Determination of the rate of aldicarb sulphoxidation in rat liver, kidney and lung microsomes

Article abstract of DOI:10.1080/004982597239877

1. The rate of sulphoxidation of aldicarb (2-methyl-2-(methylthio) propanal O-[(methylamino) carbonyl oxime], Temik) in rat hepatic, renal and pulmonary microsomes was determined by quantitating the levels of aldicarb sulphoxide and aldicarb sulphone produced during incubations. Under in vitro experimental conditions used in the present study, aldicarb sulphoxide was the only metabolite produced, and further metabolism of aldicarb sulphoxide to aldicarb sulphone was negligible. 2. The average maximal velocity μmol/min/mg protein) for the sulphoxidation of aldicarb, based on measurements of product formation, in liver, kidney and lung microsomes was 5·41, 39·51 and 2·45 respectively. The corresponding values for the Michaelis constant (μM) were 184, 1050 and 188 respectively. 3. These results imply that under in vivo conditions (1) aldicarb sulphoxidation is not likely to be saturable even at lethal doses in the kidney will be limited by the rate of blood flow and not metabolizing enzyme levels.

Full text of DOI:10.1080/004982597239877

xenobiotica , 1997, vol. 27, no. 11, 1113±1120
Determination of the rate of aldicarb sulphoxidation
in rat liver, kidney and lung microsomes
M. PELEKIS and K. KRISHNAN*
Departem ent de medecine du travail et d’hygiene du milieu, Universite de Montreal,
C.P. 6128, Succ. Centre-ville, Montreal, QC, Canada HC3 3J7
Received 17 April 1997
1. The rate of sulphoxidation of aldicarb (2-methyl-2-(methylthio) propanal O-
[(methylamino)carbonyloxime],Temik‡ ) in rat hepatic,renal and pulmonarymicrosomes
was determined by quantitating the levels of aldicarb sulphoxide and aldicarb sulphone
produced during incubations. Under in vitro experimental conditions used in the present
study, aldicarb sulphoxide was the only metabolite produced, and further metabolism of
aldicarb sulphoxide to aldicarb sulphone was negligible.
l
2. The average maximal velocity ( mol min mg protein) for the sulphoxidation of
}
}
aldicarb, based on measurem ents of product formation, in liver, kidney and lung
±
microsomes was 5 41, 39 51 and 2 45 respectively. The corresponding values for the
±
±
lm
Michaelis constant ( ) were 184, 1050 and 188 respectively.
3. These results imply that under in vivo conditions (1) aldicarb sulphoxidationis not
likelyto be saturable even at lethal doses in the rat, and (2) aldicarbclearancein rat liver and
kidney will be limited by the rate of blood ¯ow and not metabolizing enzyme levels.
Introduction
Aldicarb (2-methyl-2-(methylthio) propanal O-[(methylamino) carbonyl
oxime], Temik‡ ) is widely used to control insects, mites and nematodes (World
Health Organization1991). In mammals,it is readily absorbed and distributedto all
tissues by systemic circulation (Knaak et al. 1966, Andraweset al. 1967, Dorough et
al. 1970, Cambon et al. 1979). It is initially oxidized to aldicarb sulphoxide (ALX)
and subsequently to aldicarb sulphone; aldicarb and its metabolites are susceptible
to hydrolysis, with the subsequent dehydration giving rise to the corresponding
oximes and nitriles (Baron and Merriam 1988). Although hydrolysis destroys the
insecticidalactivity, both aldicarb and its oxidative metabolites, ALX and aldicarb
sulphone, are potent cholinesterase inhibitors (Hastings et al. 1970, Cambon et al.
1979, Baron and M erriam 1988).
Whereas the in vitro and in vivo metabolism of aldicarb has been studied in a
variety of mammalian and non-mammalian species and plants (Knaak et al. 1996,
Metcalf et al. 1966, Andrawes et al. 1967, Bull et al. 1967, Dorough and Ivie 1968,
Bartley et al. 1970, Dorough et al. 1970, Montesissa et al. 1991, 1994, 1995), the
maximal velocity (V ) and the Michaelis aænity constant (K ) for aldicarb
max
m
sulphoxidation have only been determined in ®sh. Schlenk and Buhler (1991)
determined the V and K for aldicarb sulphoxidation in rainbow trout using
max
m
liver,kidneyand gill microsomes.In all three tissues,ALX was the majormetabolite,
with trace amounts of ALX oxime being formed in the liver, and aldicarb oxime in
kidney and liver.
* Author for correspondence.
±
’
0049±8254}97 $12 00
1997 Taylor & Francis Ltd

