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OCR for page 101
Exposure to Neurotoxins
Throughout the Life Span:
Animal Moclels for Linking Neurochemical
Effects to Behavioral Consequences
Hanna Michalek and Annita Pintor
Exposure to toxic substances is a potential threat throughout the
human life span. Therefore, it is essential that risk assessment of
potentially toxic substances be carried out at critical periods over the
entire range. Although much valuable information may be obtained
from epidemiological and clinical studies, it must be supplemented
by research using animal models and the advantages of experimental
methods. This is particularly apparent when information is needed
about the modes and sites of action of toxic substances that constitute
the substrates of adverse behavioral effects. The discussion that fol-
lows uses the laboratory rat as its animal model and a class of compounds
affecting the cholinergic neurotransmitter system, organophosphorus
anticholinesterases, as examples of potentially neurotoxic substances
to which the possibility of human exposure is widespread. Examples
have been chosen to illustrate the basic characteristics of research
designs, their implementation, and the analysis and interpretation of
results. The discussion begins with consideration of normally occurring
changes in neurochemical events during early development and later
aging.
NEUROCHEMICAL CHANGES DURING
DEVELOPMENT AND AGING
Chemical substances entering the body, toxic or nontoxic, produce
their effects by altering biochemical events already underway. Many
101
OCR for page 102
102
MANNA MICHALEK AND ANNITA PINTOR
of the events affected, particularly those in the nervous system, are
involved in the behavior of an organism as an integrated whole. In
any individual the nature of these events is determined by interactions
between genetic and environmental factors, interactions that are
characterized by changes throughout the life span. In general, most
neuronal functions in the neonatal organism are incompletely devel-
oped. The evidence is that processes involved in the synthesis, stor-
age, release, and inactivation of neurotransmitter substances are less
well developed in early life than in the adult. The blood-brain barrier
is generally not as effective in the immature, developing brain, allowing
penetration of chemicals that are manifested only by peripheral effects
at later ages. Neurochemical changes during early ontogenesis have
been shown to parallel behavioral development. Furthermore, declines
in behavioral functions with normal or pathological aging suggest
that the developmental trends in the central nervous system (CNS)
are reversed during aging.
Because of its key roles in behavior, the cholinergic system pro-
vides examples of how neurochemical changes during the life span
influence behavioral effects of neurotoxic agents. A considerable number
of compounds exist that have specific cholinergic effects, some widely
used throughout the world as pesticides (Koelle, 1975). Examples from
one class of these, organophosphate (OP) compounds, serve our present
purposes of assessing neurotoxicity throughout the life span. Human
intoxication by OF may result from occupational exposure (agricultural
or industrial), adventitious contact indoors or outdoors, or consumption
of contaminated food or water. The fact that the risk of exposure
may be greater in the indoor than the outdoor environment (Reinert,
1984) places all members of the family from pregnant women and
young children to the elderly—in jeopardy. Epidemiological data
indicate that as many as 500,000 people in the world are exposed
annually to these compounds at levels requiring clinical attention
and that about 5,000 poisonings are fatal (Russell and Overstreet,
1987~. Animal models are essential for experimental analyses of the
mechanisms by which these compounds produce their effects, for
determination of threshold limit values beyond which exposures are
unacceptable, for creating therapeutic procedures to treat adverse
symptoms, and for monitoring public health programs designed to
protect against misadventures.
THE CHOLINERGIC SYSTEM IN BEHAVIOR
"Most impressive is the singular fact that ACh (acetylcholine) is
the only substance that can influence every physiological or behav-
OCR for page 103
ANIMAL MODELS
103
ioral response thus far examined" (Myers, 1974~. This statement takes
into consideration the roles of ACh as the transmitter at neuromuscular
junctions and in various pathways in the CNS (Butcher and Woolf,
1986~. Normal functioning of the cholinergic system may-be im-
paired when an individual is exposed to OP compounds. Upon en-
tering the body through any of several routes (inspiration, ingestion,
injection, transdermally), an OP is first carried to its site of action,
the "pharmacokinetic" phase of its journey. Significant molecular
modifications may occur during the transit, e.g., the relatively inactive
compound parathion is converted to its active metabolite, paraoxon,
predominantly in the liver.
