31st annual meeting of the Japanese Teratology Society. Izumo, Japan, July 11-12, 1991. Abstracts.

TERATOLOGY 44: 1B-55B

Abstracts of Papers Presented at the Thirty-First Annual Meeting

of the Japanese Teratology Society

Izumo, Japan July 11

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1 2 , 1991

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JAPANESE TERATOLOGY SOCIETY ABSTRACTS

CONTENTS Lectures by Invitation ........................... 3B Symposium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4B Workshop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5B Papers Presented From Platform . . . . . . . . . . . . . . . . . . 7B Poster Sessions ................................. 32B Behavioral Teratology Meeting . . . . . . . . . . . . . . . . . . 49B


LECTURES BY INVITATION

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unknown effect as basis for decisions on public health. In vivo experiments can't be replaced by in vitro systems, but the usefulness of in vitro models as an additional strategy in teratological research has been established.

SL- 1 SCHUMACHER, G.-H., lnstitut fur Anatomie, Wil helms-Pieck-Universita t Rostock. Findings and possibilities in experimental teratology. Congenital malformations are caused by complicated interactions between genetic and environmental factors. According to Wilson (1972) 25% of birth defects can be attributed to genetic causes and 5-10%0 to embryonal environment but the remaining 65-70% are originated by some interplay of exogenic and endogenic factors of which we don't have any detailed knowledge. For teratoepidemiological investigations the use of accepted classifications is very important. In 1982 it was proposed to classify birth defects in regard of their etiology and pathogenesis in malformation, disruption, deformation and dysplasia. The catalogue of Shepard (1986) contains more than 900 agents with teratogenetic effects in laboratory animals. In human less than 30 agents are known with corresponding actions. The use of in vivo-experiments includes the maternal metabolism and the placental transfer. Therefore only mammals are privileged to study the interactions between mother and fetus. The differences between the various species and man in regard of diaplacentary function lead to completely different results. The increasing number of chemical substances to be tested involve the need of more experimental animals. In view of this problem, the progress made in the field of in vitro techniques suggested the application of culture systems. In vitro-methods have some experimental advantages as compared with teratological in vivo test methods. On the other hand in vitro systems have also their disadvantages. Looking at the spectrum of available in vitro-models, the following classification is recommended. 1. Cultivation of cells after isolation into single cells - Monolayer cultures (ML-cultures) - Organoid cultures 2. Cultivation of tissues without isolation - Organ culture - "Whole embryo" culture When evaluating the applicability and usefulness of the in vitro-systems for teratological research it has to be differentiated between scientific and practical aspects as well as aspects related to public health. The various culture techniques have undoubtedly proved to be very fruitful in scientific research. The difficulties concern the assessment of the teratogenic risk of substances of a still

SL-2 KUROKI, Y., Division of Medical Genetics, Kanagawa Children's Medical Center, Yokohama, Kanagawa. Birth defects monitoring in Japan: An overview. Recently a sharp decrease of infant mortality and a sharp increase of the proportion of infant deaths by birth defects are clearly noticed in Japan. Birth defects monitoring program is one of the effective strategies to prevent birth defects mainly caused by environmental factors. Since 1981 two hospital-based monitoring programs, one regional and the other nationwide, and four population-based monitoring programs have been in operation in Japan. The number of births surveyed varies in each program, from 6,000 to 130,000 births annually. Total number of births monitored has reached 260,000 annually. Maximum age at diagnosis is 7 days in all programs. These programs are widely scattered in Japan, thus the data collected in these programs are the representatives of Japan. Each system has already accumulated relevant baseline data. Though statistically significant increase was observed several times in some malformations in each program, no true unusual events were detected in the last decade. However, recent progress and wide application of prenatal diagnoses has reduced the prevalences of neural tube defects and Down syndrome. In developed countries including Japan, toxicity testing systems for drugs or chemicals have been well established, thus potent teratogens like thalidomide will be rarely introduced into our environments. Besides the function to detect new teratogens, monitoring systems have another function to test the safety of our widely exposed factors such as smoking, alcohol, coffee, low dose radiation etc. An increased incidence of birth defects, especially multifactorial defects, in smoking mothers was confirmed by a case-control study. But the population attributable risk percent by maternal smoking was only 3.6%. The effects of low dose radiation on the incidence of birth defects have been studied. The exposure rate of low dose radiation was significantly elevated in the mothers of the newborns with congenital malformations. The expected dose, however, were very low. In addition, some of the

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mothers smoked, drank alcohol, or were engaged in VDT work. The episodes of high fever or skin rashes were sometimes observed. Thus the teratogenicity of low dose radiation is yet considered questionable. Further studies are needed to clarify the relationship behveen low dose radiation and the births of congenital malformations.

SYMPOSIUM

plexus of the brain and retina of the eye as well as in the liver, we speculate that TTR may perform an unknown function in neuronal systems. It may be that the variant TTR is functionally deficient and the lack of normal TTR function causes neuropathy. To elucidate physiological function of TTR, we disrupted the TTR gene by homologous recombination in cultured mouse embryonic stem cells and obtained chimeric mice with germline transmission. The biological effect of disrupting TTR gene in mice is under investigation.

S SHIONO. H., Department of Legal Medicine, Shimane Molecular Medical University, Izumo, Shimane. biologic approach to congenital anomalies. Congenital anomalies are morphologic and functional abnormalities observed at birth. Some types of anomalies are not detected until some time after birth. Congenital anomalies are caused by 1) genetic factors, 2) environmental factors and 3) interaction between genetic and environmental factors. In other words, congenital anomalies represent differences in the base sequence of DNA that is transmitted to offsprings through gonocytes. On the other hand, molecular biology has achieved remarkable progress, especially in the field of DNA. In view of this, we have invited, for comments and discussion at this symposium, four eminent researchers who are examining the causes of congenital anomalies at various levels. In their presentations, we requested that they focus on the convergence of morphologic and molecular biologic approaches to the congenital anomalies in their research.

s-1 MAEDA, S., Department of Biochemistry, Kumamoto University Medical School, Kumamoto. DNA diagnosis and molecular pathogenesis of familial amyloidotic polyneuropathy. Familial amyloidotic polyneuropathy (FAP) is a dominantly inherited systemic amyloidosis, characterized by polyneuropathy and extracellular deposition of amyloid protein. The amyloid deposits consist of a variant TTR with a single amino acid substitution. We established a DNA diagnosis of FAP and demonstrated a direct link between l T R gene mutation and FAP. To investigate the molecular pathogenesis of FAP, we constructed transgenic mice carrying and expressing the human mutant TTR gene. In these mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys 6 months after birth and extended to various other organs and tissues with advancing age. Amyloid deposition was shown to be composed of human variant TTR. These transgenic mice, however, did not develop peripheral neuropathy which is the most prominent symptom of FAP. As TTR is synthesized in choroid

s-2 NISHISHO, I., Department of Biochemistry, Cancer Institute, Tokyo. Identification of a gene responsible for hereditary tumor: Linkage analysis and positional cloning. To date, many DNA fragments that detect restriction fragment length polymorphisms (RFLPs) have been isolated. The location of the responsible genes for many hereditary diseases has been assigned to specific chromosomal regions by linkage analysis using these DNA markers. To map a disease gene by linkage analysis, highly polymorphic markers and sufficient families are needed. Localization of the gene for adenomatous polyposis coli, the most common autosomal dominant disease leading to cancer predisposition, was assigned to chromosome 5q21 by linkage analysis. To identify the responsible gene, over 40 cosmid clones located at chromosome 5q21 region were isolated. Linkage and physical map of this region was constructed. One of the cosmid clones obtained, L5.71, revealed that an approximately 200 kb segment is deleted in the gerrnline of one APC patient. The nucleotide sequence of L5.71 was highly conserved in rodent DNA and two exon like regions were determined. By exon-connection strategy using these sequences, an expressed gene from this region was identified. Further analysis revealed that the gene (MCC gene) is somatically mutated in several colorectal cancers. The MCC gene is thus a candidate for the putative colorectal tumor suppressor gene located at 5q21. But no mutations of the MCC gene in the germline of APC patients have been observed so far.

s-3 NAKAGOME, Y., National Children's Medical Research Center, Tokyo. Molecular biology of human Y chromosome. The human Y chromosome is composed of four distinct segments. Firstly, the 2.5 Mb pseudo-autoso-

JAPANESE TERATOLOGY SOCIETY ABSTRACTS mai region (PAR) is located at the distal end of the short arm. A recombination event between the Y chromosome PAR and the corresponding PAR on the X is mandatory in male meiosis. The SPY gene is located proximal to this Alu insert. Koopman et al. have recently shown that the mouse homologue of SPY is the Y chromosome-linked testis-determining gene (Tdy). The non-PAR segment of the short arm is 13-15 Mb long, as is the proximal segment of the long arm. This long arm region contains genes responsible for gonadoblastoma development and spermatogenesis. The distal portion of the long arm is quinacrine positive and consists of about 3000 copies of the DYZl repeat element. We have isolated 10 navel probes from a Y-chromosome-specific phage library. DNA samples from 60 patients with structurally abnormal Y chromosomes were used for deletion mapping of the 10 probes, as well as of six previously reported loci. These probes divide the non-fluorescent region of the Y chromosome into 23 segments. One of these segments was found to be deleted in four patients with azoospermia (this work was done in collaboration with Dept. Urol., Osaka Univ.).

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is a powerfui strategy to elucidate the relationship between gene functions and developmental phenomena.

