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Retinal toxicity of methylazoxymethanol acetate is developmentally specific
Developmental Brain Research, 1 (1981) 425-428 © Elsevier/North-Holland Biomedical Press
425
Retinal toxicity of methylazoxymethanol acetate is developmentally specific
LEE E. EIDEN, CAROLE LATKER and MARTIN ZATZ Unit on Neuroendocrinology and (M.Z.) Section on Pharmacology, Laboratory o f Clinical Science, National Institute o f Mental Health, Bethesda, Md. 20205 and (C.L.) Department of Anatomy, Uniformed Services University of the Health Sciences, Bethesda, Md. 20014 (U.S.A.)
(Accepted January 13th, 1981) Key words: methylazoxymethanol - - retinal and nervous system development -- retinal dysplasia
A single injection of the neurotoxin methylazoxymethanol administered during certain critical perinatal periods results in permanent dysplasia of retinal cytoarchitecture. Methylazoxymethanol (MAM) appears in nature as a glycoside in cycad plants. Its carcinogenic and neurotoxic effects have been studied extensively7. The mechanism of M A M toxicity has usually been studied using the more stable aglycone M A M acetate. M A M acetate is capable of methylating purine bases in vivoa, 9 and this may be the basis for its carcinogenicity and neurotoxicity. Like any agent which kills rapidly dividing cells, M A M is a potential tool for producing developmentally specific lesions in the nervous system of laboratory animals if administered in utero or in early postnatal life. M A M causes a severe cerebellar dysplasia as well as cerebrocortical microencephaly in mice and rats1,4, t0, and the developmental specificity of MAMinduced microencephaly has been described 11. We felt that an investigation of M A M toxicity in the retina as a function of time of administration during development would be valuable as a further demonstration of the value of M A M as a developmental probe. Pregnant rats (Sprague-Dawley; Zivic-Miller) received a single intraperitoneal injection of either methylazoxymethanol acetate (Aldrich Chemical, Milwaukee, Wise.) (20 mg/kg) in 0.9 ~ saline, or saline alone on one of days 11 through 20 (E 1 l-E20) of gestation (day of insemination = day zero of gestation). Litters were delivered on day 21 of gestation. Newborn rats received a single s.c. injection of MAM (20 mg/kg) or saline on one of days 1 through 7 (PE1-PE7) after birth. All pups were sacrificed at 3-4 weeks of age by decapitation. Eyes were removed and fixed in phosphate-buffered 3 ~o paraformaldehyde-glutaraldehyde. Tissue thus fixed was dehydrated in graded ethanols and embedded whole in methacrylate (JB-4, Polysciences, Warrington, Pa.) Sections of 3-4 # m were cut, stained with toluidine blue, and examined on a Zeiss photomicroscope. M A M had no readily observable effects on the cytoarchitecture of the retina when administered on days E11-El6 (compare Fig. 1A and 1B). M A M treatment on day El7 results in rosette formation in the photoreceptor layer, a thinning of the
426
Fig. 1. Photomicrographs of retinas from rats treated with MAM or saline prior to birth and sacrificed at 3 weeks of age ( x 800). A: retina from a control animal treated with saline on day 17 (El7) of gestation. B: retina from an animal treated with MAM on day 12 (El2) of gestation. The cytoarchitecture is similar to the retina seen in A. C: anterior retina from an animal treated with MAM on day 17 (El7) of gestation. The photoreceptor cells (PR) are in a linear pattern adjacent to a rosette (asterisk). Over the rosette the bipolar layer (BP)is reduced to one or two cells and the outer plexiform layer is obliterated (arrow). D: retina from an animal treated with MAM on day 20 (E20) of gestation. The cytoarchitecture is similar to the retina seen in A.
427
Fig. 2. Photomicrographs of retinas from rats treated with MAM postnatally and sacrificed at 3 weeks of age (× 800). A: retina from an animal treated with MAM on day 1 (PE1) after birth. The photoreceptor cells appear disorganized and form small irregular rosettes where occasional areas of degeneration are seen (asterisk). The outer plexiform layer is missing in some areas and wide in others (arrow). The bipolar layer is reduced to 2 or 3 cells and frequently is split by an extra fiber layer (broad arrow). B: anterior retina from an animal treated with MAM on day 4 (PE4) after birth. The photoreceptor cells abruptly change (arrow) from a linear pattern to rosettes (asterisk).
