its half-life to 0.35-fold of that of DEET alone.
Although DEET was the most stable test compound in plasma after incubation for 1 or 8 h,
combined incubation with PB, permethrin or both PB and permethrin accelerated its
ꢂ1
disappearance, with K (min ) and half-life (min) values of: 0.003, 212; 0026, 258; and 003,
2
30, respectively.
100
PB
PB+DEET
PB+Permethrin
PB+ DEET+Permethrin
8
6
4
2
0
0
0
0
0
5
10 15 20 25 30 35 40 45 50 55 60
Time (min)
Figure 2. Disappearance of PB alone and in combination with DEET and permethrin following in vitro
incubation with human plasma.

3
00
A. W. Abu-Qare and M. B. Abou-Donia
DEET
DEET+PB
DEET+Permethrin
DEET+PB+ Permethrin
1
00
9
8
7
6
0
0
0
0
0
5
10 15 20 25 30 35 40 45 50 55 60
Time (min)
Figure 3. Disappearance of DEET alone and in combination with PB and permethrin following in vitro
incubation with human plasma.
Permethrin
1
00
Permethrin + DEET
Permethrin + PB
Permethrin +PB + DEET
8
6
4
2
0
0
0
0
0
5
10 15 20 25 30 35 40 45 50 55 60
Time (min)
Figure 4. Disappearance of permethrin alone and in combination with PB and DEET following in vitro
incubation with human plasma.
In vitro human liver microsomal incubation. Disappearance data for PB, DEET, and
permethrin following human liver microsomal incubation with an NADPH-generating
system, alone or in combination, are shown in Figures 5, 6, and 7, respectively, and Table II.
The results show that the three test compounds had remarkably similar disappearance rates,

Metabolism and interactions
301

3
02
A. W. Abu-Qare and M. B. Abou-Donia
PB
100
PB+DEET
PB+Permethrin
PB + DEET + Permethrin
8
6
4
2
0
0
0
0
0
5
10 15 20 25 30 35 40 45 50 55 60
Time (min)
Figure 5. Disappearance of PB alone and in combination with DEET and permethrin following in vitro
incubation with human liver microsomes.
100
DEET
DEET + PB
DEET + Permethrin
DEET + PB + Permethrin
8
6
4
2
0
0
0
0
0
5
10 15 20 25 30 35 40 45 50 55 60
Time (min)
Figure 6. Dissipation of DEET alone and in combination with PB and permethrin following in vitro
incubation with human liver microsomes.
ꢂ1
with K (min ) and half-life (min) values of: 0.011, 65.0; 0.01, 60.0; and 0.011,
6
3 (Table II). DEET had no effect on the metabolism and disappearance of PB from
microsomal incubate, whereas permethrin moderately decreased disappearance of PB and
prolonged its half-life by 1.74-fold. Tertiary incubation of both DEET and permethrin with

Metabolism and interactions
303
100
Permethrin
Permethrin + PB
Permethrin + DEET
Permethrin + PB + DEET
8
6
4
2
0
0
0
0
0
5
10 15 20 25 30 35 40 45 50 55 60
Time (min)
Figure 7. Disappearance of permethrin alone and in combination with PB and DEET following in vitro
incubation with human liver microsomes.
PB moderately decreased PB metabolism and prolonged its half-life by 1.66-fold.
Furthermore, binary incubation of permethrin with PB or DEET, and tertiary incubation
with both PB and DEET did not significantly affect the metabolism and disappearance rate
of permethrin. In contrast, combined incubation of DEET with PB or permethrin alone
greatly decreased the metabolism of DEET, slowed its disappearance, and prolonged its
half-life by 2.64- and 3.27-fold, respectively. Furthermore, the metabolism of DEET was
further diminished by combined incubation with both PB and permethrin and its half-life
was prolonged by 5.31-fold.
Metabolic biotransformation of test compounds
Plasma esterases. N-methyl-3-hydroxy pyridinium bromide was detected as the hydrolysis
metabolite of permethrin in plasma (Figure 8). The rapid disappearance of permethrin
following incubation with human plasma was accompanied by the appearance of its
hydrolytic metabolite m-phenoxybenzyl alcohol (Figure 9).
Liver microsomal oxidases. The PB metabolite, N-methyl-3-hydroxypyridinium bromide,
and the permethrin metabolite m-phenoxybenzoic acid were detected after the incubation of
parent compounds with human liver microsomal incubations. m-Toluamide was also
identified as metabolites of DEET in human liver microsomal incubates, with K and Vmax
m
ꢂ1
ꢂ1
values of 62 mM and 112 pmol min mg of protein, respectively. Figures 10, 11 and 12
show the rate of formation of the PB metabolite, N-methyl-3-hydroxypyridinium bromide,
the DEET metabolite m-toluamide, and the permethrin metabolite m-phenoxybenzyl
alcohol with human liver microsomal enzymes.