WORKSHOP

(a)

s-4 OTANI. H., Department of Anatomy, Shimane Medical University, Izumo. Shimane. Transgenic mice in the search for development-related genes. Transgenic mice are useful to study gene function and regulation of gene expression in a whole organism. In developmental biology, it has been used to search for either unknown functions of known genes during development or unknown genes which are related with developmental events. Nine of 12 human IL-2 transgenic mouse founders commonly exhibited motor ataxic symptoms from 2 weeks of age. Histopathologically, the cerebellum, skin, testes and eyes were severely damaged by inflammation. Introduced IL-2 gene was expressed in multiple organs including those with pathological changes several days earlier than pathological changes occurred, as deduced by PCR method. Expression of the introduced IL-2 gene was suggested to be the cause of these changes, although additional modifying factors should exist. Among murine myelin-basic-protein-gene introduced transgenic mice, microtia and/or abnormal bite were occasionally observed in offspring from only one founder mouse. Developmentally, in some of the transgenic embryos, bleeding and hypoplasia were observed in the second pharyngeal arch on days 9 and 10 of gestation, respectively. It was suggested that insertional mutation of an unknown gene by the transgene caused these pathological changes. By in situ hybridization, the transgene was mapped on 61 -3 region of the 10th chromosome. Using PCR and conventional cloning methods, multiple clones covering 10 kb DNA fragment franking the transgene, which is supposed to include the preinsertional gene, have been cloned. These data suggest that the transgenic method

W ETO, K. and K. SHIOTA, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, and Faculty of Medicine, Kyoto University, Kyoto. The study of congenital anomalies in culture of cells and whole embryos. Culture techniques of cell, tissue, organ and whole embryo are practical tools for the elucidation of mechanism in congenital anomalies in vitro. In this workshop, studies using micromass culture of limb bud and midbrain cells of rodent embryos, and also whole embryo culture during the period of preimplantation, organogenesis and post organogenesis to term stages are introduced. Cell differentiation may be efficiently examined in micromass culture, however, available cell types are a few such as limb bud, midbrain and facial mesenchyma1 cells. The advantages of whole embryo culture are: 1 ) precise control of conditions (concentration of administrating chemical or biological agents, duration of exposure, site of exposure in embryo or membranes and stage of embryonic development), 2) detection of metabolic activity of embryo by sampling the medium, gas phase of culturing tissues, 3) continuous observation of culturing embryos with the possibility of microsurgery and microinjection. However, culture techniques also have disadvantages such as 1) very exacting and laborious, 2) limited culture periods. 3) undetachable functional abnormaiities and so forth. The limitations and possibilities of these culture techniques in practical experiments, and the combined method of cell and whole embryo culture, ceii and organ culture or in vivo and in vitro method are discusses.

w-1 TATEWAKI, R., Department of Biology, Shimane Medical University, Izumo, Shimane. Mouse embryo culture for chromosome analysis. Mouse embryo culture has been established and applied to study causal mechanisms of abnormal development. Mouse embryo culture can be an experimental system to examine influence of teratogen on early development. We combined mouse embryo

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culture with embryo transfer and chromosome analysis, We have studied relationship between environmental factors including maternal factors and genetic predisposition in developmental abnormalities in mouse embryos of diabetic mothers (Tatewaki et al., 1987, 1988, 1989, 1990). In mouse embryo culture from 1 cell to blastocyst stage, fertilized eggs were incubated in modified Whitten's medium and mixed gas (5%0,, 5%CO,, 90%N,) at 37°C for three days. P-mercaptoethanol 20 pM and EDTA-2Na 10 p M were added in order to overcome "2 cell block". Under these conditions, from 55 to 100% of cultured 1 cell stage embryos developed into blastocysts depending on strain. For chromosome preparation, Kamiguchi's method (1 978) was modified. This method can be applied from 1 cell to preimplantation stage embryos, and allows a higher rate of success (63% of prepared embryos) in preparing chromosome slides than rated in literature. Using improved methods of embryo culture, embryo transfer and chromosome analysis, we have obtained evidence that both environmental and genetic factors are the causes of malformation in diabetic pregnancy. Mouse embryo culture associated with chromosome analysis is a useful procedure, and offers more important data for clarification of the causal mechanisms in development abnormalities.

w-2 OSUMI-YAMASHITA. N., Section of Craniofacial Development and Anomalies, Institute of Stomatognathic Science, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo. Lineage analysis of cranial neural crest cells using the mouse whole embryo culture system. For the bases of analyzing congenital anomalies induced by abnormal behavior of cranial neural cells, information is required concerning the lineage of crest cells, that is, the pathways, destinations, developmental fates, and gene expression of the cells, in normal development of mammalian embryos. In this study, pathways and destinations of cranial crest cells were revealed by using a vital dye and whole embryo culture system. Eight-day mouse embryos were dissected from the uterus with the placenta and embryonic membranes intact. A lineage tracer, Dil, was injected into several areas of the cranial neural crest. After 24-hour culture in the rotating system, labelled descendants were observed under a fluorescent microscope as wholemount or by making serial sections. The correspondence between branchial arches and primary segments of the neural plate was clearly obtained. Pathways of crest cells were dependent on the position in the neural plate along anterior-posterior axis. Crest cells from a certain region migrated into a terminal which was different from that of crest cells originated from the adjacent region. From these results and the previous data on the gene expression of retinoic acid receptors, it is sug-

gested that lineage restriction of crest cells occurs before emigration from the neural plate according to the position along the anterior-posterior axis.

w-3 FUKIISHI, Y. and G. MORRIS-KAY, Shionogi Research Laboratories, Shionogi & Co.. Ltd., Osaka, and Department of Human Anatomy, Oxford University, Oxford. The fate of neural crest cells injected into the endocardiac tube of rat embryos. Differences of gene expression and differentiation potential are shown by neural crest cells of different levels of origins. In order to discover whether these differences are reflected in an ability to select specific environment during migration, we labelled neural crest cells with wheat germ agglutinin and injected them into the endocardiac tube of unlabelled 8 somite-stage embryos. The injected cells attached to the endocardium or to the endothelium of cranial blood vessels, passed through into the extravascular tissues and dispersed. The labelled cells were taken from (a) the midbrainlrostral hindbrain neural crest, or (b) the trunk crest. The host embryos were cultured for short periods in order to observe the processes of attachment and egress, or for 22 hours in order to analyze the positions of the labelled cells after dispersal. Ninety-nine percent of the midbrahihindbrain crest cells which left the cardiac region were located in the cranial region, but showed no preference for the areas normally populated by cells from that level. Trunk cells were also preferentially located in the cranial region, but none was found in the areas normally populated by midbrain neural crest cells, and only one was found in rostra1 hindbrain neural crest territory. These results indicates that neural crest cells injected into the heart leave the vascular system either from the heart itself, or from vessels close to it, gaining access predominantly to the head. After migration, trunk cells show a clear bias towards more caudal levels of the head compared with the generalised cranial dispersal pattern shown by the cranial neural crest cells.

w-4 SUZUE, T., Department of Physiology, School of Medicine, Tokyo Medical and Dental University, Tokyo. A novel method for maintenance of live rodent fetuses in late gestation in vitro and its application for developmental studies. A new method was developed for maintaining physiological activities of late gestation fetuses of the rat and mouse in vitro (T. Suzue, Proceedings of the Japan Academy, Vol 66 (1990) 177-181.). Fetuses that are functionally connected with uterus through

JAPANESE TERATOLOGY SOCIETY ABSTRACTS placenta and umbilical cord were isolated from pregnant rats (E14-E20) and were perfused through a uterine artery with a physiological salt solution containing general anaesthetics. After decerebration, preparations were perfused with a physiological solution without general anaesthetics. Under this condition, fetuses showed heartbeats and spontaneous body movements. Some of the body movements looked very much like respiration of postnatal animals. It was possible to evoke body movements by electrical stimulation of the skin. Both the spontaneous and stimulation-induced body movements were depressed after perfusion with general anaesthetics i.e., ether, halothane or pentobarbital, suggesting that the movements are controlled by the activities of the central neurons. The fact that the activities of the central nervous system as well as that of the heart were retained under present conditions implies that the general condition of the fetuses is good. Therefore, the present method may be useful for studying the mechanism of normal and abnormal development of mammalian fetuses.

W-5 YONEMOTO. J., National Institute for Environmental Studies, Tsukuba, Ibaraki. Teratogen prescreening using micromass culture of rat limb bud mesenchymal cells. Although there are no alternative test systems which replace in vivo mammalian teratogenicity tests because of the diversity and complexity of the causes and outcomes of teratogenicity, the need for alternative tests as a prescreening is enormous since the in vivo test is laborious, time-consuming and expensive. Among alternative tests developed so far, micromass culture of rodent limb bud mesenchymal cells (LBC) is one candidate for teratogenicity prescreening tests. LBC assesses cell differentiation and cell proliferation simultaneously which allows us to calculate a kind of "developmental hazard" estimate. A validation study of LBC was conducted using metals and reiated compounds teratogenic to mammalian experimental animals. Fifty percent inhibition concentration for cell proliferation (IP,,), 50% inhibition concentration for differentiation (IDso)and the ratio of IPS, to IP, (P/D ratio) were obtained from LBC. Among teratogens, P/D ratios were more than 3 except methylmercury, Ni and Li, while P/D ratios were around 1 among nonteratogens. Using the criteria of high PID ratio and very low ID, LBC was useful for metals and related compounds to rank the priority for further teratogenicity tests.

