Progress in Histo- and Cytochemistry, Vol. 23
W.Graumann, J.Drukker (Eds.), Histo- and Cytochemistry as aTool in Environmental Toxicology. © Fischer Verlag· Stuttgart· New York· 1991
8 Neurotoxicity 8.1 The effect of pre- and postnatal exposure to organic solvents on the development of the cerebellar cortex in the rat GISELA STOLTENBURG-DIDINGER Institute of Neuropathology, Klinikum Steglitz, Freie Universitiit, Berlin (FRG)
Introduction The cerebellum of the neonatal rat presents a unique opportunity for the study of neuronal maturation. This structure is comparatively immature at birth (ADDISON 1911) and develops rapidly during the first month of extrauterine life (ALTMAN 1972). Moreover, the adult cerebellar cortex possesses well known and characteristic anatomical features which facilitate developmental compansons. The cerebellum has been subjected to a large number of enzyme histochemical studies (FRIEDE 1957; BARGETON-FARKAS and PEARSE 1965; KUCKUK 1967; ALTMAN 1972). The enzyme maturation pattern of the cerebellum was studied as correlated with its morphology. This could best be accomplished by enzyme histochemistry which allows investigation of enzyme activities at the cellular level during development. The purpose of enzyme histochemistry is the localization of specific enzyme activities to intact tissue components. The characterization and quantitation of the enzyme activities mediating the histochemical reaction is as integral a function of this discipline as the localization of the activities themselves. Enzyme histochemical studies on normal rat cerebellum have indicated a high and early activity of enzymes involved in anaerobic metabolism, while enzymes involved in aerobic metabolism increase rapidly after birth. The aerobic pathway soon becomes quantitatively the most important one in the postnatal cerebellum (ROBINS and LOWE 1961; BARGETON-FARKAS and PEARSE 1965). The external and internal granular cells exhibit weak oxidative enzyme activity at all ages. The Purkinje cells show increasing oxidative enzyme activity from birth, reaching adult activity at the end of the 4th week in normal ratss. The aim of the present study was to investigate whether any differences occur in the enzyme maturation pattern between solvent-exposed and normal rat cerebellum as correlated with changes in its morphology, caused by pre- and postnatal exposure to hexacarbons.
228 . G. Stoltenburg-Didinger Although the neurotoxic effects of organic solvents in the adult organism have been well studied, there is a paucity of information on the impact of in utero exposure to solvents on the developing brain (ALTENKIRCH et al. 1978; 1982a, b; SPENCER and SCHAUMBURG 1975; STOLTENBURG-DJDINGER and ALTENKIRCH 1988). Animal studies demonstrate that a variety of solvents readily cross the placenta (PELKONEN 1985) and that maternal inhalation of various solvents results in neurodevelopmental deficits in the newborn rodent (Bus et al. 1979; MARKS et al. 1980; DEACON et al. 1981). In utero exposure to organic solvents may also affect the development of the human brain (TOUTANT and LIPPMANN 1979; HOLMBERG et al. 1980; HERSH et al. 1985, ESKENASI et al. 1988). The primary fissure of cerebellar vermis was used as an enzymatically and morphologically defined and homogeneous model system of an early maturing region (LARSELL 1952; KUCKUK 1967; ALTMAN 1972). A unique property of the commonly-used histochemical oxidation-reduction indicators, the tetrazolium salts, is the reversible binding in their oxidized tetrazole form to cytoplasmic components of almost all cells (ADAMS 1965). The binding of the tetrazolium salt to tissue components permits electron transport in situ to form the insoluble colored reduced product, the formazan, near the site of enzymatic activity.
