TOXICOLOGY AND APPLIED PHARMACOLOGY

ARTICLE NO.

152, 56 65 (1998)

TO988514

Gestational Exposure to Chlorpyrifos:

Apparent Protection of the Fetus?
T. L. Lassiter,*, S. Padilla, S. R. Mortensen,, S. M. Chanda,*, V. C. Moser, and S. Barone, Jr.
*Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina; Cellular and Molecular Toxicology Branch, Neurotoxicology
Division, National Health Effects and Ecological Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park,
North Carolina; and Department of Environmental Toxicology, Clemson University, Clemson, South Carolina
Received March 12, 1998; accepted June 15, 1998

al., 1992; Kishi et al., 1995; Weinbaum et al., 1997), are potent
irreversible inhibitors of serine esterases. The toxicity of these
pesticides is attributed specifically to the inhibition of the
enzyme acetylcholinesterase (EC 3.1.1.7; reviewed in Fukuto,
1990; Ecobichon, 1996). The classical role of acetylcholinesterase is to hydrolyze the neurotransmitter acetylcholine, effectively clearing it from the synapse and terminating impulse
conduction. There is, however, a growing body of literature
suggesting a novel role for both acetylcholinesterase and butyrylcholinesterase (EC 3.1.1.8) in the developing organism
(Burt, 1975; Drews, 1975; Dudai and Yavin, 1978; Kostovic
and Rakic, 1984; Robertson, 1987; Layer and Willbold, 1995;
Small et al., 1996; Greenfield, 1996; Layer, 1996). Although
the profiles of cholinesterase inhibition following exposure of
adults to various organophosphorus pesticides are often investigated, inhibition of fetal cholinesterase activity resulting from
gestational exposures has received substantially less attention.
Considering the suggested developmental role of the cholinesterases and the paucity of data regarding fetal cholinesterase inhibition, concern has been issued recently from both
the scientific and regulatory communities regarding the sensitivity of the developing embryo/fetus to anticholinesterase
pesticides (National Research Council, 1993; Food Quality
Protection Act, 1996). There is evidence in some avian and fish
species that certain organophosphorus pesticides adversely affect development. For example, when mallard eggs are exposed to the organophosphorus pesticide EPN (O-ethyl O-4nitrophenyl phenylphosphonothioate) at 12 mg/g egg, the
embryo weight, brain weight, and crownrump length are all
decreased on embryonic day 18 (Hoffman and Sileo, 1984).
Another organophosphorus pesticide, parathion (10 ppm),
caused developmental abnormalities in the circulatory system
of medaka fish (Solomon and Weis, 1979). Similarly, most
mammalian studies have been limited to assessing embryotoxic
or teratogenic effects (reviewed in Tyl, 1992). It is well known
that, for a given dosage of many organophosphorus pesticides,
brain cholinesterase is much more inhibited in young, postnatal
animals as compared to adults (Brodeur and DuBois, 1963; Lu
et al., 1965; Benke and Murphy, 1975; Pope et al., 1991;

Gestational Exposure to Chlorpyrifos: Apparent Protection of

the Fetus? Lassiter, T. L., Padilla, S., Mortensen, S. R., Chanda,
S. M., Moser, V. C., and Barone, S., Jr. (1998). Toxicol. Appl.
Pharmacol. 152, 56 65.
Previous studies have shown that, in general, young, postnatal
animals are more sensitive than adults to the toxic effects of anticholinesterase (antiChE) pesticides. Paradoxically, often fetal brain
cholinesterase (ChE) is less inhibited than maternal brain after gestational exposure to an antiChE, presumably due to placental and
fetal detoxification of the antiChE. The present investigation was
designed to study selected toxicokinetic and toxicodynamic factors
surrounding the toxicity of chlorpyrifos (CPF; [O,O*-diethyl O-3,5,6trichloro-2-pyridyl] phosphorothionate) in pregnant rats dosed repeatedly or singly during late gestation. Dams were dosed daily (po)
with CPF in corn oil (0 or 7 mg/kg) on gestational days (GD) 14 to 18.
Animals were euthanized at 2 to 120 h after the last dose and tissues
were collected for enzyme analysis. Using this dosing regimen, we
found that (1) the time of maximal ChE inhibition was the same (i.e.,
510 h after dosing) for both maternal and fetal brain, (2) the degree
of fetal brain ChE inhibition was 4.7 times less than maternal brain
inhibition, and (3) the detoxification potential (i.e., carboxylesterase
and chlorpyrifos-oxonase) of the fetal tissues was very low compared
to the maternal tissues. A separate group of experiments showed that
if pregnant dams received only one oral dose of 7 or 10 mg/kg CPF
on GD18, the degree of ChE inhibition in the fetal brain was comparable to the maternal brain ChE inhibition. Taking into consideration the net increase (more than fourfold) in fetal brain ChE activity
from GD14 to 18 in control animals, and the fact that maternal brain
ChE was inhibited more than fetal brain ChE only in a repeateddosing regimen, we conclude that the fetus is not genuinely protected
from the toxic effects of a given dose of CPF. We propose that fetal
brain ChE is simply able to recover more fully between each dose as
compared to maternal brain ChE, giving the illusion that the fetal
compartment is less affected than the maternal compartment.
Key Words: cholinesterase inhibition; chlorpyrifos; gestational
exposure; time course; carboxylesterase; chlorpyrifos-oxonase.