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W-6 TSUCHIYA, T . , Division of Medical Devices, National Institute of Hygienic Sciences, Tokyo. Method of micromass culture of midbrain cells and its application to in vitro mechanistic study. Midbrain (MB) tissue were removed from the embryos of pregnant rats on day 11, 12, or 13 of gestation, or from pregnant mice on day 10 of gestation. MB tissues were dissociated into individual cells by trypsin. The adhering MB cells formed separate micromass islands and were cultured for 5-7 days. The cultures were stained with hematoxylin for cell differentiation assay. The number of individual foci/cell islands was counted. A remarkable species difference in the teratogenicity of ethylenethiourea (ETU) was observed between rats (teratogenic) and mice (nonteratogenic). The purposes of this report are to examine the relationship between the malformations of the cultured rat embryos and the alterations of MB cells at the embryonic stage of organogenesis induced by ETU, and to examine the alterations in rat and mouse MB cells induced by ETU and by the serum samples prepared from the animals given ETU. From these experimental results, we clarify that the different sensitivities of the midbrain of these two species may be the main reason that ETU is teratogenic in rats but not in mice. Next, we report that MB are unsuitable for estimating the teratogenic potential of retinoids. The arotinoids did not specifically inhibit cell differentiation but were cytotoxic at high concentrations. Finally, embryolethality of new herbicides was not detected by the micromass teratogen test.

PAPERS PRESENTED FROM PLATFORM A-01 MURANAKA, R., Y. FUKIISHI, H. TSUlKl and Y. HASEGAWA, Shionogi Reseach Laboratories, Shionogi & Co., LTD., Toyonaka, Osaka. Teratogenic characteristics by single dosing of antineoplastic platinum complexes in rats. Antitumor platinum complexes produced severe embryolethal effects when they were consecutively applied to pregnant rats during the period of organogenesis in fetuses. No details, however, were obtained as to their teratogenic potential. It is possible that malformed embryos after dosing with the complexes were lost as resorbed ones until term. In the present study, teratogenic potential of platinum complexes was examined by single dosing in pregnant rats. Novel antitumor platinum complexes, 254-S (cis-diammine (glycolato) platinum) and cisplatin (CDDP) were select-

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JAPANESE TERATOL

ed. Pregnant rats (sperm day = DO) were given i.v. single dosing of 254-S (0.75-12 mg/kg) or CDDP (0.375-6 rngikg) on each day of D5 to D16. All rats were sacrificed on D21, and their fetuses were examined for viability and external appearance. Resuits were as follow; (a) The highest embryolethality was obtained from dams treated on D7 in 254-S groups and D6-D9 in CDDP groups, respectively. (b) External malformations in fetuses were produced in the biphasic pattern in 254-S groups treated on D6-D9 and on D14-DI5, although they were monophasic in CDDP groups treated on D5-D8. (c) Teratogenic spectrum in 254-S groups was not necessarily coincident with that of CDDP groups. (d) Tail or digital malformations were observed in 254-S groups treated on D7 and in CDDP groups treated on D5 or D6. In conclusion, two examined antitumor platinum complexes, 254-S and CDDP, have teratogenicity as well as embryolethality in rat fetuses. It is unique that digital or tail malformations were produced when complexes were given at the period before the onset of organogenesis of digits or tail.

A-02 IKEO, T., S. NAGASAWA, I.TAMURA, A. FUJITA and Y. SAKIYAMA, Department of Biochemistry, and Department of Oral Anatomy, Osaka Dental University, Osaka. External anomalies in fetal mice caused by dichloromethylidene-bisphoshonate (CI,MBP). CI,MBP was administered i.p. one time at a dose of 40 or 200 mg/kg of maternal body weight on day 7, 8, 9, 10 or 11 of gestation (Jcl:ICR, VP=O). All fetuses were examined on day 18. No difference between the controls (14.5) and experimental groups (13.1-17.3) was observed in the average number of implants. Although dead and resorbed fetuses appeared in all the 200 mg/kg experimental groups, there were none in the 40 mg/kg groups when the drug was administered on days 9 or 10. The appearance of dead and resorbed fetuses was especially marked in 200 mglkg group on day 7 (16.3%). The average body weight of the live fetuses was significantly lower in all but one of the experimental groups, compared with the controls. Fetuses with external anomalies could be categorized as those with internal hemorrhage and/or malformation (exencephalia, cleft palate, cacogenesis and so on). About 40% of the fetuses in the experimental groups had external anomalies compared with 3.7% for the controls. There were differences in the sensitivity of each dam, as well as between fetuses in a particular dam.

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A-03 WATANABE, T. and A. ENDO, Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Yamagata. Effects of maternal biotin deficiency on embryonic development in hamsters. We previously reported that biotin deficiency during gestation had teratogenic effects on mouse embryos. However, the teratogenicity of biotin deficiency in hamsters is rather equivocal as yet. In this study, effects of maternal biotin deficiency on hamster embryos were examined by adding four different amounts of avidin (0,50, 100, or 1000 mg/kg) in the powdered commercial diet (CRF-1). At midgestation (dg10.5), biotin deficiency caused a high incidence of resorbed and dead embryos. Also both crown-rump and head length of biotin-deficient embryos were less and their digit development was retarded. These embryos were characterized by pericardial cavity enlargement (40.0%) and zig-zag closure line of neural rube (44.0%). In addition, some embryos exhibited abnormalities of craniofacial region and tail. At near term (dg14.5), embryonic growth retardation and external malformation were seen in biotin-deficient groups. The common defects were cleft palate (10.6%), micromelia (8.5%) and micrognathia (7.4%). The histological examination of the placenta revealed some differences in spongiotrophoblast and labyrinth layers between the control and biotin-deficient groups. These findings indicate that maternal biotin deficiency during gestation has deleterious effects on embryonic growth and development in hamsters as well as mice.

A-04 MA, N., Y. TAKAGlSHl and H. YAMAMURA, Department of Anatomy, Mie University School of Medicine, Tsu, Mie, and Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi. Light and electron microscopic localization of glutamate immunoreactivity in the jaundiced Gunn rat cerebellum. The immunoreactivity of glutamate (Glu), a possible excitatory neurotransmitter, was examined in the cerebellar cortex from homozygous (jij) and heterozygous (+/j) Gunn rats with and without hereditary hyperbilirubinemia, respectively, from postnatal day 1 to adulthood. By light microscopy using an immunoperoxidase method, Glu immunoreactivity was found in the molecular layer from day 1 to adulthood in both j/j and +/j cerebella. Though the molecular layer was weakly stained until day 3, the staining became intense with the thickening of the layer from day 7 forward in +/j cerebella. Cells in each layer of the cortex were Glu

JAPANESE TERATOLOGY SOCIETY ABSTRACTS immunonegative at all stages. In j/j cerebella, the molecular layer was thinner than that in t / j cerebella from day 7 to adulthood, and the immunoreactivity was weak and diffuse. Cells were also imrnunonegative at all stages. Electron microscopy using an immunogold procedure revealed gold particles over almost all cells and fibers in the molecular layer. But the number of particles was small until day 12. From day 18 on, a dense accumulation of particles was found on parallel fiber synaptic boutons in both j/j and t / j cerebella. However, the density of particles on parallel fiber boutons was significantly lower in j/j than t / j cerebella at day 30 and adulthood. The present study suggests that the Glu transmission is adversely affected in j/j cerebella.

A-05 FUNAHASHI, A,. M. INOUYE and H. YAMAMURA, Institute of Environmental Medicine, Nagoya University, Abnormal serotonin-immunoNagoya, Aichi. reactive neurons in the raphe nucleus of rats with methylazoxymethanol-induced microcephaly. The majority of serotonin (5HT) neurons in the dorsal and median raphe nuclei in rats are produced on days 13 and 14 of gestation. Treatment of pregnant rats with methylazoxymethanol acetate (MAM), an anti-mitotic agent, on day 15 of gestation results in formation of severe microcephaly in the offspring. This study immunohistochernically examined possible developmental alteration of 5HT neurons in the dorsal and median raphe nuclei of the microcephalic rats (MAM-rats) at 35 days of age. 5HT-immunoreactive neurons were observed in the serial sections through the superior colliculi rostrally to the inferior colliculi caudally in both the control and MAM-rats. However, 5HT-immunoreactive neurons in the MAM-rats were reduced in number and irregularly distributed in the dorsal and median raphe nuclei compared with those in the control. Besides, in MAM-rats, the dendrites of 5HT-immunoreactive neurons in these nuclei were very short and twisted. These observations support the idea of target-dependent secondary degeneration of 5HT neurons in the dorsal and median raphe nuclei because a direct cytotoxic effect of MAM upon the majority of precursors of neurons in the dorsal and median raphe nuclei would not be severe when dams were exposed to MAM on day 15 of gestation.

A-06 TANAKA, H., M. YAMANOUCHI and M. ARIMA, Division of Mental Retardation and Birth Defect Reserch, National Institute of Neuroscience, NCNP, Kodaira. Tokyo, and Kohnodai Hospital, NCNP, Ichikawa, Chiba. The effect of maternal triethylene tetramine dihydrochloride on the mouse fetus. Triethylene tetramine dihydrochloride (lrien-2HCI) is a copper-chelating drug used clinically in the treat-

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ment of Wilson's disease. The purpose of this study is to determine the adverse effect of maternal Trien-2HCI treatment during pregnancy on the fetal mouse of C3H strain. We gave female mice throughout pregnancy four kinds of drinking fluid, a d libitum; tap water, and 3,000 ppm, 6,000 pprn and 12,000 ppm of Trien-2HCI in tap water. Pregnancies were terminated on gestational day 19 by means of cesarean section. Mother and offspring were examined for weights, litter size, gross abnormalities, viability and copper concentrations. Trien-2HCI at levels higher than 3,000 ppm caused fetal resorption, a decrease in copper concentrations of maternal liver and reduced fetal body and cerebral weights. With increasing concentrations of Trien-2HCI in maternal drinking fluid, the copper concentrations in fetal cerebrum and liver decreased and fetal abnormalities in brain such as intracerebral hemorrhage and exencephaly increased. When we compare these data on copper concentrations of mother and fetus given 12,000 ppm with those in C3H brindled model mice of Menkes disease, maternal hepatic copper was higher than that in Menkes heterozygous mouse, and fetal hepatic or cerebral copper was higher than or the the same as that in Menkes hemizygous mouse. Our results suggest that the effect of Trien-2HCI during pregnancy was much clearer in the fetus than in the mother, and fetus had a pronounced adverse effect on tissue copper concentrations rather than on body and tissue weights.

