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Apr 22, 1993 - and Neurochemical. Effects of Acute Chlorpyrifos in. Rats: Tolerance to Prolonged. Inhibition of Cholinesterase1. PHILIP. J. BUSHNELL,.
0022-3565/93/2662-1007$OO.OO/O THE JOURNAL OF PHARMACOLOGY Copyright C 1993 by The American

AND

Vol. 266, No. 2

THERAPEUTICS

EXPERIMENTAL

Society

for Pharmacology

and Experimental

Therapeutics

Behavioral and Neurochemical Rats: Tolerance to Prolonged PHILIP

J. BUSHNELL,

CAREY

Neurotoxicology Division (P.J.B., Park, North Carolina and Division Louisiana

Accepted

for publication

N. POPE

and

Effects of Acute Chlorpyrifos Inhibition of Cholinesterase1

STEPHANIE

ChE,

results

the in

street This

to chemicals

primary

degradative

et at.,

reduction

from

over

has time

to ChE

which

inhibit

enzyme

signs

of

for

toxicity

inhibition ChE

activity

inhibitors

Received for publication 1 The research described

October in this

behavioral

effects

of ChE,

and

does

recover

not

complicates 26, 1992. article

has

been

activity

typically with

associated

et a!.,

(Chippendale

in the

the

ACh,

Bignami et at., 1975; Russell been observed in experimental

1974;

despite continued for tolerance. The fact that

exposure

the

overstimulation

recovery

United States Environmental Protection Agency, Research Triangle School of Pharmacy, Northeast Louisiana University, Monroe,

April 22, 1993

exposure recovery

cholinergic

in

Overet at., 1986a). animals

of the

is taken during

classical

reviewed

1972;

as a

inhibitor, as evidence prolonged

demonstra-

by the Health

Effects

Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency or does mention of trade names or commercial products constitute endorsement or recommendation for use. A portion of these data were presented at the annual meeting of the Society of Toxicology, Seattle, WA, February 26, 1992.

ChE activity in whole blood in a dose-related manner for more than 53 days. The degree and time course of ChE inhibition in blood and brain and the downregulation of muscarinic receptors in brain after 1 25 mg/kg of CPF closely paralleled the previously reported effects of 25 daily injections of 0.2 mg/kg of DFP. In addition, CPF-treated rats were subsensitive to oxotremorineinduced hypothermia for at least 32 days after CPF. However, functional deficits (in working memory and motor function) appeared within 2 days after injection of CPF and recovered within 3 weeks, long before ChE activity and receptor density returned to control levels. Thus, the effects of CPF were neither progressive nor as persistent as those seen during daily DFP injections. This difference suggests that the DFP-induced behavioral changes observed previously cannot be attributed entirely to its effects on ChE activity and changes in [3H]qumnuclidmnyl benzilate binding.

tions ofthe

of tolerance inhibitor

as a rightward

signs (Costa

and with alterations et al., 1982a; Russell

shift

in the

dose-effect

function

(e.g., McPhillips, 1969). Therefore, tolerance to a ChE inhibitor is typically demonstrated in the form of decreased sensitivity to cholinergic agonists or increased sensitivity to cholinergic antagonists (Chippendale et at., 1972; Overstreet, 1974; Russell et aL, 1986a; Raffaele et at., 1990; Pope et at., 1992). Of several possible mechanisms by which tolerance could develop, reduced density of muscarinic receptors appears to be associated most closely with the attenuation of cholinergic in sensitivity and Overstreet,

to

cholinergic 1987; Hoskins

drugs and

Ho, 1992). Given that compensatory changes occur in the nervous system in response to repeated exposure to ChE inhibitors, it may be asked whether normal behavior can be maintained despite chronically suppressed ChE activity. The common view of tolerance

return

suggests to normal

that behavior

it can

and

in the

considerable face

ABBREVIATIONS: ChE, cholinesterase; ACh, acetyicholine; OP, organophosphate; DFP, diisopropyffluorophosphate; diethyl-O-3,5,6-tnchloro-2-pyndyl phosphorothionate); ONB, quinuclidinyl benzilate; DMTP, delayed matching-to-position; FOB, functional observational battery; IRT, inter-response time; ANOVA, analysis of variance.

of prolonged

evidence

for

a

inhibition

CPF, chiorpyrifos (0,0’VD, visual discrimination; 1007

Downloaded from jpet.aspetjournals.org at ASPET Journals on February 13, 2015

Prolonged

USA.

in

PADILLA

S.P.), Health Effects Research Laboratory, of Pharmacology and Toxicology (C.N.P.),

ABSTRACT The preponderance of studmes of tolerance to organophosphate (OP) cholinesterase (ChE) inhibitors indicates that functional recovery accompanies neurochemical compensations for the inhibited enzyme. Contrary to prediction, rats dosed with the OP diisopropylfluorophosphate (DFP) showed progressive and persistent impairment of cognitive and motor function over a 3week period of daily exposure, despite neurochemical and pharmacological evidence of tolerance to its inhibition of ChE. To determine whether these functional effects of DFP resulted from inhibition of ChE and downregulation of muscarinic cholmnergic receptors, rats were dosed with chlorpyrifos (CPF), an OP pesticide which inhibits blood and brain ChE of rats for weeks after a single injection. Long-Evans rats were trained to perform an appetitive test of memory and motor function and were then injected s.c. with 0, 60, 125 or 250 mg/kg of CPF in peanut oil and tested 5 days/week for 7 weeks. Unconditioned behavior was also rated for signs of cholmnergic toxmcity. CPF inhibited

of

Pri,Ued

Bushnell

1008

that,

ChE exists. although

of

OP

of

et al.