Organophosphorus pesticides, used agriculturally and nonagriculturally worldwide to control insect pests (e.g., Richter et
0041-008X/98

56

GESTATIONAL EXPOSURE TO CHLORPYRIFOS

Moser and Padilla, 1998). This age-related sensitivity to anticholinesterases apparently does not apply to the fetus because
the fetal brain appears to be protected from the cholinesterase inhibition in dams dosed with anticholinesterases
(Michalek et al., 1985; Santhoshkumar and Shivanandappa,
1994; Chanda et al., 1995; Chanda and Pope, 1996), however
there are a few exceptions (Kewitz et al., 1977; Bisso et al.,
1982; Meneguz et al., 1989). Our laboratory is extremely
interested in the age-related effects of pesticides, and the
apparent protection of the fetal compartment warrants further
investigation.
This study measured the cholinesterase, carboxylesterase
(EC 3.1.1.1), and chlorpyrifos-oxonase (EC 3.1.8.1) activities
of various fetal and maternal tissues following gestational
exposure to the organophosphorus pesticide chlorpyrifos (O,Odiethyl O-[3,5,6-trichloro-2-pyridyl] phosphorothionate). This
systematic investigation addressed three issues: (1) the time
course and amount of cholinesterase inhibition in the fetal and
maternal brain, (2) the detoxification potential of carboxylesterase and chlorpyrifos-oxonase in maternal and fetal tissues,
and (3) the comparison of cholinesterase inhibition after multiple doses as compared to a single dose.
METHODS
Experimental Design
This paper represents a group of experiments in which chlorpyrifos was
administered orally to pregnant dams either with a single dose on gestational
day (GD)1 18 or repeated dosing from GD14 to 18 using either 7 or 10
mg/kg/day. In pilot studies repeated gestational exposure to 7 or 10 mg/kg
chlorpyrifos gave a consistent and measurable degree of cholinesterase inhibition, without any morbidity or mortality. The most detailed analysis involved
the repeated dosing with 7 mg/kg/day and is discussed in detail below. The
experimental design for the remainder of the experiments is basically identical
to the repeated 7 mg/kg experiment; any differences are noted.
Animals. Time-pregnant primiparous LongEvans rats were obtained
from Charles River Laboratories Crl: (LE) BR (Portage, ME) and were
determined to be sperm-positive on GD0. Dams arrived on GD4 and were
housed individually on a 12:12 h light:dark cycle, with food (Rodent Diet
5001; Lab Diet, Indianapolis, IN) and water provided ad libitum. Maternal
weight was measured daily from GD4 to 21. Dams were allocated to treatment
groups on GD13 according to a ranking of body weight gain to assure balanced
body weights among time points and dose groups. Dams were dosed daily on
GD14 through 18 by gavage with 0 or 7 mg/kg chlorpyrifos (n/dose/time
point $ 4) in corn oil (1 mg chlorpyrifos/ml of corn oil). At 2, 5, 10 (GD18);
24 (GD19); and 48(GD20) h after the last dose tissue, samples were collected.
Dams and pups were also euthanized at 120 h after the last dose (postnatal day
1, P1). The day of birth was considered P0. The indicators of maternal health
status were body weight gain GD14 to 21, daily postdosing observations, and
more detailed observations immediately preceding euthanasia. Each day,
within 2 h after dosing, the animals were assessed for the common signs of
cholinergic overstimulation by cage-side observation (e.g., smacking, lacrimation, exophthalmus, diarrhea, and tremors). In addition, the dams were assessed
using an abbreviated version of a functional observational battery (Moser et
al., 1988; Moser, 1989; protocol described in McDaniel and Moser, 1993),

Abbreviations used: DFP, diisopropylfluorophosphate; GD, gestational

day; pNPA para-nitrophenyl acetate.

