Abstracts of Papers Presented at the

Eighteenth Annual Meeting of the

Congenital Anomalies Research Association of Japan

Yokohama, Japan July 13-14, 1978

TERATOLOGY (1978)18: 125-168.

CONTENTS Symposium on Mass Screening of Neonates for Metabolic Disorders .................................. 127 Symposium on Perspectives in Developmental Toxicity Testing ......................................... 129 Symposium on Animal Models of Congenital Anomaly of Man . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Symposium on Recent Developments in Medical Cytogenetics ......................................... 134 Symposium on What Did the Outbreak of Thalidomide Embryopathy Teach Us? . . . . . . . . . . . . . . . . . . . . . . . . . 136 Lectures by Invitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Papers Presented from Platform. . . . . . . . . . . . . . . . . . . . . . 139 Papers Read by Title.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Symposium on

MASS SCREENING OF NEONATES FOR METABOLIC DISORDERS Introduction by M. ARIMA Department of Developmental Disorders National Center of Neurology Musashi, Kodaira, Tokyo

Pilot studies in some selected areas in Japan revealed that the incidence of PKU was approximately half of that found in European countries. The frequency of cretinism appeared to be higher than that of PKU. With the cooperation of specialists in metabolism, obstetricians and the Ministry of Health and Welfare, nationwide screening of four metabolic disorders of amino acids and galactosemia was begun in October, 1977. This symposium was held with the aim of evaluating the results of the routine screening programs and to promote improvement in the management of patients discovered with these disorders.

NARUSE, H., National Institute of Psychiatry and Neurology, Kodaira, Tokyo. Present status of mass screening for metabolic disorders and related diseases. Mass screening for metabolic disorders was initiated as a tool for the early detection of PKU, and R. Guthrie established a system of neonatal mass screening in 1 9 6 1 . From 1964 through 1 9 6 6 , methods detecting other metabolic disorders using dried blood on filter paper were developed by R. Guthrie, E. Beutler et al. and the system of multiple screening was established. In Japan, nationwide screening for PKU, galactosemia, maple syrup urine disease, homocystinuria and histidinemia was started in October, 1 9 7 7 . The Interlaboratory Quality Control Survey on multiple screening began in November, 1 9 7 7 , and all screening centers in Japan are covered by the quality control system. At present, there are 43 screening centers in Japan. The most important next step in mass screening is for congenital hypothyroidism. In 1 9 7 4 , J. Dussault et al. in Canada succeeded in establishing a screening system for hypothyroidism based on a thyroxine assay using dried blood on filter paper, and in 1 9 7 5 a screening method for this disease based on a thyroid stimulating hormone assay in dried blood was developed by M. Irie et al. (Irie. M., Enomoto, K. and Naruse, H.: Lancet, December 2 0 , 1 9 7 5 , p. 1 2 3 3 ) . The frequency of this disease seems to be much

higher than that of PKU not only in Japan but in the USA and Europe as well. In my laboratory, 45000 samples of newborn blood were tested in 1 9 7 7 , and the following cases were confirmed and treated successfully: classical PKU, 1; galactosemia, 1; histidinemia, 6 ; and congenital hypothyroidism, 8.

KITAGAWA, T., Department of Pediatrics, Nihon University School of Medicine, Tokyo. Diagnosis and interpretation of positive screening tests in newborn infants. A positive test reported from a neonatal screening program may be interpreted in several different ways. It may indicate a variant form of the disease rather than the typical form of inborn errors of metabolism, or a prematurity of enzyme development rather than a true genetic abnormality. Further studies on patients with positive tests are always indicated, until the nature of the condition has been throughly established by the analysis of the metabolites in the blood or urine and/or enzyme determinations in blood cells or biopsy specimens, Not only laboratory tests but also clinical tests, such as dietary challenge tests at different ages or vitamin supplement tests, are sometimes required to make an accurate diagnosis. A positive screening test must, therefore, be considered only as an indicator of several

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different conditions of quite different genotypes and phenotypes, and each with a different clinical significance.

SASAKI, T., Director of the Maternal and Child Health Division, Children and Families Bureau, Ministry of Health and Welfare, Tokyo. screening program for inborn errors of metabolism. From 1970 to 1975, a screening program for phenylketonuria using the urine test was carried out by health authorities. As it was recognized that the Guthrie test was more effective, a training seminar for the Guthrie test was held in 1976 and a screening program for the five diseases, phenylketonuria, maple syrup urine disease, homocystinuria, histidinemia and galactosemia was launched in 1977 by the Ministry of Health and Welfare. The Beutler test for galactosemia and the Guthrie test for the other four diseases were adopted and one screening laboratory was established in each prefecture except for several prefectures where two or three laboratories were set up. The blood specimen of the new-born infant is mailed to the laboratory from the hospital. Quality control of the screening program is monitored and research projects concerning inborn errors of metabolism are promoted by the Ministry.

TADA, K., Department of Pediatrics. Tohoku University Medical School, Sendai, Miyagi. Treatment of inborn errors of metabolism found by mass screening. Treatment of phenylketonuria (PKU) is based on limitation of' phenylalanine (PA) intake in order to reduce blood PA levels. PA is, however, an essential amino acid even in phenylketonurics. Demand of PA is relatively greater in infancy when rapid growth takes place. Excess limitation of PA may result in development of PA deficiency syndrome, with failure to thrive, anorexia, irritability, vomiting,

diarrhea, eczema, infections, hypoproteinemia, anemia or bone change. PA deficiency syndrome occurs frequently during the first one to two months of treatment. It is therefore necessary to monitor blood PA levels frequently during this period and to decide adequate amounts of PA intake for each individual case. Successful treatment depends on teamwork among the doctor, parents and the nutritionist. Guide lines for treatment of PKU, galactosemia, maple syrup urine disease, homocystinuria and histidinemia are described.

OKUDAIRA, M., Committee on Congenital Metabolic Disorders, Kanagawa Prefectural Medical Association and Department of Pediatrics, National Yokohama Hospital, Yokohama, Kanagawa. Mass screening for congenital metabolic disorders in Kanagawa Prefecture. Since November 1, 1976 surveillance for congenital metabolic disorders has been underway using the Guthrie method in neonates born in Kanagawa, under the supervision of the Committee on Congenital Metabolic Disorders of the Kanagawa Prefectural Medical Association. This committee, composed of cooperative membership of government authorities, obstetricians, pediatricians, educational institutes, specialized medical centers and the screening center laboratory, has supervised the entire screening system, which terminates in medical intervention of infants with positive test. The cost of testing is borne by the government. During the 16 months after the onset of the survey 91225 neonates were examined and the majority of these were from the obstetrical units in this district (92.6%). 91.3% of positives at initial testing were recalled for a repeated test, and definitive testing was performed in 25 cases with a positive repeat test including 6 histidinemia, 4 hypermethioninemia and 1 heterozygote PKU. It is planned to add screening for cretinism to the present program in the future. Survey data are stored in a computer system for rapid retrieval and reporting results.

