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Organometals and brain development
G.J. Boer, M. G. P. Feenstra, M. Mirmiran, D. F. Swaab and F. Van Haaren Progress in Brain Research. Vol. 7 3 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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CHAPTER 19
Organometals and brain development Zoltan Annau The Johns H o p h University,Depamnent of Environmental Health Sciences, Baltimore. MD 21205, USA
The study of the mechanisms of neurotoxicity and their relationship to behavioral changes is intimately involved in the question of neurobiological function and behavioral expression. The field of neuroscience owes its rapid development to the fact that researchers have been willing to cross disciplinary lines to investigate the functions of the nervous system. The fields of neurotoxicology and behavioral toxicology, which have evolved during the past 20 years, also represent an attempt to extend the knowledge in neuroscience into an applied area dealing with the effects of toxic chenicals on nervous system function. Two reasons can be advanced for the rapid development of neurotoxicology: one is the pressing need to understand the mechanisms and expressions of toxicity of pharmaceutical agents and environmental chemicals, and the other, more familiar to basic neuroscience researchers, is that through the study of neurotoxic chemicals we often gain a better understanding of nervous system function, and sometimes can use experimental compounds to create models of human disease states that otherwise could not be modeled in animals. This latter approach has been largely successful because chemicals have been found that destroy specific neurochemical substrates without necessarily destroying fiber paths coursing in the vicinity of these substrates. The two most successful experimental approaches along these lines have been the use of kainic acid lesions to create an animal model of Huntingtons’s chorea (Coyle et al., 1978), and the use of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) to create a model of parkinsonism. The great interest in MPTP research comes from the fact that there was already evidence from drug abusers that the chemical was capqble of inducing parkinsonism in humans that seemed indistiguishable from the idiopathic disease (Langston et al., 1984). Work with both of these chemicals has been successful because of the previous intensive investigations on the etiology of these diseases in the basic and clinical neurosciences. In contrast to these well-recognized clinical problems, there have been few epidemics of environmental intoxication with neurotoxic chemicals and when these have occurred the causes were recognized relatively rapidly and the problems solved by eliminating or reducing the use of the chemical. The widespread exposure of large populations to chemicals such as lead and mercury has for the most part not been considered life-threatening and therefore has received limited attention from researchers. In addition, when these chemicals produce overt toxic symptoms at relatively large doses, the symptoms may be relatively diffuse, making the investigation of their mechanisms of action difficult. In this paper we shall review some of the evidence on the neurotoxicity of the organometals methyl mercury, trimethyl tin and trimethyl lead. Organometals were chosen because they may represent the most toxic forms of the heavy metals and also because they either have or may become serious environmental hazards. Methyl mercury
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was selected because it is associated with the most important examples of human environmental intoxication and also because it exerts its maximum effect during the perinatal period, which may be the most sensitive period of the nervous system to the action of neurotoxic chemicals. In addition, we shall review some of the evidence regarding the toxicity of triethyl lead and trimethyl tin, which seem to exert their toxic action primarily on the limbic system and come somewhat closer to the experimental neurotoxicants such as kainic acid. These chemicals are found in the environment, but so far have not posed a serious health threat to populations.
Metbyl mercury The first important episode of methyl mercury (MM) intoxication occurred in 1953 in Minamata, Japan. One of the striking aspects of this outbreak of mercury poisoning was that asymptomatic mothers gave birth to severely mentally retarded children, who subsequently showed only slight recovery of function (Kojima and Fujita, 1973). Subsequent examination of some of the victims who died revealed that the mercury exposure had reduced the total brain mass by up to 40% (Harada, 1976), an effect also seen in primates (Fig. 1). These striking effects of prenatal intoxication also raised the possibility that at lower doses of exposure, deterioration in mental performance could be observed, without the radical destruction of neuronal populations. In order to investigate this, animals were exposed to MM at various times during gestation. Spyker et al. (1972), using mice, showed that a single injection of MM given to the mother during the first trimester of gestation produeed behavioral changes without overt signs of toxicity in either the mother or the offspring. This demonstration of 'behavioral' teratology confirmed the human data obtained in Minamata, and led other investigators to extend these findings through both behavioral and neurobiological studies. These investigations have recently been
Fig. I. Extensive hypotrophy of the brain of an infant monkey congenitally exposed to methyl mercury. Arrows point to loss of mass at visual cortex and widening of interhemispheric sulci. Taken from Mottet et al., 1987.
reviewed extensively (Eccles and Annau, 1987; Chang and Annau, 1984), and several interesting features of prenatal MM intoxication have emerged. First, administration of MM postnatally has been shown to lead to mitotic arrest within 24 h and subsequent cell loss at 48 h in the cerebellum (Sager et al., 1982). Similar results, though somewhat less severe, were seen when the mercury was administered on day 12 of gestation in a subsequent study (Rodier et al., 1984). Khera (1973) had demonstrated in cats that prenatal exposure results in an incomplete granular layer in the cerebellum as well as alterations in the cyto-
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architecture. Delayed myelination and delayed migration of the external granule cells were also described by Khera et al. (1974) and Khera and Tabacova (1973). The delayed migration of cells in the parietal cortex has also been described recently by Choi (1986). At the electron microscopic level, Chang (1 987) has reported that in the early stages of intoxication there is an increase in neuronal lysosomes associated with the disintegration of the rough endoplasmic reticulum, followed by focal cytoplasmic degradation and neuronal vacuolation. Behavioral studies in animals have confirmed the early observations on the human victims both in Minamata and later in Iraq (Amin-Zaki et al., 1976). Thus Eccles and Annau (1982a) showed that rats treated on gestational days 8 and 15 with either 5 or 8 mg/kg of MM showed altered development of motor activity in the pre-weaning period. This alteration was reflected in changes in the dopaminergic system (Cuomo et al., 1984). When tested as adults in a two-way avoidancelearning task, MM-exposed animals demonstrated a retardation not only in acquisition, but also in retention of this task. This latter effect was greatly increased when the mercury was injected on day 15 of gestation. An additional interesting finding reported by these investigators was the altered responsivenessof the mercury-treated animals to the psychoactive agent amphetamine (Eccles and Annau, 1982b). An earlier paper by Hughes and Sparber (1978) had reported similar results with amphetamine, and these authors suggested that one of the delayed effects of prenatal intoxication may be altered drug metabolism. It was not clear from the results of Hughes and Sparber, however, whether the altered behavioral sensitivity displayed by their rats was due to central or peripheral mechanisms. While the use of pharmacological challenges has become very popular in neurotoxicology (see Walsh and Tilson, 1986) as a way of determining the potential mechanism of neural injury, very little effort has been expended on determining the reason for the altered drug sensitivity. Hughes and Sparber
reported that the effect they observed was only apparent in males, thus raising the possiblity that hormonal influences may play a role in this delayed expression of neurotoxicity. In addition to the behavioral investigations of prenatal toxicity, both electrophysiological and neurochemical measurements have been made. These have been reviewed recently (Annau and Eccles, 1987; Komulainen and Tuomisto, 1987) and, while there seems to be no doubt that high doses of mercury will cause injury to the peripheral nervous system as well, the central nervous system is by far the most sensitive target. Using the visual evoked potential technique, Dyer et al. (1978) have shown that a single prenatal exposure to MM will alter the evoked potential recorded in the adult offspring. One of the interesting findings of this study was the shortening of the latencies of some of the components of the evoked potential, which the authors speculated could have been caused by the loss of small fibers because of the mercury treatment. In terms of neurochemical measurements following MM treatment, Komulainen and Tuomisto conclude that while the mercury does have potent inhibitory effects in vitro, in vivo the effects seem considerably less impressive. Thus, perinatal administration of MM has been shown to (1) interfere with neuronal migration, (2) delay and reduce myelination, and (3) arrest mitosis and reduce the number of neurons. These effects are correlated with (1) transient alterations in the development of motor activity, (2) reduced ability to learn and retain complex tasks, and (3) altered responsiveness to pharmacological agents, as well as to altered neurophysiological responses in the visual system. These effects have been relatively well defined in many experimental models and have confirmed that this chemical even at very low doses can cause irreversible damage to the immature nervous system, as can be seen in recent literature reviews (Eccles and Annau, 1987).