A-01 NAKATSUKA, T., T. KOMATSU and T. FUJII, Development Reseach Laboratories, Banyu Pharmacological Co., Ltd., Saitama. Axial skeletal malformations induced by acetazolamide in rabbit fetuses. Acetazolarnide produces unique forelimb defects in rats, mice and hamsters. However, the teratogenic potential of the compound has not been well investigated in non-rodent mammalian species. In the present study, we evaluated the teratogenic potential of acetazolarnide in rabbits and observed drug-related axial skeletal malformations in the fetuses. Three groups of 18 artificially inseminated females were treated orally by gavage on Days 6 through 18 of gestation with acetazolamide at 50, 100 or 150 mg/kg/day. These doses produced reductions in maternal body weight gain during the treatment priod. Blood gas and serum biochemical analyses performed 4 hr. after the last dose revealed treatment-related acidosis (decreased arterial pH and HCO,-) and electrolyte changes (decreased potassium and increased chloride) which were expected pharmacological effects of the compound as a carbonic anhydrase inhibitor. There were no treatment-related effects on embryonic survival. Average fetal body weights in the drug treatment groups were dose-dependently decreased compared to controls (5%. 7% and 12% below control in the 50, 100 and 150 mgikglday groups, respectively). No treatment-related external abnormalities were seen. In the fetal skeletal examination, thoracic and lumbar vertebral malformations occurred in 0.7%, 3.9% and 6.1% of fetuses in the 50, 100 and 150 mglkglday groups, respectively, compared

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to none in the control group. In addition, missing vertebra defined as one reduction in the number of pre-sacral vertebrae was seen in a small number of fetuses in the 100 and 150 mglkglday groups. These axial skeletal malformations were, in some cases, associated with costal malformations. These results indicate that acetazolamide at maternotoxic doses can Produce axial skeletal but not limb malformations in rabbits.

A-08 KUMAZAWA, T.. I. HAYASAKA, M. INOUYE and H. YAMAMURA, Safety Assessment Laboratory, Sanwa Kagaku Kenkyusho Co., Ltd., Kasugai, Aichi, and Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi. Initial changes in the development of wavy ribs caused by furosemide in the rat fetus. This study was designed to observe the initial changes of wavy ribs induced by furosemide, a loop diuretic, in rats. Pregnant Crj:CD (SD) females were treated orally with 300 mglkg of furosemide on day 16 of gestation. The I 1 th-12th ribs of their fetuses were observed light- and electron-microscopically at different times after treatment. Morphological features of the initial calcification sites were not significantly different between treated and control fetuses during the first 6 hrs. At 12 hr, extracelluar matrix vesicles, particularly calcified ones, derived from osteoblasts in treated fetuses were fewer than those in control fetuses, although osteoblasts and chondrocytes of treated fetuses were similar in fine structure to those of controls. At 24 hr, while intramembranous and endochondral ossification were observed in control fetuses, they did not appear in treated fetuses. At 48 hr, caicification spread toward both ventral and dorsal sides from the primary ossification center in the control fetuse. Calcification started in the treated fetuses and excessive osteoid was observed in the periphery of the shaft. Bent ribs first appeared at this stage. These results suggest that defective calcification in furosemide-treated fetuses is caused by inhibition of matrix vesicle formation and/or matrix vesicle calcification.

A-09 ISHIKAWA. H., K. OMOE and A. ENDO, Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Yamagata. Digit malformations induced by cytosine arabinoside (Ara-C) in mouse fetuses: Relationship between the hour of the day (dg 10) of treatment and the incidence. We examined how the incidence and patterns of Ara-C-induced digit malformations differ when pregnant mice are treated at different times within the same gestational day. ICR female mice were housed under

a controlled IighVdark cycle of 12 hours (lighting: 0800-2000). Females were paired with a male for one hour from 0200 to 0300. The pregnant mic were injected intraperitoneally with a single dose of 5 mg/kg of Ara-C on dg 10 at either 0800, 1100, 1400 or 1700 (plug day = dg 0). All mice were killed at 1000 on dg 17. After laparotomy, implantation sites were counted and live fetuse were removed, sexed, weighted and examined for digit malformations. During the 9-hour period studied, the incidence of oligodactyly of both the forelimbs and the hindlimbs increased gradually, while the incidence of polydactyly in hindlimbs decreased gradually, when the time of the treatment grew later. At dg 10, development of digits in mouse embryos was very stage-specific, and the sensitivity of digit formation to Ara-C seemed to change even hourly. Conversely, the patterns of Ara-C-induced digit malformations can be used as sensible markers to speculate on the developmental stage of the embryos.

A-10 ISHIKAWA, H., K. OMOE and A. ENDO, Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Yamagata. "Catch-up" phenomenon of digit formation in mouse fetuses from delayed fertilization. We previously observed that digit formation in mouse embryos from early morning mating progresses to the extent that it does not differ from that from overnight mating. If such a catch-up phenomenon exists throughout the whole stage of gestation, the time-spectrum of the critical period for organogenesis must shift accordingly. In this study, to examine whether the embryos from normal and delayed matings (three, six or nine hours delay) respond differently to some acute teratogens when they are treated at the same time-point from mating, 5 mglkg of cytosine arabinoside (Ara-C) was given to pregnant mice intraperitoneally at 246, 249 or 252 hours after mating. The results showed that the patterns of Ara-Cinduced anomalies in embryos from delayed mating were of more advancen stages, when compared to the groups with the same time intervals from mating. In other words, oocytes fertilized up to nine hours after the presumed time of ovulation could grow similarly to those of normally fertilized oocytes at their timeschedule of development. This suggests that the "ovulation clock" may be governing the growth and development of oocytes rather than "fertilization clock".

A-11 OHMORI, H., T. KOBAYASHI and M. YASUDA, Department of Anatomy, Hiroshima University School of Medicine, Hiroshima. Development of mouse cerebellum following oral administration of phenytoin (PHT) in neonatal period.

JAPANESE TERATOLOGY SOCIETY ABSTRACTS Phenytoin (PHT) is a commonly prescribed anticonvulsant drug. Chronic PHT administration to epileptic patients may cause cerebellar dysfunction and degeneration. PHT is weakly acidic and slightly soluble in water. Recently, it was reported that oral administration of oil suspension of PHT results in a significant increase of its bioavailability. The purpose of this study is to examine neurotoxic effects of PHT on the development of the mouse cerebellum. PHT suspended in sesame oil was administered by gavage to newborn Jcl:ICR mice with 50 mgikg body weight once a day for the period of postnatal days 214. The mean particle size of PHT powder was 17 pm. Oral administration of PHT in neonatal period reduced total brain weight, cerebellar weight, and size of the cerebellum (transverse width and logitudinal width) measured on postnatal day 56. In reflexologic tests, placing response and negative geotaxis were poorly developed. Pyknotic cells in the external granular layer significantly increased and were prominent in the vermis area of the cerebellum from 14-dayold mice treated with PHT. These results indicate that oral administration of PHT in the neonatal period produces neurotoxic injuries in the developing cerebellum.

A-12 YAMAMOTO, Y., K. YAMAMOTO. Y. FUKUI and A. KURISHITA, Department of Legal Medicine, Faculty of Medicine, Kyoto University, Kyoto, and Department of Radiation Research, School of Medicine, Tohoku University, Sendai. Miyagi. Teratogenic effects of methamphetamine. The spread of methamphetamine (MAMP) abuse is a serious problem especially in the case of pregnant women, because this drug may have harmful effects on the fetus. There are, however, few reports on this problem. We reported that MAMP has teratogenic effects on mice and that environmental factors such as caging conditions have a profound effect on the mortality rate. In the present study, the effect of housing conditions on the rate of malformations (gross and skeletal) were examined. Pregnant Jcl:ICR mice were caged individually or in groups of three. On day 8 of gestation, they were given an intraperitoneal injection of MAMP hydrochloride at a dose of 21 mg/kg. On day 18 of gestation, they were killed and fetuses were removed and examined for gross and skeletal malformations. There was a significant difference in the malformation rate between the two groups. The animals kept in group showed a higher rate of malformation. Severe malformations such as exencephaly were observed only in the grouped animals. The same was the case with costal anomalies (defect and fusion). It was confirmed that caging conditions have a profound effect on malformation rate as well as on mortality rate.