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Symposium on

PERSPECTIVES IN DEVELOPMENTAL TOXICITY TESTING Introduction by T. TANIMURA Department of Anatomy Faculty of Medicine, Kyoto University Kyoto

Animal experiments to predict adverse effects on development in humans of various environmental agents, especially chemicals, are becoming more complicated along with the expansion of the definition of teratogenicity and the establishment of the idea of developmental toxicity. In this symposium, some relatively new methods considered to be more useful and efficient for the extrapolation of animal data to humans are selected from among the following six categories in developmental toxicity testing. 1 Species. In addition to the extensive use of rodents, the use of nonrodents is also recommended. 2 Pathophysiological states of animals. Since pharmaceutical drugs are used for control of diseases, genetically or exogenously induced diseased animals are recommended for investigation of the interaction with chemicals. 3 Dose. The maximum level a t which no effects are seen, as well as doseeffect relationships should be thoroughly investigated. 4 Route of administration. The same routes by which humans are exposed should be used. Since inhalation is a major route of exposure to environmental pollutants, the importance of inhalation tests is stressed. 5 Period of administration. Some guidelines such as the three segment system for pharmaceutical drugs and the multigeneration test for food additives are available. The significance of direct neonatal administration in rodents is discussed. 6 Observation. Establishment of a practical classification of malformations is desirable. Among various postnatal manifestations, effects on immunological development may become more important.

ESAKI, K . , C e n t r a l I n s t i t u t e f o r Experimental Animals, Kawasaki, Kanagawa. The beagle dog i n embryotoxicity tests. It i s recommended t h a t non-rodent animals be used i n a d d i t i o n t o r o d e n t s i n embryotoxicity tests. However, t h e only non-rodent t h a t has been s t u d i e d e x t e n s i v e l y from t h e reproductive, developmental and t e r a t o l o g i c a l s t a n d p o i n t s is t h e r a b b i t . The u s e f u l n e s s of t h e beagle dog i n embryotoxicity t e s t s was s t u d i e d . 1. The organogenesis s t a g e of t h e beagle dog appears t o s t a r t on t h e 1 8 t h day of pregnancy and f i n i s h on t h e 35th day, according t o

o b s e r v a t i o n of f e t u s e s on t h e 20, 25, 30, 35, 40 and 50th days of pregnancy. 2. Abnormal t a i l s and c o a r c t a t i o n of t h e a o r t a were obsenred as t h e main malformations i n groups t r e a t e d w i t h 50-800 mg/kg o f t h a l i d o mide. G a s t r o s c h i s i s and v e n t r i c u l a r s e p t a 1 d e f e c t s w e r e observed mainly i n groups t r e a t e d with 10-25 rnglkg of c o r t i s o n e a c e t a t e . 3. No remarkable malformations were observed i n groups t r e a t e d w i t h phenytoin, methotrexa t e , a s p i r i n and t e t r a c y c l i n e . There a r e some advantages of using beagle I t i s p o s s i b l e t o o b t a i n good q u a l i t y dogs.

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animals with defined gestation stages at any time; considerable data are available concerning reproduction and toxicology; external, skeletal and visceral malformations of each fetus can be examined. The disadvantages are high costs and a long estrus cycle. From the above results, the beagle dog is considered to be useful in comparative teratology and embryotoxicity tests as well as in general toxicology.

TANIMURA, T., Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto. Significance of non-human primates in developmental toxicity tests. Species difference is one of the most important factors in extrapolation of results of developmental toxicity tests to humans. Recently, there have been a growing interest in the use of non-human primates which are phylogenetically closest to homo sapiens. The main reasons for their use are 1) ontogenetic relatedness to humans, 2 ) similarities of metabolism and 3 ) susceptibility to human teratogens such as thalidomide. More than 70 reports using non-human primates are now available on the teratogenicity tests of chemicals. Macaques and baboons (Old World Monkeys) have been generally used, but recently smaller sized marmosets (New World Monkeys) have become the object of attention. In addition to tests during the period of organogenesis, their use in experiments in the fertilization or perinatal periods is becoming popular. However, it should be noted that the development of newborns in macaques is slightly advanced compared to that in humans. The use of non-human primates is expected to contribute greatly to comparative developmental pharmacology such as placental transfer and biotransformations in both the mother and fetus in order to link the widely used rodents and humans. As an example, placental transfer of acetylsalicylic acid was compared between ICR-JCL mice (day 12 and 14) and cynomolgus monkeys (day 4 5 ) . However, the non-human primates have not been fully developed as experimental animals and there are many problems including their supply. Moreover, more extensive basic researches on comparative reproductive biology and embryology are also desirable.

KANOH. S., Department of Pharmacology, National Institute of Hygienic Sciences, Osaka. Studies on the fetal toxicity of antipyretics against endotoxin fever in the rat. Bacterial endotoxin (Lipopolysaccharide; LPS) can produce fever in both animals and man. The mechanism of the fever is not yet well understood. However, various antipyretics can effectively decrease the fever. The fetal toxicity of these agents might be influenced by

both fever and LPS. In this report, the following results were obtained. 1) The pyrogenicity of LPS in the rat is weak but can produce abortion or resorption at a dose of 1 ug/kg iv at various stages of gestation. 2 ) Fetal toxicity including fetal death, resorption, abortion and SFD was weak with a dose of 250 mg/kg PO of aspirin and 50 mg/kg PO of aminopyrine, but it was increased in pregnant rats treated with LPS. 3 ) Enhancement of lethal toxicity of antipyretics was observed in normal rats by injection of LPS. 4 ) Reserpine, which can decrease normal body temperature, produced SFD when injected at a dose of 2 . 5 mg/kg ip during late gestation. 5) Antipyretics administered orally were distributed in the placenta, fetus and various maternal organs. In addition the tissue concentration was increased by injection of LPS. The ATP and cathecholamine contents were decreased by reserpine. These data suggest that permeability of the placental barrier increased and consequently the increased drug concentration may enhance toxicity.

SHIMADA, M., Department of Pediatrics, Shiga Medical College, Otsu, Shiga. Histological evaluation of long-term effects of chemicals given to the neonate. Histogenesis of various human organs continue even after birth. Some organs of newborns and infants which seem morphologically to be fully mature are still immature in their functions. Thus chemicals which are nontoxic in adults may be toxic to the newborn and infant. Most organs or tissues which have been inhibited in their growth or damaged by noxious chemicals usually catch up in growth and/or completely repair the damage. However, some organs such as the brain and retina do not catch up in growth or recover completely. In this paper, the permanent effects on the cerebellum of a radiomimetic substance, given to neonatal mice under various schedules, is presented. Three divided doses of cytosine arabinoside were injected to suckling mice either 2 to 4 , 6 to 8, or 10 to 12 days of age. All animals treated subsequently developed cerebellar abnormalities of various severity. When the chemical was injected at 2, 3 and 4 days of age, mice developed severe hypoplasia and histological abnormalities which were easily recognized by the usual histological methods. Abnormalities of the cerebellums of adult mice which had cytosine arabinoside injections at 6, 7 and 8 days of age were predominant in the Purkinje cell dendrites visualized by silver impregnation. However, the cerebellums of adult mice which were treated with the chemical at 10, 11 and 12 days of age showed an abnormality of the synapse. Abnormal synapses were found when the molecular layer was examined with the electron microscope. (Supported by a Grant from the Ministry of Education of Japan)

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KAWAI, K. and K. NOZAKI, National Institute of Industrial Health, Kawasaki, Kanagawa. Inhalation techniques for developmental toxicity testing. Several techniques are available to test the biological effects of a substance in the airborne state or in the gaseous or vapor phase. Whatever method one may choose, respiratory exposure is a kind of combined administration via the lungs and gastrointestinal tract, because an appreciable amount of materials once deposited in the respiratory organs is drained into the gastrointestinal tract through the self clearance mechanism of the respiratory organs. At present whole body exposure and head exposure are commonly used. The former has the disadvantage of skin contamination. The

latter suffers from hazards due to forced fixation of test animals. Comparison between these two methods w i l l be made for better planning of toxicity testing with inhalation techniques. There are other techniques utilizing a mask or tracheal cannula. These techniques may be less practical for developmental toxicity testing. The two major aspects of planning and operating an inhalation chamber are the accuracy of exposure dose and the maintenance of favorable conditions for test animals. Special consideration should be given to the latter when testing developmental toxicity. Several technical points will be discussed in this regard. Adequate selection of experimental conditions should be emphasized for developmental toxicity testing with inhalation techniques.