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Trimethyl tin Interest in research on the neurotoxicity of trimethyl tin (TMT) started with a paper published in 1979 by Brown et al., who described the behavioral and pathological changes seen in rats following either single or repeated doses of this organotin. The behavioral symptoms consisted of increased aggressiveness,vocalizations, tremors, seizures and hyperreactivity. These symptoms persisted for approximately two to three weeks and then subsided. Neuropathological examination of the brains of the exposed animals showed severe damage to the hippocampus (Fig. 2) and lesser damage to other parts of the limbic system such as the pyriform cortex and amygdala. Because this organotin seemed such an ideal ‘limbic system’ poison, this research paper was followed by extensive studies on the nature and mechanisms of toxicity. The observations of Brown et al. were confirmed and extended by subsequent studies (Dyer et al., 1982a). The dose-response function for this chemical was extremely steep for the rat, with the LD,, at about 7.3 mg/kg and the LD,, at 10 mg/kg. Following the injection of TMT, there was a mild hypothermia which persisted for about four days and was directly related to dose. Several behavioral alterationsoccurred within two days of injection. A fine tremor-at-rest was seen at the higher doses. Increased reactivity to handling which increased over time was also observed. Animals that showed this symptom also appeared to be likely to have seizures. The final behavioral morbidity appeared to be tail self-mutilation,with 50% of the animals showing this at the 7 mg/kg dose (Dyer et al., 1982b). Ruppert et al. (1983) showed that when placed in figure-eight maze, the animals dosed with 7 mg/kg became hyperactive after 24h and remained so up 50days after dosing. Not surprisingly, given the significant destruction of hippocampal pyramidal cells, several measures of learning and retention showed major alterations following TMT administration. h i -
mals exhibited retention deficits in a passive avoidance task which was evident at 5, 6 and 7 mg/kg and was not dose-related (Walsh et al., 1982a). At 7 mg/kg, rats were significantly impaired in a series of problem-solving tasks in the Hebb-Williams maze (Swartzwelder et al., 1982). The animals exhibited more errors, less error reduction over days and showed perseverative behavior that seemed characteristic of animals with hippocampal lesions. A further test of the hippocampal damage was also performed by
Fig. 2. Section through the hippocampus at normal rat (top) and rat exposed to 3 mgFg of TMT once a week for 3 weeks. Note extensive loss of pyramidal cells in areas CA3 and CA4. Taken from Valdes etal., 1983.
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Walsh et al. (1982b), who trained their animals in a radial-arm maze prior to TMT administration. Fifteen days after TMT dosing (6 mg/kg), the animals were retrained in the maze. There was a severe disruption of maze performance during retraining which persisted up to 70 days postdosing. Other measures of TMT-induced damage to the nervous system indicated that the pathology was not restricted to the limbic system. Dyer et al. (1982~)showed that when rats were implanted with chronic recording electrodes in the optic tract and the visual cortex, the TMT-treated rats exhibited an increase in latencies and decreases in amplitudes in the early peaks of the visual evoked potential at both electrode locations. These changes were suggestive of retinal damage. Other changes in the later components of the evoked potential were suggestive of altered arousal. A more detailed analysis of the seizure susceptibility of TMT-treated animals also showed that these animals were more susceptible to seizures induced by pentylenetetrazol, a general (not limbicsystem-specific) convulsant, indicating that the early hypothesis of TMT being a specific limbic system or hippocampal toxicant was not conh e d (Dyer et al., 1982d). Other areas of investigation included neurochemical studies of TMT-induced damage, as well as perinatal exposures. Valdes et al. (1983) showed that neurochemical alterations occured in intrinsic hippocampal neurotransmitters and not in extrinsic transmitters such as norepinephrine, and Ali et al. (1985) showed that there was a significant reduction in muscarinic cholinergic binding in whole brain and frontal cortex 2-7 days following treatment. In utero exposure, however, failed to show this neurochemical effect or the neuropathological pattern of adult TMT administration, suggesting that the immature nervous system was less sensitive to this toxicant than the fully developed nervous system (Paule et al., 1986). This was further confirmed by Lipscomb et al. (1986), who showed that the fetal brain concentration was identical to the maternal
brain concentration 96 h after exposure, although at 338 h the fetal brain levels were significantly lower than the maternal. Chang (1984) has also shown that administration of TMT up to postnatal day 4 causes relatively little pathology, but after that developmental day the pathology increases markedly, an observation also reported by Ruppert et al. (1983) Thus, while the most severe damage induced by TMT in the adult animal is clearly in the hippocampus, as revealed by behavioral and pathological studies, it has also become clear that many other parts of the nervous system are also involved in the pathology (Chang et al., 1982). It is of great interest that, unlike methyl mercury, trimethyl tin seems to be less toxic in the fetal brain than the adult brain, although during the neonatal period, at least in the rat, this lack of sensitivity to toxicity diminishes rapidly. Triethyl lead
The organo leads which have been used as gasoline additives in increasingly large quantities until recently (Grandjean and Nielsen, 1979)have been associated with many cases not only of accidental human intoxication (Beattie et al., 1972), but also of voluntary ingestion as ‘sniffing’ as an inexpensive drug of abuse (Valpey et al., 1978). The symptoms are clearly associated with central nervous system disorders and are characterized by disturbances of emotionality and memory as well as increased motor excitability, not unlike those seen after human TMT poisoning (Ross et al., 1981). A recent review of the neurotoxicology of organo leads (Walsh and Tilson, 1984), in fact, makes a strong case for the similarity in the neurobehavioral pathology between TMT and triethyl lead (TEL), the toxic metabolite of tetraethyl lead, the gasoline additive. Not only are the pathological lesions seen in the adult animal following TEL administration concentrated in the limbic system, particularly the hippocampus, but also the behavioral effects are in accordance with
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the expectations of this type of lesion. More specifically, the animals show improved two-way avoidance learning, and impaired passive avoidance and spatial learning. Further, chemical analyses have shown that the hippocampus concentrates lead and that the concentrations of essential elements such as copper and zinc are decreased following TEL exposure (Niklowitz and Yeager, 1973). The neonatal brain again appears to be exquisitely sensitive to this neurotoxic agent (Booze et al., 1983). Konat (1984) has shown that administration of TEL to neonatal rats results in very significant hypomyelination because oligodendrocytesappear to be particularly targeted. This suggests that TEL, while having many similaritiesto TMT in its effect on the limbic system, may yet have its own unique toxicity, at least in the neonatal brain.
Mechanisms of toxicity The biochemical and metabolic changes caused by methyl mercury administration have been reviewed recently by Chang (1987). Following mercury administration, the incorporation of amino acids into the nervous system is reduced significantly (Yoshimo et d., 1966) resulting in decreased protein synthesis. In addition, Verity et al. (1977) have suggested that a disruption of mitochondrial respiration may also inhibit synaptosomal protein synthesis, and further studies (Frenkel and Harrington, 1983) have shown that DNA and RNA synthesis in isolated mitochondria is inhibited. Finally, Sager et al. (1983) have reported microtubule disruption by methyl mercury. These studies indicate that mercury may disrupt all levels of the nervous system, particularly in the developing organism, and may explain the relative sensitivity of the organism to its toxicity. In both triethyl lead and trimethyl tin intoxication protein synthesis inhibition seems to play a major role in the development of pathology. Recently Costa and Sulaiman (1986) have shown that following TMT administration protein syn-
thesis is decreased significantly. Previous studies have shown that TMT inhibits oxidative phosphorylation (Aldridge and Street, 1971), and Costa and Sulaiman have suggested that this may indirectly account for the inhibition of protein synthesis. Konat (1986) has reviewed the triethyl lead toxicity literature and has indicated that this chemical also inhibits protein synthesis, not only at the neuronal level, but also at the level of myelin formation, where it inhibits proteins involved in membrane assembly. It appears therefore that all of these organic metals have certain common properties that account for their toxicities. While the neuropathologies of TEL and TMT have certain similarities in the brain regions affected, the neuropathology of MM is clearly very different, suggestingthat considerably more complex underlying biochemical mechanisms are involved in their individual toxicities.