The "pharmacodynamic" behavioral and physiological effects of
an OP compound begin with the binding of the compound to the
active site of the acetylcholinesterase (ChE) molecule, inhibiting
inactivation of the neurotransmitter ACh when released from
presynaptic neurons and producing overstimulation by the neuro-
transmitter. Although there is no universal agreement concerning
"critical levels" of brain cholinesterase (ChE), most investigators have
emphasized that symptoms of acute intoxication and changes in behavior
appear only when brain ChE activity is reduced by at least 50-60
percent (Bignami and Michalek, 1978; Bignami et al., 1975; Russell,
1977~. In the early phase of acute intoxication, behavioral distur-
bances are accompanied by reduced brain ChE and elevation of brain
ACh levels. The disappearance of the symptoms of intoxication with
return of ACh to normal levels occurs considerably earlier than the
normalization of ChE activity. Moreover, repeated administration of
anticholinesterases (antiChEs) to adult rodents induces the develop-
ment of tolerance to their toxicity; i.e., behavioral disturbances disappear
despite persisting low levels of brain ChE. In recent years a decrease
in the density of muscarinic and nicotinic receptor sites has been
recognized as one of the main adaptive mechanisms to overstimulation
by acetylcholine in adult animals (Costa et al., 1982; Russell, 1982;
Russell and Overstreet, 1987~.
It is clear from these brief comments that the neurobehavioral ef-
fects of even one class of potentially neurotoxic substances involve
complex interactions among the chemical processes it initiates upon
entry into the body and the outcome it produces in physiological and
behavioral functions. For purposes of the present discussion, examples
are chosen from research using one typical OP, diisopropyl fluoro-
phosphate (DFP), which has been used extensively as a model compound
(Michalek et al., 1978, 1981, 1988; Overstreet and Russell, 1984; Russell
and Overstreet, 1987~. Among various components involved in the
mechanisms of synthesis and degradation of ACh (Russell and Overstreet,
OCR for page 104
104
MANNA MICHALEK AND ANNITA PINTOR
1987), this chapter deals only with the following three markers, all
located pre- or postsynaptically:
1. ChE, the primary target of antiChE agents the enzyme involved
in the inactivation by hydrolysis of ACh;
2. choline acetyltransferase (ChAT), whose enzymatic activity is
responsible for the synthesis of ACh from its immediate precursors,
choline and acetylcoenzyme A; and
3. muscarinic ACh receptors (mAChRs), essential for brain cholinergic
neurotransmission and linked to second messenger systems that me-
diate a subsequent "cascade" of events leading to physiological and
behavioral effects.
Changes in these components are discussed first with regard to the
phenomena of intoxication and tolerance during critical developmental
stages of the rat, i.e., the pre- and early postnatal periods and senes-
cence.
EFFECTS OF PRENATAL EXPOSURE TO DFP:
FROM BIRTH TO WEANING
In the initial phase of prenatal subchronic intoxication, i.e., from
the sixth to the tenth day of pregnancy, DFP has been shown to
cause, in the pregnant female, a syndrome of cholinergic stimulation
(tremors, sweating, salivation, lacrimation, and diarrhea) lasting for
many hours after each injection. Results of a typical experiment are
summarized in Table 1. Maternal weight gain is significantly reduced.
The toxic syndrome appears considerably more pronounced than that
previously observed in adult males treated similarly (Michalek et al.,
1982~. Moreover, a great variability in the response of individual
dams in terms of severity and duration of the symptoms is evident.
Subsequently, the symptoms attenuate markedly in some dams, but
remained quite evident in others. Although the treatment does not
cause mortality of dams, the pups of DFP-treated litters may be still-
born or die within a few hours after birth. These cases of reproduc-
tive wastage are clearly associated with the marked depression of
weight and possibly with delayed parturition (by about 24 hours).
After prenatal exposure of mothers to DFP, the body weight in
newborns is about 6 percent lower than that of controls and there is a
slight retardation of body growth up to day 10. The postnatal pattern
of gain in brain weight is not modified by DFP treatment. Data on
brain ChE and mAChRs are presented in Table 2. The levels of brain
ChE at birth in the DFP group do not differ from those of the controls,
and both groups showed similar increases of enzymatic activity until
OCR for page 105
ANIMAL MODELS
TABLE 1 Effects of Subchronic Intoxication with DFP in Pregnant
Rats on Gestation, Birth Statistics, and Litter Survival
105
Control
DFP
Total number of dams
Length of gestation (days)
Weight gain of dams (g)
6th-lOth day
10th-20th day
Number of pups per litter
Lost at birth
Lost within 48 h
Litters surviving up to weaning 19
20
21.2 ~ 0.2
14.4 + 2.8
71.9 + 4.2
11.4 ~ 0.8
o
1
20
21.8 ~ 0.2
3.6 ~ 2.4a
71.8 ~ 5.5
10.1 + 0.6b
4
8
8
NOTE: Treatment of Wistar rats (220-240 g) on alternate days: DFP (in arachis oil)
first dose of 1.1 mg/kg (subcutaneous) on day 6 of pregnancy, subsequent doses of 0.7
mglkg until day 20 (corresponding to 25% of LD50).
