Teratogenic Mechanisms Research Articles

The teratogenic mechanism of 6-aminonicotinamide on limb formation of chick embryos: abnormalities in the biosynthesis of glycosaminoglycans and proteoglycans in micromelia.

After a dose of 10 micrograms of 6-aminonicotinamide (6-AN) was administered to day-4- chick embryo in ovo, micromelia was obviously observed in the hind limbs of 7-day chick embryos. We examined the teratogenic mechanism of 6-AN by using the normal or micromelial hind limbs (buds) from day 5 to day 7, with special attention to the biosynthesis of glycosaminoglycan (GAG) and proteoglycan as an index of limb chondrogenesis. The present study provides evidence for abnormalities in the levels of GAG or proteoglycan biosynthesis in the micromelial hind limbs (buds). 1) Both [35S]sulfate and [3H]glucosamine incorporation into GAG per 10 limbs or mg DNA of the micromelia were inhibited, suggesting a decrease of GAG synthesis. 2) The micromelial limbs synthesized low-sulfated chondroitin sulfate (chondroitin) as judged by the 35S/3H ratio, the proportion of unsulfated disaccharide (delta Di-0S), and the result of cellulose acetate electrophoresis, although there were no significant differences in the approximate molecular size of 35S-chondroitin sulfates synthesized between the normal and micromelial limbs. 3) PAPS-synthesizing activity in the micromelial limbs was markedly inhibited, and this may result in the production of low-sulfated proteoglycan. 4) The transition from mesenchymal- to cartilage-specific proteoglycan synthesis did not appear in the micromelial limbs as judged by the sedimentation profiles. 5) 6-AN caused marked reductions in the oxygen consumption and ATP level of the micromelial limbs, thereby causing the defect in PAPS formation. We suggest that these 6-AN-induced sequential molecular defects (the reduction of respiratory activity, ATP and PAPS level, and concomitant interference with GAG and proteoglycan biosynthesis) in the limbs (buds) during the critical period of limb morphogenesis must be major factors resulting in the cartilage growth retardation or disorder, i.e., micromelia.

Increased incidence of congenital malformations in the offspring of diabetic rats and their prevention by maternal insulin therapy.

Intensive care of the pregnant mother with diabetes has dramatically decreased the incidence of diabetic fetopathy. The persistently high rate of fetal and neonatal mortality in diabetic pregnancies is nowadays mainly due to the increased incidence of serious congenital malformations. However, attempts to elucidate the precise teratogenic mechanisms have been sparse, presumably because of a lack of relevant animal models. In the present study we recorded the incidence and types of skeletal malformations in live offspring of normal rats and in rats made diabetic with the B-cytotoxic agent streptozotocin (SZ) at least 2 wk before conception. In some of the diabetic animals insulin treatment was begun 1 wk after the SZ injection and continued throughout pregnancy. In addition, the fetal development was followed by assessing the calcification of the skeleton on gestational days 20 and 22 with the aid of Alazarin Red S staining. Manifest diabetes in the pregnant rat induced a decrease in fetal weight and viability and marked retardation of skeletal maturation. In addition, about 20% of 135 viable fetuses showed skeletal malformations comprising either micrognathia or caudal dysgenesis. These defects were not found in 314 offspring of the control rats. Only two cases of caudal dysgenesis and none of micrognathia were detected among 233 offspring of the insulin-treated rats. The present data underscore the importance of a strict differentiation in the offspring of the diabetic rat between transient development retardations and true malformations. They also demonstrate that correction of the maternal glucose intolerance is crucial for preventing the fetus from developing skeletal malformations. Altogether the data suggest that fetal malformations in the diabetic rat are attributable either to the hyperglycemia as such or to some accompanying metabolic consequence of insulin deficiency.

1221 MECHANISM OF DEXAMETHASONE-INDUCED CLEFT PALATE IN RATS: INHIBITION OF PROSTAGLANDIN AND THROMBOXANE SYNTHESIS IN FETAL PALATES IN VIVO

We have hypothesized that the teratogenic mechanism of glucocorticoids involves the same biochemical pathway as the mechanism of their anti-inflammatory hormonal action, i.e., inhibition of arachidonic acid release and of prostaglandin (PG) and thromboxane (TX) production. Arachidonic acid reversed the production of cleft palate in rats by dexamethasone and this reversal was blocked by indomethacin, an inhibitor of PG and TX synthesis (cyclo-oxygenase) (Fed. Proc. 3616:955, 1980). Pregnant rats were treated with dexamethasone 3.75mg/kg or vehicle on days 12 to 15 of pregnancy, a regimen giving about 80% cleft palate. Three hours after the last dose of steroid on day 15 fetuses were removed by c-section and palates from each litter were dissected, pooled, sonicated, and incubated at 37°C for 3 hours. 100μl aliquots were removed at 1 hr. and 3 hrs. and the reaction stopped with lml ethanol. Aliquots of the ethanol extracts were measured for PG and TX by radioimmunoassay. PG and TX synthesis was proportional to time of incubation. Dexamethasone inhibited significantly the production of F2α, E2, 6-keto F1α, and TX B2 by fetal palates to about 50% of control values. Thus, evidence has been presented for the precise biochemical mechanism of glucocorticoid-induced cleft palate.