However, recovery

poisoning,

Vol. 266

examination of the literature occurs for a variety of behavioral

the

recovery

time

differs

upon the index examined. For example, water normally after 3 days of treatment at., 1971), whereas recovery of operant of

Differential

a

Reinforcement

widely

depending

rats

were

with

DFP

behavior

of Low

shows indices

rates

able

to lap et (performance (Russell

schedule

or Sid-

man shock avoidance) required 10 days (Russell et at., 1969). Other more complex behaviors, including single alternation and performance of a multiple operant schedule, did not fully recover for the duration of those experiments (Overstreet et a!., 1974; Genovese et aL, 1988). This pattern resembles that which occurs

during

restoration

irreversible

muscarmnic

of

receptors

agonists

and

after has

treatment

been

interpreted

dosing

regimen

ask whether dosing

of Glow

this

pattern

regimens

ularly

earlier of the

tolerance

injections

of 0.2

mg/kg

of working

effect

occurred

DFP,

subsensitivity

muscarinic carinic

not

agonist

longed tional

inhibition cost may In

normal

function

and

DFP,

of

function

reduced

and

may

(behavior)

in the

sulted

from

the

ChE

or

face

of the

as

of ChE. whether

DFP-induced

DFP the

with

functional

(Bushnell functional

Thus, inhibition rinic

effects

if the

impairment

et aL, 1991), effects

of ChE

this

similar

during

daily

provides

ChE

of DFP on (and consequent

then

receptors),

of

CPF

re-

to inhibit

dosing

a means

inhibition

changes

with

to assess

without

function resulted downregulation

functional

DFP.

from its of musca-

should

result

from acute injection of CPF. Alternatively, if the functional effects of CPF differ from those seen previously with DFP, then those effects must result from some other action of DFP. The present study was thus designed to determine whether the prolonged inhibition of ChE produced by a single injection

of CPF observed

activity

would

induce

previously

in blood

behavioral during

and

brain

changes repeated

were

in rats injections

measured

similar of

to assess

feeding

to those DFP.

ChE

the degree

(Charles

River,

Raleigh,

in suspended

in a housing

facility

photoperiod

occurred

(All

et a!.,

in the

with

in the

light

daily and maintained (Ralston

1992);

NC

plastic

fully

and

cages on

accredited

by the

of training

and

at

onset

350

at

of the

6:00

cycle.

g b.wt.

by

all

A.M.;

Each

animal

home

scheduled

MO) after daily test sessions ad libitum in the home cage. Rats

available

was

(n

studies

light phase

St. Louis,

Purina,

water

behavioral

were 4.5 months

37)

=

14 (Experiment

of

1) Or 9 (Experiment

at the start

age

2) months

of age

Rats in the neurochemical studies (n = 62) were housed and maintained exactly as those in the behavioral studies. Apparatus. For the studies of working and reference memory (DMTP/VD tests), six modified rat operant conditioning chambers were used; they were identical to those used previously (Bushnell et aL, dosing.

On

the

front

Instruments,

wall

Lehigh

apart

and

data

2.5

cm

Equipment software

Corp.,

Tests

of

above

chamber

were

the

NJ).

were

(State

each

PA)

(model

floor,

two

MA)

(one

cup

the

complete

and

SKED

levers,

above

with a rear wall

45-mg

(PDP11/83,

interface

with

each hinged,

of the pellets

food conditions

of experimental

Kalamazoo,

conditioned

lights

in the

Coulbourn

response

by minicomputer

Maynard, Systems,

cue

A food

Control

accomplished

Ebb,

two retractable

and a houseight. door was centered were nutritionally

Frenchtown,

recording

of

Valley,

a loudspeaker transparent plastic chamber. Reinforcers (Bio-Serv,

effects is

light:dark

cage

maintain

of DFP,

shavings

testing

weighed

was

to

ChE to a similar degree and for a similar time a different compound. Pope et at. (1992) showed that a single injection of CPF (279 mg/kg s.c.) reduced brain ChE activity of rats by 95% after 2 weeks, 82% after 4 weeks and 60% after 6 weeks. Because this temporal pattern of ChE inhibition resembles that associated

12-hr:12-hr

lever),

se with

pine

behavioral

a funcChE inhi-

effect

per

tutes

that

anomalous

inhibition

As

13 cm

of ChE. This of ChE itself, which follows

rats

individually

Association for Accreditation of Laboratory Animal Care. required by this association, care and use followed National Instiof Health guidelines for laboratory rodents. Lighting followed a

pro-

neurochemical

Long-Evans

were housed

American

1991).

is

during

a direct

these

ChE

pattern

of function

changes induced to compensate for the inhibition functional cost may result from the inhibition from the downregulation of muscarmnic receptors

prolonged inhibition of unrelated to its inhibition One way to determine

of mus-

This

possible

to

direct

numbers

be

This

Male

ME)

heat-treated

at

tolerance

of the

cortex.

