The Toxicologist - Society of Toxicology

1335 IN VITRO EVALUATION OF INHIBITION OF
BUTYRYLCHOLINESTERASE AND NEUROPATHY
TARGET ESTERASE CAUSED BY THE ENANTIOMERS
OF METHAMIDOPHOS IN BLOOD OF HENS.
G. L. Emerick 1, 2 , R. V. Oliveira 2 , K. A. Belaz 2 , M. Gonçalves 1 and G. H.
DeOliveira 1 . 1 Department of Natural Active Principles and Toxicology, University
Estadual Paulista, Araraquara, Sao Paulo, Brazil and 2 Department of Chemistry,
Universidade Federal de São Carlos, Sao Carlos, Sao Paulo, Brazil.
Compounds containing an asymmetric center can exist in two isomeric forms
known as enantiomers. About 25% of pesticides in current use are chiral, and this
percentage is still increasing as more pesticides with complex structures are introduced
into application. These enantiomers usually have the same physical and
chemical properties, however, often different biological activity. Methamidophos,
which has an asymmetric center at the phosphorus atom, is one of organophosphate
pesticides in the broader spectrum of action and also the highest toxicity.
Thus, the objective of this work was to evaluate the in vitro IC50 of isoforms of
methamidophos in relation to plasma cholinesterase (BChE) and neuropathy target
esterase in lymphocytes (LNTE) of hens. For determination of BChE and LNTE
activity was used the method of Ellman et al. (1961) and Schwab and Richardson
(1986), respectively, supplemented with different concentrations of methamidophos
in order to estimate the constant of enzyme inhibition and IC50. The form
(+)-Methamidophos had lower IC50 for the BChE (2.2 ± 0.40 mM). The racemic
form showed intermediate IC50 (4.3 ± 0.23) and Ki (2.4 mM-1). The (-)-
Methamidophos had the highest IC50 (6.4 ± 0.30) and lowest Ki (1.6 mM-1),
therefore, less toxicity in vitro for the BChE. Regarding LNTE activity, the inhibitory
concentrations have shown that the form (+)-Methamidophos had lower
IC50. The positive form of methamidophos showed an IC50 value of approximately
6 times smaller than the negative form. These results showed significant differences
in toxicity among the three isoforms of methamidophos in relation to
BChE and NTE and that it is necessary to undertake further studies in order to reduce
the toxic effects on non target species. Animal Committee of Ethics,
Resolution: 24/2009. Financial support: FAPESP, grant number 09/51048-8.
1336 CLUSTERING OF SERINE HYDROLASES BY
SIMILARITIES OF INHIBITION PATTERNS TOWARD
THE 4 STEREOISOMERS OF ISOMALATHION AGREES
WITH PHYLOGENETIC PROXIMITIES INFERRED
FROM SEQUENCE ALIGNMENTS.
S. J. Wijeyesakere 1 , S. Jianmongkol 1 , J. A. Doorn 1 , B. R. Marable 1 , C. M.
Thompson 2 , I. I. Baskin 3 , V. A. Palyulin 3 , G. F. Makhaeva 4 and R. J.
Richardson 1 . 1 Toxicology Program, Department of Environmental Health Sciences,
University of Michigan, Ann Arbor, MI, 2 Department of Pharmaceutical Sciences,
University of Montana, Missoula, MT, 3 Department of Chemistry, M.V. Lomonosov
Moscow State University, Moscow, Russian Federation and 4 Institute of Physiologically
Active Compounds, Russian Academy of Sciences, Chernogolovka, Russian Federation.
The purpose of the present study was to test the hypothesis that the relative stereoselectivity
of serine hydrolases toward inhibition by the 4 stereoisomers of isomalathion
is reflected in the phylogenetic order inferred from the aligned sequences of
each hydrolase. Bimolecular rate constants of inhibition (ki) of each of the 4
stereoisomers of isomalathion were determined for human (Hu), bovine (Bo), rat
(Ra), chicken (Ck), electric eel (Ee), and Torpedo californica (Tc) acetylcholinesterase
(AChE); equine (Eq) and Hu butyrylcholinesterase (BChE); and Bo
cholesterol esterase (CholE). A scatterplot matrix (Systat 13.00.05) of normalized
ki values showed that the order of similarity of serine hydrolases was HuAChE =
BoAChE > RaAChE > CkAChE > EeAChE = TcAChE > EqBChE = HuBChE >
BoCholE. Moreover, neighbor-joining cluster analysis (Statistica 9.1) of normalized
ki values generated a dendrogram with the same ordering as that observed in the
scatterplot matrix. Finally, these orderings were in accord with the phylogenetic
order inferred from maximum likelihood sequence alignments (ClustalX2 and
GeneDoc 2.4). The results suggest that isomalathion stereoisomers may be useful
probes of active site topologies and phylogenetic relationships among diverse serine
hydrolases. Supported in part by NIH T32 ES07062.
1337 AGE-RELATED BEHAVIORAL EFFECTS OF
METHOMYL IN BROWN NORWAY RATS.
R. C. MacPhail, K. A. Jarema and P. M. Phillips. NHEERL, U.S. EPA, Research
Triangle Park, NC.