Summary and conclusion
This brief review of the neurotoxicity of three organometals has shown that each metal seems to have its own pattern of toxicity which changes as a function of developmental stage of the brain. Methyl mercury in the fetal brain may be the most indiscriminately toxic compound because of its cytotoxicity. The nature of the neurotoxicity of prenatal exposure to TEL remains to be elucidated but in the neonatal period it may have both glial and neuronal targets. Finally, TMT at this time seems relatively non-toxic in the fetal period, but becomes extremely toxic to limbic system neurons in the neonatal period. The sensitivity of the hippocampus to a variety of environmental insults has puzzled investigators for many years, and even though, as has been pointed out above, there is some evidence that it concentrates lead (both organic and inorganic forms) this does not account for the total pathology seen. The relationship between behavioral and neurobiological indices of toxicity is becoming closer as we learn to use our knowledge regarding
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brain lesions and behavioral effects and apply specific behavioral techniques derived from this literature to the testing of neurotoxicity of environmental chemicals (see Wenk and Olton, 1986). It is also quite evident that as we descend the doseresponse curve of neurotoxic chemicals, we continue to uncover additional evidence of subtle alterations of nervous system dysfunction. These subtle, often ‘latent’ effects of perinatal intoxication have rarely been investigated in human populations. Needleman (1986) was one of the first to call attention, in the case of inorganic lead exposure, to such effects by developing the techniques to measure them. As we become more concerned with both the consequences of environmental hazards and widespread drug abuse we have to increase our efforts to establish the noeffect dose for all chemicals. It is our hope that through the continued investigations of animal models of perinatal neurotoxicity and concurrent human studies, we will ultimately be able to safeguard the health of human populations. References Aldridge, W.N. and Street, B.W. (1977) The relation between the specific binding of trimethyltin and triethyltin to mitochondria and their effects on various mitochondrial functions. Biochem. J., 124: 221-234. Ali, S . F., Slikker, W., Jr., Newport, G. D. and Goad, P.T. (1985) Cholinergic and monoaminergic alterations in the mouse central nervous system following acute trimethyltin exposure. Acta Pharmacol. Toxicol., 59: 179-188. Amin-Zaki, L., Ehassani, S., Majeed, M. A., Clarkson, T. W., Doherty, R.A., Greenwood, M.R. and GiovanoliJakubczak, T. (1976) Perinatal methyl mercury poisoning in Iraq. Am. J. Dis. Child., 130: 1070-1076. Annau, Z. and Eccles, C. U. (1987) Sensory deficits caused by exposure to methyl mercury. In C.U. Eccles and Z . Annau (Eds.), The Toxicity of Methyl Mercury, The Johns Hopkins University Press, Baltimore, pp. 104-1 13. Beattie, A.D., Moore, M.R. and Goldberg, A. (1972) Tetraethyl lead poisoning. Lancet, ii: 12-1 5. Booze, R.M., Tilson, H. A., Annau, Z. and Mactutus, C.F. (1983) Neonatal triethyl lead neurotoxicity in rat pups: Initial observations and quantification. Neurobehav. Toxicol. Teratol., 5: 367-315. Brown, A. W., Aldridge, W. N., Street, B. W. and Verschoyle, R.D. (1979) The behavioral and neuropathological
sequelae of intoxication by trimethyltin compounds. Am. J. Pathol., 104: 59-82. Chang, L. W. (1984) Trimethyltin induced hippocampal lesions at various neonatal ages. Bull. Environ. Contam. 33: 295-301. Tox~co~.., Chang, L.W. (1987) The experimental neuropathology of methyl mercury. In C.U. Eccles and Z. Annau (Eds.), The Toxicity of Methyl Mercury, The Johns Hopkins University Press, Baltimore, pp. 54-72. Chang, L.W. and Annau, Z. (1984) Developmental neuropathology and behavioral teratology of methyl mercury. In J. Yanai (Ed.), Neurobehavioral Teratology, Elsevier, Amsterdam, pp. 405-432. Chang, L. W., Tiemeyer, T.M., Wenger, G. R., McMillan, D.E. and Reuhl, K.R. (1982) Neuropathology of trimethyltin intoxication. I. Light microscopy study. Environ. Res., 29: 435-444. Choi, B. H. (1986) Methylmercury poisoning of the developing nervous system: I. Pattern of neuronal migration in the cerebral cortex. Neurotoxicology, 7: 591-600. Chyle, J.T., McGeer, E. G., McGeer, P. L.and Schwarz, R. (1978) Neostriatal injections: a model for Huntington’s chorea. In E.G. McGeer, J. W. Olney and P. L. McGeer (Eds.), Kainic Acid as a Tool in Neurobiofogv,Raven Press, New York, pp. 139-159. Costa, L. G. and Sulaiman, R. (1986) Inhibition of protein synthesis by trimethyltin. Toxicol. Appl. Phannacol., 86: 189-196. Cuomo, V., Ambrosi, L., Annau, Z., Cagiano, R.,Brunello, N. and Racagni, G. (1984) Behavioral and neurochemical changes in offspring of rats exposed to methyl mercury during gestation. Neurobehav. Toxicol. Teratol.,6: 249-254. Dyer, R.S., Eccles, C.U. and Annau, Z. (1978) Evoked potential alterations following prenatal methyl mercury exposure. Phannacol. Biochem. Behav., 8: 127-141. Dyer, R.S.,Walsh, T. J., Swartzwelder, H. and SandWayner, M.J. (Eds.) (1982a) The neurotoxicology of the alkyltins. Neurobehav. Toxicol. Teratol., 4: 125-278. Dyer, R. S.,Walsh, T. J., Wonderlin, W. F. and Bercegeay, M. (1982b) The trimethyltin syndrome. Neurobehav. Toxicol. Teratol., 4: 127-133. Dyer, R. S., Howell, W.E. and Wonderlin, W.F. (1982~) Visual system dysfunction following acute trimethyltin exposure in rats. Neurobehav. Toxicol. Teratol., 4: 191-196. Dyer, R.S., Wonderlin, W.F. and Walsh, T.J. (1982d) Increased seizure susceptibility following trimethyltin administration in rats. Neurobehav. Toxicol. Teratol., 4: 203-208. Eccles, C. U. and Annau, Z. (1982a) Prenatal methyl mercury exposure: I. Alterations in neonatal activity. Neurobehav. Toxicol. Teratol., 4: 371-376. Eccles, C.U. and Annau, Z. (1982b) Prenatal methyl mercury exposure: 11. Alterations in learning and psychotropic drug sensitivity in adult offspring. Neurobehav. Toxicol. Teratol., 4: 317-382.