57

which was performed on the dams 2 min before they were euthanized. All
dams were removed from the home cage and observed in an open field for 1
to 2 min. An observer blind to the dose assignment scored the level of arousal
and activity, reactivity or excitability while being handled, and noted any
tremors, clonus, lacrimation, salivation, diarrhea, or polyuria. Pupil size and
light-induced pupillary constriction were also evaluated.
Other dosing regimens. In addition to the repeated 7 mg/kg dosing experiment outlined above, fetal and maternal brain cholinesterase activities were
also assessed subsequent to a repeated 10 mg/kg/day repeated dose level of
gestational chlorpyrifos or to a single gestational dose of chlorpyrifos. The
single exposure dams were administered either 7 or 10 mg/kg of chlorpyrifos
on GD18 by gavage (n/dose $ 4). In these additional experiments fetal and
maternal brains were collected 5 h after dosing (time of peak inhibition, as
determined in the above experiment). Control GD14 fetal and maternal brains
were also collected for determining the change in cholinesterase activity
between GD14 and 18.
Sample collection. Dams were anesthetized with CO2 and euthanized by
decapitation for collection of tissue samples. Following collection of the trunk
blood, each dam was perfused transcardially with saline. The perfusion continued until the normally dark liver turned a very pale brown. This cleared the
hepatic blood that might otherwise confound quantitation of chlorpyrifosoxonase and carboxylesterase activities (Mortensen et al., 1996). At each time
point, dam blood, liver, and brain were collected and frozen on dry ice. The
uterus was removed, placed on wet ice, and the position of any resorptions
recorded. Four fetuses were removed: upper right horn, lower right horn, upper
left horn, and lower left horn. The fetal livers and brains were collected
individually and frozen on dry ice. The placenta corresponding to each fetus
sampled was also collected and frozen on dry ice. Body and brain weights were
recorded for dams, fetuses, and P1 pups. Tissues were stored at 280C until
analysis. Litter was considered the smallest unit of analysis, meaning that
endpoints recorded for individual fetuses/pups from a single litter were averaged as replicates.
Biochemical Determinations
Sample homogenization. Total cholinesterase (acetylcholinesterase and
butyrylcholinesterase), carboxylesterase, and chlorpyrifos-oxonase activities
were measured on all the tissues collected. Samples were homogenized (larger
samples, .50 mg wet wt: 20 s, Setting 6, Polytron, Brinkman Industries,
Westbury, NY) or sonicated on ice (smaller samples, ,50 mg wet weight: 10 s,
Setting 3, Branson Sonifier 250) in 0.1 M Na phosphate buffer (pH 8)
containing 1% Triton X-100. Maternal liver was homogenized for 45 s.
Control activity was expressed as mmol of the appropriate substrate hydrolyzed/ min/g wet wt or /ml. Activity of the chlorpyrifos-treated subjects was
expressed as a percent of the age-matched control.
Cholinesterase activity. Total cholinesterase activity was determined for
dam blood, dam brain, placenta, and fetal brain using a Hitachi 911 Automatic
Analyzer (Boehringer Mannheim Corp., Indianapolis, IN) according to the
method outlined in Hunter et al. (1997). The 911 is a robot-equipped spectrophotometer that appropriately dispenses sample, buffer, chromogen, and substrate to assay cholinesterase activity according to a variation of the Ellman et
al. (1961) method. Considering the high sulfhydryl background present in the
liver, it was prohibitive to use this colorimetric technique with liver homogenates. Fetal and maternal liver cholinesterase activities, therefore, were assayed using the Johnson and Russell (1975) radiometric assay with a final
substrate concentration of 1.2 mM. The [3H]acetate produced by the hydrolysis
of [3H]acetylcholine was quantified using a Wallac 1410 liquid scintillation
counter.
Carboxylesterase activity. Carboxylesterase activity was determined using
the Chanda et al. (1997) microassay which is a variation of the Clement and
Erhardt (1990) method. Briefly, the carboxylesterase activity was determined
as the para-nitrophenyl acetate (pNPA) hydrolyzing capacity of the sample in
the presence of 1 mM EGTA, in 50 mM Tris buffer. The EGTA chelates the
Ca21, such that chlorpyrifos-oxonase hydrolysis of pNPA will not contribute

58

LASSITER ET AL.

to determination of carboxylesterase activity. Carboxylesterase cleaves pNPA,

producing para-nitrophenol. Production of para-nitrophenol increased the spectrophotometric absorbance of the reaction mixture at 405 nm using a ThermoMax Microtiter Plate Reader (Molecular Devices, Menlo Park, CA).
Chlorpyrifos-oxonase activity. Chlorpyrifos-oxonase activity was determined per the Mortensen et al. (1996) modification of the Furlong et al. (1989)
method. Tris buffer (0.1 M, pH 8.5) and solubilized CaCl2 (2 mM) were
combined with the sample. The reaction was initiated by the addition of the
substrate chlorpyrifos-oxon. Chlorpyrifos-oxonase activity was measured as
the increase in absorbance at 310 nm as a result of the generation of 3,5,6trichloro-2-pyridinol from the cleavage of chlorpyrifos-oxon using a Beckman
DU-70 spectrophotometer.
Statistics
In the repeated 7 mg/kg/day time course experiment the data for each
enzymes activity (not percent of control) in each tissue was analyzed using a
two-way analysis of variance (ANOVA) for independent samples. For a
significant dose by time interaction at an a 5 0.05, a TukeyKramer Honestly
Significant Difference (Tukey, 1953; Kramer, 1956) post hoc analysis was
used to determine the differences across time between the two dose groups.
The single and repeated gestational chlorpyrifos exposure data presented in
Fig. 8 were collapsed from three separate experiments. The statistical significance of this data was determined by performing a two-way (age vs dose)
ANOVA on the log transformation of percentage of respective control cholinesterase activities. Following the significant interaction of age with dose,
each treated group was compared to the control using Dunnetts test.