Materials and methods Animals: Virgin rats of the Wistar strain, 3 months of age, were mated with males varying in age from 4 to 8 months. The time of fertilization was determined by vaginal smears taken daily. The mode of inhalation was 23 h a day, 7 days a week. Controls were kept under the same conditions but without solvent exposure. The solvent was pumped on a glass frit through which filtered air was conducted. The hexane concentration was monitored with a continously measuring flame ionization detector. In the first solvent experiment only n-hexane was used, and the animals were exposed to low concentrations (500ppm) during prenatal development (21 days). In the second solvent experiment we used higher concentrations and exposed one group only prenatally (21 days) and a second group also postnatally (42 days) in order to include the growth spurt of the cerebellum into the exposure period. n-Hexane and MEK were studied in this experiment. In the third solvent experiment we used even higher concentrations (1000 ppm, initially 1500 ppm). There were likewise two groups: one was exposed prenatally (21 days), the other pre- and postnatally (51 days). The substances used were n-hexane (Merck, Darmstadt, nr.4367 99%), methyl-ethyl-ketone (Merck, Darmstadt, nr.6014 99%) and a mixture of both (hex/MEK = 1200:300). The newborn rats were examined for effects on body and brain maturation, weight development, fitness for survival and the possible occurrence of deformities. Preparation: All rats were decapitated. The skull was opened immidiately with scissors. Cerebellum and cerebrum were lifted out. Each cerebellar hemisphere was cut sagittally with a razor blade and each opposing half hemisphere was mounted onto a cork, then immidiately quenched in liquid nitrogen. All cryostat blocks were stored at a temperature of at least -20°C and sectioned at the same temperature in 7!!m thick sections. Sections were immidiately incubated for enzyme histochemistry of succinic dehydrogenase (SDH) and NADH tetrazolium reductase (NADH-Tr). All enzyme histochemical reactions were performed according to the methods described by NOVIKOFF (1960). Nitro Blue Tetrazolium (NBT) was used as hydrogen acceptor. All incubation solutions were freshly prepared. The chemicals were obtained from Sigma Chemical Company and from E. Merck AG, Darmstadt. All incubations were made for 30 min at a temperature of 37°C (SDH) or 20°C for NADH-Tr respectively. Paired substrate-free sections were used as negative controls in order to exclude nonspecific reduction of NBT. The enzyme histochemical activity as revealed by its formazan deposition was studied in the primary fissure of the cerebellar vermis at postnatal days 1, 9 and 21.
Organic solvents and cerebellar cortex . 229
Results Differences in formazan deposition between SDH and NADH-Tr activity could not be detected. Because of the finer granules of the reaction product figures were selected from NADHTr. Normal development. Activity of the oxidative enzymes SDH and NADH-Tr as visualized by formazan deposition was present already at birth in Purkinje cells. All activity was located in the cytoplasm. The activity at birth appeared weak, then gradually increased, by the third day strong SDH and NADH-Tr activity was present in the bodies of Purkinje cells. The layer of Purkinje cells remained the only one with marked activity until the ninth day (Fig. 2), when dendrites with marked oxidative activity grow into the molecular layer (Figs. 2 a, 3a). The external granular cells showed a low activity, which appeared as a thin rim of perikaryal activity, equal in the proliferative and premigratory zones. As the molecular layer expanded and regression of the external granular layer continued, NADH-Tr was seen closer to the subpial surface, reflecting the outgrowth of Purkinje cell dendrites, thereby providing a clear picture of the Purkinje cell dendritic tree (Fig. 3a). At day 9 the first faint trace of oxidative activity was seen in the internal granular cells. This activity remained low to moderate at all ages into adult life. Development of solvent-exposed rats. The development of SDH and NADH-Tr activity paralleled that of normal rats with a delay. After prenatal exposure only the activity of the Purkinje cell apical cones was higher at day 9 compared to normal rats, reflecting the delayed outgrowth of the Purkinje cell apical dendritic tree (Fig. 3 b). After day 21, both groups showed equal formazan deposition in the Purkinje cells. No differences in SDH and NADH-Tr activity between prenatally exposed and normal rats could be seen either in the external and internal granular cells. After pre- and postnatal exposure, the Purkinje cells of exposed rats showed a higher SDH and NADH-Tr activity at day 9 than those of normal rats. Pre- and postnatal exposure to n-hexane resulted in a persisting apical cone and delayed formation of the apical dendritic tree of the Purkinje cells in the cerebellum from a 9-day-old rat (Fig. 3c). After pre- and postnatal exposure to the mixture of n-hexane and methyl-ethyl-ketone (MEK), the retardation of cell maturation was even more pronounced (Fig. 3d). At day 9, the Purkinje cells showed persisting maximal perikaryal formazan coloration, indicating a high, concentrated NADH-Tr activity. Furthermore, the difference in width of the molecular layer between pre- and postnatally exposed and normal rats was greater as a result of the retarded apical dendrite formation (Fig. 3a-d). This could be demonstrated by enzyme histochemistry much more impressing than by conventional hematoxylin-eosin-staining (Figs. 1 and 2). All solvents studied delayed the histogenesis of the cerebellar cortex in the experimental animals at all concentrations examined (Figs. 1,2, 3a-d). Even at the lowest concentration, applied only prenatally as in the first solvent experiment, the histological preparations of frozen sections of the fissura prima of the vermis cerebelli showed a delay in migration of the outer granular cells and a persistence of Purkinje cells at a lower stage of development (Figs. 1b, 2 b, 3 b). Again it was proven that postnatal exposure aggravated the developmental delay (Figure 1c, 2c, 3c). Methylethyl-ketone (MEK) potentiated n-hexarie-neurotoxicity (Figs. 1d, 2d, 3d).