302 Eccles, C.U. and Annau, Z. (Eds.) (1987) The Toxicity of Methyl Mercury, The Johns Hopkins University Press, Baltimore. Frenkel, G. D. and Harrington, L. (1983) Inhibition of mitochondrial nucleic acid synthesis by methyl mercury. Biochem. Pharmacol., 32: 1454-1456. Grandjean, P. and Nielsen, T. (1979) Organolead compounds: environmental health aspects. Residue Rev., 72: 97-148. Harada, M. (1976) Minamata disease. Chronology and medical report. BuU. Inst. Constitut. Med. Kumamoto Univ., 25: Suppl. 1-60. Hughes, J.A. and Sparber, S.B. (1978) d-Amphetamine unmasks postnatal consequences of exposure to methyl mercury. Pharmacol. Biochem. Behav., 4: 385-301. Khera, K. S. (1973) Teratogenic effects of methyl mercury in the cat: note on the use of this species as a model for teratogenicity studies. TeratoZogy, 8: 293-303. Khera, K. S. and Tabacova, S.A. (1973) Effects of methylmercuric chloride on the progeny of mice and rats treated before or during gestation. Food Cosmet. Toxicol. 11: 243-254. Khera, K. S., Iverson, F., Hierliiy, L., Tanner, R. and Trivett, G. (1974) Toxicity of methyl mercury in neonatal cats. Teratology, 10: 69-76. Kojima, K. and Fujita, M. (1973) Summary of recent studies in Japan on methyl mercury poisoning. ToxicoZlogv, 1: 43-62. Komulainen, H. and Tuomisto, J. (1987) The neurochemical effects of methyl mercury in the brain. In C. U. Eccles and Z. Annau (Eds.), The Toxicityof MethyZ Mercury,The Johns Hopkins University Press, Baltimore, pp. 172-189. Konat,. G. (1984) Triethyllead and cerebral development: an overview. Neurotoxicology, 5 : 87-96. .angston, J.W., Forno, L.S., Rebert, C. S. and Irwin, I. (1984) Selective nigral toxicity after systemic administration of l-methyl4pheny-l,2,5,6-tetrahydropyridine (MPTP) in the squirrel monkey. Brain Res., 292: 390-394. ipscomb, J.C., Paule, M.G. and Slikker, Jr., W. (1986) Fetomaternal kinetics of ''C-trimethyltin. Neurotoxicology, 7: 581-590. Mottet, K. N., Shaw, C.-M. and Burbacher, T.M. (1987) The pathological lesions of methyl mercury intoxication in monkeys. In: C.E. Eccles and Z . Annau, (Eds.), The Toxicity of Methyl Mercury, The Johns Hopkins Press, Baltimore, pp. 73-103. Needleman, H.L. (1986) Epidemiological studies. In Z. Annau (Ed.) Neurobehavioral Toxicology, The Johns Hopkins University Press, Baltimore, pp. 279-287. Niklowitz, W. J. and Yeager, D. W. (1973) Interference of Pb with essential brain tissue Cu,Fe and Zn as main determinant in experimental tetraethyllead encephalopathy. L$e Sci., 13: 897-905. Paule, M. G., Reuhl, K., Chen, J. J., Mi, S. F. and Slikker, Jr.,
W. (1986) Developmental toxicology of trimethyltin in the rat. Toncol. Appl. Pharmacol., 84: 412-417. Rodier, P.M., Aschner, M. and Sager, P.R. (1984) Mitotic arrest in the developing CNS after prenatal exposure to methyl mercury. Neurobehav. Toxicol. Teratol., 6: 379-386. Ross, W.D., Emmett, E.A., Steiner, J. and Tureen, R.(1981) Neurotoxic effects of occupational exposure to organotins. Am. J. Psychiatry, 138: 1092-1095. Ruppert, P. H., Dean, K. R. and Reiter, L. W. (1983) Developmental and behavioral toxicity following acute postnatal exposure of rat pups to trimethyltin. Neurobehav. Toxicol. Teratol., 5: 421-429. Ruppert, P.H., Walsh, T.J., Reiter, L.W. and Dyer, R.S. (1982) Trimethyltin-induced hyperactivity: time course and pattern. Neurobehav. Toncol. Teratol., 4: 135-140. Sager, P. R., Doherty, R. A. and Olmsted, J. B. (1983) Interaction of methylmercury with microtubles in cultured cells and in vitro. Exp. Cell Res., 146: 127-137. Sager, P. R., Doherty, R. A. and Rodier, P.M. (1982) Effects of methyl mercury on developing mouse cerebellar cortex. Exp. Neurol., 77: 179-193. Spyker, J.M., Sparber, S.B. and Goldberg, A.M. (1972) Subtle consequences of methylmercury exposure: behavioral deviations in offspring of treated mothers. Science, 177: 621-623. Swartzwelder, H. S., Hepler, J., Holahan., W., King, S.E., Leverenz, H.A., Miller, P.A. and Myers, R.D. (1982) Impaired maze performance in the rat caused by trimethyltin treatment: problem-solving deficits and preservation. Neurobehav. Toxicol. Teratol., 4: 169-176. Valdes, J. J., Mactutus, C.F., Santos-Anderson, R., Dawson, Jr., R. and Annau Z. (1983) Selective neurochemical and histological lesions in rat hippocampus following chronic trimethyltin exposure. Neurobehav. Toxicol. Teratol., 5 : 357-361. Valpey, R., Sumi, S.M., Dopass, M.K. and Goble, G.J. (1978) Acute and chronic progressive encephalopathy due to gasoline sniffing. Neurology, 28: 345-350. Verity, M.A., Brown, W.J., Cheung, M. and Czer, G. (1977) Methyl mercury inhibition of synaptosome and brain slice protein synthesis: in vivo and in vitro studies. J. Neurochem., 29: 673-679. Walsh, T.J. and Tilson, H.A. (1984) Neurobehavioral toxicology of organoleads. Neurotoxicology, 5: 67-86. Walsh, T.J. and Tilson, H.A. (1986) The use of pharmacological challenges. In Z. Annau (Ed.), Neurobehavioral Toxicologv, The Johns Hopkins University Press, Baltimore, pp. 244-267. Walsh, T., Gallagher, M., Bostock, E. and Dyer, R. S. (1982a) Trimethyltin impairs retention of a passive avoidance. Neurobehav. ToncoZ. TeratoZ., 4: 163-168. Walsh, T.J., Miller, D.B. and Dyer, R.S. (1982b) Trimethyltin, a selective limbic system toxicant, impairs radial-arm maze performance. Neurobehav. Toxicol. Teratol., 4: 177-184.
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Wenk, G. L. and Olton, D. S. (1986) Lesion analysis. In Z. Annau (Ed.), Neurobehavioral Toxicology, The Johns Hopkins University Press, Baltimore, pp. 268-278. Yoshino, Y., Mozai, T. and Nakao, K. (1966) Distribution of mercury in the brain and its subcellular units in experimental organic mercury poisoning. J. Neurochem., 13: 397-406.
Discussion C.V.Vorhees: You raised an important issue about the possible changes in effects over time and the need for longitudinal analyses. Have you examined your methylmercury animals to see if the same animals that show altered activity were the same ones that showed the most disrupted avoidance performance? Z. Annau: Unfortunately at the time we performed these experiments we did not keep track of the animals over the entire period and therefore this type of analysis was not available. H.J. Romijn: Is tin as a metal just as toxic as its organic compound? If not, why have you chosen the organic compound in your studies? Z. Annau: Tin as a metal is not considered very toxic. We chose trimethyl tin because it is a widely used chemical (in anti-fouling paints on boats, and as plasticizers in the plastics industry) and because of several human accidental exposures that showed this form of tin to be extremely neurotoxic. B.E. h n a r d : Trimethyl tin is suggested to be a limbic toxin. Bernadette Easton in my laboratory has recently shown that, following a single dose of this, there is a dose-dependent
change in choline acetyltransferase and muscarinic receptor density in many regions outside the limbic areas. Regarding the behavioral defects after trimethyl tin, a major abnormality occurs in spatial learning at doses that have little effect on most other behavioral parameters. Would you like to comment on these points? Z. Annau: It is clear from the recent trimethyl tin studies that it is not a specific limbic system poison, but that it also causes lesions in many other parts of the brain. Given its effects on the cholinergic system and the input of that system into septal-hippocampal pathways, it is not too surprising that you see spatial learning task deficits. C.J. Boer: Is there any idea at what neurochemicd level the organometals are acting to exert their deleterious effects? Z. Anaru: This in one of the areas of research that has not been exploited fully. Of course, each metal has a different mechanism of toxicity, and seems to interfere at several neurotransmitter systems. T.A. Slotkin: How do you interpret changes in postsynaptic receptors and receptor-mediated responses without evaluating presynaptic function? Receptor up-regulation might represent a compensation for presynaptic hypofunction, thus producing a synapse wich operates normally. Z. Anaru: I agree that, based on our data, it is impossible to determine the exact location of the alteration, but that the postsynaptic alteration may be one such possible mechanism. P.M. Rodier (comment): We have been able to measure Hg in all our experiments and I agree with you that much (probably most) of the variance we see in response to methyl mercury is due to differences in kinetics from animal to animal. We are constantly surprised at the difference in brain levels from the same dose!