aSignificantly different from control p < 0.001 as determined by l-test.
bNot including four litters with pups stillborn or dead within a few hours after
delivery, which were often cannibalized.
SOURCE: Michalek et al. (1985).
TABLE 2 Effects of Subchronic Intoxication with DFP in Pregnant
Rats on Brain Total Cholinesterases (ChE) and [3H]Quinuclidinyl
Benzilate (QNB) Receptor Binding Sites During Postnatal
Development
Brain ChE (nmol AcThCh
hydrolyzed/min/mg protein)
13H]QNB binding (fmol/mg protein)
Age (days) Control DFP Control DFP % of Control
Newborn 22.9 + 1.4 21.8 + 2.0 102 + 7 70 + 4a 68
5 34.0+ 1.3 31.0+ 1.6 142+ 7 127+ 9 89
10 39.6 ~ 3.0 32.9 + 1.7 258 ~ 7 193 +13a 74
15 38.1 ~ 2.6 38.7 ~ 13 335 + 15 263 + 19 78
20 40.0 ~ 2.0 38.5 ~ 2.5 443 ~ 36 442 i 10 100
NOTE: For treatment see Table 1. Mean ~ SEM of 8 animals for each age (except
newborn n = 16) belonging to different litters. [3H]QNB at 1.5 nM concentration; mean
+ S.E.M, n = 10 animals for each age (except newborns n = 20). AcThCh = acetylthiocholine.
aSignificantly different from control values (p < 0.01) as determined by l-test.
SOURCE: Michalek et al. (1985).
OCR for page 106
106
MANNA MICHALEK AND ANNITA PINTOR
weaning. On the other hand, experiments on quinuclidinyl benzilate
(QNB) receptor binding show a significantly lower level of mAChRs
at birth and at 10 days in DFP pups compared to controls.
Results reported in Table 3 show that exposure to DFP at the end
of pregnancy produces a consistent depression of ChE activity in
maternal brain during a period of at least 48 hours. The enzyme
activity in fetal brain is less inhibited initially and approaches full
recovery within the period. These data on fast recovery of fetal brain
ChE are in agreement with results reported in the literature for other
OF compounds. Subacute exposure of rats to parathion during the
third trimester of pregnancy did not modify brain ChE in newborns
(Talens and Wooley, 1973~. Daily administration of dichlorvos to
pregnant rats during the same period lowered ChE levels in newborns,
but no substantial delay in postnatal development was subsequently
observed (Zalewska et al., 1977~. Prenatal exposure of mice to dicrotophos
did not alter the postnatal development of brain ChE and ChAT (Bus
and Gibson, 1974~.
What processes may be involved in these differences between ef-
fects of OF on ChE activity in fetal and maternal brain? It is well
known that pharmacokinetic factors influence the processes by which
an antiChE reaches its sites of action. For example, such compounds
bind to molecules other than acetyl-ChE (i.e., butyrylcholinesterase
and aliesterase) that produce no apparent functional effects on behavioral
or physiological variables. These enzymes, found in plasma and
erythrocytes, have been described as "scavengers" or "sinks" that
can reduce the concentration of an antiChE entering the CNS (Russell
et al., 1986~. For example, higher levels of plasma ChE in females
have been shown to result in lesser brain sensitivity to DFP, as com-
pared to males (Overstreet et al., 1979~. In fetal brains after in utero
exposure to DFP, cholinesterases present in maternal plasma, erythrocytes,
and placenta also play an important role as "scavengers." Other data
obtained in our laboratory indicate that total cholinesterases in maternal
plasma and amniotic fluid 90 minutes after DFP were inhibited by 95
percent, and those in fetal plasma by 75 percent, i.e., considerably
more than maternal and fetal brain ChE (i.e., 80 and 50 percent, re-
spectively). The fast recovery of ChE in the fetus probably depends
on the considerably higher protein synthesis rate in fetal compared
to adult brains (Gupta and Dettbarn, 1986, 1987; Gupta et al., 1984;
Lajtha and Dunlop, 1981~. These facts suggest that following exposure
to OPs, recovery to normal levels of ChE activity occurs more rapidly
in the fetus than in the adult because (1) initial reduction in ChE
activity is not as great in the former and (2) de nova synthesis of
replacement Chl? is more- rapid.
