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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

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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 elderlyin 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-

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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,

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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

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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).

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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.

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107 Ct U ._ Cal - Ct - Ct o Fat ~ ~ o cn I= Ct ~ ~ X o In o U X = o ~ ._ U ~ 7 O ol cn `0, `~_ o -U. <~) a em _ ~ X ._ o - o - x - cn U .= o be .5 - N o a U o to .5 +1 +1 o ~ ~ u~ - ~ ~ O +1 +1 ~ c~ ~ o ~ ~ u ~ o :~` - ~ o ~ u ~ v - o o u a~ - ~ ~ ~ ~ - - ~ ~ ~ ~ v , _ ~ ;n ~4 o cn +1 ~ o ~ o ~ ~ ~4 ~ ~ [ t:s ~ ts ts ts ~ ~ oo u) o ~ ~ o o o +1 +1 +1 +1 +1 +1 ~ ~ c~ o oo ~ . . . . . . O u~ ~ di ~ o ~ o o o +1 +1 +1 +1 +1 +1 ~ ~ ~ ~ o ~ - . . . . . . o ~ ~ c~ co oo u~ ~ . . ~ ~ oo ~ d~ oo co ~ u, .cn cq ct =- on o V a,, u - ~ o .= u ~ o z ~ ~ 11 u o E-l x u - ~ c o 3) .~ - - :2 ~ ~ u ~ ~ o o ~o . cn ~ ~ ~ o ~ o ~ - ~ ~0 V 11 ~ '= ~ o ~ ~ ~ - O g cn +1 :~ ~ ~ a,; ~S m~ ~ ~ ~ ~ eu o ~ ~ ~ o c (~=u) z o ~ - - o ~o - ct -

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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

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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.

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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

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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.

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113 ~4 o o Ed U x _' V) U o o _' o U) _. cn - V) _ _ Cal - ~ O a.' ~ _ Lit hi on ~ EM En Ct lo: U. Lo o 1 U. ._ a U. ._ En x - CC - ~ o _ ~ ~ ~ x.5 - ~4 - - ._ cn - o ._ be ~ ._ - ~4 - - Cal - F~ . . _ ~ U. ~ ~ _ ~ ~ ~ X .= - to ._ Ct V) - ~ ~ cr) - . . ~) z z . o z z ~ . . . ~ cO tn. ci> o i7 ~ c~ u~ ~ ~ oo N ~ . . . ~ o ~ . . . cn c~ cn . . . z z z . . . ~ o ~ ~ c~ ~ cn o cn z ~ z x ~ u) o ~ ~ u ~ CL,- v, ~: u) - ct o ct E~ E~ - x - u] X~ ._ e,~ ~ ~ _ ~ ~ .= s~ ~, - ~4 - - ~ O X a,, 6,0 ~ ~ _ ~ ~ ~ ~ .= _ - ~0 - ~ O X ~ ~ b4 ~ ~ - ~ ~ ~ ~ .= - ~ - ~4 - . . . c~ cn ~ . . . z z z too u' o 11. cn . . . . c~ uo cn Z z z z o. o v 5= ~ oo o. o di oo U~ ~ u~ ~ oo A A oo cn cr, ci, 0 . ~ Z Z Z % o O. O V ~, ~ O di ~i oo ~D ~ ~ O C~ ~ d4 ~ U] O .~ CJ C~ _ - ._ ~0 ~0 O .Q b4 ~ . . o . . . c~ un cn Z Z Z oo ~ U) o ~ ~ d4 C~ ~ ~ . . . oo ~4 O ~ Ct g ~ ,~, V ,~, =-_ CL, _ U ~ ~ ._ ._ oo oo - - o ._ oo C~ - . - . . U o ~n

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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

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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

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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. REFERENCES Austin, L., and K. A. C. James. 1970. Rates of regeneration of acetylcholinesterase in rat brain subcellular fractions following DFP inhibition. Journal of Neurochemistry 17:1705-1707. Bartus, R. T., R. L. Dean, B. Beer, and A. S. Lippa. 1982. The cholinergic hypothesis of geriatric memory dysfunction. Science 217:408-417. Bartus, R. T., R. L. Dean, M. J. Pontecorvo, and C. Flicker. 1985. The cholinergic hypothesis: A historical overview, current perspective and future directions. An- nals of the New York Academy of Science 444:322-358. Bignami, G., and H. Michalek. 1978. Cholinergic mechanisms and aversively moti- vated behaviors. Pp. 173-255 in Psychopharmacology of Aversively Motivated Behavior, H. Anisman and G. Bignami, eds. New York: Plenum. Bignami, G., N. Rosic, H. Michalek, M. Milosevic, and G. L. Gatti. 1975. Behavioral toxicity of anticholinesterase agents: Methodological, neurochemical, and neuro- psychological aspects. Pp. 155-215 in Behavioral Toxicology, B. Weiss and V.G. Laties, eds. New York: Plenum. Bus, J. S., and J. E. Gibson. 1974. Bidrin: Perinatal toxicity and effect on the development of brain acetylcholinesterase and choline acetyltransferase in mice. Food and Cos- metics Toxicology 12:313-322. Butcher, L. L., and N. J. Woolf. 1986. Central cholinergic systems: Synopsis of anatomy and overview of physiology and pathology. Pp. 73-86 in The Biological Substrates of Alzheimer's Disease, A.B . Scheibel, A. F. Wechsler, and M. A.B . Brazier, eds. New York: Academic Press. Chippendale, T. C., C. W. Cotman, M. D. Kozar, and G. S. Lynch. 1974. Analysis of acetylcholinesterase synthesis and transport in the rat hippocampus: Recovery of acetylcholinesterase activity in the septum and hippocampus after administration of diisopropylfluorophosphate. Brain Research 81:485~96. Coleman, P. D. 1989. How old is "old"? Neurobiology of Aging 10:115. Collerton, D. 1986. Cholinergic function and intellectual decline in Alzheimer's disease. Neuroscience 19:1-28 Costa, L. G., B. W. Schwab, H. Hand, and S. D. Murphy. 1981. Reduced 3H quinuclidinyl benzilate binding to muscarinic receptors in disulfoton-tolerant mice. Toxicology Applied Pharmacology 60:441-450. Costa, L. G., B. W. Schwab, and S. D. Murphy. 1982. Tolerance to anticholinesterase compounds in mammals. Toxicology 25:79-97. Cote, L. J., and L. T. Kremzner. 1983. Biochemical changes in normal aging in human brain. Pp. 19-30 in The Dementias, R. Mayeux and W. G. Rosen, eds. New York: Raven Press. Dwyer, B. E., J. L. Fando, and C. G. Wasterlain. 1980. Rat brain protein synthesis declines during postdevelopmental aging. Journal of Neurochemistry 35:74~749.

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ANIMAL MODELS 123 Talens, G., and D. Woolley. 1973. Effects of parathion administration during gesta- tion in the rat on development of the young. Proceedings of the Western Pharma- cology Society 16:141-145. Wecker, L., P. L. Mobley, and W. D. Dettbam. 1977. Central cholinergic mechanisms underlying adaptation to reduced cholinesterase activity. Biochemical Pharmacol- ogy 26:633~37. Zalewska, Z., I. Rakowska, G. Matraszek, and D. Sitkiewicz. 1977. Effect of dichlorvos on some enzyme activities of the rat brain during postnatal development. Neuropatologia Polska 15:255-262.