1114
M. Pelekis and K. Krishnan
The information on the quantitative nature (i.e. rate and aænity) of the
metabolism of aldicarb is essential to evaluate its biopersistance and pro®le of
elimination in other non-target species such as rodents and man. Accordingly, the
objective of the present study was to determine the V
and K for aldicarb
m
max
sulfoxidation in rat liver, kidney and lung microsomes.
M aterials and m ethods
Materials
Aldicarb (ALD), aldicarb sulphoxide (ALX), aldicarb sulphone (ALU) were obtained from Chem
Service (West Chester, PA, USA) and were at least 98% pure. The purity of the carbamateswas veri®ed
±
by hplc analysis (EPA method 531 1) prior to all experiments. NADPH, Tris-HCl, Tris-acetate,
potassium chloride, potassium phosphate, sucrose and EDTA were obtained from Sigma ChemicalCo.
(St Louis, MO, USA). Methanol(hplc grade), glycerol and sodium pyrophosphatewere purchased from
Fischer Chemicals (Montreal, Quebec, Canada). NaOH and o-phthalaldehyde (2-dimethylamino
ethanediol hydrochloride, OPA) were purchased from Pickering Laboratories (Mountain View,
CA, USA).
Preparation of microsomes
Male Sprague-Dawley rats weighing 180±200 g were obtained from Charles River Canada (St
Constant, Quebec, Canada) and maintained in stainless steel cages on Purina Certi®ed Rodent Chow
(Ralston-Purina Co., Ontario, Canada) and water ad libitum . Following a 4±7-day acclimatization, the
rats were euthanized (following exposure to CO ), exsanginated and the tissues (liver, kidney and lung)
from individual animals obtained. All tissues were blotted with ®lter paper (Whatman no. 1), weighed
#
±
±
and washed with ice-cold Tris-HCl buåer (0 1 , pH 7 4) containing 0 1 KCl and 1 m EDTA.Liver
±
m
m
m
±
and kidney tissues from several animalswere pooled and homogenizedin Tris-HClbuåer (0 1 , pH 7 4,
1 :4 v v) with a Te¯on homogenizer. The tissue homogenates were initially centrifuged at 10000 g for
±
m
}
20 min and the supernatant was re-centrifuged at 100000 g for 60 min. The resulting pellet was re-
suspended in the above buåer and the homogenate centrifuged at 100000 g for 60 min. The ®nal pellet
±
was suspended in 0 1 Tris-HCl containing 0 25 sucrose and 5 m EDTA at a volume equal to the
weight of the tissue. The same procedure was followed for the preparation of lung microsomes, except
±
m
m
m
±
that in the second centrifugation 0 1 potassium pyrophosphate buåer (pH 7 4) was used, and the ®nal
pellet was suspended in 0 01 Tris-acetate buåer containing 1 m EDTA and 20 % (v v) glycerol (pH
±
m
±
m
m
}
°
±
7 4) (Reitz et al. 1996). The microsomeswere stored at ®70 C and used within 2 months of preparation.
The concentration of protein in the was determined immediately after the last centrifugation with the
Bio-Rad‡ method (Bio-Rad Laboratories, Hercules, CA, USA). Brie¯y, this method involves the
incubation of an aliquot of the microsomal preparation with the Bio-Rad Dye reagent (mixture of
Coomassie Brilliant Blue, ethanol and phosphoric acid) and the subsequent determinationof the optical
density of the solution at 595 nm (Bradford 1976).
In vitro assays
The experimental approach consisted of the addition of ALD to a mixture of microsomes, 5 m
m
±
NADPH and 0 1 potassium phosphate buåer (pH 7 4) containing 5 m EDTA, in a total volume of
1 ml. The optimal NADPH concentration (5 m ) and pH (7 4) were chosen on the basis of preliminary
±
m
m
±
m
studies with rat liver and or kidney microsomes (data not shown). The rate of ALD metabolism was
assayed by measuring the production of ALX and ALU. In all assays duplicate controls were used as
references. In the ®rst one, ALD was incubated with buåer alone and was used to check for
}
contamination and or non-enzymatic degradation of ALD. In the second control experiment, all
}
componentsexcept NADPHwere added to the incubationmixtureand were used to evaluate the residual
metabolic activity of the microsomes. All incubations were conducted in 5-ml glass screw cap tubes at
°
37 C.
Time-course assays
The linearity of incubation time was determ ined by incubating ALD (5 25 or 10 5
±
concentration, in 20 l methanol) with 0 14±0 51 mg ml microsomal protein for up to 60 min (liver and
±
lm
®nal
±
±
l
}
m
lung 60 min, kidney 45 min). Microsomalprotein was added to tubes already containing5 m NADPH,
and the reaction was initiated with the addition of ALD (in 20 l methanol). At diåerent time points, the
l
±
reaction was terminated by adding 0 5 ml methanol and immersion of the assay tubes in ice. All tubes