230 . G. Stoltenburg-Didinger
2d Fig. 1. Frozen sections of rat cerebellum, sagittal view of fissura prima of vermis (early developing region), third solvent experiment, postnatal day 9, HE. - x 300. a: Control; b: MEK prenatal exposure; c: n-Heyane, prenatal exposure; d: n-Hexane/MEK, pre- and postnatal exposure Note differences in thicknes of outer granular layer and molecular layer as well as differences in cell density in inner and outer granular layer. Fig. 2. Specimen identical to Fig. 1, NADH-Tr. - x 300. Note differences in cell size and staining intensity of Purkinje cells.
Organic solvents and cerebellar cortex . 231
Discussion The biological oxidation mechanisms, whereby electrons and frequently hydrogen are transferred through one or more intermediates to hydrogen, afford the major energy source of the living cell. Within the mitochondrion the electron carrier components are bound to one another (ADAMS 1965). The activity of oxidative enzymes in a given region may be considered an approximate parameter of the intensity of the oxidative metabolism (WOODWART et al. 1969). A comparison of these patterns provides a quite reliable picture of the functional activity of a given region (FRIEDE and PAX 1961). In rats prenatally exposed to n-hexane or methyl-ethyl-ketone (MEK), a delay in the development of the high oxidative enzyme activity of the Purkinje cell apical cone was noticed. This retarded increase in enzyme activity was followed by a delayed decrease in activity of the Purkinje cell apical cone parallel to the delayed formation and differentiation of the Purkinje cell apical dendritic tree (SIMA and PERSSON 1975). It seems reasonable to suppose that the delayed increase of enzyme activity in the apical cone prior to the dendritic formation and the delayed decrease of activity in the Purkinje cell apical cone concomitant to the retarded development of the dendritic tree may be related to inhibition of glycolysis by n-hexane and its main metabolite 2,5-hexanedione (SABRI et al. 1979; SPENCER et al. 1979; SPENCER et al. 1980). This molecular mechanism is yet not the only mode of action (DE CAPRIO 1985). Toxicity of n-hexane is mediated by 2,5hexane-dione, its main metabolite, which plays a major role in the neurotoxicity of this compound. Neurotoxicity of n-hexane depends on the toxicogenic transformation in the organism, enzyme induction having an additive effect. In the immature liver of fetuses and newborn animals, the biotransformation systems are, at any rate, developed only incompletely or not at all. Thus, the newborn animals receive a loading with diketones via the placenta or the mother's milk. Since active metabolization of hexacarbons to diketones is not possible in young animals, they do not develop any clinically detectable peripheral paralyses (STOLTENBURG-DIDINGER et al. 1990). Thus the peripheral nervous system of adult rats seems to be more vulnerable to solvents than the immature one, possibly because of its paucity in neurofilaments as well. It is concluded that prenatal solvent exposure causes a retarded enzymatic development of the molecular and internal granular layers as well as of the Purkinje cells in rat cerebellum, followed by catch-up growth and maturation. This is consistent with other investigations where mild or missing developmental deficit was found after solvent exposure during pregnancy (Bus et al. 1979; MARKS et al. 1980; DEACON et al. 1981; BHATT et al. 1988). The persistent delay in enzymatic development after pre- and postnatal exposure to organic solvents parallels a morphological retarded development of cerebellar structures and can be compared to the effect of malnutrition (STOLTENBURG-DIDINGER et al. 1990). A retarded differentiation of neurons in early undernutrition has been observed in cerebellum. PERSSON and SIMA (1975) demonstrated a delay in the formation and enzymatic maturation of the Purkinje cell apical dendritic tree. After postnatal exposure to hexacarbons there is no catch-up growth because the period of the growth spurt of the cerebellum is the most vulnerable (DOBBING and SANDS 1971). It might therefore be assumed that the delayed differentiation and migration of the external granular cells as well as the delayed outgrowth of the Purkinje cell dendritic tree results in a delayed synaptogenesis and, due to permanent loss of cerebellar cortical neurons, probably also a permanent loss of synapses. To prove this by other than enzyme histochemical methods remains a considerable challenge.