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Symposium on

ANIMAL MODELS OF CONGENITAL ANOMALY OF MAN Introduction by K. BABA

Department of Pediatrics School of Medicine, Nihon Uniuersity Tokyo

It is modern medical practice to utilize animal models for the study of congenital anomalies in man. It is well known that many congenital anomalies very similar to those found in man are seen in other mammals. Among these, several animal models with widely recognized congenital anomalies have been selected and will be discussed in this symposium. I believe that this symposium contributes to the further understanding of the treatment and prevention of these disorders in humans.

KASHIWAMATA, S., Department of Perinatology, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi. Gunn rats. In 1938 C. K. Gunn reported a new mutant strain of rats with hereditary acholuric jaundice, which was observed in the breeding stock of Wistar origin at the Connaught Laboratories, Toronto, Canada. This jaundiced strain, known as Gunn rats, was first introduced to Japan by Prof. Baba in 1964. Evidence is presented that homozygotes (j/j) of Gunn rats are extremely low in hepatic bi1irubin:UDP-glucuronyltransferase activity and hence show a diminished ability to conjugate bilirubin with glucuronic acid, resulting in unconjugated hyperbilirubinemia throughout the life of the animal. The jaundiced condition has been characterized as an autosomal recessive inheritance, and the Gunn strain of rats is generally regarded as an animal model comparable to a human genetic disease, Crigler-Najjar syndrome, Type I, which is often accompanied by kernicterus in the neonatal period with rare exceptions. Hyperbilirubinemic Gunn rats have been used not only for investigating pathogenetic mechanisms of kernicterus including neurotoxicity of bilirubin on the central nervous system, but also for studies on the photochemical modification of bilirubin, the conjugation and transport systems in the liver, mechanisms of renal urinary concentration insufficiency (polyuria), cerebellar hypoplasia, behavioral disturbances and so forth. From these points of view, the Gunn strain of rats is considered to be a very useful experimental animal both in the clinical and basic research fields. It is expected that characterization of Gunn rats will provide much

valuable information for understanding fundamental processes in living organisms.

TSUJI, S., Department of Physiology, Wakayama Medical College, Wakayama. Muscular dystrophy in the mouse. Since the discovery of a strain of mice carrying an autosomal recessive gene for a form of progressive muscular dystrophy in 1955, there have been numerous studies of the pathological, biochemical and physiological aspects of the disease in the dystrophic mouse in order to demonstrate its metabolic and etiologic correlation with human muscular dystrophy. Although opinions differ regarding its gross symptomatology and histopathologic character, the mouse disease closely parallels the Duchenne type of dystrophy or the clinical myotonica dystrophica, and it is undoubtedly an exciting animal model for the investigation of muscle disease.

NAKAMLJFU,H., Department of Anatomy, Hiroshima University School of Medicine, Hiroshima. Inherited limb anomalies in mice. Limb anomalies are one of the common congenital anomalies in man, however very little is known about the pathogenesis of human limb malformations. As the use of human material for experimental study is somewhat limited, it is very important to grow mutant animals which serve as models for human limb malformations. Many mutant strains of mice with limb de-

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formities have been reported. We have kept mutant meromelia mice (gene symbol, mem) since 1974. Though the meromelia mouse shows various types of limb malformations, the mode of inheritance is autosomal recessive. The mem/mem homozygote embryo shows a hypoplastic apical ectodermal ridge (AER), lack of the AER at the site of the cleft of the foot plate, or delayed involution of the AER. These features are comparable to those of human limb malformations such as cleft hand and preaxial polydactyly. The peridermal cells covering the AER show special morphological features with an orderly arrangement when observed by scanning electron microscopy. When the AER is not distinct in meromelia mice, the orderly arrangement of the peridermal cells along the apical rim is disturbed. Delayed involution of the AER is also detectable by scanning electron microscopy. With these model animals, some experimental analyses of abnormal limb morphogenesis are now in progress.

TAKAHASHI, H., Research Center for Laboratory Animals, Fujita-Gakuen University School of Medicine, Toyoake, Aichi. Inherited bilateral polycystic kidneys in mice (KK/cy strain). A new mouse mutation was observed in a substrain of KK at the Faculty of Agriculture of Nagoya University in 1973 and is called polycystic kidneys. From the results of crossing tests, it is concluded that an autosomal recessive gene (cy, linkage not known) is responsible for occurrence of the polycystic kidneys. Homozygotes are viable and fertile. Polycystic kidneys in affected mice can be checked at autopsy at about four months of age. At that time, the average weight of the kidneys is 3.69 grams, and is about ten times the weight of normal kidneys in the mouse. In this condition, always bilateral, the kidneys become enlarged and cysts appear in the cortex or medulla. At five months of age, they have a slight anemia with a hematocrit of about 30%. The average life span of the affected mice is about 170 days and the female life span is shorter by about one month.

The finding of bilateral polycystic kidneys in the KK/cy strain provides a new model for the study of polycystic kidney in adult humans. (Supported by Grants No. 012206, 111506 and 210706 from the Ministry of Education, Science and Culture of Japan)

ISHIBASHI, M., Department of Veterinary Medicine, Azabu Veterinary College, Sagamihara, Kanagawa. A strain of rats with congenital spinal malformations. The Ishibashi rat (IS rat), originating from a cross between a wild male and a Wistar strain female rat, having congenital spinal malformations is introduced. The malformations are mostly seen in the lumbar vertebrae. They are mainly abnormalities of vertebral bodies, such as cleft, partial failure, complete or incomplete fusion, unilateral unsegmentation, etc. Owing to these abnormalities, the spines show kyphoscoliosis or kyphosis. In severe cases, the spinal canals are constricted and the spinal cords are compressed. However, function does not seem to be much affected. From results with Alizarin Red S bone staining and histological examination of the fetal specimens, both the cartilage and bone formation in abnormal lumbar vertebral bodies were found to be quite irregular. Upon biochemical testing, the plasma esterases and serum alkaline phosphatases in IS rats are found to show different patterns from those of other strains of rats. From these findings and other abnormalities seen such as skin inflammation and tooth decay, their spinal abnormalities are thought to be the result of congenital calcium metabolic lesions. These abnormalities in IS rats seem to be quite similar to those of humans, so they are very good animal models for basic studies of such diseases, especially to clarify their mechanisms of occurrence. (Supported by a grant-in-aid (No. 11506) for scientific research from the Ministry of Education, Science and Culture of Japan)

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Symposium on

RECENT DEVELOPMENTS IN MEDICAL CYTOGENETICS Introduction by

Y. NAKAGOME Department of Human Genetics National Institute of Genetics Mishima, Shizuoka

The advent of banding techniques has made it possible to detect even a very small change of a chromosome and to identify very complicated structural rearrangements. In addition, areas of new applications have opened up. In the present symposium, a few selected topics of medical cytogenetics in the light of recent developments will be reviewed in order to give, hopefully, a bird's-eye view of the field for those who are not familiar with it and to give more insight to those who are familiar.

3 ) discuss the severity and the prognosis of chromosomal syndromes; 4 ) discuss human chromosome mapping by the use of clinical cases.