Screening for teratogenic hazards: nature of the problems.

State of the art teratology is sufficient to develop a screening system of teratogenic hazard potential if one remembers the basic developmental biology underlying teratogenesis. Understanding of teratogenic mechanisms may provide better systems but such knowledge is not yet available. The system described is an interim step and may not prove valid. Hopefully Karnofsky's law will remain in our attention thereby permitting our realization that the question, "Is table salt teratogenic?" is naive and largely irrelevant. The answer is "Yes" from the viewpoint of total well-being of the conceptus. What the question is really asking is: "What is the developmental hazard potential of table salt?" This question can be answered quantitatively and it is, "No, it is not a developmental hazard because it is a coeffective teratogen with a developmental hazard index of less than 0.1." If we could all understand these basics, we could realistically proceed to safeguard your grandchildren and mine.

Absence of a role for salsolinol in the mechanism of ethanol teratogenicity.

Alcohol and its primary metabolite, acetaldehyde, are central nervous system teratogens. Acetaldehyde can further interact with endogenous biogenic amines under certain conditions to generate tetrahydroisoquinoline (TIQ) alkaloidal metabolites, some of which interfere with adrenergic neuronal function and structural integrity. The possible role of salsolinol (the TIQ derived from the condensation of acetaldehyde with dopamine) in the mechanism of alcohol teratogenicity was investigated in rats. Administration of 2 mg/kg of salsolinol to rats on the 15th day of gestation did not result in the recovery of this TIQ in the fetuses. Furthermore, acute oral administration of alcohol (3 g/kg x 3 doses) to rats on different days of gestation did not result in the synthesis of salsolinol by the fetuses despite the high maternal blood ethanol levels (124 +/- 22 mg%). These findings argue against a role for salsolinol in the mechanism of alcohol teratogenicity. Small amounts of endogenous salsolinol (3.6 ng/g) were detected in fetuses from control rats, in agreement with previous findings in neonatal rats. The physiological role (if any) of endogenous salsolinol in the fetal and neonatal rat is not known.

Cytotoxic effects of ethylnitrosourea on central nervous system of rat embryos. Special references to carcinogenesis and teratogenesis.

For the purpose of observing the cytotoxic effect of N-ethyl, N-nitrosourea (ENU), a potent carcinogen on the central nervous system (CNS) at the late period of organogenesis, the embryonal telencephalic wall was hourly examined under the light and electron microscopes. Pregnant rats on the 13th day and 15th day of gestation were given either a single intravenous injection of 40 or 80 mg/kg, of ENU. The cytotoxic effect on the embryo treated on the 15th day of gestation was severer than that treated on the 13th day of gestation. The common pathological changes in both treated groups are; (1) mitotic arrest 1 hour after administration in the most inner zone of the ventricular layer, (2) degeneration and necrosis accurred predominantly in the so-called DNA synthetic zone and resulting in cell loss of the ventricular layer, (3) elimination of degenerating products; and (4) tissue repair. The findings mentioned may indicate a cytotoxic effect inducing microencephalia, and furthermore, the teratogenic and carcinogenic mechanisms are discussed.

BOOK REVIEWS

Book reviewed in this article: Advances in the Study of Birth Defects. Volume 1—Teratogenic Mechanisms. Edited by T. V. N. Persaud Medical Imaging, CT U/S is NMR—A Basic Course. Edited by Louis Kreel Clinics in Obstetrics and Gynaecology. Volume 6, No. 1‐Contraception. Edited by E. B. Connell (Guest Editor) Craig's Care of the Newly Born Infant. By A. J. Keay and D. M. Morgan Maternal Recognition of Pregnancy. By CIBA FOUNDATION SYMPOSIUM 64 (new series) Pregnancy Metabolism, Diabetes and the Fetus. By Ciba Foundation Symposium