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CHAPTER 19
Organometals and brain development Zoltan Annau The Johns H o p h University,Depamnent of Environmental Health Sciences, Baltimore. MD 21205, USA
The study of the mechanisms of neurotoxicity and their relationship to behavioral changes is intimately involved in the question of neurobiological function and behavioral expression. The field of neuroscience owes its rapid development to the fact that researchers have been willing to cross disciplinary lines to investigate the functions of the nervous system. The fields of neurotoxicology and behavioral toxicology, which have evolved during the past 20 years, also represent an attempt to extend the knowledge in neuroscience into an applied area dealing with the effects of toxic chenicals on nervous system function. Two reasons can be advanced for the rapid development of neurotoxicology: one is the pressing need to understand the mechanisms and expressions of toxicity of pharmaceutical agents and environmental chemicals, and the other, more familiar to basic neuroscience researchers, is that through the study of neurotoxic chemicals we often gain a better understanding of nervous system function, and sometimes can use experimental compounds to create models of human disease states that otherwise could not be modeled in animals. This latter approach has been largely successful because chemicals have been found that destroy specific neurochemical substrates without necessarily destroying fiber paths coursing in the vicinity of these substrates. The two most successful experimental approaches along these lines have been the use of kainic acid lesions to create an animal model of Huntingtons’s chorea (Coyle et al., 1978), and the use of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) to create a model of parkinsonism. The great interest in MPTP research comes from the fact that there was already evidence from drug abusers that the chemical was capqble of inducing parkinsonism in humans that seemed indistiguishable from the idiopathic disease (Langston et al., 1984). Work with both of these chemicals has been successful because of the previous intensive investigations on the etiology of these diseases in the basic and clinical neurosciences. In contrast to these well-recognized clinical problems, there have been few epidemics of environmental intoxication with neurotoxic chemicals and when these have occurred the causes were recognized relatively rapidly and the problems solved by eliminating or reducing the use of the chemical. The widespread exposure of large populations to chemicals such as lead and mercury has for the most part not been considered life-threatening and therefore has received limited attention from researchers. In addition, when these chemicals produce overt toxic symptoms at relatively large doses, the symptoms may be relatively diffuse, making the investigation of their mechanisms of action difficult. In this paper we shall review some of the evidence on the neurotoxicity of the organometals methyl mercury, trimethyl tin and trimethyl lead. Organometals were chosen because they may represent the most toxic forms of the heavy metals and also because they either have or may become serious environmental hazards. Methyl mercury
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was selected because it is associated with the most important examples of human environmental intoxication and also because it exerts its maximum effect during the perinatal period, which may be the most sensitive period of the nervous system to the action of neurotoxic chemicals. In addition, we shall review some of the evidence regarding the toxicity of triethyl lead and trimethyl tin, which seem to exert their toxic action primarily on the limbic system and come somewhat closer to the experimental neurotoxicants such as kainic acid. These chemicals are found in the environment, but so far have not posed a serious health threat to populations.
Metbyl mercury The first important episode of methyl mercury (MM) intoxication occurred in 1953 in Minamata, Japan. One of the striking aspects of this outbreak of mercury poisoning was that asymptomatic mothers gave birth to severely mentally retarded children, who subsequently showed only slight recovery of function (Kojima and Fujita, 1973). Subsequent examination of some of the victims who died revealed that the mercury exposure had reduced the total brain mass by up to 40% (Harada, 1976), an effect also seen in primates (Fig. 1). These striking effects of prenatal intoxication also raised the possibility that at lower doses of exposure, deterioration in mental performance could be observed, without the radical destruction of neuronal populations. In order to investigate this, animals were exposed to MM at various times during gestation. Spyker et al. (1972), using mice, showed that a single injection of MM given to the mother during the first trimester of gestation produeed behavioral changes without overt signs of toxicity in either the mother or the offspring. This demonstration of 'behavioral' teratology confirmed the human data obtained in Minamata, and led other investigators to extend these findings through both behavioral and neurobiological studies. These investigations have recently been
Fig. I. Extensive hypotrophy of the brain of an infant monkey congenitally exposed to methyl mercury. Arrows point to loss of mass at visual cortex and widening of interhemispheric sulci. Taken from Mottet et al., 1987.
reviewed extensively (Eccles and Annau, 1987; Chang and Annau, 1984), and several interesting features of prenatal MM intoxication have emerged. First, administration of MM postnatally has been shown to lead to mitotic arrest within 24 h and subsequent cell loss at 48 h in the cerebellum (Sager et al., 1982). Similar results, though somewhat less severe, were seen when the mercury was administered on day 12 of gestation in a subsequent study (Rodier et al., 1984). Khera (1973) had demonstrated in cats that prenatal exposure results in an incomplete granular layer in the cerebellum as well as alterations in the cyto-
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architecture. Delayed myelination and delayed migration of the external granule cells were also described by Khera et al. (1974) and Khera and Tabacova (1973). The delayed migration of cells in the parietal cortex has also been described recently by Choi (1986). At the electron microscopic level, Chang (1 987) has reported that in the early stages of intoxication there is an increase in neuronal lysosomes associated with the disintegration of the rough endoplasmic reticulum, followed by focal cytoplasmic degradation and neuronal vacuolation. Behavioral studies in animals have confirmed the early observations on the human victims both in Minamata and later in Iraq (Amin-Zaki et al., 1976). Thus Eccles and Annau (1982a) showed that rats treated on gestational days 8 and 15 with either 5 or 8 mg/kg of MM showed altered development of motor activity in the pre-weaning period. This alteration was reflected in changes in the dopaminergic system (Cuomo et al., 1984). When tested as adults in a two-way avoidancelearning task, MM-exposed animals demonstrated a retardation not only in acquisition, but also in retention of this task. This latter effect was greatly increased when the mercury was injected on day 15 of gestation. An additional interesting finding reported by these investigators was the altered responsivenessof the mercury-treated animals to the psychoactive agent amphetamine (Eccles and Annau, 1982b). An earlier paper by Hughes and Sparber (1978) had reported similar results with amphetamine, and these authors suggested that one of the delayed effects of prenatal intoxication may be altered drug metabolism. It was not clear from the results of Hughes and Sparber, however, whether the altered behavioral sensitivity displayed by their rats was due to central or peripheral mechanisms. While the use of pharmacological challenges has become very popular in neurotoxicology (see Walsh and Tilson, 1986) as a way of determining the potential mechanism of neural injury, very little effort has been expended on determining the reason for the altered drug sensitivity. Hughes and Sparber
reported that the effect they observed was only apparent in males, thus raising the possiblity that hormonal influences may play a role in this delayed expression of neurotoxicity. In addition to the behavioral investigations of prenatal toxicity, both electrophysiological and neurochemical measurements have been made. These have been reviewed recently (Annau and Eccles, 1987; Komulainen and Tuomisto, 1987) and, while there seems to be no doubt that high doses of mercury will cause injury to the peripheral nervous system as well, the central nervous system is by far the most sensitive target. Using the visual evoked potential technique, Dyer et al. (1978) have shown that a single prenatal exposure to MM will alter the evoked potential recorded in the adult offspring. One of the interesting findings of this study was the shortening of the latencies of some of the components of the evoked potential, which the authors speculated could have been caused by the loss of small fibers because of the mercury treatment. In terms of neurochemical measurements following MM treatment, Komulainen and Tuomisto conclude that while the mercury does have potent inhibitory effects in vitro, in vivo the effects seem considerably less impressive. Thus, perinatal administration of MM has been shown to (1) interfere with neuronal migration, (2) delay and reduce myelination, and (3) arrest mitosis and reduce the number of neurons. These effects are correlated with (1) transient alterations in the development of motor activity, (2) reduced ability to learn and retain complex tasks, and (3) altered responsiveness to pharmacological agents, as well as to altered neurophysiological responses in the visual system. These effects have been relatively well defined in many experimental models and have confirmed that this chemical even at very low doses can cause irreversible damage to the immature nervous system, as can be seen in recent literature reviews (Eccles and Annau, 1987).