NAKAGOME, Y., National Institute of Genetics, Mishima, Shizuoka. Few banding techniques. In addition to the standard Q-, G-, C- a d R-techniques, a variety of newer methods have been introduced. They include T-, Cd-, G11and N-techniques as well as the BUdR-acridine technique for DNA-replication study; ethidiumbromide treatment to improve resolution of Gbanding and immunofluorescence and/or immunoenzyme techniques. On the other hand, there are efforts aimed in a different direction, i.e., the development of simpler, easier and less expensive techniques. In the present communication, two new techniques developed by the author, LBA-technique and a method of Q-banding not necessitating a fluorescence microscope, are presented. The latter includes the use of a new interference filter specifically designed for the purpose. In addition, some of the new banding techniques are reviewed.

KUROKI, Y., Division of Medical Genetics, Kanagawa Children's Medical Center, Yokohama, Kanagawa. New chromosomal syndromes. I n the last decade, a great advance in banding techniques has enabled easy identification of each chromosome and establishment of many new chromosomal syndromes. In reviewing these new chromosomal syndromes in our hospitals and in the literature, I have tried to focus on the following areas: 1) . .present several new chromosomal syndromes; 2 ) discuss the karyotype-phenotype relationship;

YOSHIDA, M. C., Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo. The current status of human gene mapping. In this symposium, I would like to review the somatic cell genetic contribution to human gene mapping by outlining the methodological aspects of the subject and by providing an upto-date account of the human gene map. Somatic cell genetics has provided a highly productive approach to chromosome mapping. The first step in the method is fusion of human somatic cells with those from another species, usually rodent cells, by using Sendai virus o r a chemical agent for cell fusion. In subsequent cell division there is a progressive and preferential loss of human chromosomes from humanrodent hybrid cells. The resulting hybrid clones have only certain human chromosomes. Gene mapping in cell hybrids depends on the detection of discrete human gene products of specific enzymes in cultured cells. In the gene assignment, the location of a specific gene locus on a specific human chromosome is demonstrated by concordance between the presence or absence of the specific chromosome and the specific human phenotype in many hybrid clones. The information is based on four International Workshops on Human Gene Mapping. genetic traits are known A total of about 1300 to exist in human chromosomes. For many of

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the 200 specially assigned genes on the 24 chromosomes (22 autosomes plus the X and the Y chromosomes) some information on regional localization in a particular chromosome is available.

OSHIMURA, M., Department of Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University. Tokyo. Cytogenetical aspects of human cancer and leukemia. The use of modern chromosomal banding techniques in malignant disorders has afforded evidence regarding the possible nonrandom distribution of chromosomal abnormalities. Of these conditions, the most convincing are: 1) a t 9 - 1/22 translocation in chronic myelocytic leukemia; 2) a 1 8 - 1\21 translocation in acute

myeloblastic leukemia; 3) partial or total loss of a 1/22 chromosome in meningioma; 4 ) the presence of an extra band of medium fluorescence at the terminal end of the long arm of one of the 1/14 chromosomes in malignant lymphoma; and 5 ) frequent occurrence of hyperdiploidy in acute lymphoblastic leukemia. These findings not only indicate that some particular etiologic agent or similar mechanisms may possibly be responsible for bringing about the karyotypic changes that appear to be specific for each of the conditions mentioned, but also suggest their diagnostic and prognostic usefulness. In the present symposium, the author suggests how useful the cytogenetic data are for a better understanding of malignant disorders, when combined with careful evaluation of the clinical aspects of these conditions.

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Symposium on

WHAT DID THE OUTBREAK OF THALIDOMIDE EMBRYOPOATHY TEACH US? Introduction by K. TSUCHIYA Department of Orthopedic Surgery Yokohama City University School of Medicine Yokohama, Kanagawa

Thalidomide drugs were first distributed in Japan on January 20, 1958 and the decision was made to withdraw them from the market on September 13, 1962.The withdrawal was thought to be actually completed by the end of 1963. The thalidomide malformations began to appear in 1959,reached a peak in 1962 and ceased in 1964.Studies carried out by t h e Ministry of Health and Welfare in recent years reveal t h a t there are 303 thalidomide children thus far recognized in this country. This is the third largest number, next to West Germany and England. Thalidomide embryopathy is the only exogenous malformation definitely caused by the intake of a drug during pregnancy. We have not had a similar experience in the past and we should not have a similar episode again in the future. I t was a n unfortunate event for human beings which has happened only once and should not be repeated. The purpose of this symposium is to study the malformation from the various standpoints of medical science, to leave the important and necessary records for the next generation and to establish the policy from the medical standpoint that this tragedy will not be repeated. YASUDA, M . , Department of Anatomy, Hiroshima U n i v e r s i t y S c h o o l of Medicine, Hiroshima. Embryology o f t h a l i d o m i d e embryopathy. S i n c e t h e f i r s t s u s p i c i o n of t h e t e r a t o g e n i c i t y of t h a l i d o m i d e i n man, numerous s t u d i e s have r e v e a l e d v a r i o u s a s p e c t s of t e r a t o g e n e s i s with thalidomide. The c r i t i c a l p e r i o d f o r i n d u c t i o n of m a l f o r m a t i o n s i n man h a s been e s t a b l i s h e d t o r a n g e from t h e 3 4 t h t o 5 0 t h d a y s a f t e r t h e l a s t menstrual period. This critical period c l o s e l y c o r r e s p o n d s w i t h t h a t o f morphogenesis of t h e t a r g e t o r g a n s . The mode of t e r a t o g e n i c a c t i o n of t h a l i d o m i d e , however, s t i l l r e m a i n s unclear. Lash and Sax& ( ' 7 2 ) s u g g e s t e d t h a t t h e Yrimary s i t e a f f e c t e d by t h a l i d o m i d e might be t h e mesodermal t i s s u e of t h e mesonephros of t h e embryo and t h i s might l e a d s e c o n d a r i l y t o t h e d e f e c t s of t h e limb bud. McCredie ( ' 7 3 ) s p e c u l a t e d t h a t t h a l i d o m i d e might c a u s e an emb r y o n i c s e n s o r y p e r i p h e r a l n e u r o p a t h y by damagi n g n e u r a l crest c e l l s and t h a t t h e limb def o r m i t i e s might b e s e c o n d a r y t o t h e n e u r o p a t h y . A s f o r t h e chemical s t r u c t u r e of t h e u l t i m a t e compound which induced t h e m a l f o r m a t i o n s , t h e d i s c o v e r i e s of a t e r a t o g e n i c d e r i v a t i v e of t h a -

l i d o m i d e (EM12) and a n o n - t e r a t o g e n i c d e r i v a t i v e (EM87) h a v e c o n t r i b u t e d g r e a t l y t o s o l v i n g t h e problem. One o f t h e i m p o r t a n t l e s s o n s which t h a l i d o mide h a s t a u g h t t e r a t o l o g i s t s i s t h a t t h e human embryo may be i n o r d i n a t e l y s e n s i t i v e to a subs t a n c e t h a t c a u s e s l i t t l e o r no t o x i c i t y i n human a d u l t s o r i n c e r t a i n t e s t a n i m a l s . T h i s f a c t s t r o n g l y i n d i c a t e s t h a t t h e c u r r e n t method of t e s t i n g t e r a t o g e n i c i t y of drugs i n experimental animals is i n s u f f i c i e n t f o r evaluation o f t h e s a f e t y of d r u g s i n man. E x p e r i m e n t a l t e r a t o l o g i s t s s h o u l d aim a t d e v e l o p i n g more e f f e c t i v e measures t o e v a l u a t e t h e t e r a t o g e n i c p o t e n t i a l o f d r u g s i n man t h r o u g h i n v e s t i g a t i o n i n t o t h e mechanism of t e r a t o g e n e s i s w i t h thalidomide.