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A-13 TAKANO, T., M. OHNO, T. YAMANO and M. SHIMADA, Department of Pediatrics, Shiga University of Medical Science, Ohtsu, Shiga. Congenital hydrocephalus in suckling hamsters caused by transplacental infection with parainfluenza virus type 3. The possible occurrence of congenital hydrocephalus by viral infection of the mother was examined by inoculating pregnant hamsters intravenously with parainfluenza virus type 3 (PIV-3). Female Syrian hamsters 9-12 weeks of age were mated with breeder males for 24 hours. Seven pregnant hamsters were inoculated with 0.5 ml of 1 .O x 10' pfuiml of PIV-3 into the left cervical vein on either day 11, 12, 14 or 15 of gestation. Brains of offspring born to the mother hamsters were examined 13 to 17 days after birth to confirm the possible occurrence and incidence of congenital hydrocephalus. When PIV-3 was inoculated on the 11th or 12th day of gestation, all mothers aborted. The hamsters which had been inoculated on the 14th or 15th day of gestation gave birth on the day of term, i.e. the 16th day of gestation. Sixteen offspring of 28 alive at birth survived until 13 days of age. Three of these survivors showed a centro-occipital prominence of the skull. Macroscopic examination of the brains showed a bulged cerebral hemisphere. Histological examination on the brains of these hydrocephalic hamsters revealed marked ventricular dilatation with thinning of the cerebral cortex and aqueductal forking. Results obtained in this experiment may indicate that a virus inoculated into the cervical vein can arrive in the fetuses after passing through the placenta and infect the central nervous system.

A-14 TSUTSUI Y., A. KASHIWAI. C . KADOTA and N. KAWAMURA, Department of Morphology, Institute for Developmental Reserch, Aichi Prefectural Colony, Kasugai, Aichi. Analysis of susceptibility of mouse embryos to murine cytomegalovirus infection in early and middle gestational stages. Cytomegalovirsu (CMV) is the most significant infectious cause of congenital anomalies of the central nervous system. However, little is known about the timing of gestational stage and site of embryos. In the present study, we tried to infect mouse embryos with murine CMV (MCMV) in the early and middle gestational stages. For early embryos. we injected MCMV into blastocysts from BDF1 mice using a micromanipulator and returned them to the uteri of the pseudopregnant ICR mice. On day 11 of gestation, embryos were fixed in Bouin's solution, embedded in paraffin and subjected to serial sections for immunohistochemical detection of viral antigen-positive cells. We produced no infected embryo using this system. Then, the conceptus on day 8.5 of gestation were injected with MCMV by the Jaenisch method (1985). Embryos from day 10.5 to 12.5 of gestation were

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analyzed immunohistochemically. Viral antigen positive cells were first observed in the placenta, then endothelial cells and mesodermal cells in the embryos. After day cells were first observed in the placenta, then endothelial cells and mesodermal cells in the embryos. After day 12.5 of gestation, viral antigen-positive cells were observed even in the neuroectoderm and eye balls.

A-15 SAITO, K., K. SUZUKI and S. MOTOYOSHI, Department of Veterinary Physiology, and Department of Veterinary Internal Medicine, Nippon Veterinary and Animal Science University, Tokyo. Lethal and teratogenic effects of radio frequency radiation on developing chick embryo. Developing chick embryos were exposed to 428MHz radio frequency (RF) radiation at a power density of 5.5 mW/cm2 for more than 20 days. The calculated SAR using both values of the electric and magnetic fields ranged between 3.1 and 47.1 mW/kg. Temperature change in eggs of which inlet was replaced by 25% gelatin, was not detected by fluoroptic thermometer in the RF field. Ten eggs were incubated in each of seven experiments. The 1st and 4th experiments were sham operated for control. The average hatchability was 84.2% and 38.0% in control and exposed eggs, respectively. In the RF exposed group, embryonic development was stopped within 10 days after the start of incubation, while the remainder (76.6%) died in the egg shell because of inability to hatch. Since the control eggs hatched on days 21 or 22. and the exposed ones on days 21 -23, it was clear that prolongation of the incubation period occurred in the exposed groups. A functional abnormality consisting of creeping movement and inability to stand was found in 89% of the hatched embryos in the exposed group, but was not in the controls. It was concluded that 428 MHz RF radiation to developing chick embryos induced embryolethal and/or teratogenic effects and delayed hatching. It was also demonstrated that these adverse biological effects were due to a direct action of the RF radiation and not its inherent thermal effect.

A-16 INOUYE, M.,M. TAMARU, H. YAMAMURA and T. AZAKAMI, Reserch Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi, Fujita Health University School of Medicine, Toyoake, Aichi, and Nagoya Institute of Technology, Nagoya, Aichi. Combined effects of microwave and ionizing radiations on the developing mouse brain. Pregnant Slc:ICR mice were exposed to single microwave and gamma-radiations, and both of them in combination, on day 13 of pregnancy: (1) 2.45 GHz

microwaves at 46.0 mW/cm' for 3 min (rectal temperature 42.3 ? 1.3"C); (2) 0.24 Gy gamma-rays; (3) gamma-radiation first and then microwave radiation within a half hour, or 2 or 3 hours later, (4) microwave pretreating 1-1 5 hours before gamma-radiation, and (5) sham-treatment. To examine the effect of microwaves alone, dams were killed and their fetuses were sampled 1-12 hours after exposure. For the combined effects, fetuses were recovered 8 hours after exposure to gamma-rays. The fetal brains were histologically examined for cell death (apoptosis) in the ventricular zone of telencephalon. Levels of cell death following exposure to sham, microwaves alone, and gammaradiation alone were 0.12, 0.5 and 8.3%, respectively. Exposure to microwaves 1 hour before or 2 hours after gamma-radiation caused respectively 8.8 and 8.9% cell mortality, much the same as the sum of the levels for microwaves alone and gamma-radiation alone. Microwave exposure within a half hour after gammaradiation, on the other hand, caused 10.4% mortality, more than the sum of their respective levels. Whereas, microwave pretreating 12 or 15 hours before gammaradiation resulted in slightly, but significantly, lower mortality than that for gamma-radiation alone. Thus, enhanced or adaptive radioresponses were induced by different exposure regimens to microwaves in the developing mouse brain.

A-17 MORISHIMA, M., H. YASUI, S. MIYAGAWA-TOMITA, M. ANDO, M. NAKAZAWA and K. MOMMA, Research Division, and Department of Pediatric Cardiology, the Heart Institute of Japan, Tokyo Women's Medical College, Tokyo. Cardiovascular anomalies induced by hyperthermia in fetal rats. Previously we reported the teratogenicity of hyperthermia in fetal mice, especially cardiovascular anomalies. However, in mice it is difficult to control any increases in body temperature, so the present study was done in rats. Wistar-lmamichi pregnant rats at day 9 of gestation were exposed to a temperature of 43.0-43.8% for 12-18 minutes by immersing in a water bath twice a day. On day 18 of gestation, the fetuses were obtained to examine cardiovascular anomalies. Fetuses were divided into 2 groups: Group A included fetuses whose dams had shown an increase in body temperature to more than 42.8"C, and group B those of dams whose body temperature was less than 42.8%. The incidence of cardiovascular anomalies (CVA) in group A was higher than in group B (27134, 79% vs. 31/67. 46%). Fifteen fetuses in group A and 2 in group B had CVA with heterotaxia, including AV canal defect with double-outlet right ventricle (DORV) (10/17) or transposition of the great arteries (TGA) (3/17), DORV (2/17),AV canal defect with tetralogy of Fallot (1/17), and single left ventricle with TGA (li17). The cases with heterotaxia showed left isomerism or a tendency to left isomerism of the visceroarterial situs, and ten of them showed the lev0 type of ventricular loop.

JAPANESE TERATOLOGY SOCIETY ABSTRACTS These results suggest that maternal hyperthermia induced cardiovascular anomalies with heterotaxia including left isomerism in fetal rats, and it is a useful model to investigate the differentiation of sidedness.

A-18 SHOJI, R., R. KIMURA, F. MATSUI, N. MAEDA and A. OOHIRA, Department of Embryology, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi. Effects of Tween 80 on reaggregation from dissociated fetal rat brain cells in vitro. Dissociated cell aggregation culture technique was applied to develop an in vitro preliminary screening test system for environmental teratogens. Fetal brains of Slc:SD rats on day 14.5 of gestation (vp day = 0) were dissociated into single cell suspension with collagenase treatment. The initial cell suspension, containing 5 x lo6 cells, was diluted with 3 ml of culture medium in 20 ml Erlenmeyer flasks. The flasks were gassed with a mixture of 5% CO, in air, and were incubated for 48 hours at 37°C on an orbital shaker at 70 rpm. Inhibiting effects on the cell aggregate formation were examined in various concentrations of Tween 80. The number of cell aggregates was not changed in 0.01 and 0.025% of Tween 80, but increased in 0.05 to 2.0%. The size of aggregates decreased to 63% in 0.05% and suddenly around 20% in 0.075 to 2.0% groups in the major axis compared with that of the control specimen, respectively. The shape of aggregates was abnormal in 0.05% or more treatment groups. The cell viability examined after 24 hours of incubation was almost the same as the control level with about 75% in groups treated with 0.05% or less of Tween 80. In the concentration of 0.075% half of the treated cells survived, but only a few in treatments with 0.1% over. Treatments with over 2.5% of Tween 80 completely inhibited aggregate formation. The results suggest that the critical dose may be around 0.05% of Tween 80 in the in vitro tissue reconstruction with dissociated fetal rat brain cells.