not

the daily

deterio-

for

effect

recovery

it

to

in rats.

evidence

and

predicted

words,

by

particthat we

In contrast that repeated

hypothermic

hippocampus

became of DFP

of ChE and further suggested accrue to tolerance to OP-induced

other

given

a progressive

motor

oxotremorine in

with

bition.

induced

presence

to the

question effects

1991). we found

model, of DFP

the

receptors

consistent

et at.,

memory in

OP,

et aL (1967). Thus, it is also fair to of recovery might generalize to other

(Bushnell

prediction

ration

a single

with other OPs. This in light of anomalous

germane

reported

using

Subjects.

Portage,

and

Digital SKED-il

MI).

behavior.

DMTP/VD

tests

were

conducted

et a!. (1991). A complete session consisted of 100 matching trials and 100 discrimination trials, presented in random order. To start a matching trial, one lever (chosen randomly for each trial) was inserted into the test chamber as the “sample.” The rat was as described

in Bushnell

given

to press

15 sec

it,

after

which

it retracted

for

the

duration

of a

variable delay interval (1, 5, 10, 15 or 20 see). To prevent rehearsal of the correct response during the delay, the rat was required to make nosepoke responses by pressing the door of the food cup during the

delay. Nosepokes were required to occur at a rate of at least 0.5/sec and the last nosepoke had to be made within 3 sec after the end of the nominal to be

(predetermined) extended;

A correct

response the

pellet

into

lever) respond

turned

timeout

the

period

was required to indicate

was

(i.e.,

on

food

off the

within

the

delay. rat

This

then the

of the

10 sec lever)

an

incorrect

for a 5-sec

any of the response and a repeat ofthe trial. for visual discrimination location

nosepoke

sample

cup, whereas houselight

last

allowed

correct

caused

both levers one of them.

to press

caused

delivery

response

timeout

of a food

(on

period.

the other Failure

to

time

criteria caused the same The same sequence of responses trials, which utilized cue lights response

at the

end

of the

delay.

Thus, the cue light above the sample lever was lit at the beginning of the trial and turned off during the delay. One cue light was lit at the end of the delay to indicate the correct lever; a food pellet was then

delivered to the rat for pressing that completed trials, 50 response failures Behavioral measures. Analysis

lever. A session or 105

mm

had

of DMTP/VD

ended

when

200

elapsed.

responding

has

Downloaded from jpet.aspetjournals.org at ASPET Journals on February 13, 2015

the

results

Methods

as

preferential recovery of more basic physiological functions before recovery of sensory, motor and cognitive capacities (Russell et at., 1986b, 1989). Furthermore, the current model of tolerance is based primarexperimental

1988).

with

reflecting

ily upon

and time course of direct effects of CPF, for comparison with effects of DFP. Evidence of tolerance was evaluated pharmacologically by challenges with the direct muscarinic agonist oxotremorine and neurochemically by assays of maximal [3H] QNB binding, to measure downregulation of muscarinic receptors in the brain. The same behavioral task used previously with DFP (DMTP/VD) was used to assess cognitive and motor function. In addition, rats were observed for signs of cholinergic poisoning, using selected components of a FOB (Moser et at.,

1993

and ToleranCe

ChE InhibItion

and

described in detail (Bushnell, 1990; Bushnell et aL, 1991). Matching accuracy required memory for the sample information during the delay, which changed from trial to trial; thus matching accuracy was taken as an index of working memory. On discrimination trials, the cue light always indicated the correct response at the end of the delay; been

because

this

information

discrimination

remained

accuracy

was

constant

taken

as

for

an

the

index

entire

of

(-80#{176}C)before

at 25C using et a!., 1961).

Rectal

experiment,

reference

frozen

measured (Ellman

memory.

temperatures.

injected methyl

i.p. 30 mm after bromide (Sigma).

serting

a telethermometric

assay. ChE activity 0.5 mM acetylthiocholine

1009

in whole blood was as the substrate

A dose of 0.2 mg/kg of oxotremorine was an i.p. injection of 1.0 mg/kg of scopolamine Colonic temperatures were obtained by inprobe

(Yellow

Springs

Yellow

Instruments,

This usage of the concepts of working and reference memory follows that of Honig (1978) and Olton et a!. (1979). Matching accuracy was defined as the proportion of correct choice responses on matching trials averaged across delays. Least-squares linear regressions of matching accuracy against delay were used to generate retention gradients for each animal. The intercept of the gradient averaged across animals was interpreted as the accuracy of encoding the sample information and its slope as retention or the rate

Springs, OH) 2.5 cm past the anal sphincter with the animal restrained gently by hand. The hypothermic effect of oxotremorine was taken as the difference in temperature between the reading taken after methyl-

at

which

information

and Milar,

1984;

as the

proportion

Because

the zero,

interval

and type

the the

choice

was

calculated

in a session

during

all delay scored

were

by

using

Each rated

by an two

treatment.