232 . G. Stoltenburg-Didinger
3c
Organic solvents and cerebellar cortex . 233
References ADAMS, C. W. M.: Neurohistochemistry. - Elsevier, Amsterdam 1965. ADDISON, W. H. F.: The development of the Purkinje cells and of the cortical layers in the cerebellum of the albino rat. - J. compo Neurol. 21, 459-488 (1911). ALTENKIRCH, H., STOLTENBURG, G., WAGNER, H. M.: Experimental studies on hydrocarbon neuropathies induced by Methyl-Ethyl-Ketone (MEK). - J. Neurol. 219, 159-170 (1978). ALTENKIRCH, H., WAGNER, M., STOLTENBURG, G., SPENCER, P. S.: Nervous system responses of rats to subchronic inhalation of n-hexane and n-hexane and methyl-ethyl-ket"llc mixtures. - J. Neurol. Sci. 57, 209-219 (1982 a). ALTENKIRCH, H., WAGNER, H. M., STOLTENBURG-DIDINGER, G., STEPPAT, R.: Potentiation of hexacarbonneurotoxicity by methyl-ethyl-ketone (MEK) and other substances: Clinical and experimental aspects. Neurobehav. Toxicol. Teratol. 4, 623-627 (1982b). ALTMAN, J.: Postnatal development of the cerebellar cortex in the rat II Phases in maturation of Purkinje cells and of the molecular layer. - J. compo Neurol. 145, 399-464 (1972). BARGETON-FARKAS, E., PEARSE, A. G. E.: Aspects histo-enzymologiques de la maturation du systeme nerveux. - J. Neurol. Sci. 2, 213 (1965). BHATT, A., KHAN, S., PANDYA, K. P., SABRI, M. 1.: Effect of hexacarbons on selected lipids in developing rat brain and peripheral nerves. - J. appl. Toxicol. 8, 53-57 (1988). Bus, J. S., WHITE, E. L., TYL, R. W., BARROW, C. S.: Perinatal toxicity and metabolism of n-hexane in Fischer-3H-rats after inhalation exposure during gestation. - Toxicol. appl. Pharmacol. 51, 295-302 (1979). DEACON, M. M., PILNY, M. D., JOHN, J. A., SCHWETZ, B. A., MURRAY, F. J., YAKEL, H. 0., KUNA, R. A.: Embryo- and fetotoxicity of inhaled methyl-ethyl-ketone in rats. - Toxicol. appl. Pharmacol. 59, 620-622 (1981). DE CAPRIO, A. P.: Molecular mechanisms of diketone neurotoxicity. - Chern. BioI. Interact. 54, 257-270 (1985). DOBBING, J., SANDS, J.: Vulnerability of developing brain. IX. The effect of nutritional growth retardation on the timing of the brain growth spurt. - BioI. Neonat. 19, 363-378 (1971). ESKENASI, B., GAYLORD, L., BRACKEN, M. B., BROWN, D.: In utero exposure to organic solvents and human neurodevelopment. - Dev. Med. Child Neurol. 30,492-501 (1988). FRIEDE, R. L.: Die histochemische Reifung des Kleinhirns der Ratte dargestellt durch das Verhalten der Succinatdehydrogenase. - Arch. Psych. Nervenkr. 196, 196-204 (1957). FRIEDE, R. L.: Topographic Brain Chemistry. - Academic Press, New York 1966. FRIEDE, R. L., PAX, R. A.: Mitochondria and mitochondrial enzymes. A comparative study of localization in the eat's brainstem. - Histochemie 2, 186-191 (1961). HERSH,J. H., PODRUCH, P. E., ROGERS, G., WEISSKOPF, B.: Toluene embryopathy. - J. Pediatr.106, 922-927 (1985).