Methomyl is a cholinesterase-inhibiting carbamate pesticide that is used in the field
on cotton and a variety of fruits and vegetables. Concerns have been raised generally
about age-related differences in susceptibility to cholinesterase-inhibiting pesti-
286 SOT 2011 ANNUAL MEETING
cides, especially for children. This experiment, part of a larger program on life-stage
susceptibility, compared methomyl effects on the behavior of adolescent, youngadult
and middle-age male Brown Norway rats (Charles River, N=24/age) that were
initially 1, 4 or 12 months of age, respectively. Another group of 12-month rats
(NIA) was included to evaluate vendor differences. Testing occurred at weekly intervals
in photocell devices that recorded horizontal and vertical locomotor activity
during 30-min sessions. Methomyl (ChemService) was dissolved in deionized water
and given orally in doses of 0 (i.e., vehicle), 0.5, 1.25, 2.0 and 2.75 mg/kg, 15-min
before a test session. Order of dosing was counterbalanced so that each rat received
all treatments. Following vehicle, horizontal activity was highest in young adults
and roughly equal in adolescent and middle-age rats; vertical activity was highest in
young adults, slightly lower in adolescents, and considerably lower in middle-age
rats. Methomyl produced dose-related decreases in both horizontal and vertical activity.
Adolescent rats were least affected. ED50s for horizontal activity were ca.
5.72, 2.43 and 2.19 mg/kg for the adolescent, young-adult and middle-age rats, respectively.
Corresponding ED50s for vertical activity were 4.41, 2.20 and 1.85
mg/kg. Middle-age rats from NIA were similarly affected (horizontal ED50=1.96
and vertical ED50=1.61 mg/kg). These results indicate aging increases susceptibility
to methomyl in Brown Norway rats; they also emphasize the importance of evaluating
pesticide effects at multiple life stages in order to make informed inferences
regarding susceptibility and risk. (This is an abstract of a proposed presentation and
does not reflect US EPA policy.)
1338 AGE-RELATED DIFFERENCES IN ACQUISTION,
STEADY-STATE PERFORMANCE AND CARBARYL
EFFECTS ON THE OPERANT BEHAVIOR OF BROWN
NORWAY RATS.
K. A. Jarema, P. M. Phillips and R. C. MacPhail. NHEERL, U.S. EPA, Research
Triangle Park, NC.
The rapid increase in older adults in the population highlights the importance of
understanding the role of aging in susceptibility to environmental contaminants.
As part of a larger program on life-stage susceptibility, this experiment determined
the effect of the carbamate pesticide carbaryl on the operant performance of youngadult,
middle-age and senescent male Brown Norway rats. Rats (Charles River)
were 4, 12, and 23-24 months old at the start of the experiment, weight-maintained,
and trained to perform under a multiple variable-interval (VI) 10-sec variable-interval
100-sec schedule of food reinforcement during 45-min test sessions.
Once performances stabilized, one group (n=10-12/age) received each week a dose
of carbaryl (3, 6.5 and 10 mg/kg) or corn oil, p.o., 30-minutes before testing. Doses
were administered in a mixed order. A second group (n=11-12/age) received weekly
carbaryl (10 mg/kg) and corn oil, p.o., at 30, 60, 120 or 240 minutes before testing;
treatment times were also arranged in a mixed order. During acquisition, initial response
rates for the senescent rats were lower than the young-adult rats, but increased
to rates higher than young adults as performance stabilized. Acquisition for
middle-age rats resembled young-adult rats early in training, and senescent rats late
in training. Carbaryl produced dose-dependent decreases in responding, with the
exception of 6.5 mg/kg in the middle-age rats. Senescent rats were overall most affected
by carbaryl, especially under VI 100- sec following the lowest dose.
Differences in performance due to age after the highest dose (10 mg/kg) were statistically
insignificant. Senescent rats, however, took the longest to recover following
10 mg/kg. Age-related differences in carbaryl effects demonstrate the importance
of using a range of ages in order to make informed inferences about toxicant
susceptibility and risk. (This is an abstract of a proposed presentation and does not
reflect US EPA policy.)
1339 CARBARYL NEUROTOXICITY ACROSS THE LIFE-SPAN
OF THE BROWN-NORWAY RAT.
V. C. Moser, P. M. Phillips and K. L. McDaniel. Toxicity Assessment Division, U.S.
EPA, Research Triangle Park, NC.
Demographics show that the proportion of older adults is increasing every year.
While there has been considerable attention paid to potential sensitivity of the
young to environmental chemicals, there is much less known about the relative vulnerability
of the aged. Differences in response could be due to altered sensitivity as
a result of toxicokinetic and/or toxicodynamic changes. We systematically compared
the dose-response data across the age continuum for neurotoxicity produced
by carbaryl, an N-methyl carbamate that produces acute toxicity via inhibition of
acetylcholinesterase (ChE). Male Brown-Norway rats were tested on postnatal day
18 (PND18), and at 1, 4, 12, and 24 months of age (n=9-10/dose/age), and were
orally gavaged with vehicle (corn oil), 3, 7.5, 15, or 22.5 mg/kg carbaryl.
Horizontal (all ages) and vertical (rearing; adults only) motor activity was measured
for 20 min beginning 15 min after dosing. Within 5 min after the activity session,
rats were sacrificed and brain and RBC were collected for subsequent assay of ChE