5)

head

forelimbs

and

and an

Chemicals. s.c.

in

CPF

oil

at

respectively.

of

MO)

ice

in a dark

CPF

dosing.

was

trials. in

nosepokes

delay

group =

treatment

2

conditions

et aL, 1988). absorbent paper and

a FOB

(Moser

frequency/intensity 11 and

3)

alert,

tenseness;

movement and jaws;

mild

fme

active and

motor

categories 2) normal

tremor,

disturbance,

6)

14 after

seen 5)

exploration; high:

extreme

included 1) quivering of typically in the

myoclonic

jerks;

6 represent

whereas

a rough

category

Service,

West

of 30,

62.5

yielding

Chester, or

doses

sesquifumarate

prepared

fresh

on

PA) was dissolved

125

mg/ml

of 60,

125

day

of

injected

250 mg/kg, Chemical Co., St.

(Sigma

the

and

they

that

they were trained

unrelated

were

divided

to

into

dose/time).

removed

7.4).

Hypothalamus

of

the

brain

striatum

and kept

vial.

treatment

three

in Experiment

to perform experiments.

2 [CPF

groups

into 120

treatment

two

days

of age;

groups

half

between

the

optic

cortical

chiasm

rostral

tissue

the

optic

the tissues

to the optic

mammillary chiasm),

bodies), forebrain

were thawed

and homogenized

at

120

mM

NaC1,

5 mM

Brain

ChE

assay.

KC1,

2 mM

ChE

CaCl2

activity

and

in

1 mM

MgCl2.

membrane (Johnson

samples from and Russell,

delineated

concentration zyme activity et a!., 1951).

conditions

necessary was

Muscarinic

for

calculated

of both incubation

linear

rates

relative

receptors:

time and tissue

of substrate

hydrolysis.

to protein

concentration

binding.

Muscarinic

[3HJQNB

receptor

density was measured by the method of Yamamura and Snyder (1974), modified by Pope et aL (1992). Samples of the homogenates were centrifuged at 48,000 x g for 10 min and the resulting pellets were

by the samples

tissues

incubated

week

for

7 weeks.

performance Drug

was

challenges

subsequently (not

to

measured

be

reported

5 days/ here)

were

with

washed binding

aL,

(2.0

percentage

Blood rats

all

in

rats

i.p.). ChE

Samples

assays. 1 on

Experiment

in Experiment

2 on

was collected from the tail (Metofane; Pittman-Moore, 1:20 in 0.5 mM

Days

of whole 4, 15, 32,

Days vein

sodium-phosphate

blood 52 and

4, 31 and under

light

Mundelein, buffer

were

obtained

74 after

53 after

CPF.

methoxyflurane

IL). The containing

from

all

CPF and from Each

sample

anesthesia

blood was 1% Triton

diluted X-100

buffer at

37’C

and (3 ml, 3 times) over Whatman GF/C paper by using a receptor harvester (Brandel, Gaithersburg, MD). Specificity was deter-

QNB

administered as follows: Day 11, scopolamine HBr (0.056 mg/kg i.p.); Day 16, pilocarpine HC1 (0.56 mg/kg i.p.; Experiment 2 only); Day 18, nicotine ditartrate (0.18 mg/kg s.c.); and Day 25, mecamylamine HC1 mg/kg

En(Lowry

mined

0. DMTP/VD

each

brain region was assayed radiometrically 1975) by using ACh iodide spiked with [3Hjacetylcholine iodide (73.7 mCi/ mmol, New England Nuclear, Boston, MA) as a substrate. Preliminary

paired

Day

=

32,000

rpm for 20 sec on ice with a Polytron PT-3000 (Brinkmann Instruments, Westbury, NY) in 50 mM Tris(hydroxylmethyl)amino methane buffer (1:30, w:v), pH 7.4, at 25#{176}C. This Tris-salts buffer also contained

on

=

(the

chiasm)

washed twice by resuspension in equivalent volumes ofTris-salts and recentrifugation. The tissues were incubated for 60 mm

of age

of each dosing (a

and hippocampus free-hand using Paxinos and Watson (1982) as a guide. were weighed and frozen (-80C) until assay. On the

were dissected These regions day of the assay,

rostral

and

to the

In Experiment 1, rats were divided into two groups at and given s.c. injections of either peanut oil vehicle (n = 7) or CPF at 125 mg/kg (n = 8). In Experiment 2, rats were divided into three groups at 9 months of age and given s.c. injections of either peanut oil (n 6), 60 mg/kg of CPF (n 8) or 250 mg/kg of CPF (n = 8). All rats were injected within 1 hr after behavioral testing 14 months

and

doses

7 days and the other All rats were sacrificed

(caudate-putamen,

remaining

present

half 21 days after by decapitation; their brains and placed in ice-cold phosphate buffer (50 mM, pH (a 2.0 mm3 cube removed from the ventral surface

was sacrificed

6 per

the

of 0 (peanut s.c.]. Eighteen rats were sacrificed 7 days dosing (n = 6 or 7 per dose/time). Twenty-

were

or

administration

of age,

except

tasks,

with the animals 60 or 250 mg/kg,

experiments

1 reflects

of dyskinesia.