Fig. 3. Frozen sections of rat cerebellum, sagittal view of fissura prima of vermis, third solvent experiment (1500-1000 ppm), postnatal day 9, NADH-Tr. - x500. a: Control. Purkinje cells of normal rat cerebellum with clear resolution of the apical cones and advancing apical dendrite formation. The dendrites divide into tertiary and quartenary branchlets. The perikaryal formazan deposition is submaximal. b: n-Hexane., prenatal exposure. Note the persisting apical cone with maximal activity. Retarded development of apical dendrites is seen, resulting in a delayed decrease of the apical cone NADH-Tr activity c: n-Hexane, pre- and postnatal exposure. Intense NADH-Tr activity confined to the perikarya of the Purkinje cells. Note the absence of apical cone formation and persistence of numerous somatic dendrites of the Purkinje cells, contrary to the finding in normal rats d: n-Hexane/MEK, pre- and postnatal exposure. Purkinje cells demonstrating high to moderate activity mostly confined to the apical cone and perikaryon. Badly developed dendritic tree, very thin molecular layer
234 . G. Stoltenburg-Didinger HOLMBERG, P. c., NURMINEN, M.: Congenital defects of the central nervous system and occupational factors during pregnancy. A case referent study. - Amer.]. into Med. 1, 167-176 (1980). KUCKUK, B.: Dber die Entwicklung und Chemodifferenzierung des Kleinhirns der Ratte. - Histochemie 9, 217-255 (1967). LARSELL, 0.: The morphogenesis and adult pattern of lobules and fissures of the cerebellum of the white rat.J. compo Neurol. 97, 281-356 (1952). MARKs, T. A., FISHER, P. W., STAPLES, R. E.: Influence of n-hexane on embryo and fetal development in mice. - Drug Chern. Toxicol. 3,393-406 (1980). NOVIKOFF, A. B.: Biochemical and staining reactions of cytoplasmic constituents. - In: Developing Cell Systems and Their Control. - Ronald Press, New York 1960. PELKONEN, 0.: Fetoplacental chemistry - xenobioptic metabolism and pharmacokinetics. - In: Occupational Hazards and Reproduction (HEMMINKI, K., SORSA, M., VAINIOO, H., eds.). - Hemisphere, Washington 1985. PERSSON, L., SIMA, A.: The effect of pre- and postnatal undernutrition on the development of the cerebellar cortex in the rat II Histochemical observations. - Neurobiology 5, 151-166 (1975). ROBINS, E., LOWE, P.: Quantitative histochemical studies of the morphogenesis of the cerebellum. 1. Total lipid and four enzymes. - J. Neurochem. 8, 81-95 (1961). SABRI, M. 1., MOORE, C. L., SPENCER, P. S.: Studies on the biochemical basis of distal axonopathies. - J. Neurochem. 32,683-689 (1979). SIMA, A., PERSSON, L.: The effect of pre- and postnatal undernutrition on the development of the cerebellar cortex of the rat I Morphological observations. - Neurobiology 5, 23-34 (1975). SPENCER, P. S., SCHAUMBURG, H. H.: Experimental neuropathy produced by 2,5- hexanedione. - A major metabolite of the neurotoxic industrial solvent methyl-n-buryl ketone. - ]. Neurol. Neurosurg. Psych. 38, 771 (1975). SPENCER, P. S., SCHAUMBURG, H. H., SABRI, M., VERONESI, B.: The enlarging view of hexacarbon neurotoxicity. - Crit. Rev. Toxicol. 7, 279-356 (1980). STOLTENBURG-DIDINGER, G., ALTENKIRCH, H.: Neurotoxic effects of hexacarbons (n-hexane, methyl-nbutyl-ketone, 2,5-hexanedione, 1,4-diketones). - In: Nervous System OONES, T. c., MOHR, V., HUNT, R. D., eds.) pp. 32-41. - Springer, Berlin-Heidelberg-New York-London-Paris-Tokyo 1988. STOLTENBURG-DIDINGER, G., ALTENKIRCH, H., WAGNER, H. M.: Neurotoxicity of organic solvent mixtures: Embryotoxicity and Fetotoxicity. - Neurotox. Teratol. 12, 585-589 (1990). TOUTANT, C., LIPPMANN, S.: Fetal solvents syndrome. - Lancet 1, 1356 (1979). WOODWARD, D. J., HOFFER, B. J., LAPHAM, L. W.: Postnatal development of electrical and enzyme histochemical activity in Purkinje cells. - Exp. Neurol. 23, 120-139 (1969).