OCR for page 107
107
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OCR for page 108
108
MANNA MICHALEK AND ANNITA PINTOR
Because pharmacodynamic processes leading to behavioral effects begin
with the binding of the neurotransmitter ACh to its receptor sites,
effects of the above changes in ChE activity on mAChRs are of spe-
cial interest in the context of the present discussion. Analyses of
receptor binding (Table 3) have shown decreases in brain from both
DFP-treated dams and 21-day fetuses. The overall pattern for the
latter is a significant decrease in levels of mAChR at birth persisting
through postnatal day 10, with recovery at subsequent developmental
stages. Results of other experiments in which animals were examined
before delivery of the young have also shown decreases in numbers
of mAChR binding sites in both fetal and maternal brain. Because
decreases in numbers of receptor sites appear to be an adaptive mechanism
to overstimulation by endogenous ACh, it can be postulated that
high levels of ACh in fetal brain must have occurred in spite of the
relatively rapid recovery of ChE after treatment. This conclusion is
supported by a report (Kewitz et al., 1977) that a single administration
of a sublethal dose of DFP to a 20-day pregnant dam elevated free
ACh in the fetal brain that lasted considerably longer than in the
maternal brain. The temporary delay in the postnatal development
of mAChR may indicate that in this time period, the tolerance induced
by prenatal OF treatment is gradually being reversed. The time taken
by muscarinic receptor sites for recovery (i.e., about three weeks) is
similar to that found by Costa and coworkers (1981) in adult rats
treated with another OF (disulfoton). A major feature of information
now available about prenatal exposure to OPs is the finding of a fetal
reduction of mAChRs and a postnatal delay in their development
well after the complete recovery of brain ChE inhibition.
EFFECTS OF EARLY POSTNATAL
EXPOSURE TO DFP
Effects of exposure to DFP during early postnatal development are
summarized in Figure 1. Repeated treatment causes only a weak and
short-lasting behavioral syndrome characteristic of cholinergic stimulation,
without reduction in body or brain weight gain or modification of
protein content in brain tissue. Neurochemically some significant
effects of DFP are clearly observable. Brain ChE activity in control
animals shows a systematic increase. Levels of ChE in those treated
with DFP are consistently lower than controls, being reduced at 14
days by about 45 percent and at 28 days by about 70 percent. Recovery
occurs following the end of treatment, but levels are still some 30
percent below control levels after 12 days of withdrawal. These findings
OCR for page 109
ANIMAL MODELS
50
' 40
E
C 20
E
to
o
a' I
_ ~
109
Total ChE
, _
~ ,
ChAT
16
14
12
10
8
6
4
2
o
1400
1200
1000
800
600
400
200
o
- ~ Control mACh~
- ~ DFP
7 14 20 28 ~ 40
AGE (days)
FIGURE 1 Effects of subchronic intoxication by DFP in 7- to 27-day Wistar rats on
development of brain ChE, ChAT, and mAChRs of pups belonging to four litters and
nursed by their mothers up to weaning. Treatment on alternate days: DFP (in arachis
oil) 0.45 mg/kg (subcutaneous) from day 7 up to day 19, and subsequently 0.70 mg/kg
up to day 27 (25 percent of LD50). Animals killed 24 hours after the last treatment.
Each column represents mean + SEM from eight animals (two from each litter). Aster-
isks indicate a significant difference from control (* p < 0.05, ** p < 0.01) as determined
by l-test. AcThCh = acetylthiocholine.
SOURCE: Michalek et al. (1985), adapted.
OCR for page 110
11~/
MANNA MICHALEK AND ANNITA PINTOR
are generally consistent with reports of investigators using adult ani-
mals as subjects, who have reported median recovery times within
the range of 10-12 days (Austin and lames, 1970; Chippendale et al.,
1974; Ehlert et al., 1980~. That full recovery may not be complete
even after four weeks of withdrawal has also been demonstrated in
adult animals (Russell et al., 1989~.