Aldicarb sulphoxidation
1115
°
were centrifuged for 15 min at 3200 g (4 C) to remove the protein precipitate. The supernatant was
transferred to 2-ml glass vials sealed with Te¯on-coated rubber septa and analysed immediatelyfor levels
of ALX.
Protein- course assay
The linear range of microsomal protein concentration was determined by incubating ALD (®nal
±
±
concentration 5 25 or 10 5
10 min and measuring the concentrations of ALX.
±
) with various amounts of protein (®nal concentration 0 06±12 mg ml) for
lm
}
Kinetic analyses
The kinetic parameters for ALD sulphoxidation were determined by adding various quantities of
l
ALD (dissolved in 20 l methanol; ®nal concentrations 36±3700
(corresponding to 0 18±0 32 mg protein per ml), cofactor (5 m NADPH) and of 0 1
lm
) to a mixture of microsomes
±
±
±
m
m
potassium
°
±
phosphate buåer (pH 7 4, 1 ml ®nal volume) at 37 C and determ ining the concentration of ALX at the
end of a 10-min incubation.
Analytical methods
For the separation and quantitation of ALD and its metabolites, the EPA method 531 1 was used
±
(USEPA1989). A Varian‡ hplc system equippedwith an autosampler(Model9100), and a programmable
¯uorescencedetector (Model 9070) linked to a Varian Star LC workstation was used. A dual post-colum n
derivatizationsystem (PCX-5100, Pickering Laboratories,Mountain View, CA, USA)was connected to
the hplc system. The post-column reaction unit consisted of two reagent pum ps, an hplc column
°
°
thermostat controlled at 42 C, and two reaction coils. The ®rst reaction coil was heated to 100 C for
NaOH hydrolysisof ALD,ALX and ALU and the second one was kept at ambienttemperature for OPA
derivatization of the methyl amine resulting from the hydrolysis of the carbamates.
±
The separation was achieved with a Pickering C18 column (250¬4 6 mm i.d., 5-mm packing)placed
°
in the thermostat of the post-column reaction unit and maintainedat 42 C. The mobile phase employed
a simple water:methanol gradient. The initial composition was 8 % methanol:92% water, which was
maintainedfor a 1-min hold, after which a 20-min linear gradientprogram to 20 % methanol: 80 % water
was begun. The mobile phase composition was then changed to 50 :50 and an 8-min gradient to 80 %
methanol:20% water was initiated.Subsequently,the mobilephase was set at 100% methanolfor 2 min
to provide column clean-up, before returning to the initial condition.The ¯ow rate was 1 ml min. Under
}
±
±
±
these conditions, ALX elutes ®rst (14 5 min) followed by ALU (16 5 min) and ALD (25 5 min). The
separated carbamates were derivatized with OPA to improve sensitivity and selectivity, and the
¯uorescence of the resulting 1-methylthio-2-methylisoindolewas quanti®ed. Both NaOH solution and
±
the OPA reagent in the post-colum n reaction unit were constantly pumped at a ¯ow rate of 0 3 ml min
}
l
during the whole sequential cycle. The injection volume was 10 l. Excitation and emission wavelengths
of the ¯uorescence detector were set at 330 and 466 nm respectively. Calculationsof the concentrations
of carbamates in samples were based on area measurement.
Data analysis
The metabolic constants (V
and K ) for aldicarb sulphoxidation in rat liver, kidney and lung
m
microsomeswere determ inedfrom Hanes±Woolfplots of the data on ALX concentrationobtained at the
max
end of incubation with the corresponding initial concentrations of ALD.
Results
The initial series of studies focused to determine the linear range of incubation
time and protein concentration with respect to ALD sulphoxidation in rat tissue
microsomes. Figure 1 shows the time-course of ALX formation in rat liver, kidney
±
and lung microsomalpreparationsfor an initialALD concentrationof 5 25 lm (liver
±
and kidney)and 10 5 lm (lung). With the choice of 10 min from the linearpart of this
curve, the in¯uence of protein concentration on the rate of ALX formation was
elucidated. The eåect on ALD sulphoxidation was linear for microsomal protein
±
concentrations of up to 1 mg ml in liver, 0 6 mg ml in kidney, and 0 4 mg ml in
±
}
}
}
lung microsomes respectively (Figure 2).
The ®nal series of experiments involved the determination of the rate of ALX