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Trimethyl tin Interest in research on the neurotoxicity of trimethyl tin (TMT) started with a paper published in 1979 by Brown et al., who described the behavioral and pathological changes seen in rats following either single or repeated doses of this organotin. The behavioral symptoms consisted of increased aggressiveness,vocalizations, tremors, seizures and hyperreactivity. These symptoms persisted for approximately two to three weeks and then subsided. Neuropathological examination of the brains of the exposed animals showed severe damage to the hippocampus (Fig. 2) and lesser damage to other parts of the limbic system such as the pyriform cortex and amygdala. Because this organotin seemed such an ideal ‘limbic system’ poison, this research paper was followed by extensive studies on the nature and mechanisms of toxicity. The observations of Brown et al. were confirmed and extended by subsequent studies (Dyer et al., 1982a). The dose-response function for this chemical was extremely steep for the rat, with the LD,, at about 7.3 mg/kg and the LD,, at 10 mg/kg. Following the injection of TMT, there was a mild hypothermia which persisted for about four days and was directly related to dose. Several behavioral alterationsoccurred within two days of injection. A fine tremor-at-rest was seen at the higher doses. Increased reactivity to handling which increased over time was also observed. Animals that showed this symptom also appeared to be likely to have seizures. The final behavioral morbidity appeared to be tail self-mutilation,with 50% of the animals showing this at the 7 mg/kg dose (Dyer et al., 1982b). Ruppert et al. (1983) showed that when placed in figure-eight maze, the animals dosed with 7 mg/kg became hyperactive after 24h and remained so up 50days after dosing. Not surprisingly, given the significant destruction of hippocampal pyramidal cells, several measures of learning and retention showed major alterations following TMT administration. h i -
mals exhibited retention deficits in a passive avoidance task which was evident at 5, 6 and 7 mg/kg and was not dose-related (Walsh et al., 1982a). At 7 mg/kg, rats were significantly impaired in a series of problem-solving tasks in the Hebb-Williams maze (Swartzwelder et al., 1982). The animals exhibited more errors, less error reduction over days and showed perseverative behavior that seemed characteristic of animals with hippocampal lesions. A further test of the hippocampal damage was also performed by
Fig. 2. Section through the hippocampus at normal rat (top) and rat exposed to 3 mgFg of TMT once a week for 3 weeks. Note extensive loss of pyramidal cells in areas CA3 and CA4. Taken from Valdes etal., 1983.
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Walsh et al. (1982b), who trained their animals in a radial-arm maze prior to TMT administration. Fifteen days after TMT dosing (6 mg/kg), the animals were retrained in the maze. There was a severe disruption of maze performance during retraining which persisted up to 70 days postdosing. Other measures of TMT-induced damage to the nervous system indicated that the pathology was not restricted to the limbic system. Dyer et al. (1982~)showed that when rats were implanted with chronic recording electrodes in the optic tract and the visual cortex, the TMT-treated rats exhibited an increase in latencies and decreases in amplitudes in the early peaks of the visual evoked potential at both electrode locations. These changes were suggestive of retinal damage. Other changes in the later components of the evoked potential were suggestive of altered arousal. A more detailed analysis of the seizure susceptibility of TMT-treated animals also showed that these animals were more susceptible to seizures induced by pentylenetetrazol, a general (not limbicsystem-specific) convulsant, indicating that the early hypothesis of TMT being a specific limbic system or hippocampal toxicant was not conh e d (Dyer et al., 1982d). Other areas of investigation included neurochemical studies of TMT-induced damage, as well as perinatal exposures. Valdes et al. (1983) showed that neurochemical alterations occured in intrinsic hippocampal neurotransmitters and not in extrinsic transmitters such as norepinephrine, and Ali et al. (1985) showed that there was a significant reduction in muscarinic cholinergic binding in whole brain and frontal cortex 2-7 days following treatment. In utero exposure, however, failed to show this neurochemical effect or the neuropathological pattern of adult TMT administration, suggesting that the immature nervous system was less sensitive to this toxicant than the fully developed nervous system (Paule et al., 1986). This was further confirmed by Lipscomb et al. (1986), who showed that the fetal brain concentration was identical to the maternal
brain concentration 96 h after exposure, although at 338 h the fetal brain levels were significantly lower than the maternal. Chang (1984) has also shown that administration of TMT up to postnatal day 4 causes relatively little pathology, but after that developmental day the pathology increases markedly, an observation also reported by Ruppert et al. (1983) Thus, while the most severe damage induced by TMT in the adult animal is clearly in the hippocampus, as revealed by behavioral and pathological studies, it has also become clear that many other parts of the nervous system are also involved in the pathology (Chang et al., 1982). It is of great interest that, unlike methyl mercury, trimethyl tin seems to be less toxic in the fetal brain than the adult brain, although during the neonatal period, at least in the rat, this lack of sensitivity to toxicity diminishes rapidly. Triethyl lead
The organo leads which have been used as gasoline additives in increasingly large quantities until recently (Grandjean and Nielsen, 1979)have been associated with many cases not only of accidental human intoxication (Beattie et al., 1972), but also of voluntary ingestion as ‘sniffing’ as an inexpensive drug of abuse (Valpey et al., 1978). The symptoms are clearly associated with central nervous system disorders and are characterized by disturbances of emotionality and memory as well as increased motor excitability, not unlike those seen after human TMT poisoning (Ross et al., 1981). A recent review of the neurotoxicology of organo leads (Walsh and Tilson, 1984), in fact, makes a strong case for the similarity in the neurobehavioral pathology between TMT and triethyl lead (TEL), the toxic metabolite of tetraethyl lead, the gasoline additive. Not only are the pathological lesions seen in the adult animal following TEL administration concentrated in the limbic system, particularly the hippocampus, but also the behavioral effects are in accordance with
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the expectations of this type of lesion. More specifically, the animals show improved two-way avoidance learning, and impaired passive avoidance and spatial learning. Further, chemical analyses have shown that the hippocampus concentrates lead and that the concentrations of essential elements such as copper and zinc are decreased following TEL exposure (Niklowitz and Yeager, 1973). The neonatal brain again appears to be exquisitely sensitive to this neurotoxic agent (Booze et al., 1983). Konat (1984) has shown that administration of TEL to neonatal rats results in very significant hypomyelination because oligodendrocytesappear to be particularly targeted. This suggests that TEL, while having many similaritiesto TMT in its effect on the limbic system, may yet have its own unique toxicity, at least in the neonatal brain.
Mechanisms of toxicity The biochemical and metabolic changes caused by methyl mercury administration have been reviewed recently by Chang (1987). Following mercury administration, the incorporation of amino acids into the nervous system is reduced significantly (Yoshimo et d., 1966) resulting in decreased protein synthesis. In addition, Verity et al. (1977) have suggested that a disruption of mitochondrial respiration may also inhibit synaptosomal protein synthesis, and further studies (Frenkel and Harrington, 1983) have shown that DNA and RNA synthesis in isolated mitochondria is inhibited. Finally, Sager et al. (1983) have reported microtubule disruption by methyl mercury. These studies indicate that mercury may disrupt all levels of the nervous system, particularly in the developing organism, and may explain the relative sensitivity of the organism to its toxicity. In both triethyl lead and trimethyl tin intoxication protein synthesis inhibition seems to play a major role in the development of pathology. Recently Costa and Sulaiman (1986) have shown that following TMT administration protein syn-
thesis is decreased significantly. Previous studies have shown that TMT inhibits oxidative phosphorylation (Aldridge and Street, 1971), and Costa and Sulaiman have suggested that this may indirectly account for the inhibition of protein synthesis. Konat (1986) has reviewed the triethyl lead toxicity literature and has indicated that this chemical also inhibits protein synthesis, not only at the neuronal level, but also at the level of myelin formation, where it inhibits proteins involved in membrane assembly. It appears therefore that all of these organic metals have certain common properties that account for their toxicities. While the neuropathologies of TEL and TMT have certain similarities in the brain regions affected, the neuropathology of MM is clearly very different, suggestingthat considerably more complex underlying biochemical mechanisms are involved in their individual toxicities.