MATSUNAGA, E . , Department of Human G e n e t i c s , N a t i o n a l I n s t i t u t e o f G e n e t i c s , Mishima, Shizuoka. A g e n e t i c - e p i d e m i o l o g i s t ' s view. I n Japan, t h e thalidomide d i s a s t e r has a number of a s p e c t s t h a t u r g e upon u s a b i t t e r

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reflection. Particularly painful is that, while in Western Europe thalidomide was withdrawn from the market soon after the warning by W. Lenz, Japan is the only country where the withdrawal lingered on as long as nine months, resulting in an appreciable number of births of affected children. In order that such a disaster not be repeated, it is necessary not only to test for possible teratogenicity of new drugs, but also to systematically collect information concerning the adverse effects of drugs currently being used, to establish a monitoring program for birth defects, and to educate medical students to become alert practitioners as stressed by R. W. Miller.

SUGIURA, Y., Department of Orthopaedic Surgery, Nagoya University School of Medicine, Nagoya, Aichi. Roentgenographic classification of reduction anomalies of the upper limbs in thalidomide embryopathy. To date, 303 cases of thalidomide embryopathy have been ascertained in Japan by the Ministry of Welfare. Among the 303 cases, 2 4 5 had upper limb anomalies of longitudinal deficiency types, and others had ear anomalies with deafness of various degrees. The author classified roentgenographically the upper limb anomalies due to thalidomide into 5 types according to the severity of the anomalies; 1) thenar muscle hypoplasia-type, 2 ) thumb ray hypoplasia-type, 3) radial clubhand-type, 4 ) phocomelia-type, and 5) ameliatype. The order of frequency of these types was In the radial as follows: 3 ) > 2 ) > 1 ) > 4 ) > 5). clubhand-type, about half of the cases showed complete aplasia of the radius and the other half showed partial aplasia of the radius with or without radioulnar synostosis of various degrees. In the thumb ray hypoplasia-type, some cases showed triphalangeal thumb, while others showed floating thumb. There were many cases which showed an intermediate type among the 5 types mentioned above. Therefore, it is the author's opinion that upper limb anomalies of the longitudinal deficiency type in thalidomide embryopathy can be

arranged sequentially from the mildest type to the most severe type. Roentgenograms as well as the external appearances-of these 5 types and the intermediate cases among them are illustrated and discussed in detail. The pattern of anomalies presented here can be used as a general pattern of the longitudinal deficiency type of upper limb malformations.

KIDA, M., Department of Pediatrics, Teikyo University School of Medicine, Tokyo. Medical background of thalidomide-embryopathy. The thalidomide tragedy raised two principal points. The first is the necessity of preventing outbreaks of congenital anomalies. Mothers who took thalidomide preparations during early pregnancy delivered babies with a definite symptom complex consisting of ear aplasia, difficulty in hearing, phocomelia, radial ray defect,' triphalangia, and other features. These patients were able to be differentiated from similar conditions such as Holt Oram syndrome and radial aplasia thrombocytopenia. When accurate diagnosis of congenital malformation syndromes is possible, and when all newborns in a certain area are examined for congenital malformation syndromes continuously, we may be able to detect an increase in frequency of autosomal dominant mutations and to estimate influences of environmental mutagens on mankind. The second point relates to the practical aspects of health care systems and the medical examination of patients. Making careful observations on developing young patients and their conditions may lead us into a more dynamic diagnostic methodology.

ICHIBANGASE, Y.. Department of Social Welfare, Faculty of Literature and Humanities, Japan Women's University, Tokyo. Social problems of thalidomide cases. Research on the educational and vocational training of three hundred thalidomide cases in Japan was carried out. This study is for the social rehabilitation of thalidomide children.

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LECTURES BY INVITATION SUGAWA, Y.,*Kanagawa Childred's Medical Center, Yokohama, Kanagawa. The sysrem of maternal and child health directed at the prevention of congenital anomalies. Among current problems of maternal and child health in Japan, one of the most important is prevention of congenital anomalies. This concern has become one of the most important in the Ministry of Health and Welfare of Japan. In order to make the program successful, the births of children with congenital anomalies should be monitored. Various measures for the prevention of congenital anomalies are discussed, together with estimated incidence of congenital anomalies. (*Former President)

SMITH, D. W., Department of Pediatrics, School of Medicine, University of Washington, Seattle, Washington. Biomechanics in normal and abnorma1 morphogenesis. Biomechanical factors exert an Important role in both normal and abnormal morphogenesis. The basic precept I s that the magnitude of force effects the form of a tissue. Studies have indicated that physical forces affect a multitude of features in craniofacial development. The major forces are the consequence of the growth, form and function of the brain. Examples to be shown include the impact of the brain on development of the face, the calvarium and its sutures and on scalp hair directional patterning. Also the indirect effect of brain function on muscle function and thereby facial and external ear form will be shown. External forces may also affect craniofacial form, especially the effects of uterine constraint. The application of these principles will be illustrated by new modes of management for craniostenosis and for plagiocephaly, as examples. ~~

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PAPERS PRESENTED FROM PLATFORM HAYASHI, Y., K. HOSHINO and Y. KAMEYAMA, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi. Early pathological changes of matrix cells of the telencephalon following low-dose X-irradiation in mouse embryos: Effects of 10 R irradiation on day 13. In a previous study, the pathologicalchanges in matrix cells of the telencephalon in CF#l mouse embryos exposed to 25 R of X-irradiation on day 13 of gestation were reported. In this study, the effects of 10 R of X-Irradiation were examined. The pregnant mothers were killed 1 - 5 hours after irradiation and the telencephalon of embryos was examined by both light and electron microscopy. The number of pyknotic cells and cells with cytoplasmic inclusions were counted by light microscopy. The number of cells with cytoplasmic inclusions at 3 hours after irradiation was 3 per 500 cells. That of pyknotic cells at 5 hours after irradiation was 3 per 500 cells. Those in the control group numbered less than 1 per 500 cells. In 25 R irradiated embryos, the number of inclusions was 8 per 500 cells and the number of pyknotic cells was 20 per 500 cells after 5 hours. On electron microscopic observation, cytoplasmic degeneration observed at 2 hours after irradiation was not coated by membrane and was thought to be ribosomal degeneration. Cytoplasmic inclusions observed at 3 hours after irradiation were necrotic cells phagocytosed by the neighboring cells. Compared with the effects of 25 R X-ray irradiation in the previous study, 10 R irradiated embryos showed cytoplasmic degeneration similar to that which was observed in the 25 R irradiated group. Phagocytosis of necrotic cells appeared at 3 hours after irradiation in 10 R irradiated embryos, but at 5 hours after in those which were irradiated at 25 R.