(Chem

studies,

operant

and 20 rats 21 days after four additional untrained rats were divided and dosed with 0 or 125 mg/kg of CPF at

emitted

Rats in Experiment

4) normal:

mouth

limbs;

Oxotremorine

Louis,

delay

on completed

months

dosed along oil vehicle),

was

the

across

1, 2, 3, 4, 7, 8, 9, 10,

Involuntary

of 2 ml/kg,

on

latency

cumulated

animals’

from

excitement,

concentrations

a volume

trials.

have been defined previously (Moser et scores included 1) stupor: no movement; 2) without exploration; 3) somewhat low: some

dimension

in peanut

to the

exploration;

of increasing

independent

appetitive

averaged

of

head; 4) severe whole-body tremor; convulsions. Categories 2 through

6) clonic

continuum

behavior.

blind

slight

and

in the behavioral

other At 9

scales

movements

vibrissae,

those

session.

on Days

excitement.

repetitive

only

on a bench top covered with scales for level of arousal and

slow

high:

(Heise defined

of

trials,

measured

categories

These

with

alertness,

matching

72

was

did not differ

nosepoke

32

time

response

last

as the total time total number

observer

Briefly, arousal slow movement,

somewhat

Choice

by the

movements

movement

on

in that

behavioral

of involuntary

aL, 1988). low: some

delay

of unconditioned

rat was placed on two 6-point

CPF

discrimination

across

the

was

divided

intervals

Observations

on

accuracy delays.

response Latency

over accuracy

responses

between

outcome.

IRT

Nosepoke

intervals

across

elapsed

deteriorates

Discrimination

choice

averaged time

and

sample

of discrimination

it was as

trial

of correct

slope

defined

the 1990).

lenges

densities

(0.75

were

nM

final

inclusion

and in calculated

concentration)

of atrophic calculated the

presence

relative

as

and

(10

mM

rapidly

fmal

the difference and to

absence protein

filtered

concentration)

in binding of

atropine.

concentration

in

between Receptor (Lowry

et

1951).

ChE activity in whole blood was converted to a of the concurrent control mean. The hypothermic effect of oxotremorine was indexed by difference scores (post-oxotremorine minus pre-oxotremorine) on Days 8, 32 and 52 after CPF; the Day 74 data were not analyzed because only the 125-mg/kg CPF group was challenged then. Separate two-way ANOVAS with CPF dose and day of treatment (a repeated measure) were used to assess the significance of changes in these measures. ChE activity and muscarinic receptor Data

analysis.

Downloaded from jpet.aspetjournals.org at ASPET Journals on February 13, 2015

from

about Bushnell,

and that taken after oxotremorine. Oxotremorine chalwere administered to all rats in Experiments 1 and 2 on Days 8, and 52 after CPF; rats in Experiment 1 were challenged also on Day after CPF. Neurochemistry. Rats were housed and maintained identically to

scopolamine

Bushnell

1010 density

in each

with

brain

separate

in the

(CPF

modeling

Moser

et a!., 1988)

analysis

to evaluate

ditioned

behavior

observation

Vol. 266

region

two-way

Categorical

For

et al

was

used

the

SAS

as

version

6.06;

analyzed

significantly density did

SAS,

1990;

changes

and

arousal)

in uncon-

across

days

of

the

effect

of

2.

obtained

from

CPF dose and the day by CPF

the DMTP/VD

interaction

test,

were

tested

with

to 80 to 85% of control not change significantly

by Day 21 after this

dosing-,

after dose

in

and hypothalamus. After 125 mg/kg of CPF, density in these three regions reached levels of 70 to control at both time points. After 250 mg/kg of CPF, density in these regions dropped to 70 to 75% of control after CPF and dropped further to about 60% of control after

two-way

dosing.

Changes

in

hypothalamic

receptors

were

much

measures analyses of covariance (SAS Version 6.06, General Model: SAS, 1990), by using baseline (pre-CPF) scores as covariates and days of treatment as the repeated measure. The Day

smaller in magnitude were not significant

effect

Oxotremorine-induced hypothermia. Each CPF group became significantly less hypothermic than controls when challenged on Days 8 and 32 after CPF (fig. 1B); no group differed significantly from control on Day 52. Rats in the 250-mg/kg CPF group became hyperthermic in response to oxotremorine on Day 8. Neither CPF nor methylscopolamine alone affected

repeated

Linear

was

assessed

gradients

were

CPF

of

again

using

measures

effect by

time

point

criterion

Details

CPF

using

11 after

on

on

the

to analysis

working

slopes

and

of covariance,

as covariates.

of asymmetrical

for

of treatment

each

scores

treatments

In all repeated-

variance-covariance

Greenhouse-Geisser

dl

mat-

corrections;

the

() is reported after each F CPF dose by Day interactions were analyzed with of the covariate-adjusted means at each time point.

ratio. Significant one-way ANOVAs

The

of

subjected

Retention

7, 9 and

Days

of

effects

baseline

the

covariate.

on

effects

were

of df correction

Comparisons

The

gradients

appropriate

the

performance

the

1990).

these

minimized

amount

at

across

characterize

analyses, was

omitting

means

of statistical

with

by using

made

significance

in

analyses

are

the

control

Dunnett’s

all

statistical

given

in the

group

test

(Myers,

tests figure

mean

was

1966).

a

0.05.

=

legends.