Embryotoxic, teratogenic, and metabolic effects of ribavirin in mice

To gain comparative perspective on the teratogenic mechanisms of action of drugs that are precursor analogs or antimetabolites of nucleic acids, we employed a new antiviral agent—ribavirin—which is known in other systems to inhibit the biosynthesis of guanine nucleotides. Pregnant ICR mice were injected at 10th–13th days of gestation (stages 14–20) with a single ip dose of ribavirin in the range of 10–200 mg/kg. All dosages in excess of 25 mg/kg were teratogenic. The optimal teratogenic dose varied with the stage of development, being higher at advanced stages of development. Depending on the dose and stage of treatment, virtually all parts of the skeleton including the craniofacial and limb bones were susceptible to ribavirin. Both the frequency and multiplicity of skeletal defects increased as the dose was raised. The stage dependency of defects in the orofacial bones was markedly apparent. Treatment on Day 10.5 resulted in shortened maxilla in all survivors, while treatment on either Day 11 or 11.5 resulted in a high frequency of reduction in the length of both upper and lowerjaws. Treatment on the 12th day resulted in a very low incidence of effect on the maxilla (4%) but a high frequency (88–100%) of reduction and deformation of the mandible. This enhanced susceptibility of individual facial bones at different stages of development with virtually no overlap provides an experimental model to study cellular phenomena underlying normal and abnormal facial development. Ribavirin, both in vivo and in vitro, inhibited embryonic DNA synthesis. The inhibition was transitory and did not seem to be directly related to the embryolethal activity of the drug. Although the role of metabolic inhibition in precipitating teratogenesis is not clear, cytotoxic action of ribavirin against proliferating limb bud mesenchymal cells is directly associated with the origin of limb deformities.

Book Reviews

Book reviewed in this article:Human Growth, Vols. 1, 2 and 3 Edited by Frank Falkner and J. M. TannerScanning Electron Microscopy of Human Reproduction, Vol. 4 Edited by E. S. E. HafezCurrent Pediatric Therapy 8 Edited by Sydney S. Gellis and Benjamin M. KaganTreatment of Status Epilepticus By Walter Fr&scherNeonatal Medicine By Malcolm L. ChiswickIntroduction to Biochemistry, 2nd. Edn. By John W. SuttieAdolescent Medicine in Primary Care By Walter R. AnyanEducational Handicap, Public Policy, and Social History: A Broadened Perspective on Mental Retardation By Seymour B. Sarason and John DorisAdvances in the Study of Birth Defects Vol. 1: Teratogenic Mechanisms Edited by T. V. N. PersaudThe Use of Drugs to Modify Behaviour in Retarded Persons A Practical Guide for Parents and Advocates By Roger D. Fréeman Downsview

The stilbestrol-adenosis-carcinoma syndrome.

For the development of the DES carcinoma syndrome, two different mechanisms may be assumed. 1. Indirect effect via teratogenesis: squamous cell carcinoma develops from adenosis vaginae with the assistance of cofactors (Fig. 6). Till now, a conversion of adenosis to clear cell carcinoma has never been observed (Hilgers, 1977). Adenosis is probably a precancerous lesion only for squamous cell carcinoma. 2. Transplacental terato-oncogenesis: clear cell carcinoma as a mesonephric carcinoma develops from persisting wolffian ducts with a latent period of 15 to 20 years. DES here acts only as a promoter by way of the transplacental teratogenic mechanism, thus as a terato-co-carcinogenic factor (Fig. 6).

Chemical-biological reactions common to teratogenesis and mutagenesis

Cytotoxic chemicals have in common the ability to act specifically on cells in cycle. Bacteria are more sensitive in the exponential growth phase than when growing slowly in media. Similar observations have been made on a variety of systems ranging from bacteria, yeast, higher plants and invertebrates to vertebrates including primates. The embryo and fetus are highly susceptible to cytotoxic agents because they have continuous groups of cells in the growth phase. Acutely toxic doses may cause cellular death and result in developmental defects or fetal death. Cytotoxic agents can be grouped as alkylating agents, electrophilic reactants, antimetabolites, intercalating agents, amino acid antagonists, spindle poisons, and an additional group of chemicals which covalently bind to DNA. These cytotoxic groups of chemicals may also be mutagenic by interacting with DNA to produce changes in sequences of nucleotides resulting in heritable defects either in a somatic cell line or in a germinal cell line. The mechanisms of chemical-induced teratogenicity and mutagenicity are similar. This commonality is further discussed in the text.