RESULTS

Enzyme Activities of Control Animals between GD18 and P1

There were many changes in cholinesterase, carboxylesterase, and chlorpyrifos-oxonase activities in both the maternal
and fetal compartments during the last 4 days of pregnancy and
first 2 days after birth. The late gestational time course of
A-esterase is presented in Fig. 1. The time course of the control
activities of cholinesterase and carboxylesterase in each tissue
is presented as an inset in the time course profiles for each
treated tissue (see Figs. 27).
A-esterases. The chlorpyrifos-oxonase activity was analyzed
in the treated and control animals at all time points, and the results
were compared statistically. As expected, there was no difference
in the chlorpyrifos-oxonase activity due to treatment with chlorpyrifos. Therefore, the values for the treated and control activities
were combined for each time and tissue and are presented in Fig.
1. Note that no chlorpyrifos-oxonase activity was detected in
either maternal or fetal brain, which agrees with our previous
results in neonatal and adult male rat brain (Mortensen et al.,
1996). In general, most of the time-dependent changes in chlorpyrifos-oxonase activity of the maternal and fetal/neonatal tissues
occurred after birth (Fig. 1). Placental activity showed no change
at all from GD18 to 20. Maternal blood chlorpyrifos-oxonase
activity was variable before birth, but decreased on the day after
birth (Fig. 1). The opposite pattern was noted in the liver: fetal
liver exhibited minimal activity during gestation, but at P1 the
fetal liver showed an eightfold increase in chlorpyrifos-oxonase
activity; maternal liver chlorpyrifos-oxonase increased slightly
after birth (Fig. 1).

FIG. 1. Late gestational ontogeny of chlorpyrifos-oxonase activity. The

age-dependent changes in chlorpyrifos-oxonase activity in the maternal and
fetal/neonatal tissues occurred primarily after birth. Maternal liver had the
most activity, and the time course of this activity was variable, but ultimately
demonstrated a slight postpartum increase. Maternal blood chlorpyrifos-oxonase activity was variable before birth, but decreased on the day after birth.
Placenta had a small amount of chlorpyrifos-oxonase activity, but did not
change from GD18 to 20. Fetal liver exhibited minimal activity during gestation, but the P1 pup liver showed an eightfold increase in chlorpyrifos-oxonase
activity. Brain, either maternal or fetal, had no detectable activity (not shown).

B-esterases. From GD18 through P1 cholinesterase activity in the fetal/neonatal brain showed a threefold increase.
Carboxylesterase activity in the fetal/neonatal brain did not
change GD18 to P1. Maternal brain cholinesterase and carboxylesterase activities also remained basically unchanged
during the same period (insets: Figs. 2 vs 7). Cholinesterase
activity in the fetal liver decreased by nearly 40% during this
same time, whereas fetal liver carboxylesterase activity increased more than twofold (insets: Figs. 4 vs 5). The maternal
liver showed yet another pattern of change for these two
enzymes: cholinesterase activity was variable while maternal
liver carboxylesterase activity remained unchanged. Placenta
and maternal blood also demonstrated distinct activity profiles:
placental cholinesterase activity nearly doubled, but maternal
blood cholinesterase activity was variable, while carboxylesterase activity in both tissues decreased 40 to 50% (insets: Figs.
3 vs 6).
Summary. To recapitulate, the pattern of changes for a
particular esterase (A-esterase, cholinesterase, or carboxylesterase) in a particular tissue was not predictive of that same
enzyme in other tissues. Similarly, the activity profile of one
esterase was not indicative of the profile of another esterase,
and the maternal and fetal compartments did not necessarily
show changes at the same time or in the same direction.
Enzyme Activity in the Animals Exposed Repeatedly to
Chlorpyrifos (7 mg/kg/day)
Overt toxicity. The increase in maternal gestational weight
was similar for control and chlorpyrifos-treated dams. There
were no overt signs of toxicity in any of the animals when
assessed by a cage side observer 2 h after each dose. Prior to

GESTATIONAL EXPOSURE TO CHLORPYRIFOS

FIG. 2. Fetal and maternal brain cholinesterase inhibition after repeated

dosing with 7 mg/kg chlorpyrifos. The time of maximal cholinesterase inhibition was the same in maternal and fetal brain: 510 h after the last dose of
chlorpyrifos. There was 4.7-fold less inhibition in the fetal brain than in the
maternal brain. Fetal brain cholinesterase inhibition recovered by 24 h after the
last dose, but maternal brain activity did not recover completely within 5 days.
The inset shows the cholinesterase activity in untreated fetal and maternal
brains. There was no net change in maternal cholinesterase activity, but fetal
brain activity nearly tripled during this period of time (GD18 P1).

euthanasia the dams were assessed using a functional observational battery and no statistically significant, treatment-related
effects were observed. There were, however, a few individual
animals that showed some signs of toxicity: two treated rats
showed miosis (one at 2 h, one at 5 h), three showed exophthalmus (one at 2 h, two at 5 h), and three had scores indicating
abnormally low reactivity scores when handled (two at 5 h, one
at 10 h). Therefore, considering that weight gain was unaffected and these observational assessments revealed only a few
rats with these mild signs of exposure, this repeated gestational
exposure to 7 mg/kg/day chlorpyrifos GD14 to 18 was not
determined to be overtly toxic to the pregnant dams. Similarly,
there were no treatment-related effects on resorptions, fetal/
pup body weights, fetal/pup brain weights, litter size, or number of live births (data not shown).
Cholinesterase activity. The time course of cholinesterase
inhibition in fetal and maternal brain was similar with maximal
inhibition 5 to 10 h after the last dose (Fig. 2). There was a
substantial difference in the maximal degree of cholinesterase
inhibition in the fetal vs maternal brain: at the time of peak
effect, fetal brain cholinesterase activity was slightly depressed
(75% of control), while maternal brain cholinesterase activity
was markedly reduced (16% of control). This represents a
4.7-fold difference in the degree of cholinesterase inhibition
between the maternal and fetal brain at the time of peak
inhibition after the fifth dose of chlorpyrifos. Maternal brain
cholinesterase activity still had not recovered completely
within 5 days after the last chlorpyrifos dose (56% of control),
but fetal brain activity had recovered to control levels by 24 h
after the last dose of chlorpyrifos (Fig. 2). Maternal blood
cholinesterase activity, similar to maternal brain, was maximally inhibited (10% of control) at 2 to 10 h posttreatment and

