Enhancement of chlorcyclizine teratogenicity in the rat by coadministration of calcium chelating agents.

Chlorcyclizine and structurally related drugs induce a high incidence of cleft palate and skeletal malformations in fetal rats. We have shown previously that these teratogens bind tightly and reversibly to chondroitin sulfate of cartilage and compete with calcium for binding. Experiments reported here demonstrate that co-administration of calcium chelating agents with chlorcyclizine significantly increases both the frequency of malformations and retention of [14C] chlorcyclizine by embryos. Retention of radioactive teratogen by embryos is inverse to retention of [45Ca]calcium. These findings suggest that drug binding to embryonic glycosaminoglycans is involved in the pathogenesis of malformations produced by chlorcyclizine.     

Molecular basis for skeletal variation: insights from developmental genetic studies in mice

Skeletal variations are common in humans, and potentially are caused by genetic as well as environmental factors. We here review molecular principles in skeletal development to develop a knowledge base of possible alterations that could explain variations in skeletal element number, shape or size. Environmental agents that induce variations, such as teratogens, likely interact with the molecular pathways that regulate skeletal development.     

Multiple Complex Congenital Malformations in a Rabbit Kit (Oryctolagus cuniculi)

Congenital malformations may occur during early embryogenesis in cases of genetic abnormalities or various environmental factors. Affected subjects most often have only one or 2 abnormalities; subjects rarely have several unrelated congenital defects. Here we describe a case of a stillborn New Zealand white rabbit with multiple complex congenital malformations, including synophthalmia, holoprosencephaly, gastroschisis, and a supernumerary hindlimb, among other anomalies. There was no historical exposure to teratogens or other known environmental causes. Although not confirmed, this case was most likely a rare spontaneous genetic event.Congenital malformations occur when there is derangement of the embryologic developmental process. Neural development and organogenesis is a critical time of development that occurs during early embryogenesis.^2,37 Congenital malformations that manifest at this stage of development may occur in association with various genetic abnormalities, such as point mutations and chromosomal abnormalities.^25,29 In addition, environmental factors, including maternal health status, nutritional deficiencies, and exposure to teratogenic drugs or chemicals, may play a role in the development of congenital malformations.^12,25 However, in 65% to 75% of human cases, the cause is unknown, resulting from a complex set of events such as polygenic or multifactorial genetic disorders, spontaneous genetic errors, and synergistic interactions of teratogens.^3,25 Approximately 78% of human cases demonstrate only a single developmental malformation, with cardiovascular defects accounting for approximately 30% to 35% of organ defects. Cases of more than 2 or 3 malformations in a single person are extremely rare.²⁸Here we describe a New Zealand white rabbit that was stillborn with numerous complex developmental abnormalities, including synophthalmia, a supraoptic proboscis, holoprosencephaly with other associated craniofacial deformities, Chiari malformation type I, gastroschisis, a supernumerary hindlimb, a fused (horseshoe) kidney with a supernumerary kidney, and male pseudohermaphroditism.     

The use of in vitro procedures in teratology.

The capabilities of investigators in the fields of teratology and toxicology are greatly enhanced by the use of tissue culture procedures in unraveling the mechanisms of drug action. Techniques currently available for the culture of postimplantation mammalian embryos permit evaluation of their metabolic responses to potential teratogens even when the length of time embryos survive and develop in culture is too short to allow a conventional teratologic survey of malformations. A simple procedure for culturing mouse embryos during early organogenetic stages is described in this report that will be of value to teratologists; it avoids the requirements of special glassware and equipment by using ordinary capped test tubes which are rotated tomaintain and efficient nutritional and gaseous evnironment. Some studies concucted with this procedure to monitor the metabolism of embryo during the first 24 h of culture are summarized. Another aspect of tissue culture, organ culture, provides further manipulative capability by which embryonic organs can be maintained for long periods of time during which they develop and differentiate to an extent that their morphological and biochemical responses to a teratogen can usually be made. Comparative effects of several teratogenic agents and the relative concentration of each that produces a similar degree of response are summarized. It is concluded that organs are more sensitive to teratogens in culture than they are in vivo, and that different teratogens possess enough specificity to isolate their simple growth-retarding effect from the role they play in distrubing other specific developmental events.     

Theoretical and applied aspects of experimental teratology]

The dependence of teratogenic effect from the agent specific properties, dose and exposition and the genotype of embryo and maternal organism is considered. The stage specificity of teratogens and the concepts of critical developmental periods are analyzed. The data on general mechanisms of embryonic defects related to the mutations and their phenocopies induced by teratogens are evaluated. The applied aspects of experimental teratology and, in particular, the testing of drugs' teratogenicity and the development of mathodical bases for the establishment of teratogens among the chemical pollutions are intimately connected with and depend on more profound studies of the theoretical bases of teratology. A new method of testing the chemical substances is proposed: search for embryotoxic and teratogenic factors in the blood of animals which were in contact with teratogens. With this aim the cleaving postimplantation mouse and rat embryos are cultivated in the medium with the blood serum from the animals treated with teratogens. This allows to detect in the blood not only the substance in question, but also the toxic products of its metabolism and the toxic substances formed in the maternal organism under the effect of this teratogen. The approaches to the express methods of estimation of teratogenicity are evaluated and the grounds of many steps testing the chemical polutions for teratogenicity are provided.     