RESULTS General

signs

and

study

treated

rochemical eating,

exhibited

hindquarters)

and

obvious other after

signs

group

reaching 60

self-mutilation No

Maximum

differed

levels

mg/kg

(Experiment in

and

euthanized.

ited 60 to 75%, depending (fig. 1A). Activity slowly after

of

from

in the CPF

neu-

stopped (tail

other

animal

weight

loss

and

showed among

all

rat on the 1st day average weight of by a maximum of 12

control

5 after CPF. of ChE activity in of CPF, ChE activity

injection

after,

animal

mg/kg

on Day

Inhibition after

was

One

250

18 g (-5.1%), observed in one with 250 mg/kg of CPF. The

high-dose

g (-3.4%)

tremor

of poisoning.

rats was treatment

the

with

indistinguishable

(Experiment

Experiment

reached

90%

control

mean.

2)

53, the

2, activity

of control,

Inhibition

of

blood.

from

Four

blood

was

days inhib-

control

by Day

53

and by Day 74 after 125 mg/kg time of the last sample taken

in the

but

group

still

given

differed

activity

ChE

dosing, ChE activity tum and hypothalamus

whole in whole

upon the dose of CPF administered returned to normal with time there-

1). On Day

in

250

mg/kg

significantly

brain.

from

Seven

days

in the frontal cortex, hippocampus, was inhibited 60 to 80% (60

mg/kg

had

the after striaof

CPF), 85 to 90% (125 mg/kg of CPF) and 80 to 95% (250 mg/ kg of CPF) (table 1). Between Day 7 and Day 21, partial recovery occurred in the hippocampus, striatum and hypothalamus

striatum

ChE

60

after

after

of

CPF

mg/kg

was

was evident

and

of CPF.

in No

the

hippocampus

significant

and

recovery

in

21 after 250 mg/kg of CPF. Muscarinic receptor in brain. Binding studies showed that muscarmnic density (maximal [3H]QNB binding) decreased with increasing dose and time after CPF treatment in the frontal cortex, hippocampus and striatum (table 1). Receptor density in the hippocampus and frontal cortex

activity

mg/kg 125

normal

by Day density receptor

7 days

after

time

60

mg/kg

of

CPF,

but

fell

after

after

250

mg/kg

of CPF.

temperature.

Unconditioned

behavior.

Excepting

the

one

moribund

rat

the

in

with and

neurochemical study (above), the only sign consistent cholinergic overstimulation was a fine tremor in the head limbs. No salivation, lacrimation, excessive urination or

diarrhea

were

observed.

The

intensity

of tremor

(involuntary

movements in the FOB) was related both to the dose of CPF and the time after dosing (fig. 2A), reaching a peak on Day 9 after CPF. As scored with the FOB, arousal was increased by 60 mg/kg

weighL

body

severe

body

analysis

group

were

for

each

with

in the other regions (less than 20%), 60 or 125 mg/kg of CPF and did not

of CPF

and

decreased

reaching a minimum unconditioned behavior

on

by 250

Day returned

mg/kg

4 after CPF. to normal

of CPF

(fig.

2B),

All changes in by Day 14 after

dosing.

Conditioned behavior. Rats dosed with CPF showed increased frequencies of response failure in the DMTP/VD task, reducing the number of trials completed (fig. 3A). Most incomplete trials resulted from failure to initiate a trial with the required press on the sample lever; a smaller number resulted from failure to complete the nosepoke requirement during the delay. Response failure was both dose- and time-related, with significant

decreases

kg of CPF.

The

in trial

125-mg/kg

group

differed

250-mg/kg

group

did

60-mg/kg

completion

group

from

so on

did

not

control

Days

after

125

differ

from

on Days

2 to 35, excluding

and

250

mg/

control,

the

2 to 4 and

the

Day

23.

The prevalence of response failure in the 250-mg/kg group precluded statistical analysis of their choice accuracy, because a minimum of 100 trials (50 matching, 50 discrimination) is necessary for estimation of accuracy. Thus, analyses of accuracy involved only the 0-, 60- and 125-mg/kg groups. Matching accuracy was reduced in the rats given 60 or 125 mg/kg of CPF, whereas discrimination accuracy remained unaffected (fig. 3B). Matching accuracy was reduced by 60 mg/kg of CPF on Days 7 and 11 and by 125 mg/kg of CPF on Days 2 through 16 after treatment. Choice response latency (fig. 4A) was increased by CPF in a dose- and time-dependent manner. The maximal increase in the 250-mg/kg group occurred on Day 3 after treatment; peak effects

in

the

other

groups

occurred

on

Day

9. Latency

increased significantly in the 60-mg/kg group on through 9, in the 125-mg/kg group on Days 2 through in the 250-mg/kg group on Days 2 through 14. Nosepoke

response treatment.

IRT

(fig.

4B)

was

increased

latency. Maximal effects CPF did not significantly

by

CPF

was

Days 3 11 and

similarly

to

were seen on Day 3 after change the IRT of the 60-

Downloaded from jpet.aspetjournals.org at ASPET Journals on February 13, 2015

rices

to

(Bushnell,

intercepts

ANOVA,

averaged

and used

memory

by

fall

than

the

receptor 85% of receptor 7 days 21 days

striatum

repeated-measures

of CPF-induced movements

were

ANOVAs.

a nonparametric

significance

(involuntary

measure

study

between-group

(CATMOD:

in Experiment

each

neurochemical

by day)

ChE Inhibition

1993

A.

