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 follows 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
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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 developed. The evidence is that processes involved in the synthesis, storage, 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 provides 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 OP 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 behavi
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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 impaired when an individual is exposed to OP compounds. Upon entering 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 neurotransmitter. 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 disturbances 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 development 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 effects 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 fluorophosphate (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,
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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 mediate 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 senescence.
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 stillborn or die within a few hours after birth. These cases of reproductive 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
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TABLE 1 Effects of Subchronic Intoxication with DFP in Pregnant Rats on Gestation, Birth Statistics, and Litter Survival
Control DFP
Total number of dams 20 20
Length of gestation (days) 21.2 ± 0.2 21.8 ± 0.2
Weight gain of dams (g)
6th–10th day 14.4 ± 2.8 3.6 ± 2.4a
10th–20th day 71.9 ± 4.2 71.8 ± 5.5
Number of pups per litter 11.4 ± 0.8 10.1 ± 0.6b
Lost at birth 0 4
Lost within 48 h 1 8
Litters surviving up to weaning 19 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 mg/kg until day 20 (corresponding to 25% of LD50).
a Significantly different from control p < 0.001 as determined by t-test.
b Not 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) [3H]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 ± 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.
a Significantly different from control values (p < 0.01) as determined by t-test.
SOURCE: Michalek et al. (1985).
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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 OP 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 effects of OP 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 compared 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, respectively). 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 novo synthesis of replacement ChE is more rapid.
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TABLE 3 Effects of Subchronic Intoxication of Pregnant Rats by DFP on Maternal and Fetal Brain aChE and Maximal Number of [3H]-QNB Binding Sites (Bmax) at the End of Pregnancy
Brain ChE (nmol AcThCh
hydrolyzed/min/mg protein) mAChRs (Bmax) (fmol/mgprotein) Interval
Between Last Treatment and Sacrifice (h)
Control DFP %
Control Activity
Control DFP
Dams 1.5 40.9 ± 2.4 7.6 ±
0.1a 18
24 41.7 ± 0.2 8.7 ±
1.4a 20 1,152 ±
41 780 ±
64a
48 42.1 ± 1.6 9.2 ±
1.8a 22
Fetuses 1.5 13.9 ± 0.5 7.0 ±
0.5a 50
24 18.0 ± 0.1 11.8 ±
0.3a 65 187 ± 21 153 ±
11b
48 18.1 ±
0.4c 16.5 ±
0.5a 91
NOTE: For treatment see Table 1. Binding constants (Bmax) of [3H]QNB binding calculated from Scatchard analysis. Mean ± SEM of five animals. Mean ± SEM of 10 pools of two fetuses each. AcThCh = acetylthiocholine.
a Significant difference at p < 0.01.
b Significant difference at p = 0.05.
c Newborns (in three cases delivery occurred 24 h earlier than in DFP group).
SOURCE: Michalek et al. (1985), modified.
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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 special 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 OP 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 OP (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
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