1116
M. Pelekis and K. Krishnan
Figure 1. Aldicarb sulphoxide (ALX) produced by the sulphoxidation of aldicarb by rat liver (E ,
±
protein concentration 0 5 mg ml, ALD 5 25
±
±
±
lm
lm
), kidney (_ , 0 47 mg ml, 5 25
) and lung (+ ,
) microsomes as a function of incubation time. The symbols represent
}
}
±
±
lm
0 14 mg ml, 10 5
}
experimental data (mean³SE, n ¯ 3).
Figure 2. Aldicarb sulphoxide (ALX) produced by the sulphoxidation of aldicarb by rat liver (E ,
±
5 25
±
±
lm
lm
lm
) and lung (+ , 10 5 ) microsomesas a function of the concentration
), kidney(_ , 10 5
of microsomal protein. The experimental data (symbols, mean³SE, n ¯ 3) correspond to the
amount of ALX measured at the end of a 10-min incubation.
formation by liver, kidney and lung microsomesfollowinga 10-min incubationwith
36±3700 lm ALD (®nal concentrations). From the measurement and analysis
(Hanes±Woolfplot) of ALX concentrations at the end of ALD incubations during
this series of experiments,the maximalvelocity for metabolism(V ) and Michaelis
max
aænity constant (K ) for ALD sulphoxidation in rat liver, kidney and lung
m
microsomes were estimated (Figures 3±5). The V
(lmol min mg protein) for
}
}
max
ALD metabolism in liver, kidney and lung microsomes were 5 41, 39 51 and 2 45
±
±
±
respectively, with the correspondingK ’s (lm) being 184, 1050 and 188. Under the
m
experimental condition of the present study, (1) incubation of ALD with liver,
kidney and lung microsomes resulted exclusively in the formation of ALX, and (2)
the oxidation of ALX to ALU by either liver, kidney or lung microsomes was
negligible (data not shown).

Aldicarb sulphoxidation
1117
Figure 3. Hanes±Woolfplot of aldicarbsulphoxidationin rat liver microsomes.v, initial rate of reaction
lm
l
( mol min mg protein); [S], initial aldicarb concentration ( ).
}
}
Figure 4. Hanes±Woolfplot of aldicarb metabolism in rat kidney microsomes.
Discussion
Aldicarb sulphoxidation is considered to be a bioactivation process since the
primaryoxidativemetabolite(ALX) is more potent than the parent chemical(ALD)
as an acetylcholinesterase inhibitor(W orld Health Organization 1991). The in vitro
metabolism of ALD has been investigated using subcellularfractions or whole cells
isolated from rat, rabbit, sheep, cattle, goat, chicken and ®sh (Andrawes et al. 1967,
Montesissaet al. 1991, 1994, 1995, Schlenkand Buhler1991, Venkateshet al. 1991).
All of these studies except that of Schlenk and Buhler (1991) and Venkatesh et al.
(1991) are at best semiquantitative in nature. In general, these latter studies have
shown that (1) ALX is the major product of ALD sulphoxidation and (2) ALD
sulphoxidation could be mediated both by cytochrome P450 and ¯avin-containing
monooxygenases (FMO). The experimental designs used in these latter studies