59

FIG. 3. Placental and maternal blood cholinesterase inhibition. Placental

cholinesterase activity was 66% of control 2 h after the last dose of chlorpyrifos and demonstrated considerable variability during the entire time course.
Maternal blood cholinesterase activity was only 10% of control at 2 to 10 h
after the last dose and did not recover within 5 days. The inset shows the
cholinesterase activity in untreated placentae and maternal blood. There was a
doubling of cholinesterase activity in control placentae during the last 3 days
of gestation, but the time course of cholinesterase activity for control dam
blood decreased slightly (GD18), but then was stable over the period of
examination (GD18 P1).

still had not recovered to control levels by 5 days after dosing

(Fig. 3). Cholinesterase activity in placentae from chlorpyrifostreated dams was 66% of control, 2 h after the last dose, and
demonstrated a variable degree of inhibition thereafter (74
91% of control, Fig. 3). Maternal liver cholinesterase activity
was less than 20% of control activity at 2, 5, and 10 h after the
last chlorpyrifos treatment and recovered to control levels by 2
days after the last dose (Fig. 4). There was less cholinesterase
inhibition in the fetal liver (50% of control) than maternal liver

FIG. 4. Fetal and maternal liver cholinesterase inhibition. Fetal liver

cholinesterase was maximally inhibited to 57% of control activity 2 to 5 h after
the last dose of chlorpyrifos, while maternal liver cholinesterase was less than
20% of control at 2, 5, and 10 h after the last treatment. Fetal liver cholinesterase activity recovered by 10 h after the last dose, but maternal liver did not
recover until 48 h after the last chlorpyrifos exposure. The inset shows the
cholinesterase activity in untreated fetal and maternal liver. The control fetal
liver cholinesterase activity decreased, but the activity in the control maternal
liver was variable.

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LASSITER ET AL.

FIG. 5. Fetal and maternal liver carboxylesterase inhibition. Liver carboxylesterase activity in chlorpyrifos exposed dams was less than 18% of
control 2 to 10 h after the last dose. Fetal liver carboxylesterase was less than
50% of control values for the entire time course postdosing. Fetal liver
carboxylesterase did not recover, but the maternal liver carboxylesterase activity was recovering by the 5-day time point (i.e., P1). The inset shows the
carboxylesterase activity in untreated fetal and maternal liver. The control fetal
liver carboxylesterase activity increased, but the activity in the control maternal livers demonstrated no change.

during the first 2 to 5 h after dosing, and recovery occurred

more quickly (i.e., 10 h after dosing) than in the maternal liver
(Fig. 4).
Carboxylesterase activity. Liver carboxylesterase activity
in chlorpyrifos-treated dams was less than 20% of control 2 to
10 h postdosing and did not recover until 5 days after the last
dose (Fig. 5). Fetal liver carboxylesterase demonstrated
marked inhibition over the entire time course in chlorpyrifostreated subjects with activity less than 50% of control values
for at least 5 days after the last dose of chlorpyrifos (Fig. 5).
There was no inhibition of carboxylesterase activity in either
maternal blood or placenta (Fig. 6) or fetal brain (Fig. 7). Dam
brain carboxylesterase activity, however, was inhibited at a

FIG. 6. Placental and maternal blood carboxylesterase inhibition. There

was no inhibition of carboxylesterase in either placenta or maternal blood,
suggesting that no protective detoxification was provided by carboxylesterase
in these two tissues. The inset shows that carboxylesterase activity in untreated
placentae and maternal blood decreased during late gestation.

FIG. 7. Fetal and maternal brain carboxylesterase inhibition. Fetal brain

carboxylesterase activity was not inhibited in the chlorpyrifos-exposed subjects, but maternal brain activity was approximately 70% of control activity
across the entire time course. The inset shows that carboxylesterase activity in
untreated fetal brains increased slightly while maternal brain carboxylesterase
activity did not change.