A-19 OOSHIMA, Y. and K. SHIOTA, Reseach and Development Division, Takeda Chemical Industries, Ltd., Yamaguchi, and Faculty of Medicine, Kyoto University, Kyoto. Congenital malformations in genetically diabetic mice, yellow KK. Yellow KK mice have the AYallele which was introduced into KK mice. They spontaneously developed non-insulin dependent diabetes mellitus (NIDDM). Female yellow KK mice were mated at 7 and 13 weeks of age (blood glucose: ca. 260 and 500 mg/dl, respectively) and their fetuses were examined for external and skeletal features on day 18 of gestation. The frequency of external malformations was significantly higher in the fetuses from 13-wk-old dams

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than in those from 7-wk-old dams (20.7% vs 12.5%) and microtia increased in the former (18.1% vs 10.2%). In KK mice (blood glucose: ca. 170 mgidl), external malformations and microtia were few (2.3% and 0.7% respectively). Skeletal malformations occurred at low frequency in all groups. The frequency of skeletal variations was high in all groups (93.6-95.0%) and major variations were bifurcation of cervical arch, cervical rib and lumbar rib. The lumbar rib increased with the blood glucose levels in dams: 38.6% and 29.8% in 13- and 7-wk-old dams of yellow KK mice, respectively, and 12.0% in KK mice. Yellow KK mice mated at 13 weeks of age were treated with an oral-hypoglycemic agent, ciglitazone, as an 0.1% dietary admixture during gestation period. Glucose level in dams on day 18 gestation and frequency of microtia in their fetuses were lower in the treated group than in the untreated control. From these results, some types of malformations increased with blood glucose level in dams in NIDDM model similar in IDDM model.

A-20 MIURA, S., N. NATSUME, R. HORIUCHI, H. FURUKAWA, H. KINOSHITA, S. KONDO, T. NARUKAWA and T. KAWAI, Second Department of Oral and Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi. Role of maternal factor on cleft lip and/or palate in A/J mice. There are maternal and embryonic factors concerning cleft lip/palate in N J mice. It was hypothesized that the maternal factor may be changed. We carried out the following experiment. Mice of the NJ strain were used. Skins of the B1O.A strain mice were grafted into N J mice aged 21 days. After that, females aged 70 days were mated overnight with males, (plug day = day 0). All pregnant females were sacrificed on gestation day 18. Implantation sites, resorptions, and dead fetuses were noted, and then the fetuses were removed and examined for gross anomalies. The data for the incidence of cleft lip and palate and that of isolated cleft palate were analysed with MannWhitney's U test. As a result, the incidences of cleft lip and palate in experimental groups were significantly reduced. Their fetal mortality rates were unchanged. Our results suggested that the maternal factor in NJ mice may be changed.

A-21 YOKOYAMA, A. and M. AKITA, Toxicology Reserch Lab., Japan Tobacco Inc., and Kamakura Woman's College, Kanagawa. Toxic effects of methylazoxymethanol on cultured rat embryos.

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The effect of methylazoxymethanol (MAM) on head development was studied using rat whole embryo culture system. Embryos were explanted at the stage of total somite 33, cultured in 100% rat serum containing various concentrations of MAM and examined at the 33-48 somite stage for development of the head. The technique is based on the method developed by New and Cockroft (1978). 11 .O-day-old rat embryos were cultured in one serum bottle containing 5 ml rat serum in a rotating culture system (20 r.p.m.) at 38.0"C for 48 hr. MAM treatment resulted in a dosedependent increase in the incidence of microcephaly with a maximum incidence of 100%. Each part of the head length was reduced on the MAM treated groups (2501000 pgiml). Our results suggested that the MAMinduced microcephaly was involved in the direct effect of MAM in the head of developing rat embryos.

A-22 OSUMI-YAMASHITA, N., S. KATO, H. MANO, S. MASUSHIGE and K. ETO, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo, and Faculty of Agriculture, Tokyo University of Agriculture, Tokyo. Abnormal anterior CNS development in rat embryos cultured in vitamin A-free serum. Vitamin A (VA) and its derivatives are essential for cell proliferation and differentiation in adult and for development in the embryo. Retinoids are also wellknown teratogens inducing abnormalities in the central nervous system (CNS), face, limb, etc. The purpose of this experiment was to observe the developing embryos in VA deficient environment in order to examine how VA is involved in the organogenesis of the mammalian embryo. We cultured rat embryos of the neural plate stage (day 9 P.c.) in VA-free rat serum obtained from VAdeficient male rats, or in normal rat serum as controls, for 48 hours. VA was not detected in VA-free serum (less than 0.4 pgidl) by HPLC, while normal serum contained 30 pg/dl of VA. At the end of culture the embryos were developed as weil as day 11 embryos in vivo, and general morphology was not so different between two groups. However, histological observation revealed abnormality of anterior CNS in the embryos cultured in VA-free serum. Extra bulges of the neural epithelium were anteriorly to optic vesicles, making the brain cavity narrower. No abnormalities were found in other parts of CNS. From these results, it is suggested that VA and/or its derivatives are involved in development of early CNS. Possibly retinoic acid may give positional information for subdivision of the neural plate.

A-23 NINOMIYA. H., K. KISHIDA, Y. OHNO and K. ETO, Reserch Laboratories, Nippon Shinyaku Co., Ltd., Kyoto, Division of Pharmacology, National Institute of Hygienic Sciences, Tokyo, and Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo. Development of rabbit whole embryo culture techniques. Rabbits as well as mice and rats are used in teratological study for species specificity. Mouse and rat embryo cultures are widely used as techniques in teratogenicity studies. However, the whole embryo culture of rabbits has not been sufficiently developed. Thus, we tried further development of the culture technique of rabbit embryo. Rabbit embryos of Japanese white strain on day 9, 10, or 11 of gestation were explanted and cultured for 24 or 48 hours. Embryos from day 9 of gestation were cultured in 100% rabbit serum with gas mixture containing 20%0,, 5%CO,, and 75%N, for the first 24 hours, and 95%0, and 5%CO, for the following 24 hours. Embryos from day 10 or 11 of gestation were cultured in loo%, 80%, or 60% rabbit serum respsctively with 95%0, and 5%CO, for 48 hours. At the end of the culture, crown rump length, head length, somite number and protein contents were examined in the embryos. Embryos cultured for 48 hours from day 9 and 10 with 100% rabbit serum, and 24 hours from day 11 with 100% and 80% rabbit serum showed good growth and development as well as those of in vivo embryos, but the protein contents of the embryos in each group were less than those of in vivo embryo. These results suggest that rabbit embryo cultures from day 9, 10 and 11 are suitable as a technique in teratogenicity studies. (supported by Japan Health Sciences Foundation)

A-24 AOYAMA, H., S. FUJII, H.HOJO and S. TERAMOTO, Mitsukaido Laboratories, Institute of Environmental Toxicology, Mitsukaido, Ibaraki. Alterations in bronchial branching and distribution of extracellular matrices in rat embryos with heritable pulmonary lobation anomalies. Rat embryos having heritable pulmonary lobation anomalies (@//fpb were examined for bronchial branching and distribution of extracellular matrices (ECMs) on day 12-14 of gestation (plug day = day 0) after staining lung serial sections with AZAN, alcian blue or aldehyde fuchsin. Entodermal cells of the lung buds on day 12 of gestation formed the right and left main bronchial buds. These cells were separated from surrounding mesenchymal cells by ECMs except for the distal end of the

JAPANESE TERATOLOGY SOCIETY ABSTRACTS bronchial buds. No morphological differences were found between fpl/fp/ embryos and phenotypically normal @//t littermates at this stage. In fp//+ embryos, the right main bronchial buds ramified the cranial, middle and intermediate lober bronchial buds by day 13 of gestation, and segmental bronchial buds appeared by day 14. ECMs disappeared at the tips of all lobar and segmental bronchial buds, suggesting the existence of entodermal-mesenchymal interactions in these regions. In fp//fp/embryos, on the other hand, the right main bronchial buds did not ramify the middle and intermediate lobar bronchial buds from their lateral region, but swelled ventrally on gestation day 13. The main bronchial buds remained covered with ECMs on the lateral side, but uncovered at the ventral sweliing from which the middle and intermediate lobar bronchial buds arose on gestation day 14. These observations may indicate that alterations in distribution of ECMs cause branching abnormalities of lobar bronchial buds in fp//fp/embryos.

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Kawasaki, Kanagawa. Molecular genetic analysis of the transgene integration locus in microtic transgenic mice. Dysmorphic mouse mutants are important to analyse the molecular mechanism of mammalian development and teratogenesis. We investigated mice with microtia which is caused by the insertional mutation on chromosome 10. To isolate the gene(s) which corresponds to the mutated locus, we cloned the transgene integration site from a microtic mouse. Southern blot analysis revealed that a transgene was inserted in the mouse genomic DNA with slight deletion. For cloning of the integration site of the transgene, the genomic library created from DNA isolated from a microtic mouse was screened with a part of the transgene as a probe. On the other hand, inverse PCR (IPCR) was carried out to amplify the transgene integration site directly from the mouse genomic DNA. Finally, we obtained 10 kb mouse genomic DNA adjacent to the transgene from the IPCR products and 3 phage clones. Now we are isolating the preintegration site from a wild-type mouse genomic library with the flanking region DNA as probes.

A-25 FUJIWARA, M., T. WATANABE, N. OSUMIYAMASHITA and K. ETO, Safety Research Laboratories, Yamanouchi Pharm. Co., Ltd., and Tokyo Medial and Dental University, Tokyo. Analysis of tissue interaction in lens differentiation of Uchida Rat embryos using tissue recombination. Uchida rat embryos have a characteristic phenotype, lacking both eyes and nose. Histological observations indicate that the abnormal development was caused by failure of nasal and lens placode formation. In the present study tissue recombination experiments were performed to examine the inductive tissue interaction involved in lens differentiation in Uchida rat embryos. Dispase-separated presumptive lens ectoderm from day 11 embryo was recombined with optic vesicle and cultured for 4 days in vitro. When the presumptive lens ectoderm from normal embryos was cultured in recombination with the normal or mutant optic vesicle, a developed lens was induced in the epithelium in 13 out of 24 cases (54%) or in 14 out of 27 cases (51%). However, none of the ectoderm from mutant embryos recombined with normal or mutant optic vesicles showed any lens differentiation. This may suggest that ectoderm has no potential to differentiate into the lens in response to an appropriate inducer.