1118
M. Pelekis and K. Krishnan
Figure 5. Hanes±Woolfplot of aldicarb metabolism in rat lung microsomes.
could additional provide a qualitative characterization of the pro®le of metabolites
found at the end of incubation,but not quantitativeinformation(V , K ) on ALD
max
metabolism. Schlenk and Buhler (1991) and Venkatesh et al. (1991) on the other
m
hand reported the V and K for ALD sulphoxidationusing microsomesfrom ®sh
max
m
organs and puri®ed renal and hepatic FMO from mouse respectively. Since such
quantitative information on ALD oxidation in rat tissues is not available in the
literature, the present study estimated the aænity and maximal velocity of ALD
sulphoxidation using microsomes isolated from rat liver, kidney and lung.
The M ichaelis aænity constant for ALD oxidation in rat liver and kidney
microsomes are comparable with those reported by Venkatesh et al. (1991) using
puri®ed FMO from mouse tissues. Regardless of the preparation and species, the
aænity constant for metabolism of a substrate is anticipated to be the same, as long
as the same isoenzyme(s) is involved. This has formed the very basis of some,
current default approaches for in vitro to in vivo and interspecies extrapolations of
xenobiotic metabolism (Krishnan and Andersen 1994). The fact that the K ’s
m
estimated in the present study are comparable with those reported by Venkatesh et
al. (1991) (liver 196 lm, kidney 385 lm) adds further support to the preceding
practice.
The results of the present study indicate that the K for ALD sulphoxidationis
m
comparable in liver and lung (184 versus 188 lm). Such a similarity in K for the
m
sulphoxidationof several FMO substrates has been reported previously (Venkatesh
et al. 1991). Based on the K obtained in the present study, it may be suggested that
m
ALD oxidation is not saturable even at lethal doses in the rat (LD E 1 mg kg ;
}
&!
World Health Organization1991). Therefore, the rate of ALD oxidation in rat liver,
kidney and lung can be described as a ®rst-order process. The intrinsic clearance
±
K ) for ALD sulphoxidation in rat liver, kidney and lung are 7 06,
m
values (V
}
max
±
Õ
±
"
1 02 and 0 051 l[min respectively. For the ®rst-order conditions, the clearance of
ALD in each of these tissues can be calculated as:
[V
(mg min) K (mg l)]*Q (l min)
m t
}
}
}
}
max
,
[V
(mg min) K (mg l)]-Q (l min)
}
}
}
}
max
m t