W.Graumann, J.Drukker (Eds.), Histo- and Cytochemistry as aTool in Environmental Toxicology. © Fischer Verlag· Stuttgart· New York· 1991
8 Neurotoxicity 8.1 The effect of pre- and postnatal exposure to organic solvents on the development of the cerebellar cortex in the rat GISELA STOLTENBURG-DIDINGER Institute of Neuropathology, Klinikum Steglitz, Freie Universitiit, Berlin (FRG)
Introduction The cerebellum of the neonatal rat presents a unique opportunity for the study of neuronal maturation. This structure is comparatively immature at birth (ADDISON 1911) and develops rapidly during the first month of extrauterine life (ALTMAN 1972). Moreover, the adult cerebellar cortex possesses well known and characteristic anatomical features which facilitate developmental compansons. The cerebellum has been subjected to a large number of enzyme histochemical studies (FRIEDE 1957; BARGETON-FARKAS and PEARSE 1965; KUCKUK 1967; ALTMAN 1972). The enzyme maturation pattern of the cerebellum was studied as correlated with its morphology. This could best be accomplished by enzyme histochemistry which allows investigation of enzyme activities at the cellular level during development. The purpose of enzyme histochemistry is the localization of specific enzyme activities to intact tissue components. The characterization and quantitation of the enzyme activities mediating the histochemical reaction is as integral a function of this discipline as the localization of the activities themselves. Enzyme histochemical studies on normal rat cerebellum have indicated a high and early activity of enzymes involved in anaerobic metabolism, while enzymes involved in aerobic metabolism increase rapidly after birth. The aerobic pathway soon becomes quantitatively the most important one in the postnatal cerebellum (ROBINS and LOWE 1961; BARGETON-FARKAS and PEARSE 1965). The external and internal granular cells exhibit weak oxidative enzyme activity at all ages. The Purkinje cells show increasing oxidative enzyme activity from birth, reaching adult activity at the end of the 4th week in normal ratss. The aim of the present study was to investigate whether any differences occur in the enzyme maturation pattern between solvent-exposed and normal rat cerebellum as correlated with changes in its morphology, caused by pre- and postnatal exposure to hexacarbons.
228 . G. Stoltenburg-Didinger Although the neurotoxic effects of organic solvents in the adult organism have been well studied, there is a paucity of information on the impact of in utero exposure to solvents on the developing brain (ALTENKIRCH et al. 1978; 1982a, b; SPENCER and SCHAUMBURG 1975; STOLTENBURG-DJDINGER and ALTENKIRCH 1988). Animal studies demonstrate that a variety of solvents readily cross the placenta (PELKONEN 1985) and that maternal inhalation of various solvents results in neurodevelopmental deficits in the newborn rodent (Bus et al. 1979; MARKS et al. 1980; DEACON et al. 1981). In utero exposure to organic solvents may also affect the development of the human brain (TOUTANT and LIPPMANN 1979; HOLMBERG et al. 1980; HERSH et al. 1985, ESKENASI et al. 1988). The primary fissure of cerebellar vermis was used as an enzymatically and morphologically defined and homogeneous model system of an early maturing region (LARSELL 1952; KUCKUK 1967; ALTMAN 1972). A unique property of the commonly-used histochemical oxidation-reduction indicators, the tetrazolium salts, is the reversible binding in their oxidized tetrazole form to cytoplasmic components of almost all cells (ADAMS 1965). The binding of the tetrazolium salt to tissue components permits electron transport in situ to form the insoluble colored reduced product, the formazan, near the site of enzymatic activity.
Materials and methods Animals: Virgin rats of the Wistar strain, 3 months of age, were mated with males varying in age from 4 to 8 months. The time of fertilization was determined by vaginal smears taken daily. The mode of inhalation was 23 h a day, 7 days a week. Controls were kept under the same conditions but without solvent exposure. The solvent was pumped on a glass frit through which filtered air was conducted. The hexane concentration was monitored with a continously measuring flame ionization detector. In the first solvent experiment only n-hexane was used, and the animals were exposed to low concentrations (500ppm) during prenatal development (21 days). In the second solvent experiment we used higher concentrations and exposed one group only prenatally (21 days) and a second group also postnatally (42 days) in order to include the growth spurt of the cerebellum into the exposure period. n-Hexane and MEK were studied in this experiment. In the third solvent experiment we used even higher concentrations (1000 ppm, initially 1500 ppm). There were likewise two groups: one was exposed prenatally (21 days), the other pre- and postnatally (51 days). The substances used were n-hexane (Merck, Darmstadt, nr.4367 99%), methyl-ethyl-ketone (Merck, Darmstadt, nr.6014 99%) and a mixture of both (hex/MEK = 1200:300). The newborn rats were examined for effects on body and brain maturation, weight development, fitness for survival and the possible occurrence of deformities. Preparation: All rats were decapitated. The skull was opened immidiately with scissors. Cerebellum and cerebrum were lifted out. Each cerebellar hemisphere was cut sagittally with a razor blade and each opposing half hemisphere was mounted onto a cork, then immidiately quenched in liquid nitrogen. All cryostat blocks were stored at a temperature of at least -20°C and sectioned at the same temperature in 7!!m thick sections. Sections were immidiately incubated for enzyme histochemistry of succinic dehydrogenase (SDH) and NADH tetrazolium reductase (NADH-Tr). All enzyme histochemical reactions were performed according to the methods described by NOVIKOFF (1960). Nitro Blue Tetrazolium (NBT) was used as hydrogen acceptor. All incubation solutions were freshly prepared. The chemicals were obtained from Sigma Chemical Company and from E. Merck AG, Darmstadt. All incubations were made for 30 min at a temperature of 37°C (SDH) or 20°C for NADH-Tr respectively. Paired substrate-free sections were used as negative controls in order to exclude nonspecific reduction of NBT. The enzyme histochemical activity as revealed by its formazan deposition was studied in the primary fissure of the cerebellar vermis at postnatal days 1, 9 and 21.