Cholinesterase

Activity

in Whole

Blood

B.

Oxotremorine-Induced

1011

and Tolerance

Hypothermia

‘-4

0

1

U

4.)

5

0 U

0

0

.,-4

4J

0) 0

a) o

.c U

c: (0 0)

(0 0)

__________

z

x

0

20

Days

40

after

60

80

Chiorpyrifos Ccntrol

-0---A--

CPF

-0--

CPF

CFF

Days

40

after

-..-

60 12 00

60

80

Chiorpyrifos Control

-0--

CPF

-A-

OFF

-0--

CPF

:o

Fig. 1. A, inhibition by CPF of ChE activity in whole blood, expressed as a percentage of control. ChE activity was inhibited significantly by 125 mg/ kg of CPF in Experiment 1 [main effect of CPF F(1 ,13) = 140.72, P < .0001], with significant change across time after CPF [CPF by Day interaction F(4,52) = 17.67, = 0.73, P < .0001]. ChE activity was inhibited significantly by 125 mgfkg of CPF at all time points except Day 74. In Experiment 2, ChE activity was also inhibited significantly [main effect of CPF F(2,20) = I 38.52, P < .00011 and changed significantly after dosing [CPF by Day interaction F(4,40) = 19.16, #{128}= 0.82, P < .0001]. ChE activity was inhibited significantly by 60 mg/kg of CPF on Days 4 and 31 and by 250 mg/kg of CPF on Days 4, 31 and 53. B, effect of CPF on oxotremonne-induced hypothermia. Points are mean (±S.E.M.) difference scores of colonic temperature (Tc) Obtained 30 mm after oxotremorine, subtracted from the Tc Obtained 30 mm after methylscopolamine. Data for Day 74 were not anaiyzed, because only the 125-mg/kg group was challenged then. CPF aigniticantly reduced the hypothermic effect of oxotremorine [main effect of CPF F(3,34) = 8.19, P < .0003] and the hypothermic response returned toward control levels after CPF [CPF by Day interaction F(6,68) = 2.42, = 0.95, P < .038]. Dunnett’s tests at each time point showed that all doses of CPF significantly reduced the oxotremonne-induced hypothermia on Days 8 and 32, whereas no dose did so on Day 52.

1 EffeCt of CPF on ChE activity and muscarlnic receptor densfty (maximal [H]ONB binding) Rats were dosed with ci’s on Day 0 and sacrificed for neurochemicai assays on Days 7 and 21 Values

TABLE

.

measure).

COntrOl

in four brain regions are means (±S.E.M.) percentage

hE Acbv 8n Re

Madmal 1HJQNB Bng Dayl

Day2l

0

Cortex

60 125 250 0

Hippocampus

60 125 250 Stnatum

0

± 11.8 (57.8) 36.8 ± 2.2 11.3±1.0* 1 1 .7 ± 1 .0*

± 10.5 (50.7) 43.1 ± 7.1 16.5±3.0* 16.7 ± 3.0

100.0

100.0

± 3.9 (33.8) 43.1 ± 5#{149}9* 30.7±3.4* 11.5±2.3*

250

Hypothalamus

0 60 125 250

(P


1

0

E

(0

0 U

:1 0 0 .cl:

(I) ‘-4

(0

0.

-1 I-i Co

0)

x (0

a)

x

1

40 1

Days

after

10

30

20

40

Chiorpyrifos Days

-..-

Cor,trol

-0--A-0---

CPF

after

61’

35

Control

-0--0---

60

25

Chiorpyrifos

-.--

Control

CPF

20

]:0 15

Chiorpyrifos

-.--.

Control

CIF

120

-0--

CPP

CPE

203

-A-

CPF

60 125

Fig. 3. A, number of trials completed per 200-trial session in the DMTP/VD task in Experiments 1 and 2. Both the main effect of CPF [F(3,34) = 23.3, P < .0001] and the CPF by Day interaction [F(51,578) = 7.39, ( = 0.267, P < .0001] were significant in the overall covanance analysis. The 60-mg/ kg group never differed from control; the 1 25-mg/kg group differed significantly from control on Days 2 to 4; and the 250-mg/kg group differed significantly from control on Days 2 to 18 and 25 to 35. B, accuracy of matching (lower curves, left ordinate) and discnmination (upper curves, nght ordinate) in the DMTP/VD task, for the control, 60and I 25-mg/kg CPF groups. Values are covaiiate-adjusted means from separate analyses of covariance on each measure. Neither dose of CPF affected discrimination accuracy significantly [main effect of CPF F(2,26) = 1 .97, NS; CPF by Days interactin F(34,442) = 0.79]. Matching accuracy was reduced significantly by CPF [main effectofCPFF(2,26)= 11.10, P< .00031; the CPF effect differed across days after treatment [CPF by Days interaction F(34,442) = 2.12, = 0.29, P < .028]. Matching accuracy was significantly reduced on Days 7 and 1 1 after 60 mg/kg of