Chemical-Biological Reactions Common to Teratogenesis and Mutagenesis

Cytotoxic chemicals have in common the ability to act specifically on cells in cycle. Bacteria are more sensitive in the exponential growth phase than when growing slowly in media. Similar observations have been made on a variety of systems ranging from bacteria, yeast, higher plants and invertebrates to vertebrates including primates. The embryo and fetus are highly susceptible to cytotoxic agents because they have continuous groups of cells in the growth phase. Acutely toxic doses may cause cellular death and result in developmental defects or fetal death. Cytotoxic agents can be grouped as alkylating agents, electrophilic reactants, antimetabolites, intercalating agents, amino acid antagonists, spindle poisons, and an additional group of chemicals which covalently bind to DNA. These cytotoxic groups of chemicals may also be mutagenic by interacting with DNA to produce changes in sequences of nucleotides resulting in heritable defects either in a somatic cell line or in a germinal cell line. The mechanisms of chemical-induced teratogenicity and mutagenicity are similar. This commonality is further discussed in the text.

Drug-induced limb dysplasias in fetal rabbits.

Pregnant New Zealand White rabbits were treated on gestation day 12 with 19.2 mg/kg methotrexate (MTX), 750 mg/kg hydroxyurea (HU), or 1,500 mg/kg acetazolamide. Rabbits were killed either 2-32 hours posttreatment for histological analysis of embryos or at day 29 for gross and skeletal examination of fetuses. tmtx produced cleft palate, hydrocephalus, and fore- and hindlimb reduction defects. Histological analysis revealed pyknosis and edema in mesenchymal tissues at four to eight hours following treatment. The apical ectodermal ridges (AER) of treated embryos permanently lost their characteristic pseudostratified organization. By 32 hours the limb buds had regained their normal appearance except for the AER. HU affected all fetuses with skull and facial anomalies as well as severe reduction deformities of all limbs. Histologically HU-treated embryos had numerous, basophilic, intercellular granules (presumably cell debris) which appeared within two to four hours in the limb bud mesenchyme, neural tube, and dorsal root ganglion. The architecture of the AER was unchanged. Acetazolamide produced bilateral retarded ossification or possible aplasia of the first metacarpal and talus in nearly 80% of fetuses. Microscopic examination disclosed no apparent alterations in limb-bud morphology. Methyl green-pyronin Y staining called attention to green intracellular droplets within the endoderm of the trachea and bronchi at two hours posttreatment. It was concluded that the three drugs do not produce limb dysplasias by a common teratogenic mechanism.

Mechanisms and pathogenesis of malformation.

The etiology and pathogenesis of fetal malformation are discussed with a view toward their management. Hereditary physical and social conditions can contribute to fetal malformation. Mutations chromosomal aberrations and major gene defects have often been mistaken as causes rather than the genesis of birth defects. Hereditary causal mechanisms of malformation can be initiated at the subcellular cellular and tissue levels and may be similar to or identical with environmental teratogenic mechanisms. The site of action of malformation mechanisms and teratogenic mechanisms acting on cells established tissues and developing organs are discussed. The identification of the pathogenesis of specific patterns of malformation could make it possible to establish primary causes and facilitate the prevention diagnosis and treatment of birth defects.

Rib anomalies in myelodysplasia. An approach to embryologic inference.

An embryologic teratogenic mechanism is reconstructed by utilizing the distribution of anomalous ribs in children with lumbosacral myelomeningocele. This methodology may help to coalesce information from experimental models of teratogenesis and from descriptive human embryology. It provides an approach to deductive reasoning about human structural anomalies based on dynamic embryonic models of teratogenesis. Availability of a developmental time scale, based for example on the distribution of defective ribs, may be useful in the study of other teratogenic and developmental processes.

Teratogenic screening methods and their application to contraceptive products.

ABSTRACT After an analysis of the basic principles of teratogenic drug testing and the complexity of factors involved in the production of congenital malformations, the following topics are considered: the mechanisms of the teratogenic action the role of the placenta in drug transfer the choice of an animal model for teratogenic testing of hormonal sex steroids the influence of oral contraceptives upon various phases of reproduction. To compete with the various mechanisms which control the normal intrauterine development, multiple conditions have to be fulfilled. Such a situation occurs only occasionally in humans. Much data are available but little knowledge exists regarding the teratogenic mechanisms and the precise role of the placenta in drug teratogenicity. Great differences of reactions to drugs exist among various species. They can be related to differences in the enzymatic systems involved in the metabolic turnover of a drug, but in many cases no explanation is yet available. It seems that preliminary screening of sex steroids could be done in rodents. This can contribute to the selection of interesting therapeutical agents and may aid in discovering potential side effects. Since the maintenance of pregnancy in rodents requires functioning ovaries, while in women and in primates the placenta takes over very early the hormonal control of pregnancy, it is suggested to use primates for the safety evaluation of contraceptive drugs. The results obtained in these species are likely to have good predictive value. Oral contraceptives have no mutagenic effects, they do not produce chromosomal aberrations and seem to be devoid of teratogenic action. Experimental and clinical data suggest that oral contraceptives have no adverse effects on the perinatal and postnatal development.