constant level (;70% of control) during the entire time of

assessment (GD18 P1; Fig. 7).
Cholinesterase Activity in Single vs Repeatedly Exposed
Animals (Either 7 or 10 mg/kg/day)
A separate group of experiments was also conducted to
compare the fetal and maternal brain cholinesterase inhibition
in animals dosed either once or repeatedly with chlorpyrifos.
Similar to the dams treated repeatedly with 7 mg/kg, the dams
treated repeatedly with 10 mg/kg showed limited signs of
toxicity in cage-side observations 2 h after dosing (exophthalmus). There were similarly no effects on fetal brain or body
weight (data not shown). The cholinesterase activity is presented in Fig. 8. When pregnant rats were dosed repeatedly
with either 7 or 10 mg/kg/day from GD14 to 18, maternal brain
cholinesterase activity was much more inhibited than fetal
brain cholinesterase activity (Fig. 8, right). In contrast to this
differential degree of cholinesterase inhibition between fetal
and maternal brain subsequent to repeated chlorpyrifos exposure, a different pattern emerged between the fetal and maternal brain 5 h (the time of peak effect) after a single dose of
either 7 or 10 mg/kg chlorpyrifos (Fig. 8, left). The single 7
mg/kg dose did not produce statistically significant inhibition
of brain cholinesterase in either the dam or the fetus; however,
the single 10 mg/kg dose did produce inhibition (about 60% of
control activity) of both the fetal and maternal brain cholinesterase. Interestingly the degree of maternal and fetal brain
cholinesterase inhibition was comparable. That is, there was no
difference in the degree of inhibition in either maternal or fetal
brain cholinesterase after a single dose of 10 mg/kg chlorpyrifos, a pattern that is in stark contrast with the differential
degree of inhibition after the repeated dosing with chlorpyrifos
(Fig. 8, compare left with right). Note that the control fetal
brain showed a mammoth fourfold increase in cholinesterase
activity between GD14 and 18 (Fig. 8, inset).

GESTATIONAL EXPOSURE TO CHLORPYRIFOS

61

FIG. 8. Cholinesterase activity in maternal and fetal brain following single or repeated exposure. After a single GD18 dose of 7 mg/kg chlorpyrifos, brain
cholinesterase activity was not significantly inhibited in either maternal or fetal brain. After a single dose of 10 mg/kg chlorpyrifos, however, the degree of
cholinesterase inhibition was comparable in fetal and maternal brain 5 h later (left). This pattern was very different from the differential degree of inhibition
measured after five doses of chlorpyrifos: the repeated dosing schedule caused a marked decrement of maternal brain cholinesterase activity, but considerably
less cholinesterase inhibition in fetal brain (right). (Inset) In controls, maternal brain cholinesterase activity does not change from GD14 to 18, but fetal brain
cholinesterase activity increases 4.3-fold.

DISCUSSION

Many studies have addressed the potential embryotoxicity

and teratogenicity of anticholinesterase compounds (Kimbrough and Gaines, 1968; Staples et al., 1976; Martson and
Voronina, 1976; Murray et al., 1979; Short et al., 1980; Lechner and Abdel-Rahman, 1984; Machin and McBride, 1989;
Clemens et al., 1990; reviewed by Tyl, 1992; LaBorde et al.,
1996), but, curiously, few of these studies measured cholinesterase inhibition in either the fetal or maternal compartments.
From the above studies, one may conclude that, in general, the
dosages of the anticholinesterases that were not maternally
toxic also produced no embryotoxicity or teratogenicity. These
results are informative, but the literature seems to be lacking in
studies of the developmental neurotoxicity of cholinesterase
inhibitors administered during gestation. One universal yardstick of both exposure and neurotoxicity is inhibition of cholinesterase activity in brain tissue: brain cholinesterase inhibition is considered a biomarker of exposure and is also
recognized as an adverse effect. A review of the gestational
anticholinesterase toxicity studies that compare the degree of
cholinesterase inhibition in the fetal and maternal brain reveals

a prevalent pattern. Generally, at a given dosage and a given

time after dosing, there is less cholinesterase inhibition in the
fetal brain as compared to the maternal brain (Michalek et al.,
1985; Santhoshkumar and Shivanandappa, 1994; Chanda et al.,
1995; Chanda and Pope, 1996), although there are a few
exceptions where the same degree of inhibition were noted
(Kewitz et al., 1997; Bisso et al., 1982; Meneguz et al., 1989).
The repeated, gestational chlorpyrifos oral exposure schedule
used in the current study also caused markedly less cholinesterase inhibition in the fetal brain than the maternal brain (Figs.
2 and 8, right). The finding that the maternal brain cholinesterase was inhibited 4.7-fold more than the fetal brain cholinesterase following repeated gestational exposure to chlorpyrifos
suggests that the fetal brain may be impervious to or protected
from cholinesterase inhibition. Interestingly, a single gestational oral dose of chlorpyrifos produced comparable fetal and
maternal brain cholinesterase inhibition (Fig. 8, left).
Why does the comparable cholinesterase inhibition between
fetal and maternal brain subsequent to a single dose of chlorpyrifos contrast so sharply with the differential degree of inhibition
following repeated gestational chlorpyrifos exposure (Fig. 8)?

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LASSITER ET AL.