bipolar (inner nuclear) layer and obliteration of the outer plexiform layer in the regions of rosette formation (Fig. 1C). Rosette formation was also prominent after M A M treatment on days El8 and El9. Rosettes were scattered from anterior to posterior in the retina (Latker et al., in preparation). M A M treatment on day E20 had no observable effect on the retinal cytoarchitecture (Fig. 1D). Cytoarchitectural effects of postnatal M A M treatment were most pronounced on day PE1 (Fig. 2A). Rosettes over the entire retinal cup were more numerous than on days E17-E19 but the dysplastic lesion was qualitatively similar at both periods. Lesions after M A M administration on days PE2-PE7 were progressively less extensive than those seen on day PE1. On days PE3-PE5 (Fig. 2B) areas of rosette formation were found increasingly in the anterior retina while the cytoarchitecture of the remaining retina appeared normal. M A M administration on day PE6 or thereafter resulted in an essentially normal adult retina. The cytoarchitectural lesions of the MAM-treated retina during development may be compared to those seen after X-irradiationS, 3. X-irradiation (150-200 rem) also causes rosette formation on day El7 (although not on El8 or El9), has no discernible effect on day E20 and results in rosette formation and a double bipolar
428 layer in the retina when administered 1-5 days after birth. X-irradiation is believed to cause its effects by killing germinal cells undergoing proliferation or final (differentiating) division 3. The dysplasia induced by X-rays correlates with the mitotic index of the r e t i n a l Since the dysplastic effects o f M A M after day E17 are similar to the effects o f X-irradiation, its mechanism o f action m a y be similar, i.e. arrest or destruction o f mitotic cells. It is noteworthy that M A M has no effect on retinal cytoarchitecture prior to day 16, while X-irradiation on day E11 results in pups b o r n without eyes. Thus, M A M m a y be selectively toxic to neuroblastic cells undergoing final division immediately prior to differentiation c o m p a r e d to undifferentiated germinal cells undergoing proliferative mitoses. The suggestion that M A M is selectively toxic to neuroblasts undergoing final division has been advanced by Coyle and his colleagues 5. If M A M induced dysplasia o f nervous tissue can in fact be attributed to a selective insult to neuroblasts during differentiating mitosis, then M A M should become an extremely powerful probe for studying C N S development. L.E.E. is a Research Associate in the Pharmacology-Toxicology P r o g r a m o f the N a t i o n a l Institute o f General Medical Sciences, Unit on Neuroendocrinology, Laboratory o f Clinical Science, National Institute o f Mental Health, Bethesda, Md. 20205, U.S.A.
1 Hartkop, T. H. and Jones, M. Z., Methylazoxymethanol-induced aberrant Purkinje cell dendritic development, J. Neuropath. exp. Neurol., 36 (1977) 519-532. 2 Hicks, S. P. In T. J. Haley and R. S. Snider (Eds.), Response of the Nervous System to Ionizing Radiation, Academic Press, New York, 1962, pp. 157-162. 3 Hicks, S. P. and D'Amato, C. J., Effects of ionizing radiation on mammalian development. In D. W. M. Woolham (Ed.), Advances in Teratology, Vol. 1, Academic Press, New York, 1966, pp. 196-250. 4 Hirono, I., Carcinogenicity and neurotoxicity of cycasin with special reference to species differences, Fed. Proc., 31 (1972) 1493-1497. 5 Johnston, M. V., Grzanna, R. and Coyle, J. T., Methylazoxymethanol treatment of fetal rats results in abnormally dense noradrenergic innervation of neocortex, Science, 203 (1979) 369-371. 6 Jones, M., Yang, M. and Mickelsen, O., Effects of methylazoxymethanol glycoside and methylazoxymethanol acetate on the cerebellum of the postnatal Swiss albino mouse, Fed. Proc., 31 (1972) 1508-1511. 7 Lang, M. G., Kobayashi, A. and Mickelsen, O., Bibliography of cycad research, Fed. Proc., 31 (1972) 1543-1546. 8 Matsumoto, H., Spatz, M. and Laquer, G. L., Quantitative changes with age in the DNA content of methylazoxymethanol-induced microcephalic rat brain, J. Neurochem., 19 (1972) 297-306. 9 Nagata, Y. and Matsumoto, H., Studies on methylazoxymethanol : methylation of nuclei acids in fetal rat brain, Proc. Soc. exp. Biol. (N. Y.), 132 (1969) 383-385. 10 Shimada, M. and Langman, J., Repair of the external granular layer of the hamster cerebellum after prenatal and postnatal administration of methylazoxymethanol, Teratology, 3 (1970) 119-134. 11 Spatz, M. and Laquer, G. L., Transplacental chemical induction of microencephaly in two strains of rats. I, Proc. Soc. exp. biol. (N. Y.), 129 (1968) 705-710.