Summary and conclusion
This brief review of the neurotoxicity of three organometals has shown that each metal seems to have its own pattern of toxicity which changes as a function of developmental stage of the brain. Methyl mercury in the fetal brain may be the most indiscriminately toxic compound because of its cytotoxicity. The nature of the neurotoxicity of prenatal exposure to TEL remains to be elucidated but in the neonatal period it may have both glial and neuronal targets. Finally, TMT at this time seems relatively non-toxic in the fetal period, but becomes extremely toxic to limbic system neurons in the neonatal period. The sensitivity of the hippocampus to a variety of environmental insults has puzzled investigators for many years, and even though, as has been pointed out above, there is some evidence that it concentrates lead (both organic and inorganic forms) this does not account for the total pathology seen. The relationship between behavioral and neurobiological indices of toxicity is becoming closer as we learn to use our knowledge regarding
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brain lesions and behavioral effects and apply specific behavioral techniques derived from this literature to the testing of neurotoxicity of environmental chemicals (see Wenk and Olton, 1986). It is also quite evident that as we descend the doseresponse curve of neurotoxic chemicals, we continue to uncover additional evidence of subtle alterations of nervous system dysfunction. These subtle, often ‘latent’ effects of perinatal intoxication have rarely been investigated in human populations. Needleman (1986) was one of the first to call attention, in the case of inorganic lead exposure, to such effects by developing the techniques to measure them. As we become more concerned with both the consequences of environmental hazards and widespread drug abuse we have to increase our efforts to establish the noeffect dose for all chemicals. It is our hope that through the continued investigations of animal models of perinatal neurotoxicity and concurrent human studies, we will ultimately be able to safeguard the health of human populations. References Aldridge, W.N. and Street, B.W. (1977) The relation between the specific binding of trimethyltin and triethyltin to mitochondria and their effects on various mitochondrial functions. Biochem. J., 124: 221-234. Ali, S . F., Slikker, W., Jr., Newport, G. D. and Goad, P.T. (1985) Cholinergic and monoaminergic alterations in the mouse central nervous system following acute trimethyltin exposure. Acta Pharmacol. Toxicol., 59: 179-188. Amin-Zaki, L., Ehassani, S., Majeed, M. A., Clarkson, T. W., Doherty, R.A., Greenwood, M.R. and GiovanoliJakubczak, T. (1976) Perinatal methyl mercury poisoning in Iraq. Am. J. Dis. Child., 130: 1070-1076. Annau, Z. and Eccles, C. U. (1987) Sensory deficits caused by exposure to methyl mercury. In C.U. Eccles and Z . Annau (Eds.), The Toxicity of Methyl Mercury, The Johns Hopkins University Press, Baltimore, pp. 104-1 13. Beattie, A.D., Moore, M.R. and Goldberg, A. (1972) Tetraethyl lead poisoning. Lancet, ii: 12-1 5. Booze, R.M., Tilson, H. A., Annau, Z. and Mactutus, C.F. (1983) Neonatal triethyl lead neurotoxicity in rat pups: Initial observations and quantification. Neurobehav. Toxicol. Teratol., 5: 367-315. Brown, A. W., Aldridge, W. N., Street, B. W. and Verschoyle, R.D. (1979) The behavioral and neuropathological
sequelae of intoxication by trimethyltin compounds. Am. J. Pathol., 104: 59-82. Chang, L. W. (1984) Trimethyltin induced hippocampal lesions at various neonatal ages. Bull. Environ. Contam. 33: 295-301. Tox~co~.., Chang, L.W. (1987) The experimental neuropathology of methyl mercury. In C.U. Eccles and Z. Annau (Eds.), The Toxicity of Methyl Mercury, The Johns Hopkins University Press, Baltimore, pp. 54-72. Chang, L.W. and Annau, Z. (1984) Developmental neuropathology and behavioral teratology of methyl mercury. In J. Yanai (Ed.), Neurobehavioral Teratology, Elsevier, Amsterdam, pp. 405-432. Chang, L. W., Tiemeyer, T.M., Wenger, G. R., McMillan, D.E. and Reuhl, K.R. (1982) Neuropathology of trimethyltin intoxication. I. Light microscopy study. Environ. Res., 29: 435-444. Choi, B. H. (1986) Methylmercury poisoning of the developing nervous system: I. Pattern of neuronal migration in the cerebral cortex. Neurotoxicology, 7: 591-600. Chyle, J.T., McGeer, E. G., McGeer, P. L.and Schwarz, R. (1978) Neostriatal injections: a model for Huntington’s chorea. In E.G. McGeer, J. W. Olney and P. L. McGeer (Eds.), Kainic Acid as a Tool in Neurobiofogv,Raven Press, New York, pp. 139-159. Costa, L. G. and Sulaiman, R. (1986) Inhibition of protein synthesis by trimethyltin. Toxicol. Appl. Phannacol., 86: 189-196. Cuomo, V., Ambrosi, L., Annau, Z., Cagiano, R.,Brunello, N. and Racagni, G. (1984) Behavioral and neurochemical changes in offspring of rats exposed to methyl mercury during gestation. Neurobehav. Toxicol. Teratol.,6: 249-254. Dyer, R.S., Eccles, C.U. and Annau, Z. (1978) Evoked potential alterations following prenatal methyl mercury exposure. Phannacol. Biochem. Behav., 8: 127-141. Dyer, R.S.,Walsh, T. J., Swartzwelder, H. and SandWayner, M.J. (Eds.) (1982a) The neurotoxicology of the alkyltins. Neurobehav. Toxicol. Teratol., 4: 125-278. Dyer, R. S.,Walsh, T. J., Wonderlin, W. F. and Bercegeay, M. (1982b) The trimethyltin syndrome. Neurobehav. Toxicol. Teratol., 4: 127-133. Dyer, R. S., Howell, W.E. and Wonderlin, W.F. (1982~) Visual system dysfunction following acute trimethyltin exposure in rats. Neurobehav. Toxicol. Teratol., 4: 191-196. Dyer, R.S., Wonderlin, W.F. and Walsh, T.J. (1982d) Increased seizure susceptibility following trimethyltin administration in rats. Neurobehav. Toxicol. Teratol., 4: 203-208. Eccles, C. U. and Annau, Z. (1982a) Prenatal methyl mercury exposure: I. Alterations in neonatal activity. Neurobehav. Toxicol. Teratol., 4: 371-376. Eccles, C.U. and Annau, Z. (1982b) Prenatal methyl mercury exposure: 11. Alterations in learning and psychotropic drug sensitivity in adult offspring. Neurobehav. Toxicol. Teratol., 4: 317-382.