Several explanations could account for the differential degree of

cholinesterase inhibition observed between fetal and maternal
brain following repeated gestational chlorpyrifos including: (1)
intrinsic differences in sensitivity of cholinesterase molecules to
inhibitors; (2) differences in biotransformation and/or disposition
of chlorpyrifos; (3) protection of fetal brain cholinesterases by
placental and/or fetal chlorpyrifos-oxonase and carboxylesterase;
and (4) dynamics of recovery for fetal vs maternal brain cholinesterase. Intrinsic differences, biotransformation, and disposition are
considered in lesser detail below followed by a more detailed
consideration of protection and cholinesterase recovery.
Some investigators have suggested that the fetal brain cholinesterase is less inhibited compared to the maternal brain
cholinesterase because the fetal brain cholinesterase is simply
less sensitive to inhibition by chlorpyrifos oxon than is adult
brain cholinesterase. All evidence comparing the intrinsic sensitivity of neonatal or preweanling brain cholinesterase vs adult
brain cholinesterase indicates that there is no difference in
sensitivity to anticholinesterase pesticides (Benke and Murphy,
1975; Wang and Murphy, 1982; Volpe et al., 1990; Mortensen
et al., 1998) or to chlorpyrifos-oxon (Chanda et al., 1995;
Mortensen et al., 1996; 1998), so this does not appear to be a
viable explanation for the differential inhibition.
Another possible reason for the differences in inhibition
between the dam and fetal brain cholinesterase may be the
reduced capacity of the fetal compartment to convert chlorpyrifos to the much more potent inhibitor of cholinesterase, chlorpyrifos-oxon. This scenario presumes that it is primarily chlorpyrifos that enters the fetal compartment and not the oxon. The
highly lipophilic nature of chlorpyrifos (Chambers and Carr,
1993) renders it fully capable of crossing the placental membranes and, in fact, placental transfer of other organophosphorus pesticides has been documented (Ackerman and Engst,
1970; Villeneuve et al., 1972; Budreau and Singh, 1973; Harbison, 1975; Berge and Nafstad, 1986; Tsoukali-Papadopoulou
and Njau, 1987). Although activation is important for the
effects of other developmental toxicants (reviewed by Juchau,
1989), there appears to be no specific information on oxidative
desulfuration of organophosphorus pesticides by fetal rat tissues. There is, however, evidence that cytochrome P450s are
present in the fetal liver and placenta, albeit at very low levels
(see reviews by Pelkonen, 1977; Raucy and Carpenter, 1993;
Juchau et al., 1980; Kitada and Kamataki, 1994; Miller et al.,
1996; Juchau et al., 1992; Goodman et al., 1982).
The explanation for the differential degree of inhibition that
was most thoroughly investigated in this present group of experiments concerns the question of protection of the fetal brain
cholinesterase. Two enzymes that are capable of affording protection from chlorpyrifos intoxication include chlorpyrifosoxonase and carboxylesterase. Chlorpyrifos-oxonase is a Ca21dependent hydrolase that cleaves chlorpyrifos-oxon (Furlong et
al., 1988). Carboxylesterase activity consists of a group of several
related isozymes that detoxify by stoichiometrically binding the
chlorpyrifos-oxon, thus effectively reducing the concentration of

FIG. 9. (A) Summary of chlorpyrifos-oxonase control activity. This summarizes the chlorpyrifos-oxonase activities from GD18 to 20 in the untreated
dams and fetuses. Activity is the mean of the control activity values collapsed
across the GD18 to 20 time points; values are expressed as mmol of chlorpyrifos-oxon hydrolyzed/ min/ g wet wt. (B) Summary of carboxylesterase control
activity. This summarizes the carboxylesterase activities from GD18 to 20 in
the untreated dams and fetuses. Activity is the mean of the control activity
values collapsed across the GD18 to 20 time points; values are expressed as
mmol of pNPA hydrolyzed/ min/ g wet wt.

chlorpyrifos-oxon available to inhibit cholinesterase (reviewed in

Maxwell, 1992). An important distinction to appreciate regarding
the activities of chlorpyrifos-oxonase and carboxylesterase in this
gestational exposure study is the subtle difference between detoxification and protection. Detoxification of chlorpyrifos may
occur in both the fetal and maternal compartments. Genuine
protection of the fetal brain (nervous system), however, occurs
exclusively in the fetus or placenta. Thus, fetal brain cholinesterase is protected from chlorpyrifos-oxon by detoxification enzymes
in tissues available only to the fetus and not to the mother.
Placenta, fetal liver, and fetal brain, however, contained limited
detoxifying enzymes for the protection of fetal brain cholinesterase (Fig. 9). Of these three, the only tissue that had chlorpyrifosoxonase activity was placenta; fetal liver and brain had no activity.
Although chlorpyrifos-oxonase activity in the placenta was low