425
Retinal toxicity of methylazoxymethanol acetate is developmentally specific
LEE E. EIDEN, CAROLE LATKER and MARTIN ZATZ Unit on Neuroendocrinology and (M.Z.) Section on Pharmacology, Laboratory o f Clinical Science, National Institute o f Mental Health, Bethesda, Md. 20205 and (C.L.) Department of Anatomy, Uniformed Services University of the Health Sciences, Bethesda, Md. 20014 (U.S.A.)
(Accepted January 13th, 1981) Key words: methylazoxymethanol - - retinal and nervous system development -- retinal dysplasia
A single injection of the neurotoxin methylazoxymethanol administered during certain critical perinatal periods results in permanent dysplasia of retinal cytoarchitecture. Methylazoxymethanol (MAM) appears in nature as a glycoside in cycad plants. Its carcinogenic and neurotoxic effects have been studied extensively7. The mechanism of M A M toxicity has usually been studied using the more stable aglycone M A M acetate. M A M acetate is capable of methylating purine bases in vivoa, 9 and this may be the basis for its carcinogenicity and neurotoxicity. Like any agent which kills rapidly dividing cells, M A M is a potential tool for producing developmentally specific lesions in the nervous system of laboratory animals if administered in utero or in early postnatal life. M A M causes a severe cerebellar dysplasia as well as cerebrocortical microencephaly in mice and rats1,4, t0, and the developmental specificity of MAMinduced microencephaly has been described 11. We felt that an investigation of M A M toxicity in the retina as a function of time of administration during development would be valuable as a further demonstration of the value of M A M as a developmental probe. Pregnant rats (Sprague-Dawley; Zivic-Miller) received a single intraperitoneal injection of either methylazoxymethanol acetate (Aldrich Chemical, Milwaukee, Wise.) (20 mg/kg) in 0.9 ~ saline, or saline alone on one of days 11 through 20 (E 1 l-E20) of gestation (day of insemination = day zero of gestation). Litters were delivered on day 21 of gestation. Newborn rats received a single s.c. injection of MAM (20 mg/kg) or saline on one of days 1 through 7 (PE1-PE7) after birth. All pups were sacrificed at 3-4 weeks of age by decapitation. Eyes were removed and fixed in phosphate-buffered 3 ~o paraformaldehyde-glutaraldehyde. Tissue thus fixed was dehydrated in graded ethanols and embedded whole in methacrylate (JB-4, Polysciences, Warrington, Pa.) Sections of 3-4 # m were cut, stained with toluidine blue, and examined on a Zeiss photomicroscope. M A M had no readily observable effects on the cytoarchitecture of the retina when administered on days E11-El6 (compare Fig. 1A and 1B). M A M treatment on day El7 results in rosette formation in the photoreceptor layer, a thinning of the
426
Fig. 1. Photomicrographs of retinas from rats treated with MAM or saline prior to birth and sacrificed at 3 weeks of age ( x 800). A: retina from a control animal treated with saline on day 17 (El7) of gestation. B: retina from an animal treated with MAM on day 12 (El2) of gestation. The cytoarchitecture is similar to the retina seen in A. C: anterior retina from an animal treated with MAM on day 17 (El7) of gestation. The photoreceptor cells (PR) are in a linear pattern adjacent to a rosette (asterisk). Over the rosette the bipolar layer (BP)is reduced to one or two cells and the outer plexiform layer is obliterated (arrow). D: retina from an animal treated with MAM on day 20 (E20) of gestation. The cytoarchitecture is similar to the retina seen in A.
427
Fig. 2. Photomicrographs of retinas from rats treated with MAM postnatally and sacrificed at 3 weeks of age (× 800). A: retina from an animal treated with MAM on day 1 (PE1) after birth. The photoreceptor cells appear disorganized and form small irregular rosettes where occasional areas of degeneration are seen (asterisk). The outer plexiform layer is missing in some areas and wide in others (arrow). The bipolar layer is reduced to 2 or 3 cells and frequently is split by an extra fiber layer (broad arrow). B: anterior retina from an animal treated with MAM on day 4 (PE4) after birth. The photoreceptor cells abruptly change (arrow) from a linear pattern to rosettes (asterisk).