302 Eccles, C.U. and Annau, Z. (Eds.) (1987) The Toxicity of Methyl Mercury, The Johns Hopkins University Press, Baltimore. Frenkel, G. D. and Harrington, L. (1983) Inhibition of mitochondrial nucleic acid synthesis by methyl mercury. Biochem. Pharmacol., 32: 1454-1456. Grandjean, P. and Nielsen, T. (1979) Organolead compounds: environmental health aspects. Residue Rev., 72: 97-148. Harada, M. (1976) Minamata disease. Chronology and medical report. BuU. Inst. Constitut. Med. Kumamoto Univ., 25: Suppl. 1-60. Hughes, J.A. and Sparber, S.B. (1978) d-Amphetamine unmasks postnatal consequences of exposure to methyl mercury. Pharmacol. Biochem. Behav., 4: 385-301. Khera, K. S. (1973) Teratogenic effects of methyl mercury in the cat: note on the use of this species as a model for teratogenicity studies. TeratoZogy, 8: 293-303. Khera, K. S. and Tabacova, S.A. (1973) Effects of methylmercuric chloride on the progeny of mice and rats treated before or during gestation. Food Cosmet. Toxicol. 11: 243-254. Khera, K. S., Iverson, F., Hierliiy, L., Tanner, R. and Trivett, G. (1974) Toxicity of methyl mercury in neonatal cats. Teratology, 10: 69-76. Kojima, K. and Fujita, M. (1973) Summary of recent studies in Japan on methyl mercury poisoning. ToxicoZlogv, 1: 43-62. Komulainen, H. and Tuomisto, J. (1987) The neurochemical effects of methyl mercury in the brain. In C. U. Eccles and Z. Annau (Eds.), The Toxicityof MethyZ Mercury,The Johns Hopkins University Press, Baltimore, pp. 172-189. Konat,. G. (1984) Triethyllead and cerebral development: an overview. Neurotoxicology, 5 : 87-96. .angston, J.W., Forno, L.S., Rebert, C. S. and Irwin, I. (1984) Selective nigral toxicity after systemic administration of l-methyl4pheny-l,2,5,6-tetrahydropyridine (MPTP) in the squirrel monkey. Brain Res., 292: 390-394. ipscomb, J.C., Paule, M.G. and Slikker, Jr., W. (1986) Fetomaternal kinetics of ''C-trimethyltin. Neurotoxicology, 7: 581-590. Mottet, K. N., Shaw, C.-M. and Burbacher, T.M. (1987) The pathological lesions of methyl mercury intoxication in monkeys. In: C.E. Eccles and Z . Annau, (Eds.), The Toxicity of Methyl Mercury, The Johns Hopkins Press, Baltimore, pp. 73-103. Needleman, H.L. (1986) Epidemiological studies. In Z. Annau (Ed.) Neurobehavioral Toxicology, The Johns Hopkins University Press, Baltimore, pp. 279-287. Niklowitz, W. J. and Yeager, D. W. (1973) Interference of Pb with essential brain tissue Cu,Fe and Zn as main determinant in experimental tetraethyllead encephalopathy. L$e Sci., 13: 897-905. Paule, M. G., Reuhl, K., Chen, J. J., Mi, S. F. and Slikker, Jr.,
W. (1986) Developmental toxicology of trimethyltin in the rat. Toncol. Appl. Pharmacol., 84: 412-417. Rodier, P.M., Aschner, M. and Sager, P.R. (1984) Mitotic arrest in the developing CNS after prenatal exposure to methyl mercury. Neurobehav. Toxicol. Teratol., 6: 379-386. Ross, W.D., Emmett, E.A., Steiner, J. and Tureen, R.(1981) Neurotoxic effects of occupational exposure to organotins. Am. J. Psychiatry, 138: 1092-1095. Ruppert, P. H., Dean, K. R. and Reiter, L. W. (1983) Developmental and behavioral toxicity following acute postnatal exposure of rat pups to trimethyltin. Neurobehav. Toxicol. Teratol., 5: 421-429. Ruppert, P.H., Walsh, T.J., Reiter, L.W. and Dyer, R.S. (1982) Trimethyltin-induced hyperactivity: time course and pattern. Neurobehav. Toncol. Teratol., 4: 135-140. Sager, P. R., Doherty, R. A. and Olmsted, J. B. (1983) Interaction of methylmercury with microtubles in cultured cells and in vitro. Exp. Cell Res., 146: 127-137. Sager, P. R., Doherty, R. A. and Rodier, P.M. (1982) Effects of methyl mercury on developing mouse cerebellar cortex. Exp. Neurol., 77: 179-193. Spyker, J.M., Sparber, S.B. and Goldberg, A.M. (1972) Subtle consequences of methylmercury exposure: behavioral deviations in offspring of treated mothers. Science, 177: 621-623. Swartzwelder, H. S., Hepler, J., Holahan., W., King, S.E., Leverenz, H.A., Miller, P.A. and Myers, R.D. (1982) Impaired maze performance in the rat caused by trimethyltin treatment: problem-solving deficits and preservation. Neurobehav. Toxicol. Teratol., 4: 169-176. Valdes, J. J., Mactutus, C.F., Santos-Anderson, R., Dawson, Jr., R. and Annau Z. (1983) Selective neurochemical and histological lesions in rat hippocampus following chronic trimethyltin exposure. Neurobehav. Toxicol. Teratol., 5 : 357-361. Valpey, R., Sumi, S.M., Dopass, M.K. and Goble, G.J. (1978) Acute and chronic progressive encephalopathy due to gasoline sniffing. Neurology, 28: 345-350. Verity, M.A., Brown, W.J., Cheung, M. and Czer, G. (1977) Methyl mercury inhibition of synaptosome and brain slice protein synthesis: in vivo and in vitro studies. J. Neurochem., 29: 673-679. Walsh, T.J. and Tilson, H.A. (1984) Neurobehavioral toxicology of organoleads. Neurotoxicology, 5: 67-86. Walsh, T.J. and Tilson, H.A. (1986) The use of pharmacological challenges. In Z. Annau (Ed.), Neurobehavioral Toxicologv, The Johns Hopkins University Press, Baltimore, pp. 244-267. Walsh, T., Gallagher, M., Bostock, E. and Dyer, R. S. (1982a) Trimethyltin impairs retention of a passive avoidance. Neurobehav. ToncoZ. TeratoZ., 4: 163-168. Walsh, T.J., Miller, D.B. and Dyer, R.S. (1982b) Trimethyltin, a selective limbic system toxicant, impairs radial-arm maze performance. Neurobehav. Toxicol. Teratol., 4: 177-184.
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Discussion C.V.Vorhees: You raised an important issue about the possible changes in effects over time and the need for longitudinal analyses. Have you examined your methylmercury animals to see if the same animals that show altered activity were the same ones that showed the most disrupted avoidance performance? Z. Annau: Unfortunately at the time we performed these experiments we did not keep track of the animals over the entire period and therefore this type of analysis was not available. H.J. Romijn: Is tin as a metal just as toxic as its organic compound? If not, why have you chosen the organic compound in your studies? Z. Annau: Tin as a metal is not considered very toxic. We chose trimethyl tin because it is a widely used chemical (in anti-fouling paints on boats, and as plasticizers in the plastics industry) and because of several human accidental exposures that showed this form of tin to be extremely neurotoxic. B.E. h n a r d : Trimethyl tin is suggested to be a limbic toxin. Bernadette Easton in my laboratory has recently shown that, following a single dose of this, there is a dose-dependent
change in choline acetyltransferase and muscarinic receptor density in many regions outside the limbic areas. Regarding the behavioral defects after trimethyl tin, a major abnormality occurs in spatial learning at doses that have little effect on most other behavioral parameters. Would you like to comment on these points? Z. Annau: It is clear from the recent trimethyl tin studies that it is not a specific limbic system poison, but that it also causes lesions in many other parts of the brain. Given its effects on the cholinergic system and the input of that system into septal-hippocampal pathways, it is not too surprising that you see spatial learning task deficits. C.J. Boer: Is there any idea at what neurochemicd level the organometals are acting to exert their deleterious effects? Z. Anaru: This in one of the areas of research that has not been exploited fully. Of course, each metal has a different mechanism of toxicity, and seems to interfere at several neurotransmitter systems. T.A. Slotkin: How do you interpret changes in postsynaptic receptors and receptor-mediated responses without evaluating presynaptic function? Receptor up-regulation might represent a compensation for presynaptic hypofunction, thus producing a synapse wich operates normally. Z. Anaru: I agree that, based on our data, it is impossible to determine the exact location of the alteration, but that the postsynaptic alteration may be one such possible mechanism. P.M. Rodier (comment): We have been able to measure Hg in all our experiments and I agree with you that much (probably most) of the variance we see in response to methyl mercury is due to differences in kinetics from animal to animal. We are constantly surprised at the difference in brain levels from the same dose!