Cytogenetic studies in the offspring of LSD users.

The presumed carcinogenic and chromosome-breaking potential of d-lysergic acid diethylamide (d-LSD 25) in human somatic cells has been reviewed by Fernandez et al. (1973). Reports concerning its teratogenic effect in experimental animals and man have been either contradictory or inconclusive. The following possible teratogenic mechanisms may be considered: (1) A direct effect on organogenesis of LSD ingested by the mother during early pregnancy; (2) A similar disturbance of organogenesis by a persistent metabolite of LSD ingested by the mother at some time prior to the pregnancy; (3) Chromosome damage or point mutations in parental germ cells exposed to LSD before initiation of a pregnancy.

Coenzyme competition and precursor specificity during teratogenesis induced by 6-aminonicotinamide

The teratogenic mechanism of the nicotinamide analog 6-aminonicotinamide (6-AN), is thought to be inhibition of energy exchange reactions which in turn selectively interfere with synthesis of mucopolysaccharides. Nicotinamide and 6-AN were administered singly or in combination for studying micromelia in the embryonic chick limb. These coenzyme competition, protection, and reversibility experiments suggest that cells of 4-day embryos readily accept either nicotinamide or 6-AN. Preloading with nicotinamide 1–24 hours prior to exposure to 6-AN prevented teratogenicity of 6-AN. An excess of nicotinamide up to 2 times the maximal protective dose given 2 hours or more after 6-AN did not offset the teratogenicity of 6-AN. Once exposed either to 6-AN or to nicotinamide the cells do not readily accept either alternate molecule. This mutual exclusivity enabled demonstration that 6-AN is 80% effective as a teratogen in 4-day chick embryos within 2 hours after its administration. Duration of the effectiveness of a standard dose of 6-AN given to a 4-day embryo was studied by a 30-minute terminal label with 35S-sulfate. Maximum inhibition was seen 3 days after treatment and recovery to control values occurred in about 4 1 2 days. Because limb teratogenicity is a result of atypical chondrogenesis and because the experiments above were performed on mixed tissues of whole embryos or whole limbs, early rudiments of tibia at 9, 10, and 11 days were isolated and tested with 6-AN and labeled precursors. It was found that 2 hours of exposure of bone rudiments to 6-AN was sufficient to demonstrate selective inhibition of 35S incorporation while amino acid incorporation remained similar to controls. The effect of 6-AN in the limb of the chick embryo is prompt, demonstrable in 2 hours and temporarily irreversible. Recovery is seen in 4 to 5 days, but distortion of the cartilage model results in micromelia.

Histological changes in the skeletal elements of developing rat foetuses following treatment with sodium salicylate

Abnormal vertebrae of foetuses from CFE rats treated with sodium salicylate by the oral, subcutaneous or intraperitoneal route as a single dose of 500 mg/kg on day 8, or in five separate doses of 100 mg/kg on days 7–11 of pregnancy, were examined histologically with a view to elucidating the mechanism of teratogenicity. Foci of abnormal cartilage were seen in the vertebral centra and neural arches. The most obvious defects were a reduced deposition of cartilage matrix and a precocious hypertrophy of the chondrocytes. Defects in the preosseous cartilage were related to abnormalities in the formation of neural arches and in the patterns of ossification. Malformation was such that in many foetuses the brain and spinal cord were exposed dorsally.

Studies on congenital rubella. Preliminary results on the frequency and significance of presence of rubella virus in the newborn and the effect of gamma-globulin in preventing congenital rubella.

IN 1941, Gregg1pointed out the existence of congenitally acquired rubella. There have since been many investigations into this problem in an effort to determine its scope, to define the teratogenic mechanisms, and to devise methods to prevent it. Today, 25 years later, we have available the necessary laboratory tools to seek answers in a more definitive manner.2,3The epidemic which occurred in Cincinnati and Dayton, Ohio, in the spring of 1964 provided an opportunity to study some aspects of congenital rubella. This report will present preliminary findings. Materials and Methods The 1,549 infants born at the Cincinnati General Hospital from Oct 15, 1964, to April 1, 1965, were studied. Cord bloods for antibody determination were obtained at delivery. The infants were examined and a pharyngeal swab for viral study was taken within 12 hours of birth. All infants were reexamined at least once before their hospital discharge.