Organic solvents and cerebellar cortex . 229
Results Differences in formazan deposition between SDH and NADH-Tr activity could not be detected. Because of the finer granules of the reaction product figures were selected from NADHTr. Normal development. Activity of the oxidative enzymes SDH and NADH-Tr as visualized by formazan deposition was present already at birth in Purkinje cells. All activity was located in the cytoplasm. The activity at birth appeared weak, then gradually increased, by the third day strong SDH and NADH-Tr activity was present in the bodies of Purkinje cells. The layer of Purkinje cells remained the only one with marked activity until the ninth day (Fig. 2), when dendrites with marked oxidative activity grow into the molecular layer (Figs. 2 a, 3a). The external granular cells showed a low activity, which appeared as a thin rim of perikaryal activity, equal in the proliferative and premigratory zones. As the molecular layer expanded and regression of the external granular layer continued, NADH-Tr was seen closer to the subpial surface, reflecting the outgrowth of Purkinje cell dendrites, thereby providing a clear picture of the Purkinje cell dendritic tree (Fig. 3a). At day 9 the first faint trace of oxidative activity was seen in the internal granular cells. This activity remained low to moderate at all ages into adult life. Development of solvent-exposed rats. The development of SDH and NADH-Tr activity paralleled that of normal rats with a delay. After prenatal exposure only the activity of the Purkinje cell apical cones was higher at day 9 compared to normal rats, reflecting the delayed outgrowth of the Purkinje cell apical dendritic tree (Fig. 3 b). After day 21, both groups showed equal formazan deposition in the Purkinje cells. No differences in SDH and NADH-Tr activity between prenatally exposed and normal rats could be seen either in the external and internal granular cells. After pre- and postnatal exposure, the Purkinje cells of exposed rats showed a higher SDH and NADH-Tr activity at day 9 than those of normal rats. Pre- and postnatal exposure to n-hexane resulted in a persisting apical cone and delayed formation of the apical dendritic tree of the Purkinje cells in the cerebellum from a 9-day-old rat (Fig. 3c). After pre- and postnatal exposure to the mixture of n-hexane and methyl-ethyl-ketone (MEK), the retardation of cell maturation was even more pronounced (Fig. 3d). At day 9, the Purkinje cells showed persisting maximal perikaryal formazan coloration, indicating a high, concentrated NADH-Tr activity. Furthermore, the difference in width of the molecular layer between pre- and postnatally exposed and normal rats was greater as a result of the retarded apical dendrite formation (Fig. 3a-d). This could be demonstrated by enzyme histochemistry much more impressing than by conventional hematoxylin-eosin-staining (Figs. 1 and 2). All solvents studied delayed the histogenesis of the cerebellar cortex in the experimental animals at all concentrations examined (Figs. 1,2, 3a-d). Even at the lowest concentration, applied only prenatally as in the first solvent experiment, the histological preparations of frozen sections of the fissura prima of the vermis cerebelli showed a delay in migration of the outer granular cells and a persistence of Purkinje cells at a lower stage of development (Figs. 1b, 2 b, 3 b). Again it was proven that postnatal exposure aggravated the developmental delay (Figure 1c, 2c, 3c). Methylethyl-ketone (MEK) potentiated n-hexarie-neurotoxicity (Figs. 1d, 2d, 3d).
230 . G. Stoltenburg-Didinger
2d Fig. 1. Frozen sections of rat cerebellum, sagittal view of fissura prima of vermis (early developing region), third solvent experiment, postnatal day 9, HE. - x 300. a: Control; b: MEK prenatal exposure; c: n-Heyane, prenatal exposure; d: n-Hexane/MEK, pre- and postnatal exposure Note differences in thicknes of outer granular layer and molecular layer as well as differences in cell density in inner and outer granular layer. Fig. 2. Specimen identical to Fig. 1, NADH-Tr. - x 300. Note differences in cell size and staining intensity of Purkinje cells.