The major change in activity of the synthetic enzyme ChAT occurs
as a significant increase postnatally, approaching an asymptotic level
within two weeks for both DFP and control animals. This pattern has
been reported in adult rats made tolerant to DFP and other OPs (Russell
et al., 1975; Stavinoha et al., 1969; Wecker et al., 1977~.
Changes in mAChR binding in brain tissue from control animals
follow a general course similar to that of ChE; i.e., binding increases
systematically as postnatal age increases (Figure 1~. Receptor binding
also increases with chronological age in DFP-treated animals. How-
ever, the increases are consistently less than in the controls after one
week of treatment, the difference between treatment groups being
maximal at 28 days; 12 days after withdrawal from DFP the difference
is no longer statistically significant. This general pattern has also
been observed in another rodent model (Levy, 1981~.
The information discussed above points to some important analo-
gies in mechanisms underlying the effects of DFP on functioning of
the cholinergic system. The analogies hold despite the considerable
differences in age-related effects of OPs on brain ChE activity.
EFFECTS OF EXPOSURE TO DFP: SENESCENT RATS
Genetic (Strain-Specific) Differences in Effects
Except for data reported by Pintor et al. (1988), the development of
tolerance to an OF compound late in the life span has not been
investigated. This would seem to be a matter of particular interest
because, in spite of some controversial data, most investigators report
declines in the density of mAChRs in various brain regions of senes-
cent rodents. Most research results have indicated a decrease of
cholinergic markers in the rat striatum, but data concerning age-re-
lated alterations in the cerebral cortex and hippocampus are contro-
versial (Bartus et al., 1982, 1985; Michalek et al., 1988~. One of the
major factors responsible for such discrepancies could be the different
genetic strains of rats used in these studies. In fact, behavioral and
neurochemical studies of mice, utilizing multiple strain comparisons,
have shown that the patterns of age differences are influenced by
genotype (Michalek et al., 1988~. These findings are important for
OCR for page 111
ANIMAL MODELS
111
understanding aging as a product of gene-environment interaction
and for identifying strains that offer the greatest potential for study-
ing the interaction. Most investigations of biochemical changes in
neurotransmitter systems of aging rats have been performed on ani-
mals of only one strain Wistar, Fischer 344, or Sprague-Dawley be-
ing the strains most frequently used. However, knowledge about
strain differences is important in defining "How old is old?" (Coleman,
1989).
Results of a recent series of experiments serve as an example of the
kinds of information generated by studies of interactions between
genetic factors and the aging-of neurochemical events in the brain
(Michalek et al., 1988; Pintor et al., 1988~. The experiments involved
comparisons of age-related differences in AChE, ChAT, and mAChRs
in tissues from three brain areas of Wistar and Fischer 344 male rats
at ages 3 and 24 months. It should be noted that the 50 percent
survival rates of the two strains are very similar, i.e., 28-30 months.
Results of the experiments are presented in two forms: graphically
as histograms and statistically as two-way analyses of variance ANOVAs.
The former provide information about each neurochemical variable
measured. The latter test the statistical significances of two main factors,
i.e., age and strain, and of interactions between them.
Inspection of the upper part of Figure 2 shows an overall similar-
ity between the two strains in levels of ChE activity and in decreases
in activity with aging. This conclusion receives general support from
results of ANOVA presented in Table 4A. Strain differences are significant
only in the hippocampus, Fischer animals having lower levels of activity.
Age-related declines in ChE activity are significant in all brain regions,
varying from 25 to 40 percent in both strains. Nonsignificant interactions
indicate that the aging factors are not strain dependent.
The histograms in the lower part of Figure 2 suggest that levels of
ChAT activity in the Fischer strain are consistently higher than those
in the Wistar animals at both ages, an observation supported by ANOVAs
(Table 4A). Inspection of this figure also suggests that, with one
exception, ChAT activity decreases with aging. The ANOVAs reported
in Table 4A establish that this trend is statistically significant only in
the striatum (approximately 30 percent). The only significant strain
x age interaction occurs in the cortical ChAT activity, where decreases
(approximately 15 percent) are noted with aging in the Fischer but
not in the Wistar strain.
The data on mAChR binding are given in Table 5. In all instances,
binding (BmaX) is higher in Fischer than in age-matched Wistar ani-
mals, indicating a larger population of receptor sites. The ANOVAs
(Table 4) show that both strain and age differences are highly significant.