3
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A. W. Abu-Qare and M. B. Abou-Donia

Metabolism and interactions
305
2
2
2
1
1
8
4
0
6
2
8
4
0
10
20
30
40
50
60
70
80
90
100
PB Concentration (mM)
Figure 8. Representative plot for the formation of N-methyl-3-hydroxypyridinium bromide following
PB incubation in human plasma.
2
2
2
1
1
8
4
0
6
2
8
4
0
10
20
30
40
50
60
70
80
90
100
Permethrin Concentration (µM)
Figure 9. Representative plot for the formation of m-phenoxybenzyl alcohol following permethrin
incubation in human plasma.
Butyrylcholinestrase inhibition studies
The selective inhibitor of butyrylcholinestrase, tetra-isopropyl pyrophosphoramide
(iso-OMPA) inhibited PB and permethrin disappearance in human plasma (Figures 13
and 14), indicating that their metabolism is primarily mediated via aliesterases such as

3
06
A. W. Abu-Qare and M. B. Abou-Donia
2
2
1
1
4
0
6
2
8
4
0
10
20
30
40
50
60
70
80
90
100
PB Concentration (µM)
Figure 10. Representative plot for the formation of N-methyl-3-hydroxypyridinium bromide following
PB incubation in human liver microsomes.
1
1
1
40
20
00
8
6
4
2
0
0
0
0
0
10
20
30
40
50
60
70
80
90
100
DEET Concentration (µM)
Figure 11. Representative plot for the formation of m-toluamide following FEET incubation in human
liver microsomes.
butyrylcholinesterase enzyme The extent of iso-OMPA-dependent inhibition as reflected by
the IC50 values was determined to be 42 and 56 mM following incubation with PB and
permethrin, respectively. The results show that BChE plays a major role in the metabolism
of PB and permethrin. The results also show that permethrin is moderately more efficiently
metabolized by BChE than PB.

Metabolism and interactions
307
2
1
1
1
1
1
00
80
60
40
20
00
8
6
4
2
0
0
0
0
0
10
20
30
40
50
60
70
80
90
100
Permethrin Concentration (µM)
Figure 12. Representative plot for the formation of m-phenoxybenzyl alcohol following permethrin
incubation in human liver microsomes.
2
1
1
0
5
0
5
0
10
20
30
40
50
60
70
80
90
100
Iso-OMPA Concentration (µM)
Figure 13. Inhibition of PB metabolism by iso-OMPA.
Discussion
This study presents the profile of in vitro human plasma and hepatic metabolism and kinetic
disappearance of PB, DEET and permethrin, alone and in combination, and an attempt to
identify specific enzymes involved in their metabolic pathways. The results indicate that PB

3
08
A. W. Abu-Qare and M. B. Abou-Donia
1
1
1
1
60
40
20
00
8
6
4
2
0
0
0
0
0
20
40
60
80
100
Iso-OMPA Concentration (µM)
Figure 14. Inhibition of permethrin metabolism by iso-OMPA.
and permethrin are metabolized by both human plasma and liver microsomal enzymes and
that DEET is mainly metabolized by liver cytochrome oxidase enzymes.
Incubation of test compounds with plasma showed the following:
.
.
.
.
.
.
.
PB and permethrin were efficiently metabolized by human plasma. However, DEET
metabolism was very slow even after longer incubation.
Binary incubation with permethrin decreased the metabolism of PB and its disappearance
from plasma, but it also seemed to enhance the metabolism of DEET in plasma.
Binary incubation with PB greatly decreased the metabolism of permethrin and its
disappearance rate, but it increased the metabolism of DEET in plasma.
Binary incubation of DEET had no effect on metabolism of either PB or permethrin in
plasma.
Tertiary incubation with PB and DEET greatly decreased the metabolism of permethrin
and its disappearance from plasma.
Tertiary incubation of DEET and permethrin decreased the metabolism of PB and
delayed its disappearance.
Tertiary incubation with PB and permethrin increased the metabolism of DEET in
plasma.
PB, a carbamate, and permethrin, an ester, rapidly disappeared following 60-min
incubation with plasma as the result of hydrolysis via esterase enzymes to yield the hydrolysis
products N-methyl-3-hydroxypyridinium bromide and m-phenoxybenzyl alcohol, respec-
tively. These results are in agreement with previous studies that reported the formation
of such metabolites in vivo: the m-phenoxybenzyl alcohol metabolite of permethrin
was detected in plasma and in tissues up to 48 h after an oral dose in rats (Anadon et al.
1
991). Furthermore, the PB metabolite N-methyl-3-hydroxypyridinium bromide has
been identified in human and rat plasma and urine (Taylor et al. 1991; Abu-Qare &
Abou-Donia 2000).