A-26 NAORA, H., M. KIMURA. S. TAKAGI, M. YOKOYAMA, M. KATSUKI and 0. TANAKA, Department of Anatomy, Shimane Medical University, Izumo, Shimane, Department of DNA Biology, School of Medicine, Tokai University, Isehara. Kanagawa, NTT Laboratory, Tokyo, and Central Institute for Experimental Animals,

A-27 IWASAKI, K., H. ITO, I. SHIMOKAWA, T. MATSUO and T. IKEDA, First Department of Pathology, Nagasaki University School of Medicine, Nagasaki. The distribution of tenascin in the heart and great vessels with anomalies induced by bis-diamine. Tenascin was reported to be distributed in the migratory pathway of neural crest cells, and a previous study confirmed the localization of tenascin in the conotruncal ridge and aorto-pulmonary septum. In this study we examined the distribution of tenascin in anomalies of the heart and great vessels induced by bis-diamine. Wistar strain rats were treated with bis-diamine, 200 mg/day, on day 9 and 10 of gestation. The embryos removed on day 12, 13 and 14 of gestation were fixed by the AMeX method and embedded in paraffin. The thoracic region of the embryo was sectioned serially and immunohistochemically stained with anti-tenascin antibody by the ABC method. In normal rat embryos, positivity for tenascin was noted in the distal mesenchyme of truncus on day 12 of gestation, and localization in the mesenchyme of the conotruncal ridge and aorto-pulmonary septum on day 13 and 14 of gestation. In the treated rat embryos, which showed conotruncal anomalies including persistent truncus arteriosus and pulmonary hypoplasia, there were no apparent changes in the positivity for tenascin on day 12 of gestation, but on day 13 and 14 staining for tenascin tended to decrease in correlation to the poor development of the mesenchyme in the conotruncal ridge and aorto-pulmonary septum. These results suggest that tenascin is distributed with migrating neural crest cells in the heart and great vessels and is involved in the formation and devision of the truncus.

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A-28 EMA, M., T. ITAMl and H. KAWASAKI, National Institute of Hygienic Sciences, Osaka Branch, Osaka. Teratogenicity of butyl benzyl phthalate in rats. In previous studies, we have shown that complete resorption of all implanted embryos was found in pregnant rats given plasticizer butyl benzyl phthalate (BBP) at a dose of 2.0% in the diet on day 0 through day 20 of pregnancy. The object of the present study was to determine if period of exposure would modify the developmental toxicity of BBP. Wistar rats were used throughout this study. Pregnant rats were given BBP at a dose of 2.0% in the diet on days 0-7,7-16 or 16-20 of pregnancy (sperm = day 0). Pair-fed pregnant rats were given the same amount of feed consumed by the rats fed 2.0% of BBP on days 0-20 of pregnancy. Preimplantation loss in the BBP-treated groups was comparable to control and pair-fed groups. Postimplantation loss in pregnant rats given BBP on days 0-7 or 7-16 was higher than that of control and pair-fed pregnant rats. Marked teratogenicity was detected in fetuses of dams given BBP on days 7-16. Cleft palate and fusion of the sternebrae were predominantly observed. Sixty eight of 73 fetuses had cleft palate in this group. Pregnant rats were dosed orally with BBP dissolved in olive oil at a dose of 0.5, 0.75or 1 .O g/kg on days 7-1 5 of pregnancy. In the 1 .O g/kg group, no live fetuses were derived. Twelve of 25 fetuses had cleft palate in the 0.75 g/kg group. Fusion of the sternebrae and dilatation of the renal pelvis were also frequently observed in this group. BBP was dosed orally to ratson days 7-9, 10-12 or 13-15 of pregnancy. Cleft palate was detected in 34 of 73 fetuses and 45 of 55 fetuses of dams given BBP on days 13-15 at doses of 0.75and 1 .Og/kg, respectively. It is concluded that BBP has teratogenicity in rats.

A-29 NAKATSU, T., H. IRIE, K. SHIOTA, K. ARISHIMA, T. INOMATA and M. YAMAMOTO, Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto, and Department of Anatomy, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa. Effects of retinoic acid on fetal mouse palates cultured in vitro. Retinoic acid produces a variety of malformations including cleft palate in many species. Explanted palates of day-I2 mouse fetuses were cultured in a chemically-defined medium (Shiota et al., 1990) and the effects of all-trans-retinoic acid (RA) were examined. RA was added to the culture medium at concentrations 3-3000 ng/ml. RA inhibited palatal fusion dependently on its concentration, although palatal shelves of normal size formed and made contact at 330 ngiml. Scanning electron microscopic studies showed that the medial cells of RA-treated palates failed to undergo programmed cell death which occurred in control palates. The oral epithelium of control palates lost microvilli and became squamous after palatal fusion,

but RA-exposed cells remained ciliated and simulated nasal-like epithelial cells. We have shown that RA alters the temporal and spatial sequence of expression of epidermal growth factor receptor (EGF-R) in vitro (Nakatsu et al., 1990). Thus, it seems that abnormal differentiation of the medial epithelium which may be related to altered EGF-R expression is responsible for RA-induced cleft palate and subsequently inhibits the differentiation of oral epithelium. (Supported by grants-in-aid from the Japanese Ministry of Education, Science and Culture.)

A-30 YASUDA, M., T. J. SATO and N. INOUE, Department of Anatomy, Hiroshima University School of Medicine, Resistance of exencephalic mouse Hiroshima. fetuses to cleft palate induction with dioxin. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent cleft palate inducing teratogen in mice. In a series of experiments using Jcl:ICR mice, in which exencephaly occurs spontaneously at a low rate, we incidentally found in a litter treated with TCDD at 40 pg/kg at day 12.5 of gestation (VP = day 0) that an exencephalic fetus had a closed palate whereas the remaining non-exencephalic litter mates had cleft palates. This experience prompted us to examine whether exencephalic fetuses are really resistant to the cleft palate inducing action of TCDD or not. Pregnant Jcl:ICR mice were pre-treated with CdCI, once at 6 mg/kg at day 7.5 (group A) intraperitoneally (ip) or at 2 mg/kg three times at days 7.5,8.5, and 9.5 (group B) ip to induce exencephaly in embryos. Then the dams were treated with TCDD at 40 pg/kg at day 12.5 by gavage and fetuses were examined for malformations at day 18.5. In group A, 58% of live fetuses had exencephaly. None of the exencephalic fetuses had cleft palate, whereas 50% of the fetuses without exencephaly had cleft palate. In group B, 29% of live fetuses had exencephaly without any case of cleft palate, whereas 84% of non-exencephalic fetuses had cleft palate. Thus resistance of exencephalic fetuses to the cleft palate inducing action of TCDD was demonstrated in mice. Exencephaly induced by maternal hyperthermia applied at day 8.5 was also effective for prevention of cleft palate induction with TCDD. (Supported by Grant in Aid for Scientific Research on Priority Areas #I02202128 from the Ministry of Education, Science and Culture, Japan.)

A-31 SATO, T. J., M. YASUDA and A. HORIMOTO, Department of Anatomy, Hiroshima University School of Medicine, Hiroshima. Preventive mechanism in exencephalic mouse fetuses against cleft palate induction with dioxin.

JAPANESE TERATOLOGY SOCIETY ABSTRACTS 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been suggested to inhibit programmed cell death in the medial epithelium on palatal shelves, and to induce cleft palate in mice. We showed that exencephalic fetuses were resistant to cleft palate induction with TCDD. We examined how exencephaly altered palatogenesis in mouse fetuses exposed to TCDD. On day 7.5 (VP = 0) of gestation, a 6 mg/kg dose of CdCI, was intraperitoneally injected into Jcl:ICR mice, and on day 12.5, a 40 pg/kg dose of TCDD was orally administered. Fetuses were collected from day 12.5 to 15.5, and processed for scanning electron (SEM) or light microscopy (LM). In macroscopic observation, the palatal shelves in the fetuses with exencephaly first contacted in the posterior part of the medial edge, while those in the fetuses without exencephaly contacted in the middle part. In coronal sections, the base of the skull became narrower in the fetuses with exencephaly than those without exencephaly. In SEM and LM observations, peridermal cell death was noted only in the firmly contacted part of the medial edge in the TCDD-treated fetuses with exencephaly. From the above findings, peridermal cell death in the medial edge of palatal shelves seems to begin in the posterior part in the exencephalic fetuses exposed to TCDD, and to proceed anteriorly. The difference in the part of the first contact between fetuses with and without exencephaly is related to structural changes in the base of the skull. It is suggested that these structural changes lie at the base of the preventive mechanism against cleft palate induction with TCDD.

A-32 RASJAD, C., A. R. DATU, M. YASUDA and K. YAMASHITA, Department of Surgery, and Department of Anatomy, Faculty of Medicine, Hasanuddin University, Ujung Pandang, Indonesia, and Department of Anatomy, Hiroshima University School of Medicine, Hiroshima. Patterns and pathogenesis of limb malformations in mice induced by methoxyacetic acid. Methoxyacetic acid (MAA) is one of the metabolites of phthalate esters which are commonly used as plasticizers of plastics. Jcl:ICR mice at gestational day 10.5, 11.O or 11.5 (VP = day 0) were orally treated with 0, or 10 mmol/kg of MAA. Their embryos were examined at various intervals after the MAA treatment. Observations at day 15.5 showed that forelimbs had greater susceptibility to M M than hindlimbs, and that limbs treated at day 11.5 were more severely affected than those treated at day 10.5 or 11.0. MAA produced various patterns of limb malformations according to the gestational day of administration. Under a light microscope and a transmission electron microscope, excessive cell death was observed in the mesenchyme as well as in the apical ectodermal ridge (AER) 2 h after the MA4 treatment, and became maximum at 6 h. The distribution of cell death 6 h after treatment was not consistently different in limbs treated either at day 10.5, 11.O or 11.5, however, the final

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pattern of limb defects was systematically different in fetuses treated at different stages. These results indicate that variations in patterns of limb defects induced by MA4 are due to differences in the amount and distribution of cell death, together with the different regenerative capacity of surviving cells. (Supported by JSPS Ronpaku Program.)