OCR for page 113
113
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OCR for page 114
114
MANNA MICHALEK AND ANNITA PINTOR
TABLE 5 Age-Related Differences in Binding Parameters of Muscarinic
Receptor Sites in Brain Regions of Wistar and Fischer Rats
Age (months)
Wistar
24
Fischer
24
Cerebral cortex Bmax 1,207 + 6 1,106 + 6 1,970 + 75 1,409 +36a
KD 163 +14 152+15 263+ 9 193 + 7
Hippocampus Bmax 1,111 + 76 682 +55a 1,723 + 20 1,326 + 88a
KD 140+24 110+24 253+ 10 215+ 2
Striatum Bmax 1,292 +59 856 +32a 1,874 + 116 1,211 + 72a
KD 182 +17 110 +19 110+ 16 231 + 7
NOTE: BmaX is expressed as femtomoles per milligram of protein; KD as picomolar.
Values are means + SEM of six experiments.
aSignificant difference p < 0.01, as assessed by post hoc analysis using Student's t-
test with Bonferroni correction.
SOURCE: Michalek et al. (1989).
ated with a lower affinity (higher KD) of the [3H]QNB ligand for mAChR
sites; i.e., the tendency for the ligand to bind to the receptor was less
than in the Wistar strain. The ANOVAs supported this conclusion,
strain differences being significant at p < 0.001 and age differences at
p < 0.01 (Michalek et al., 1988~. There was no significant strain x age
interaction.
Such results clearly indicate that the outcomes of studies in
neurobehavioral toxicology are likely to be affected significantly by
genetic or aging variables, both of which have effects on neurochemical
processes that are involved in behavior. The results also suggest
hypotheses about the mechanisms by which such effects are mediated.
For example, the findings described above are consistent with a loss
during aging of pre- and postsynaptic cholinergic neurons in the Fischer
344 strain. Some of the same age-related changes have been reported
by other investigators working with the same strain (Lippa et al.,
1981; Pedigo and Polk, 1985; Pedigo et al., 1984; Sherman et al., 1981~.
There also is good agreement between the results described here and
those reported by others using Wistar rats as models (Ingram et al.,
1981; London et al., 1985; McGeer et al., 1971; Roman et al., 1984~.
It is of considerable interest that marked differences in the concen-
trations of mAChRs have been described by Overstreet et al. (1984)
for two of the above regions (hippocampus and striatum) in two
selectively bred lines, Flinders sensitive line (FSL) and Flinders resistant
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ANIMAL MODELS
115
line (FRL) rats. Such studies again suggest the importance of genetic
factors in major effects on cholinergic function for some strains or
lines (Fischer 344 or FSL rats) compared to others (Wistar and FRL
rats): an increased cholinergic function may be an important factor
contributing to increased sensitivity to DFP (Russell and Overstreet,
1987) and thus influence the rate of aging in terms of deficit of corti-
cal ChAT and mAChRs. Although there are inherent limitations to
extrapolations from rodents to humans, some reports indicate defi-
cits in cortical, hippocampal, and striatal ChAT and mAChRs in eld-
erly humans (Bartus et al., 1982; Collerton, 1986; Cote and Kremzner,
1983).
Subchronic Intoxication During Senescence
One approach to testing the hypothesis that hyperfunctioning of
the cholinergic system results in greater sensitivity to DFP and to an
accelerated rate of aging is to subject senescent Fischer 344 rats to
repeated administration of DFP (Pintor et al., 1988~. In the initial
phase of subchronic intoxication (i.e., treatment 1 to 4), DFP caused a
typical syndrome of cholinergic stimulation (tremors, sweating, sali-
vation, lacrimation, and diarrhea) lasting for many hours after each
injection. In its severity and duration, the toxic syndrome appeared
more pronounced in senescent than in young animals. In particular,
tremors in the former lasted until the next DFP injection 48 hours
later, whereas they disappeared within 2-3 hours in the latter. At the
end of the treatment period the symptoms were attenuated in both
young and senescent animals. The mortality rate was significantly
higher (p < 0.02) among the senescent (60 percent) than among the
young rats (14 percent). The ANOVA for repeated measures showed
that differences in body weight were significant both for age [F(2,30)
= 28.12, p < 0.001] and for treatment [F(1,20) = 43.48, p < 0.0013. A1-
though both age groups lost weight during the treatment period, the
decrease was greater for the senescent (-94 g) than for the young (-30
g) animals. Body weight is a general measure of capability to main-
tain caloric intake and water balance.