Organic solvents and cerebellar cortex . 231
Discussion The biological oxidation mechanisms, whereby electrons and frequently hydrogen are transferred through one or more intermediates to hydrogen, afford the major energy source of the living cell. Within the mitochondrion the electron carrier components are bound to one another (ADAMS 1965). The activity of oxidative enzymes in a given region may be considered an approximate parameter of the intensity of the oxidative metabolism (WOODWART et al. 1969). A comparison of these patterns provides a quite reliable picture of the functional activity of a given region (FRIEDE and PAX 1961). In rats prenatally exposed to n-hexane or methyl-ethyl-ketone (MEK), a delay in the development of the high oxidative enzyme activity of the Purkinje cell apical cone was noticed. This retarded increase in enzyme activity was followed by a delayed decrease in activity of the Purkinje cell apical cone parallel to the delayed formation and differentiation of the Purkinje cell apical dendritic tree (SIMA and PERSSON 1975). It seems reasonable to suppose that the delayed increase of enzyme activity in the apical cone prior to the dendritic formation and the delayed decrease of activity in the Purkinje cell apical cone concomitant to the retarded development of the dendritic tree may be related to inhibition of glycolysis by n-hexane and its main metabolite 2,5-hexanedione (SABRI et al. 1979; SPENCER et al. 1979; SPENCER et al. 1980). This molecular mechanism is yet not the only mode of action (DE CAPRIO 1985). Toxicity of n-hexane is mediated by 2,5hexane-dione, its main metabolite, which plays a major role in the neurotoxicity of this compound. Neurotoxicity of n-hexane depends on the toxicogenic transformation in the organism, enzyme induction having an additive effect. In the immature liver of fetuses and newborn animals, the biotransformation systems are, at any rate, developed only incompletely or not at all. Thus, the newborn animals receive a loading with diketones via the placenta or the mother's milk. Since active metabolization of hexacarbons to diketones is not possible in young animals, they do not develop any clinically detectable peripheral paralyses (STOLTENBURG-DIDINGER et al. 1990). Thus the peripheral nervous system of adult rats seems to be more vulnerable to solvents than the immature one, possibly because of its paucity in neurofilaments as well. It is concluded that prenatal solvent exposure causes a retarded enzymatic development of the molecular and internal granular layers as well as of the Purkinje cells in rat cerebellum, followed by catch-up growth and maturation. This is consistent with other investigations where mild or missing developmental deficit was found after solvent exposure during pregnancy (Bus et al. 1979; MARKS et al. 1980; DEACON et al. 1981; BHATT et al. 1988). The persistent delay in enzymatic development after pre- and postnatal exposure to organic solvents parallels a morphological retarded development of cerebellar structures and can be compared to the effect of malnutrition (STOLTENBURG-DIDINGER et al. 1990). A retarded differentiation of neurons in early undernutrition has been observed in cerebellum. PERSSON and SIMA (1975) demonstrated a delay in the formation and enzymatic maturation of the Purkinje cell apical dendritic tree. After postnatal exposure to hexacarbons there is no catch-up growth because the period of the growth spurt of the cerebellum is the most vulnerable (DOBBING and SANDS 1971). It might therefore be assumed that the delayed differentiation and migration of the external granular cells as well as the delayed outgrowth of the Purkinje cell dendritic tree results in a delayed synaptogenesis and, due to permanent loss of cerebellar cortical neurons, probably also a permanent loss of synapses. To prove this by other than enzyme histochemical methods remains a considerable challenge.
232 . G. Stoltenburg-Didinger
3c
Organic solvents and cerebellar cortex . 233
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Fig. 3. Frozen sections of rat cerebellum, sagittal view of fissura prima of vermis, third solvent experiment (1500-1000 ppm), postnatal day 9, NADH-Tr. - x500. a: Control. Purkinje cells of normal rat cerebellum with clear resolution of the apical cones and advancing apical dendrite formation. The dendrites divide into tertiary and quartenary branchlets. The perikaryal formazan deposition is submaximal. b: n-Hexane., prenatal exposure. Note the persisting apical cone with maximal activity. Retarded development of apical dendrites is seen, resulting in a delayed decrease of the apical cone NADH-Tr activity c: n-Hexane, pre- and postnatal exposure. Intense NADH-Tr activity confined to the perikarya of the Purkinje cells. Note the absence of apical cone formation and persistence of numerous somatic dendrites of the Purkinje cells, contrary to the finding in normal rats d: n-Hexane/MEK, pre- and postnatal exposure. Purkinje cells demonstrating high to moderate activity mostly confined to the apical cone and perikaryon. Badly developed dendritic tree, very thin molecular layer
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