K., Y. HAYASHI and Y. KAMEYAMA, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi. Effects of low dose X-radiation on the cortical architecture of the brain in mice irradiated on day 13 of gestation. Previously we reported mitotic delay and an increase in pyknotic cells among the matrix cells in the telencephalon of mouse fetuses produced by low dose X-radiation (10 25R level). In the present study, the cortical architecture of the brain in 4 week old mice irradiated in utero was examined to ascertain whether the above changes after radiation may have effects on the histogenesis of the cerebral cortex. CF#l mice were exposed to a single whole

HOSHINO,

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body X-irradiation at a dose of 25R or l O O R on day 13 of gestation. Immediately after irradiation, they were injected with 3H-thymidine and allowed to give birth. The offspring were autopsied at 4 weeks of age and histoautoradiograms of their brains were obtained. The position of heavily labeledneurons was mapped on photomicrographs of the brain. In the untreated control group, 50% of the heavily labeled neurons were distributed in the deeper half of layer VI (A), 35% in the superficial half of layer VI (B) and the rest in layer V and more superficial layers (C). In the 25R group, heavily labeled neurons were distributed deeper than the control group; 65% of heavily labeled neurons were found in (A), 30% in (B) and the rest in (C). In the lOOR group, although the reduction of cortical thickness was extensive, the distribution of heavily labeled neurons was not markedly different from that in the 25R group; the majority of heavily labeled neurons were distributed in (A). (Supported by Scientific Research Grant No. 258070 from the Ministry of Education, Japan)

INOUYE, M. and U. MURAKAMI, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi. A detailed description of the cerebellar malformations in prenatally X-irradiated rats. Irregularities of the folial pattern in prenatally X-irradiated rats have often been mentioned, but a detailed analysis of the pattern has not been undertaken. This study was performed to observe the folial pattern in detail. Pregnant WKA/Hok inbred rats were exposed to a single 100 or 200 R X-irradiation on one of gestation days 16 - 19 (VP: day 0). The offspring were anesthetized and perfused with 10% formalin at age 30 or 60 days. The cerebellum was removed, stained with thionine, examined under a dissecting microscope, and drawn with the aid of camera lucida. In the cerebellum of rats exposed to 200 R, the vermis was reduced in size whereas the hemispheres were comparatively well developed, and the lobulus simplex extended anteriorly to the culmen. The crura I and I1 (Larsell, '52) extended somewhat horizontally and anteroposteriorly instead of the normal vertical and lateral arrangement. The imbalance In size between the vermis and hemispheres was most severe in rats exposed on day 16, but there was only slight irregularity in the folial pattern. The anterior portion of the lobulus simplex and lobules IV and V were foliated vertically, crus I was irregularly subdivided, and crus I1 and the paramedian lobule were poorly subfoliated in most cases exposed on day 17 or 18. Folial abnormalities were most striking in rats exposed

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on day 19. Lobules I to VII, and the lobulus simplex to the paramedian lobule were irregularly foliated. The anterior surface of the cerebellum was narrowly and vertically foliated into small fragmented lobules. In the cerebellum of rats exposed to 100 R, the reduction in size was mild and folial irregularity was minimal.

ity of mothers on day 18 of pregnancy. The types of gross malformations and eye abnormalities in mouse fetuses did not differ among the X, X+U-1.5 and X+U-3 groups. The results in mouse fetuses irradiated on day 7 of gestation suggest that the effects of X-ray irradiation are enhanced by ultrasound.

OKUMA, S., H. TANIGAWA and M. KAWASHIMA,

KIYONO, S., M. SEO and M. SHIBAGAKI, Department of Physiology, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi. Effects of prenatal X-irradiation and postnatal environmental.conditions upon the Hebb-Williams maze test: in rats. Forty male Sprague-Dawley rats were used. Experimental animals were microcephalic offspring from females which received 200 R of Xirradiation on the 17th day of gestation. Control animals were prenatally sham-irradiated. They were maintained on the standard colony condition (SC) o r enriched condition (EC) from weaning for 30 days. The Hebb-Williams test was then used to study the effects of prenatal X-irradiation and the influence of the environment. The following results were obtained. 1) The X-irradiated group had more errors than controls. 2) Among controls, EC rats had less errors than SC rats. 3) In X-irradiated animals no difference in the number of errors was found between the SC and EC groups. The results suggest that the effects of enrichment are difficult to detect in prenatally X-irradiated microcephalic rats.

SHIRAI, S., Department of Ophthalmology,Nagoya City University Medical School, Nagoya, Aichi. The teratogenic effects of simultaneous X-irradiation and ultrasound in mouse fetuses. It is thought that the effects of X-irradiation are enhanced by ultrasound. The teratogenic effects of simultaneous X-irradiation and administration of ultrasound however have not been studied in experimental teratology. On day 7 of pregnancy ddN mice were X-irradiated and/or received ultrasound as follows: 1. 150 R of X-irradiation (30 R/min) [X group1 2 . Ultrasound at 1.5 or 3 W/cm2 for 5 min (continuous ultrasonic waves at a frequency of 1 MHz) [U-1.5 o r U-3 group] 3. 150 R of X-irradiation combined with ultrasound at 1.5 or 3 W/cm2 for 5 min [X+U-1.5 o r X+U-3 group] All mice were killed on day 18 of pregnancy and the offspring were grossly examined. The eyes were then serially sectioned and examined by light microscopy. No significant difference in the frequency of malformed and dead fetuses was found among the U-1.5, U-3 and unirradiated control groups, o r between the X and X+U-1.5 groups. Fetal mortality in the X+U-3 group was significantly higher than in the other groups. In the X+U-3 group, implantation was not found in the major-

Research Laboratories, Morishita Pharmaceutical Co., Ltd.. Yasu, Shiga. Effects of food restriction during various stages of pregnancy on fetal development in rats. In reproduction studies, a decrease in food intake has been frequently observed in pregnant rats associated with high drug dosages. In order to examine the effects of food deprivation on fetal development, pregnant SD rats were restricted during various stages of gestation. The groups were as follows: 1) ad libitum (unrestricted); 2) 25% and 50% restriction of the ad libitum level on days 0 to 7 (A-25 and A-50); 3) 25% and 50% on days 8 to 17 (B-25 and B-50); 4 ) 2.5% and 50% on days 18 to 21 (C-25 and C-50). Pregnant females were sacrificed at day 21 of gestation and the body weight and skeletal development of the fetuses were examined. The fetal weights of groups B-25, B-50, C25 and C-50 were lower than in the unrestricted group and were 97%, 86%, 92% and 90% of the unrestricted fetal weight, respectively. Groups A-25 and A-50 were similar to the unrestricted group. In the proximal phalanges of the fore- and hindlimb, the appearance rates of ossification centers of groups B-50 and C-50 were lower than those of other groups. The number of sacrococcygeal bodies was slightly decreased in group C-50, but not in the other groups. In the cervical vertebral bodies, there was no difference between the restricted and unrestricted groups, It is concluded that maternal food restriction during pregnancy resulted in reduction of fetal weight but not in retardation of skeletal development.

MORIKAWA, Y., M. MINO, H. YOSHIOKA, Y. KASUBUCHI, T. KUSUNOKI. K. YAMANO and M. SHIMADA, Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, and Department of Pediatrics, Shiga Medical College, Otsu, Shiga. Effect of early undernutrition on granular cell production in the cerebellum. Recently many reports have been published on the effects of early undernutrition on brain development. Most of these are in agreement with the proposition that undernutrition inhibits increase in the number of cells in the brain. This study was undertaken to investigate the effect of early undernutrition on the time o f production of granular cells in the cerebellum using tritiated thymidine (3H-TdR) autoradiography. Newborn mice, ICR-JCL strain, were divided into two groups, the undernourished and the

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control group. In order to label all of the granule cells which were produced after 15 days of age, the suckling mice in both groups were injected with 3H-TdR successively at 8 hour intervals from 15 to 20 days of age. The cerebellar external granular layer of the undernourished group disappeared late compared with that of controls. In the undernourished group there was a 5 hour lag in the time from initial injection of 3H-TdR to the appearance of labeled cells in the molecular layer. The percentage of labeled granular cells in the internal granular layer at 20 days of age was 8 - 10% in controls and 20 - 30% in the undernourished group. Results indicate that production of granular cells in the cerebellum was delayed by undernutrition.