A-33 YASUI, H., M. MORISHIMA, S. MIYAGAWA-TOMITA, M. ANDO, M. NAKAZAWA and K. MOMMA, Department of Pediatric Cardiology, and Research Division, the Heart Institute of Japan, Tokyo Women's Medical College, Tokyo. The morphology of transposition of the great arteries induced by all-trans retinoic acid. Transposition of the great arteries is when the aorta arises from the right ventricle and the pulmonary artery from the left. In order to characterize the morphology of transposition of the great arteries VGA) induced by retinoic acid (RA), a single dose of 40, 60, or 70 mg/kg body weight of RA in 0.1 ml DMSO was given to pregnant Jcl:ICR mice intraperitoneally on day 8.5 of gestation (plug day = 0 ), respectively. Fetuses were examined on day 17.5 to determine relationship between the aorta and pulmonary artery, the angle consisting of atrioventricular valves and semilunar valves (semilunar angle) was measured according to the method described by Becker et al. Sixteen percent (40 mg/kg). 48% (60 mg/kg), 78% (70 mgikg) of fetuses had TGA. Among these fetuses with TGA, 38/101 (38%) were with intact ventricular septum, 62/101 (61%) with ventricular septa1 defect (VSD), and 1/101 (1%) with VSD and pulmonary atresia. Double outlet right ventricle (DORV), VSD, tetralogy of Fallot, or persistent truncus arteriosus were also detected. Hypoplasia of the non-facing cusp of aortic valves, and interruption of the aortic arch (type B or C) and/or aberrant origin of the right subclavian artery were coexistent frequently. The semilunar angie in TGA was widely distributed, on the other hand, it showed two peaks in DORV. We propose RA-treated mice as a useful model for morphogenetic study of TGA.

A-34 YASUDA, Y., H. KONISHI, T. MATSUO, T. KIHARA and T. TANIMURA, Department of Anatomy, Kinki University School of Medicine, Osakasayama, Osaka. Reduced level of basic fibroblast growth factor in yolk sac by retinoic acid induced embryonic death. Basic fibroblast growth factor (bFGF) not only plays a major role in mesodermal induction but also in embryogenesis. Lack of bFGF leads to embryonic death. We have shown that maternal administration of all-trans retinoic acid (RA) induced fetal death at term in rats and mice and that bFGF existed in the visceral

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yolk sac (VYS) in mouse embryos. Thus, the present study was planned to determine whether RA affects bFGF in the W S leading to embryonic death. Pregnant ICR mice were given orally 60, 40 or 0 mgikg of RA suspended in olive oil on day 8 of gestation and sacrificed three days later. Embryos were removed from uteri and examined under a dissecting microscope. W S s were removed and processed either for light microscopy and immunocytochemistry or bioassay for bFGF. The incidence of degenerating or dead embryos in litters and embryos showed a dose-relation to RA. Morphologically, development of yolk sac was suppressed and positive immunostainlg of bFGF was less pronounced in the W S from degenerating or dead embryos than in control embryos. The bFGF content of VYSs from control, RA-exposed and RA-exposed dead embryos were 13.7-20.6, 13.1-19.7 and 5.6-8.3 nglml, respectively, confirming the morphological findings in W S of degenerating or dead embryos. These findings indicate that RA reduced bFGF level in the W S and the level of bFGF for embryonic survival requires at least 13.1 ngiml of purified bFGF in the VYS.

A-35 KAJIWARA, Y., I. NARUSE, A. YASUTAKE and K. HIRAYAMA, Pathology Section, and Biochemical Section, National Institute for Minamata Disease, Minamata, and Kumamoto University College of Medical Science, Kumarnoto. Methylmercury transport across the placenta. It is well documented that methylmercury (MeHg) penetrates the placental barrier, and affects the developing fetuses in the uterus. However, little is known about the translocation mechanism of MeHg. The present experiment clarified the MeHg transport via a neutral amino acid carrier system. Pregnant Wistar rats on day 20 of gestation were i.v. injected with MeHgCl at a dosage of 4 pmolikg under pentobarbital anesthesia. At 30 or 60 min after injection, fetal and maternal blood was collected from the umbilical cord and the mesenteric vein, respectively. Mercury levels in blood cells and blood plasma were measured separately. To observe the competitive effects of neutral amino acids on MeHg uptake, dams were preinjected with 2.0 mmolikg of L-Met, L-Phe. D-Phe and then injected with MeHg. MeHg levels of both maternal blood cells (40.0 2 8.6 pgiml) and maternal plasma (0.10 f 0.02 pg/ml) were not different in any group during the experimental period. MeHg levels in fetal blood cells (1.92 r 0.33 pgiml 60 min after the injection) were suppressed by pretreatments with neutral amino acids, and reduced to 72%, 22% and 47% by L-Met, L-Phe and D-Phe, respectively. No MeHg was detected in the fetal blood plasma. The results suggest that MeHg-Cys conjugates in the maternal blood plasma are transported into the fetal blood through the placenta via L-type amino acid carrier system.

A-3 6 NARUSE, I. and Y. KAJIWARA, Pathology Section, National Institute for Minamata Disease, Minamata, Kumamoto. Effects of deuterium oxide on the development of preimplantation mouse embryos in vitro. We have very little knowledge of the effects of D,O on the development of mammalian embryos. D,O has an anti-proliferative effect, and induces chromosomal aberrations In cultured mammalian cells. Xenopus embryos treated with D,O for a few minutes just after fertilization develop into excessively dorsalized embry0s and conjoined twins. From these, we speculated that some developmental disorders might be found after D,O exposure in early mouse embryos. 2-cell embryos were obtained by flushing out of the oviduct of Jcl:ICR mice (day 1 after copulation). Brinster's media using 100% D,O (MSD ISOTOPES) and 100% H,O were mixed in various percentages. The embryos were cultured in this mixed medium in a 5% CO, incubator. The embryos cultured with 50% D,O medium showed swelling at 4 hrs. after incubation, and cleavage was inhibited. Especially, when cultured with 75% and 100% D,O medium, two severely swelled cells adhered tightly in the zona pellucida. The embryos cultured with 25% D,O medium developed to morula or blastocyst during 48-96 hrs. after cultivation. The 2-cell embryos swelled with the 75% D,O medium for 4 hrs. were reintroduced to a 100% D,O medium. These embryos continued to develop to blastocyst. Present experiments showed that a high concentration of D,O induced cell swelling and inhibited cell division in preimplantation mouse embryos. However, embryos exposed to D,O for short periods can be restored to normal development.

A-37 KIM, W.-S. and T.-K. CHOI, Department of Anatomy, and Department of Microbiology, School of Medicine, Chungnam National University, Taejon, Korea. Effect of aflatoxin B i on rat fetuses and interaction of vitamin A. The effect of aflatoxin Bi on fetuses and interactions of retinol palmitate, the active form of vitamin A, were studied. Spraque-Dawley rats (B.W. 200-250 gm) were used. On the 8th of pregnancy, aflatoxin Bi (Sigma Chemicals) 1.5 mgikg was administrated intraperitoneally. and retinol palmitate (Sigma Chernicals) 50,000 IU/kg was administrated orally dally until a day before sacrifice. On the 16th and 18th day of gestation, rats were sacrificed and fetuses were removed under ether anesthesia after examination of pregnant sacs. The body weight of fetuses was measured, and congenital anomalies of fetuses were investigated under stereoscope. The body weight of fetuses decreased markedly in the aflatoxin B i treated group, but not in the aflatoxin B i and vitamin A treated group compared with control. And, in the aflatoxin B i treated group, several congenital anomalies such as tumorous formations on the

JAPANESE TERATOLOGY SOCIETY ABSTRACTS head, congenital umbilical hernia and severe growth retardations were found. Therefore, it is considered that vitamin A could inhibit the aflatoxin Bi-induced growth retardation of rat fetuses, but could not inhibit aflatoxin Bi-induced teratogenesis.

A-38 YAMASAKI, T., S. OKU, Y. MATSUDA, T. TSURUTA, M. NAKAMURA and K. MIYATA, Department of Pediatrics, Faculty of Medicine, Kagoshima University, Kagoshima. Difference in laterality of experimental renal anomalies in Wistar rats. In previous reports the number of naturally occurred hydronephrosis in the Slonaker-addis rats was higher in the right side than in the left, but we recognized in our experiment that renal anomalies occurred in the left side rather than in the right. We use a protease inhibitor (leupeptin) to produce experimental renal anomalies and evaluated which was the dominant side for renal anomalies. Pregnant Wistar rats were injected once with 50 mg/kg of leupeptin on days 8 (L8), 9 (L9), 10 (L10) and 11 (L11) of gestation (groups; L8, L9, LIO, L11) and without leupeptin (control; c). Renal weight, renal size and renal anomalies were examined. Hydronephrosis, hypoplasia and agenesis were the dominant anomalies. There was no remarkable difference in renal weight when with other groups and between control and each group. Incidences of renal anomalies in each group were as follows, c:l%, L8:10%, L9:8%, L10:5%, L11: 4% and there were significant differences between c and L8 (p

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