CPF and on Days 2 through mg/kg of CPF. mg/kg

group; the IRT of the 125-mg/kg group was elevated Day 2 through Day 9; the IRT of the 250-mg/kg group was elevated from Day 2 through Day 25 after treatment. Analysis of retention gradients (accuracy as a function of delay, averaged across test sessions on Days 7, 9, 11 and 14 after CPF) showed that CPF lowered the intercept, but did not affect the slope, of the retention gradient (fig. 5); this change was significant after 125 mg/kg of CPF (fig. SB), but not after 60 mg/kg of CPF (fig. SA). (The data for the two experiments are shown separately due to differences in slope between the control groups. Experiment 2 rats had performed the task from

longer than from longer

Experiment

training

1 rats;

on the task.)

their

shallower

slope

resulted

16 after 125

Discussion Acute CPF produced neurochemical changes very much like those observed during repeated injections of DFP (Bushnell et at., 1991). Specifically, CPF inhibited ChE activity in the blood and brain, reduced the sensitivity of rats to oxotremorine challenges and reduced the density of muscarmnic receptors in the brain to a degree and for a time comparable to that of repeated DFP. In contrast, the transient behavioral changes after CPF differed markedly in duration from the progressive deterioration of function observed during DFP treatment. The fact that the pattern of neurochemical changes caused by DFP may be reproduced by CPF without its progressive functional

Downloaded from jpet.aspetjournals.org at ASPET Journals on February 13, 2015

Days

10

Fig. 2. A, involuntary movement scores from Experiment 2. An overall analysis was not possible with these data. Univanate tests at each time point indicated that rats given 250 mg/kg of CPF showed significantly more tremor than controls on Days 4, 7, 8, 9 and 10 after CPF; rats given 60 mg/kg of CPF did not differ significantly from control at any time. B, arousal scores from Experiment 2. Arousal was aftered significantly by CPF; the overall analysis showed significant effects of CPF [x2(2) 16.41 , P < .0003], of Days [2(9) = 44.70, P < .0001] and of the CPF by Days interaction Ex2(18) = 119.76, P < .0001]. Arousal was significantly increased by 60 mg/kg of CPF on Day 8 and decreased by 250 mg/kg of CPF on Days 2, 4, 7 and 9 after treatment.

ChE Inhibition

1993

A.

Choice

Response

Latency

B.

Nosepoke

Inter-Response

Fig. 4. A, choice response latency in the DMTP/VD task for the control, 60-, 125and 250-mg/kg CPF groups. Values are covariate-adjusted means from analysis of covariance. CPF significantly lengthened response latency [main effect F(3,33) =

Time

1.

3 0

3.69,

0)-)

0

(J).).

> 0 t::

-

Cal

2.

a

4_C

4-4

c: 1. 0. 1. 10

Days

20

after

1

10

-0--

vs. 60 mg/kg

-0--A-0--

B.

Control

1

30

40

.0001];

Chiorpyrifos -.--

Chiorpyrifos

vs. 125 mglkg

0 0

0

0.

0.

(0 (0

a) x

a) 0.

x 0.

0.

5

01

10

Delay . U 0 0

deficits

20

(sec)

Delay

Cc’rrol Cr1r CPF

M1cn . 2cr rnr3 f. M:tchirn

CF

t’’

0 0

cations follow. The neurochemical effects of CPF confirm and extend those of Pope et aL (1992), who reported prolonged inhibition of ChE activity in plasma and brain of rats given a single s.c. injection of 279 mg/kg of CPF. The present findings show similar, although less severe, inhibition of ChE activity in the blood (fig. 1A) and brain (table 1) after 60 to 250 mg/kg s.c. of CPF. The means by which CPF exerts this long-lasting effect on ChE activity is not fully understood, but several factors are consistent with this time course. First, the s.c. injections used here probably slowed release of the parent compound into

slowly

circulation.

mg/kg

entirely

to

receptors.

The

of CPF

slow

(Pope

these

DFP-induced

inhibition Details

recovery

of ChE of this

argument

of plasma

et at., 1992)

is

deficits cannot and downregulation

(sec)

Cc’-1rcl P3tc.:t.. Ccr:ro] Cis:r inirn CF 120 MalOlng Cr1’ 120 D1scriBno11on

bility.

muscarinic

that

. ,

be atof impli-

tributed

means

15

ChE

consistent

and

activity

with

its

after

this

279

possi-

CPF rapid

this

changed

increase

with

time

Fig. 5. Retention gradients obtained from rats during the 2nd week (Days 7, 9 and 1 1 averaged) after dosing with CPF at 60 mg/kg (A, Experiment 2) or 1 25 mg/kg (B, Experiment 1). Accuracy of discrimination (upper curves) and matching (lower curves) are PlOtted as a function of delay; matching accuracy declines with delay whereas discrimination accuracy does not. Analyses of covanance showed a significant change in the intercept of the matching fUnCtion after I 25 mg/kg of CPF [F(1 ,13) = 8.52, P < .012], but not after 60 mg/kg of CPF [F(1 ,1 2) = 2.1 7, NS]. The slopes of the matching functions were not affected significantly by CPF (both F values < 1). Neither the slopes nor the intercepts of the discrimination functions were affected significantly by CPF.

0.