Metabolism and interactions
309
The results show that permethrin and PB compete for certain metabolism enzymes in
human plasma because both compounds diminished the rate of disappearance of the other
compound following binary incubations. The effect of permethrin on PB disappearance was
more pronounced, indicating that permethrin was a better substrate for human plasma
esterases than PB, and effectively competed with it for metabolism. This is consistent with
the previous report of Choi et al. (2004) that hydrolysis of permethrin is a main route of
detoxification and a potential site for interactions with compounds metabolized through
similar pathways. Furthermore, Baynes et al. (2002) reported that PB modulates the dermal
disposition of permethrin in isolated perfused porcine skin flap. Further, Hoy et al. (2000)
reported an increase in the concentration of permethrin in the serum of male and female
rats following combined administration of PB with permethrin compared with permethrin
alone. The inhibition of permethrin and PB hydrolysis by the addition of the selective
butyrylcholinesterase inhibitor iso-OMPA indicates that butyrylcholinesterase plays a major
role in the metabolism of permethrin and PB metabolism, with permethrin being a better
substrate than PB.
DEET, an amide, did not significantly disappear following 1 or 8 h of incubation with
human plasma indicating that DEET was not efficiently metabolized by human plasma
esterases in vitro. This conclusion is also supported by the results that PB and permethrin
that were efficiently hydrolysed by plasma esterases did not compete with DEET for
metabolism enzymes. In contrast, either compound alone or in combination increased the
metabolism of DEET and enhanced its disappearance from human plasma. PB and
permethrin may enhance amidase activity by increasing its expression or its stability by
inhibiting specific proteases that breakdown amidases. The present result that DEET did
not alter the in vitro metabolism of PB or permethrin in human plasma indicates that
esterases are not involved in the metabolism of DEET. The lack of inhibition of esterases by
DEET is consistent with earlier findings that DEET synergized the toxicity to German
cockroaches, of acetyl cholinesterase inhibitors, malathion, carbaryl, and PB, but not
of bendiocarb, or chlorpyrifos indicating that DEET has a mechanism other than
acetylcholinesterase inhibition responsible for the toxic interactions between DEET
and the tested chemicals (Moss 1996). Furthermore, DEET antagonized the toxicity
of PB against Escherichia coli by inhibiting P-glycoprotein and PB uptake (El-Masry &
Abou-Donia 2006).
Incubation of each of the three compounds, alone or in combination with human liver
microsomes in the presence of an NADPH-generating system, revealed the following:
.
.
PB, DEET, and permethrin are metabolized to a similar extent with human liver
microsomal enzymes.
Binary incubation with permethrin, moderately decreased PB metabolism, but greatly
diminished the metabolism of DEET and prolonged their disappearance from
microsomal incubates.
.
Binary incubation of PB had no effect on the metabolism of permethrin and its
disappearance rate but decreased the metabolism of DEET and delayed its disappearance.
Binary incubation of DEET had no effect on the metabolism of either PB or permethrin.
Tertiary incubation with PB and DEET had no significant effect on the metabolism
of permethrin and its disappearance from plasma.
.
.
.
.
Tertiary incubation of DEET and permethrin moderately decreased the metabolism
of PB and delayed its disappearance.
Tertiary incubation with PB and permethrin significantly decreased the metabolism
of DEET.