Aldicarb sulphoxidation
1119
±
where Q is the rate of blood ¯ow to tissue t (liver¯ 0 016 l min, kidney¯
}
t
±
±
0 013 l min, lung ¯ 0 090 l min) (ILSI 1994). Since the value of (V
K ) is very
}
m
}
}
max
large with respect to Q in rat liver and kidney, Q in the denominator of the above
t
t
equation becomes negligible, making organ clearance of ALD equal to Q . The
t
pulmonaryclearanceof ALD,however, is not limitedsolely by Q . Therefore, in the
t
case of lung, both intrinsic clearance parameters and Q are critical determinantsof
t
ALD clearance. This is principallydue to the fact that Q for lung is very large (i.e.
t
equal to cardiac output), and the volume of the lungs is small relative to other
metabolizing tissues. Given that the volume of liver is greater than that of kidney
and lung, the former is likely to be the most important tissue metabolizingALD in
the rat. The rate of enzymatic sulphoxidation of ALD in rat organs, and its
dependence on blood ¯ow rates, elucidated in the present study, have important
implications for predicting the in vivo kinetics of ALD in the rat and subsequent
extrapolation to man for risk assessment purposes.
References
ndrawes
orough
indquist
A
B
B
, N. R., D
, H. W. and L
in rats. Journal of Agricultural and Food Chemistry , , 979±987.
, D. A., 1967, Degradationand eliminationof Temik
60
aron
erriam
, T. L. 1988, Toxicologyof aldicarb.Reviews of Environmental Contamination
, R. L. and M
and Toxicology ,
105
, 1±70.
artley
ndrawes
hancey
agley
purr
, H. W., 1970, The
, W. J., A
metabolism of Temik aldicarb pesticide [2-methyl-2-(m ethylthio) propionaldehydeO-(methyl-
18
, N. R., C
, E. L., B
, W. P. and S
carbamoyl)oxime] in the cotton plant. Journal of Agricultural and Food Chemistry , , 446±453.
radford
B
B
, M. M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of
72
protein utilizing the principle of protein-dye binding. Analytical Biochemistry , , 248±254.
indquist oppedge
, J. R., 1967, Metabolism of 2-methyl-2-(methyl-
ull
, D. L., L
thio)propionaldehyde O-(methylcarbamoyl) oxime (Temik, UC-21149) in insects. Journal of
15
,
D. A. and
C
Agricultural and Food Chemistry , , 610±616.
ambon eclume erache
, R., 1979, Eåect of the insecticidal carbamate derivatives
C
, C., D
(carbofuran, prim icarb, aldicarb) in the activity of acetylcholinesterase in tissues from pregnant
49
, C. and D
rats and fetuses. Toxicology and Applied Pharmacology , , 203±208.
orough vie $&
D
D
, H. W. and I , G. W., 1968, Temik-S in a lactating cow. Journal of Agricultural and Food
16
Chemistry , , 460±464.
orough avis
vie
, R. B. and I , G. W., 1970, Fate of Temik-carbon-14 in lactatingcows during
, H. W., D
18
a 14-day feeding period. Journal of Agricultural and Food Chemistry , , 135±142.
ILSI, 1994, Physiological Parameter Values for PBPK Models (Washington: International Life Sciences
Institute , Risk Science Institute ).
astings
ain
verson
, F., 1970, Carbamylation and aænity constants of some
H
, F. L., M
carbamate inhibitors of acetylcholinesterase and their relation to analogous substrate constants.
18
, A. R. and I
Journal of Agricultural and Food Chemistry , , 497±502.
naak allant ullivan
, L. J., 1966, The metabolism of 2-methyl-2-(m ethylthio)
K
, J. B., T
propionaldehyde O-(methylcarbamoyl)oxime in the rat. Journal of Agricultural and Food
14
, M. J. and S
Chemistry , , 573±578.
rishnan ndersen
, M. E., 1994, Physiologically based pharmacokinetic modeling in
K
,
K. and
toxicology. In Principles and Methods of Toxicology , edited by A. W. Hayes (New York: Raven).
etcalf ukuto ollins orck urk eynolds sman
A
M
, R. L., F
F., 1966, Metabolism of 2-methyl-2-(methylthio)-propionaldehydeO-(methylcarbamoyl) oxime
14
, T. R., C
, C., B
, K., B
, J. M., R
, H. T. and O
, M.
in plant and insect. Journal of Agricultural and Food Chemistry , , 579±584.
ontesissa morena iguoro ucisano
, A., 1991, Aldicarb sulfoxidative
e
M
M
M
, C., A
, M., D
L
, M. and L
suppl. 87
pathway in broilers: in vitro and in vivo evaluation. Acta Veterinaria Scandinavica ,
396±398.
,
ontesissa
e
iguoro
morena
ucisano
arli
, S., 1995, In vitro comparison
, C., D
of aldicarboxidationin various food-producinganimalspecies. Veterinary and Human Toxicology ,
37
L
, M., A
, M., L
, A. and C
, 333±336.
ontesissa uveneers
oogenboom
morena
e
iguoro
, M. and
, C., H
, M. B. M., H
, A., 1994, The oxidative metabolism of aldicarb in pigs: In vivo±in vitro comparison.
11
, L. A. P., A
, M., D
L
ucisano
L
Drug Metabolism and Drug Interactions , , 127±138.
argas endrala chuman
, A. M., 1996, In vivo and in vitro studies
eitz
R
, R. H., G
, M. L., M
, A. L. and S

1120
Aldicarb sulphoxidation
of perchloethylenemetabolism for physiologicallybased pharmacokineticmodeling in rats, mice
136
and humans. Toxicology and Applied Pharmacology ,
chlenk uhler
, 289±306.
, D. R., 1991, Role of ¯avin-containing monooxygenase in the in vitro
21
S
, D. and B
biotransformation of aldicarbin rainbow trout (Oncorhynchus mykiss). Xenobiotica , , 1583±1589.
USEPA,1989, 531.1. Measurementof N-methycarbamatesin water by direct aqueous injection HPLC
with post column derivatization, Environmental Monitoring System s Laboratory, Oæce of
Research and Development, Cincinnati, Ohio.
enkatesh
evi
odgson
V
, K., L
kidney, Biochemical Pharmacology , , 1411±1420.
orld ealth rganization
, P. E. and H
, E., 1991, The ¯avin-containing monooxygenase of mouse
42
, 1991, Aldicarb, Environmental Health Perspectives ,
121
, 20.
W
H
O