bipolar (inner nuclear) layer and obliteration of the outer plexiform layer in the regions of rosette formation (Fig. 1C). Rosette formation was also prominent after M A M treatment on days El8 and El9. Rosettes were scattered from anterior to posterior in the retina (Latker et al., in preparation). M A M treatment on day E20 had no observable effect on the retinal cytoarchitecture (Fig. 1D). Cytoarchitectural effects of postnatal M A M treatment were most pronounced on day PE1 (Fig. 2A). Rosettes over the entire retinal cup were more numerous than on days E17-E19 but the dysplastic lesion was qualitatively similar at both periods. Lesions after M A M administration on days PE2-PE7 were progressively less extensive than those seen on day PE1. On days PE3-PE5 (Fig. 2B) areas of rosette formation were found increasingly in the anterior retina while the cytoarchitecture of the remaining retina appeared normal. M A M administration on day PE6 or thereafter resulted in an essentially normal adult retina. The cytoarchitectural lesions of the MAM-treated retina during development may be compared to those seen after X-irradiationS, 3. X-irradiation (150-200 rem) also causes rosette formation on day El7 (although not on El8 or El9), has no discernible effect on day E20 and results in rosette formation and a double bipolar
428 layer in the retina when administered 1-5 days after birth. X-irradiation is believed to cause its effects by killing germinal cells undergoing proliferation or final (differentiating) division 3. The dysplasia induced by X-rays correlates with the mitotic index of the r e t i n a l Since the dysplastic effects o f M A M after day E17 are similar to the effects o f X-irradiation, its mechanism o f action m a y be similar, i.e. arrest or destruction o f mitotic cells. It is noteworthy that M A M has no effect on retinal cytoarchitecture prior to day 16, while X-irradiation on day E11 results in pups b o r n without eyes. Thus, M A M m a y be selectively toxic to neuroblastic cells undergoing final division immediately prior to differentiation c o m p a r e d to undifferentiated germinal cells undergoing proliferative mitoses. The suggestion that M A M is selectively toxic to neuroblasts undergoing final division has been advanced by Coyle and his colleagues 5. If M A M induced dysplasia o f nervous tissue can in fact be attributed to a selective insult to neuroblasts during differentiating mitosis, then M A M should become an extremely powerful probe for studying C N S development. L.E.E. is a Research Associate in the Pharmacology-Toxicology P r o g r a m o f the N a t i o n a l Institute o f General Medical Sciences, Unit on Neuroendocrinology, Laboratory o f Clinical Science, National Institute o f Mental Health, Bethesda, Md. 20205, U.S.A.
1 Hartkop, T. H. and Jones, M. Z., Methylazoxymethanol-induced aberrant Purkinje cell dendritic development, J. Neuropath. exp. Neurol., 36 (1977) 519-532. 2 Hicks, S. P. In T. J. Haley and R. S. Snider (Eds.), Response of the Nervous System to Ionizing Radiation, Academic Press, New York, 1962, pp. 157-162. 3 Hicks, S. P. and D'Amato, C. J., Effects of ionizing radiation on mammalian development. In D. W. M. Woolham (Ed.), Advances in Teratology, Vol. 1, Academic Press, New York, 1966, pp. 196-250. 4 Hirono, I., Carcinogenicity and neurotoxicity of cycasin with special reference to species differences, Fed. Proc., 31 (1972) 1493-1497. 5 Johnston, M. V., Grzanna, R. and Coyle, J. T., Methylazoxymethanol treatment of fetal rats results in abnormally dense noradrenergic innervation of neocortex, Science, 203 (1979) 369-371. 6 Jones, M., Yang, M. and Mickelsen, O., Effects of methylazoxymethanol glycoside and methylazoxymethanol acetate on the cerebellum of the postnatal Swiss albino mouse, Fed. Proc., 31 (1972) 1508-1511. 7 Lang, M. G., Kobayashi, A. and Mickelsen, O., Bibliography of cycad research, Fed. Proc., 31 (1972) 1543-1546. 8 Matsumoto, H., Spatz, M. and Laquer, G. L., Quantitative changes with age in the DNA content of methylazoxymethanol-induced microcephalic rat brain, J. Neurochem., 19 (1972) 297-306. 9 Nagata, Y. and Matsumoto, H., Studies on methylazoxymethanol : methylation of nuclei acids in fetal rat brain, Proc. Soc. exp. Biol. (N. Y.), 132 (1969) 383-385. 10 Shimada, M. and Langman, J., Repair of the external granular layer of the hamster cerebellum after prenatal and postnatal administration of methylazoxymethanol, Teratology, 3 (1970) 119-134. 11 Spatz, M. and Laquer, G. L., Transplacental chemical induction of microencephaly in two strains of rats. I, Proc. Soc. exp. biol. (N. Y.), 129 (1968) 705-710.