Effects of the subchronic DFP treatment on enzyme activities in
three brain areas are presented in Figure 3: ANOVA (2 ages x 2 treat-
ments) confirms the striking difference in both age- and treatment-
related effects on ChE (Table 4B). There are no significant interaction
factors, indicating that the groups were similarly affected. The ANOVA
of the results for ChAT showed a quite different state of affairs: age
is the only significant variable.
Effects of the DFP treatment on mAChR binding are summarized
OCR for page 116
116
MANNA MICHALEK AND ANNITA PINTOR
~ 25
i:
Cal
o
75
s So
a' 25
E
-
o
~ l
i'
Control
DFP
:
200
100
:
,:(
~ .
~ .
''
'' .
''
,,,
1
CEREBRAL HIPPOCAMPUS STRIATUM
CORTEX
FIGURE 3 Effects of subchronic intoxication by DFP on brain ChE and ChAT of young
and senescent Fischer 344 rats (for treatment see Table 6). Numbers above the col-
umns indicate age in months. Numbers in parentheses show percentage inhibition.
Each column represents mean + SEM from six animals (except for 24-month DFP-
treated rats, n = 4). For factorial analysis of variance (2 ages x 2 treatments ANOVA),
see Table 4 and text. Asterisks indicate significant differences for age (I p < 0.01, ** p <
0.001) and treatment (p < 0.001). AcThCh = acetylthiocholine.
SOURCE: Pintor et al. (1988~.
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ANIMAL MODELS
1 1 7
TABLE 6 Effects of Subchronic Intoxication by DFP on mAChRs in
Brain Regions of Young and Senescent Fischer Rats
Brain Age Bmax KD
Region Treatment (months) (fmol/mg protein) (pM)
Cerebral cortex Control 3 2,028 ~ 67 257 + 19
DFP 3 1,231 + 61 165 ~ 9
Control 24 1,483 + 20 193 + 10
DFP 24 938 + 59 120 + 5
Hippocampus Control 3 1,696 + 31 267 + 17
DFP 3 1,238 + 57 185 + 3
Control 24 1,398 + 45 220 + 10
DFP 24 932 + 65 150 + 8
Striatum Control 3 1,903 + 125 278 ~ 25
DFP 3 1,394 + 41 260 + 16
Control 24 1,334 + 16 255 +35
DFP 24 811 + 78 200 + 3
NOTE: Treatment on alternate days: DFP fin arachis oil) first dose 1.6 mg/kg
(subcutaneous); subsequent doses 1.1 mg/kg for two weeks. Means + SEM from six
animals (except for 24-month DFP-treated rats, n = 4). Factorial analysis of variance (2
ages x 2 treatments ANOVA, Table 4) showed significant differences In BmaX for age and
treatment In all areas (p ~ 0.001). BmaX and KD expressed as means + SEM from six
animals (except for 24-month DFP-treated rats, ~ = 4).
SOURCE: Cantor et al. (1988).
in Table 6. Two-way ANOVAs (Table 4B) confirm what is apparent
in the table: i.e., DFP produced down-regulation in all three brain
areas assayed. It is also clearly apparent that mAChR levels in the
brains of the senescent animals are significantly lower than in the
brains of young rats. Despite this difference, percentage decreases in
mAChR receptor binding induced by the DFP treatment were very
similar for both age groups, as reflected in the lack of significant
interaction for any of the three brain areas.
Such comparative effects of subchronic DFP intoxication in young
and senescent animals have several implications of potential interest
to neurobehavioral toxicologists. For example, the much higher mortality
in senescent animals would appear to be inconsistent with the fact
that the percent down-regulation of mAChRs during DFP treatment
was not significantly different from that in young animals. As discussed
earlier, plasticity of mAChRs appears to be an important compensatory
mechanism for decreased ChE activity and elevated ACh levels, e.g.,
in the development of behavioral tolerance to OPs. How is it possible
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118
MANNA MICHALEK AND ANNITA PINTOR
to account for the differences in mortality when receptor plasticity as
measured by mAChR binding did not differ? Several hypotheses can
be offered. One that is particularly obvious is the possibility that age-
related differences in peripheral events (e.g., cardiovascular, respira-
tory) may be the underlying mechanisms. The hypothesis that age-
related changes in permeability of the blood-brain barrier may be
involved is not supported by the experimental data showing a lack of
significant age x treatment interactions in brain ChE or ChAT activi-
ties. One theoretical model has recently been proposed that could
account for the present mortality data and have broader application
to behavioral and physiological functions (Russell, 1988; Russell et
al., 1986~. The central theme of the concept is that behavioral and
physiological processes are differentially receptor dependent; i.e., they
require different densities of receptor occupancy to function normally.