embryos in the early gestational stage were examined histologically and the fetuses in the later stages were examined in cartilage-stained and cleared specimens. Extra digits tended to be formed in the preaxial portion of the foot, especially at or by the first digit, regardless of the dose. Polydactyly types were mostly duplicated metatarsal /metacarpal and duplicated proximal phalangeal. The first detectable changes in the formation of polydactyly were preaxial extension of the apical ectodermal ridge (AER) and its delayed involution in the foot plate, Also, cell necrosis and pyknosis could be observed i n the mesoderm under the abnormally thickened AER and around the dilated marginal sinus. The AER was hypertrophic and protruded irregularly toward the mesodermal tissue. Hypertrophic AER was strongly stained by pyronine. Those changes were similar to 5-FU induced polydactyly. In rats, atypical polydactyly in which the extra digit developed on the palm was observed in the group treated on day 10. AER-like thickening of the palmar epithelium was detected at the presumptive sites of the ectopic extra digit. The results suggest a significant role of the ectoderm in the limb bud and plate for the manifestation of Ara-C induced polydactyly.

MUTO, Y., S. IT0 and I. NARITA, Department of Biology, Aichi University of Education, Kariya, Aichi. The relationship between polydactylous regeneration and temperature in toad larvae. Previously Muto et al. (1977) reported that when the hindlimb of the toad larva was amputated at the end of the limb bud stage, there was a high incidence of polydactylous regeneration. This report describes further experiments to help define this phenomenon, including anatomical and histological examination. Hindlimbs of toad larvae were amputated at five different stages as reported previously, but this time the amputation was made at the basal portion of the femur. Larvae whose left hindlimbs were amputated were cultured at 20"C, 25OC or 28"C, until metamorphic climax. The incidence of polydactylous regenerates was highest in every series of larvae when the amputation was made at the end of the limb bud stage. But, differing from previous results, there was a low incidence of polydactylous regenerates when the amputation was made at the early limb bud stage. When larvae were cultured at 28'C. there was a low incidence of polydactylous regenerates in larvae whose limb amputation was made at the late foot plate stage. In general, the incidence of polydactylous regenerates increased with the rise in culture temperature. It seems probable that these phenomena are related in some way to accelerated tissue differentiation due to the rise in temperature.

TAKEHIRA, Y., Y. HAYASHI and Y. KAMEYAMA, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi. Mor hogenesis of cytosine arabinoside induced poly actyly in mice and rats. Dieital malformations in mouse and rat fetuses-resulting from maternal administration of Ara-C were compared with those induced by 5-FU, in order to elucidate the morphogenesis of polydactyly. CF#1 mice were injected intraperitoneally with Ara-C on day 10 of pregnancy at a dose of 3-10 mg/kg, and Wistar rats were injected on day 10 o r 11 at a dose of 50-150 mg/kg. The

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WATARI, S . , S. YAMAMOTO, T. SEO and K. TSUGE, Department of Orthopedic Surgery, Hiroshima University, School of Medicine, Hiroshima. Differences between observation methods in experimental teratological study of limbs. In most experimental studies of limbs, alizarin red translucent specimens of the extirpated foetus just before delivery has been recommended as the method of observation. However this method was not satisfactory in determining the precise condition of the bones and joints in our series and we were unable to diagnose each malformation correctly. The diagnosis of simple polydactyly or oligodactyly is not sufficient clinically but more detailed diagnosis was not possible using this method. Therefore we attempted to raise the malformed offspring to one month of age and then examine their limbs radiologically. The X-ray findings showed very clear relationships among each component of the hand and foot, and correct diagnosis of malformations became possible. We were able to subdivide preaxial polydactyly into different types, and oligodactyly into developmental arrest, fusion o f neighboring digital rays and even split hand.

HAYASHI, H., K. ONO, Y. KURATA and K. EZAKI, Department of Orthopaedic Surgery, Osaka University Medical School, Osaka, and Central Institute for Experimental Animals, Kawasaki, Kanagawa. A hereditary peroneal muscle atrophy in the house mouse. Mice unable to use their legs properly were discovered in one of the inbred C F # l strains at the Central Institute for Experimental Ani-

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mals in Kawasaki. The cause of the unusual gait proved to be atrophic limb muscles. One of the authors (K. E.) established that the new trait was hereditary and breeding testing revealed it to be an autosomal recessive trait. The pathological anatomy of the legs and feet were studied, and the leg muscles, peripheral nerves and spinal cord were examined histologically. The affected limbs were small and some had tarsal fusions. The peroneal muscle group exhibited evidence of neurogenic atrophy. The primary site of this abnormality proved histologically to be in the lower motor neuron. This study establishes a new hereditary peroneal muscle atrophy in the mouse.

YAMAMURA, H. and Y. TAKAGISHI, Department of Anatomy, Mie University School of Medicine, Tsu, Mie. Correlation of Purkinje cell iniury with cerebellar size in postnatally developing homozygous Gunn rats. Homozygous (jj) Gunn rats with lifelong hyperbilirubinemia exhibit cerebellar hypoplasia of varying degrees. The cerebellar Purkinje cells ( P cells) have been found to be affected almost selectively. The present study attempted a quantitative analysis of cortical layers and medulla in relation to Purkinje cell injury in the developing cerebellum of these model animals. The area and/or length of the cortical layers and medulla were measured and the P cell number was counted on 1.5 p thick sagittal sections prepared from the culmen of the cerebellum. Heterozygous (Jj) Gunn rats without hyperbilirubinemia were used as controls. At 3 and 7 days after birth, although slight abnormalities of the P cell cytoplasm were recognized in jj animals, there was no distinct difference between jj and Jj animals in the mean values of the P cell number and the area andlor length of the layers in the culmen. However, at 1 2 days and thereafter, when the P cell degeneration was far advanced and some of the P cells had disappeared, these mean values were lower in jj than in Jj animals. At 18 to 30 days after birth the total area and circumferential length of the culmen, the areas of the molecular and internal granular layers and of the medulla and the length of the P cell layer correlated significantly with the number of P cells surviving in the culmen. Furthermore, the degree of cerebellar underdevelopment almost paralleled the degree of degeneration of the surviving P cells. The present findings strongly suggest the presence of a causal relationship between P cell injury and cerebellar hypoplasia.

OOA, S., Research Institute of Environmental Medicine, Nagoya University, Nagoya. Costovertebral dysplasia, a dumpy mutation (dup) in the mouse. In June 1976, a litter of three normal mice (two females and a male) and four with short

and curved tail and short trunk (two females and two males) was encountered in the 13th brother-sister generation of albino-like stock (PPSSW"~~). Mating experiments proved the abnormal animals to be homozygous for a new autosoma1 recessive gene, which the author proposes because of its to call "dumpy" (symbol, +) characteristic phenotype. Besides the short trunk and tail, the dumpy mouse showed a prominent chest and protuberant abdomen. The viability of dumpy homozygotes was low and most of them died within a month in this albino-like strain. The body weight was 30 - 40% less than that in controls of the same age. The length of the head and body was reduced to about 61% and that of the tail to about 16.5% of controls. The viscera were generally lighter than in controls. The kidney length was reduced to 80% of normal, but no change in width was noted. The gross anatomy of the dumpy skeleton was examined with cleared specimens. The most striking differences from controls were found throughout the spinal column and in the ribs, while no abnormality was found in the long bones. The shape of the vertebrae was very abnormal, with kyphosis, lordosis and scoliosis occurring frequently. It was difficult to determine the exact number of vertebrae because of the severe deformity. The abnormalities of the axial skeleton were similar to those in costovertebral dysplasia in the human.