3
10
A. W. Abu-Qare and M. B. Abou-Donia
Thus, whereas combined incubation with DEET did not affect the metabolism of PB or
permethrin, both PB and permethrin, alone or in combination, greatly decreased the
metabolism DEET and its disappearance via liver microsomal enzymes. These results
suggest that liver microsomal enzymes that metabolize DEET might also metabolize PB
and permethrin. In contrast, liver microsomal enzymes that predominately metabolize PB
and permethrin did not significantly affect metabolism of DEET.
In the present study, the DEET oxidative metabolite m-toluamide was identified following
incubation with human liver microsomal enzymes indicating that DEET was metabolized
through oxidative N,N-deethylation. Previous reports indicated that DEET was metabolized
through N-dealkylation, ring hydroxylation and ring dealkylation following incubation with
rat liver microsomes (Constantino & Iley 1999). These results are in agreement with
previous studies that reported the detection of the DEET metabolite m-toluamide following
DEET incubation in rat liver microsomes (Taylor 1986; Yeung & Taylor 1988), and
following dermal administration of DEET in rats (Schoenig et al. 1996), and in cattle
(
Taylor et al. 1994). An in vitro study using human liver microsomes indicated that DEET
was metabolized by the following P450 enzymes: CYPs1A2, 2B6 and 2D6*1 (Val.374), and
E1 to yield N,N-diethyl-m-hydroxymethylbenzamide, with CYP2B6 being the principal
CYP responsible its production (Usmani et al. 2002). On the other hand, CYPs2A6, 3A4,
A5, and 2C19 produced an N-deethylated metabolite, N-ethyl-m-toluamide, with 2C19
2
3
exhibiting the greatest activity.
The addition of PB and permethrin to DEET with human liver microsomes decreased the
rate of disappearance of DEET. This is in agreement with Usmani et al. (2002) who
reported that pre-incubation of human or rodent microsomes with PB or permethrin can
lead to either stimulation or inhibition of DEET metabolism, respectively. The differential
metabolic interactions between PB, DEET and permethrin might explain the regional
selective neurotoxicity induced by test compounds in rat brains when tested alone or in
combination (Abdel-Rahman et al. 2001). Thus, dentate gyrus and hippocampal regions
exhibited a significantly greater neuronal cell death from each chemical alone than exposure
to combined DEET and permethrin. In contrast, in the Purkinje cell layer of the cerebellar
hemisphere, exposure to combined DEET and permethrin caused more injury than
exposure to both chemicals when given singly. Decreased neurotoxicity or antagonism might
result from increased DEET metabolism via amidases, with combined exposure to PB and
permethrin, whereas increase toxicity or synergism might result from competition of the
three chemicals for oxidase enzymes.
The permethrin metabolites m-phenoxybenzyl alcohol and m-phenoxybenzoic acid
were identified in the liver microsome incubates, indicating oxidation of permethrin.
Previous studies reported the involvement of cytochrome P450 enzymes in permethrin
metabolism; Vulule et al. (1999) reported an increased toxicity of permethrin when
co-administered with the cytochrome P450 inhibitor piperonyl butoxide. The findings of
the present study suggest that in vitro combined incubation of PB, DEET and permethrin
with human liver microsomes might have significantly altered the rate but not the pattern
of their transformation.
In summary, human esterases and oxidases are involved in the metabolism of PB and
permethrin in vitro. The metabolism of both PB and permethrin is catalysed by
butyrylcholinesterase in human plasma as shown from the inhibition of this enzyme by
iso-OMPA, with permethrin being more efficiently metabolized than PB. Both of these latter
chemicals also undergo oxidative metabolism presumably via the cytochrome P-450 oxidase
system. Metabolism of DEET, however, is mainly catalysed by cytochrome P450 enzymes.

Metabolism and interactions
311
The results predict that combined exposure to test compounds results in increased
neurotoxicity than when given singly by: competition of PB and permethrin for plasma
butyrylcholinesterase; and PB, permethrin, and DEET can also compete for presumably, the
cytochrome P450 oxidase system-catalysed metabolism of the three compounds.
In conclusion, this study explains, at least in part, the reported increased neurotoxicity of
the compounds examined following their combined exposure by US military personnel
during the Persian Gulf War. Combined exposure to test chemicals increases their
neurotoxicity by impeding the body’s ability to rid itself of them because of the competition
for detoxifying enzymes by these chemicals, leading to their persistence in the body and
enhancing their delivery to the nervous system and increasing their concentration
at neurotoxicity target.
Acknowledgements
This study was supported, in part, by the US Army Medical Research and Material
Command under Contract No. DAMD 17-99-1-9020. The views, opinions and/or findings
contained in this paper are those of the author(s) alone and should not be construed as an
official Department of the Army position, policy or decision unless so designated by other
documentation.
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