It follows that there are "basal thresholds" in receptor populations
below which abnormalities in behavioral and physiological functions,
including mortality, occur. Reexamination of Table 6 shows that
populations of receptors (as defined by [3H]QNB binding) character-
istic of the normal young animal had decreased by 40-55 percent in
the DFP-treated senescent rats. Such decrements could significantly
affect functions involving cholinergic innervation. Put in more general
terms, understanding of the effects of pathologically or xenobiotically
induced insult to the nervous system on behavior can benefit sigrulicantly
by knowledge about the mechanisms of action involved. Animal
models are indispensable in generating that knowledge.
Time-Course Recovery of mAChRs Following
Down-Regulation in Brain of Senescent Rats
It is well established that following termination of repeated treat-
ment with an antiChE agent in newborn and young animals, down-
regulation of mAChRs gives way to almost complete recovery, re-
quiring about two weeks in early postnatal life (Michalek et al., 1985)
and three weeks in adulthood (Costa et al., 1981~. Given that protein
synthesis in the brain declines with age (Dwyer et al., 1980), it may
be predicted that muscarinic receptor recovery is slower in senescent
DFP-tolerating rats.
Preliminary experiments to test this hypothesis are now in progress
in our laboratory, with Sprague-Dawley male 3- and 24-month-old
rats serving as subjects. To reduce the mortality rate of aged rats
during treatment, lower dosages of DFP are being utilized (first dose
1.1 mg/kg, followed on alternate days by two doses of 0.7 mg/kg
and four doses of 0.35 mg/kg). The ChE and ChAT activity and
OCR for page 119
ANIMAL MODELS
119
mAChR binding in the cerebral cortex, hippocampus, and striatum
are being assayed at weekly intervals up to five weeks after termina-
tion of DFP treatment. Most previously reported findings are being
confirmed. Differences in mortality rate, although smaller, are still
present, i.e., 15 percent for young rats and 40 percent for senescent
rats. No differences are detected in brain ChE inhibition or in ChAT
activity in any area. The down-regulation of mAChR density (without
changes in affinity) in surviving senescent rats at the end of treatment
is still present and, in terms of percentage of age-matched control
values, is similar in the two age groups. However, the influence of
age on the rate of recovery is evident: both brain ChE activity and
mAChR density reach pretreatment values in young rats within two
weeks, compared to almost five weeks in senescent rats. These results
indicate that the synthesis of both ChE and mAChR molecules is
impaired in brain tissues as a consequence of aging.
CONCLUSION
It is the basic contention of the present discussion that a complete
science and technology of neurobehavioral toxicology cannot be written
without knowledge of neurochemical events intervening between ex-
posure to a neurotoxin and the consequent effects on behavior. Although
behavioral assays in and of themselves have significant contributions
to make to risk assessments, neurobehavioral toxicology has a much
broader agenda. Toxins bind to molecules on biolo~icallv active ti~
within the body in order to produce their effects. Understanding
how a toxic compound is transported to its site of action and the
nature of the cascade of events it initiates is more than a matter of
academic interest. Such knowledge can provide bases for anticipating
and identifying molecular structures that are potentially noxious. It
can give some insight into means for protecting against toxic risks
and into therapeutic procedures by which untoward exposures may
be managed.
The specific examples discussed here were chosen to illustrate how
neurochemical changes throughout the life span can influence the
effects of toxic exposures. Clearly, both risk assessment and clinical
procedures, as well as basic knowledge, must take such interactions
into consideration. Over 40 years ago, Professor C.L. Hull (1943)
commented
it _ , . _ _
. . . any theory of behavior is at present, and must be for some time to
come, a molar theory. This is because neuroanatomy and physiology
have not yet developed to a point such that they yield principles which
may be employed as postulates in a system of behavior theory....
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120
MANNA MICHALEK AND ANNITA PINTOR
These comments have relevance to neurobehavioral toxicology to-
day. It is important for neurobehavioral toxicologists never to forget
that the living organism is not empty. It is filled with a multitude of
events that are involved whenever toxic exposures induce behavioral
malfunctions. In assessing the roles of animal models In neurobehavioral
toxicology, it is apparent that they have been, and will continue to
be, essential to our understanding of the nature of these events.
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Representative terms from entire chapter:
brain che