TUTIKAWA, K. and Y. HARADA, Department of Induced Mutation, National Institute of Genetics, Mishima, Shizuoka. The effects of chemical teratogens on the expression of genes responsible for sacrovertebral abnormalities in mice. Even though the genetic background is unchanged, the genes responsible for sacrovertebra1 abnormalities produce variable manifestations. The abnormalities usually appear in the sacral and the first two or three caudal vertebrae. Sometimes the sacral or caudal vertebrae are interrupted in one or more places. In the abnormal selected line, the frequency of abnormals increased gradually until the 27th generation, after that the incidence plateaued at 40 - 50% levels. To test the combined effects of genes and chemical teratogens, several possible combinations among abnormals and phenotypic normals, between abnormals and normals within the abnormal selected line, and between abnormal and normal selected lines were conducted, and the females were given i.p. doses of 1.5 glkg of urethane or 3 mglkg of thio-TEPA on the 8.5th day of pregnancy. Results reveal that both of the chemical teratogens markedly increase the penetrance of the genes. For example, in a case of mating among phenotypic normals in the abnormal line, the frequencies of abnormals in untreated, urethane- and thio-TEPA-treated groups were 40, 72 and 77% respectively. Furthermore, thio-TEPA also increases the expression of abnormalities. For instance, in matings among phenotypic normals, the frequen-

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cies of more severe malformations in which the vertebral column ends near the root of the tail were 5/18 (0.281, 14/46 (0.30) and 19/25(0.76) in untreated, urethane- and thio-TEPA-treated groups respectively.

HOSHINO, K., S . ODA and Y. KAMEYAMA, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi. Embryological observations on the lethal effects of Tail anomaly lethal (TaZ) in rats. The mutant gene Tail anomaly lethal ( T a l ) was found in a descendant of trypan blue-treated rats and the phenotype was similar to that of Flocus mutants in mice. In this report, the lethal effect of homozygous TaZ gene was examined morphologically in the embryonic stages of the BDIX-TaZ/+ strain of rats. Heterozygous females (Tat/+) were mated to heterozygous males overnight and those with vaginal plugs were considered to be in day 0 of pregnancy. Pregnant females were killed o n day 8, 9, 10, 1 2 , 14 or 20 of gestation. The embryos of days 8, 9 and 10 were examined microscopically and those of days 1 2 , 14 and 20 were observed grossly. On day 8 of gestation, most of the live embryos were in the late egg cylinder stage. In some embryos, degenerative signs such as pyknosis and cytoplasmic inclusions were observed in the embryonic ectoderm. On day 9 of gestation, about 70% of the live embryos formed mesoderm, and the rest showed developmental retardation, remaining at the late egg cylinder stage. These retarded embryos also showed marked degenerative changes such as pyknosis and cytoplasmic inclusions in the embryonic ectoderm. The embryonic mortality was about 10% on days 8 and 9, and showed a rapid increase on day 10 (about 40%). Thereafter, it increased gradually until day 20. These results indicate that the lethal effect of homozygous TaZ gene begin to appear in the embryonic ectoderm of the egg cylinder and that most would die during days 9 and 10 without formation of the mesoderm.

NAKANE, K., K. HOSHINO and Y. KAMEYAMA, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi. Effects of hypervitaminosis A on the development of eyes in genetic microphthalmia (mic) in mice. Previously we reported an early manifestation of genetic microphthalmia (mic), namely, blockage of the contact between the optic vesicle and the head ectoderm. The consequent reduction of the lens vesicle was also nored to bring about later growth inhibition of the eyeball. The purpose of the present study was to investigate how the developmental disturbance of the facial mesodermal tissue resulting from hypervitaminosis A affects the manifestation of genetic microphthalmia (mic).

Water miscible vitamin A was injected intraperitoneally to mic mice (mic/mic) at a dose of 10,000 or 15,000 IUlanimal on day 8 or 9 of pregnancy. Embryos and fetuses were obtained on day 11 and 18 of gestation, and examined externally and histologically. In 11-day embryos, the hypoplastic maxillary process did not cover the inferior portion of the orifice of the optic cup. The lens vesicle was fairly well formed. In 18-day fetuses, a short snout and micrognathia were evident, but the eyelids were closed. The diameter of the eyeball ranged from 1.1 to 2.0 mm, and was greater than those of nontreated controls (0.8 1.7 mm). The markedly small and malformed lens characteristic of mic mice was virtually absent in the vitamin A treated mice, and the incidence of persistent lens stalk and retinal folding was less than in the control. The results suggest that developmental retardation of the maxillary process by hypervitaminosis A reduces the degree of microphthalmia in mic mice.

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TAKEUCHI, I. K. and U. MURAKAKI, Department of Embryology, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi. Influence of cysteamine on the teratogenic action of 5-azacytidine. Cysteamine (CA) is a vell-known radioprotective agent in various chemical and biological systems. Recently, it has been reported that CA can prevent the cytotoxic or oncogenic action of some chemical agents. The present report describes the preventive effect of CA against the teratogenic action of 5-azacytidine (5-AC). On day 8 of gestation, pregnant H p 1 rats were assigned to the following six groups: 1. Untreated control. 2. 5-AC (1 mg/kg, ip). 3. CA (100 mg/kg, ip) 30 minutes before 5-AC. 4. CA 3 hours before 5-AC. 5. CA 30 minutes after 5-AC. 6. CA 3 hours after 5-AC. On day 20 of gestation, the uterus was opened and the number of implantation sites, fetal deaths and live fetuses were noted. The live fetuses were weighed and examined for external malformations. The fetal death rate, weights of live fetuses and malformation rate were calculated for each litter and the mean rates in each group were statistically analysed using Wilcoxon's rank sum test. In group 2 , 5-AC caused significant increases in the mean fetal death rate and the mean malformation rate, and significant reduction in the mean weight of live fetuses, as compared with group 1. The typical malfonnations were exencephaly or encephalocele, and bi- or unilateral microphthalmia. The mean fetal death rate in groups 3 . 4 , 5 and 6 were all significantly higher than in group 1, but no significant differences were found among groups 3-6. The mean weight of live fetuses in group 3 was significantly increased compared with group 2, but decreased compared with group 1. The mean malformation rates in groups 3 , 4 and 5 were all significantly low compared with group 2.

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peared in a stage specific manner. Simultaneous administration of CA significantly increased the frequency of malformed fetuses induced by MC alone, particularly on days 8, 9 and 11. 3) the number of fetuses with skeletal malformations was also significantly increased in the MC plus CA groups. These results indicate that CA potentiated the embryocidal and teratogenic actions of MC in mice when both compounds were given simultaneously not only on day 11 of gestation but also other days of organogenesis. The types of embryopathy observed were similar to those previously reported by Tanimura (1968).

ISA, Y., Y. SAKAMOTO, S . KIMURA, T. KANZAKI, H. TANAKA and T. NOMURA, Institute for Cancer Research, Hospital Pharmacy, and Department of Fundamental Radiology, Osaka University Medical School, Osaka. Enhancement effect of barbital and phenobarbital on aminopyrine-initiated teratogenesis in mice. Aminopyrine and its molecular compound with barbital (Pyrabital) showed potent teratogenicity (0.35 mg/g body weight; 40/101, 39.6%), whereas an equivalent dose of the molecular compound with cyclobarbital (Cyclopyrabital) showed very weak teratogenicity (0.5 mg/g; 31 153, 1.9%, p