GESTATIONAL EXPOSURE TO CHLORPYRIFOS

compared to the other maternal tissues (Figs. 1 and 9A), even this
low activity may be protective by hydrolyzing chlorpyrifosoxon that crossed the placenta. We assume that the chlorpyrifosoxonase activities of maternal tissues were valuable for detoxification, but did not contribute to an explanation of the differential
degree of cholinesterase inhibition between fetal and maternal
brain, because they would be available to both compartments.
Carboxylesterase activity detoxifies chlorpyrifos by the stoichiometric sponging of chlorpyrifos-oxon. The detoxification potential of the carboxylesterases in a given tissue is related to the
amount of enzyme and the affinity of that enzyme for the inhibitor
(Chanda et al., 1997). Figure 9B is a cartoon summarizing the
amount of carboxylesterase in various fetal and maternal tissues
during late gestation. Maternal liver had the highest specific
activity followed a distant second by fetal liver and maternal
brain; maternal blood, placenta, and fetal brain all possessed low,
but detectable levels. The inhibition of carboxylesterase activity
after dosing is an excellent barometer of the detoxification performed by that tissue. After repeated dosing with chlorpyrifos
only three tissues showed carboxylesterase inhibition: maternal
liver, fetal liver, and maternal brain. As for protection of the fetal
brain, only the fetal liver carboxylesterase activity was inhibited
(Fig. 5). Because the placenta and fetal brain carboxylesterase
activity was not inhibited in the treated animals, these two tissues
probably did not afford any protection of the fetal brain cholinesterase (Figs. 6 and 7). It should be noted that an alternative
explanation for the absence of carboxylesterase inhibition could
be exceptionally rapid turnover of carboxylesterase molecules,
thus obscuring inhibition. This explanation is unlikely considering
the recovery of carboxylesterase activity was previously shown to
be as slow or slower than the recovery of cholinesterase activity in
adults (Chanda et al., 1997). In summary, although there are
detoxification enzymes present in the placenta and fetal liver,
there are limitations regarding the degree of protection afforded
by these enzymes.
Another perspective from which to view the question of
potential protection of the fetus is to consider the capacity of
the fetus to recover after exposure to an anticholinesterase. In
fact, other investigators have hypothesized that the greater
synthetic capacity of the postnatal brain may explain less
cholinesterase inhibition and receptor down-regulation in
young (postnatal) animals, as compared to adults (Chakraborti
et al., 1993). Our present data indicate that the fetal brain also
possesses an exquisite synthetic capacity, more than quadrupling the amount of brain cholinesterase activity from GD14 to
18 (Fig. 8, inset). Therefore, considering this increase in specific activity during the dosing period, it may be that the new
synthesis of uninhibited cholinesterase molecules may dilute
the inhibited molecules such that the fetal brain cholinesterase
activity recovers more quickly than the maternal brain. To test
this hypothesis empirically, one should administer a quickacting, potent, irreversible cholinesterase inhibitor, which
would be cleared quickly from the system, and then recovery
of the enzyme activity could be followed over time. This would

63

FIG. 10. Fetal brain recovered faster than maternal brain following a
single dose of DFP. Following a single gestational dose of DFP on GD20 fetal
brain, cholinesterase activity recovered from 35 to 75% of control activity
between 1.5 and 24 h after exposure and returned to control levels by 48 h after
DFP exposure. The maternal brain cholinesterase inhibition demonstrated
virtually no recovery within 48 h after dosing. This graph was adapted from the
data contained in Meneguz et al. (1989).

provide a means to determine if fetal brain cholinesterase

resynthesis/turnover was more rapid than maternal brain. Traditionally, diisopropylfluorophosphate (DFP) has been used to
study the resynthesis/turnover of cholinesterase in many systems. Interestingly, a search of the literature reveals that this
type of experiment has already been done using DFP administration on GD20 to Wistar rat dams by Meneguz and coworkers (1989) and the relevant data are regraphed in Fig. 10.
These data show clearly that the fetal brain cholinesterase
recovers much faster (as a percentage of control) than maternal
brain cholinesterase: from 1.5 to 24 h after a single dose of
DFP, fetal brain cholinesterase recovers from 35 to 75% of
control activity (i.e., 40% recovery), while the maternal brain
cholinesterase showed less than 10% recovery over the same
time period (Fig. 10). Additionally, this expeditious synthesis
of cholinesterase molecules during early brain development is
supported by other data showing high levels of acetylcholinesterase mRNA in the newborn rat brain (Brimijoin and Hammond, 1996; Koenigsberger et al., 1998). The findings outlined
above and the data presented in the current paper suggest that
the resynthesis dynamics of brain cholinesterase are important
in explaining the differential inhibition observed after repeated
dosing with chlorpyrifos (Fig. 8, right and inset; Fig. 10). In
summary, the marked gestational increase in fetal brain cholinesterase activity (Fig. 8, inset), the comparative degrees of
inhibition following a single dose of chlorpyrifos (Fig. 8, left),
and the expeditious recovery of fetal brain cholinesterase
activity following a single dose of DFP (Meneguz et al., 1989;
Fig. 10) support the recovery explanation while simultaneously
eroding the importance of protection.
These results challenge the prevailing opinion that the fetus is
protected from chlorpyrifos toxicity during gestation. These
data indicate that the fetal brain is not less susceptible to inhibition
by this compound when given gestationally, but that the fetal

64

LASSITER ET AL.

brain cholinesterase is just able to recover more quickly between

dosages. The exceptional recovery capacity of the fetal brain
cholinesterase may represent an alternative form of protection, but
ultimately the pesticide is still inhibiting cholinesterase activity in
the fetal brain. Beyond the traditional toxicological context of
cholinesterase inhibition, the inhibition of fetal brain cholinesterase may be deleterious to the coordinated development of the
brain given the postulated, novel role for the cholinesterases in
nervous system development.
ACKNOWLEDGMENTS
We thank Drs. S. Brimijoin, S. McMaster, C. N. Pope, and L. Sheets for
thoughtful critique of earlier drafts of this manuscript. We also thank Kay
Riggsbee, Debbie Hunter, Renee Marshall, Pam Phillips, Kathy McDaniel,
Najwa Haykal-Coates, Dennis House, and Kaberi Das for assisting in this
study. We thank Dow Chemical Corporation for the chlorpyrifos-oxon used in
the chlorpyrifos-oxonase assays. T. L. Lassiter is supported by NIEHS Training Grant T32 ES07126. This research has been reviewed by the National
Health and Environmental Effects Research Laboratory, U.S. EPA, and approved for publication. Approval does not signify that the contents necessarily
reflect the views and policies of the Agency, nor mention of trade names or
commercial products constitute endorsement or recommendation for use.

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