The multifactorial/threshold concept -- uses and misuses.

The common congenital malformations have familial distributions that cannot be accounted for by simple Mendelian models, but can be explained in terms of a continuous variable, "liability," with a threshold value beyond which individuals will be affected. Both genetic and environmental factors determine liability, making the system multifactorial. Cleft palate is a useful experimental model, illustrating a number of factors that contribute to palate closure, the nature of a developmental threshold, and how genes and teratogens can alter the components of liability to increase the probability of cleft palate. The nature of the genetic component to liability in human malformations in not clear, and various possibilities, ranging from polygenic in the strict sense to a major gene with reduced penetrance are compatible with the data -- but the important feature is the threshold. Much of the confusion over the concept results from inconsistent use of terminology. The term "multifactorial" should be used for "determined by a combination of genetic and environmental factors," without reference to the nature of the genetic factor(s). "Polygenic" should be reserved for "a large number of genes, each with a small effect, acting additively." When several genes, with more major effects are involved, "multilocal" can be used. When it is not clear which of these is applicable the term "plurilocal" is suggested, in the sense of "genetic variation more complex than a simple Mendelian difference." Since teratological data often represent threshold characters the concept also has important implications for the interpretation of data on dose-response curves, synergisms, and strain differences in response to teratogens.     

The interference of naloxone hydrochloride in the teratogenic activity of opiates

Diamorphine hydrochloride, methadone hydrochloride, and the synthetic enkephalin analogue FK 33-824 are potent teratogens for the central nervous system in mouse embryos. They induce the "neurotropic syndrome of malformations," which is restricted to the central nervous system if administered during the critical period of neural tube closure. Pretreatment with corresponding equimolecular doses of the antagonist naloxone hydrochloride applied 30 minutes before treatment with the opiate agonists abolishes the major severe malformations, i.e., exencephaly, craniorachischisis, and brachyury, and reduces the number of cases of kinking of the spinal cord. Dilation of the fourth brain ventricle remains unaffected. It is suggested that the mechanism of interference in the teratogenicity of the opiates by naloxone hydrochloride reported here is based on competition for opiate receptors. In general, these observations are regarded as evidence that the pharmacological affinity of opiate agonists to receptors in the central nervous system is responsible for the malformations caused by them in this system.     

Prenatal Stress and Development: Beyond The Single Cause and Effect Paradigm

Our awareness of the causes of stress‐induced developmental dysfunction has increased dramatically over the past decade, and it is becoming increasingly clear that a number of factors can have considerable impacts on the developing fetus. Although there is a tendency in investigations of developmental teratogens to attribute specific causes to adverse fetal outcomes, it is important we recognize that for most developmental dysfunctions it is unlikely a single cause, but yet a series of environmental insults combined with genetic predisposition that ultimately leads to a disease state. Nonetheless, a number of developmental teratogens, such as maternal psychological stress and chemical exposures, have been shown to increase the likelihood of developmental defects. These defects can manifest during development, leading to observable birth defects, or could become evident long after birth, even into adulthood. In addition, epigenetic mutations in the germline can alter the phenotype of successive generations through transgenerational inheritance, and in this way environmental factors can alter the developmental outcomes and disease predispositions of future generations. Understanding this complexity is essential to interpretations of causality in the studies of stress‐induced developmental dysfunction and needs to be fully considered to more effectively interpret potential outcomes. Birth Defects Research (Part C) 96:289–298, 2012. © 2013 Wiley Periodicals, Inc.     

DNA repair disorders causing malformations

DNA damage contributes significantly to the abnormal development or demise of the conceptus. The widely differing phenotypes that result from mutations in DNA repair genes suggest that these genes play critical roles during development, even in the absence of exogenous DNA-damaging agents. Molecules that sense DNA damage and regulate DNA repair, cell cycle checkpoints and apoptosis act as teratogen suppressor genes, protecting the conceptus against insult from DNA damaging teratogens.     

Teratogens as Anticancer Drugs

Most anticancer drugs are teratogens, merely because they target vital cellular functions. Conversely, some plants produce agents that intentionally target embryonic signaling pathways, precisely to cause birth defects if pregnant animals eat such plants. Cyclopamine, a teratogen produced by a flowering plant, inhibits the Hh/Gli pathway, causing developmental defects such as cyclopia (one eye in the middle of the face). In theory, selective teratogens may suppress cancer cells that re-activate embryonic pathways, while sparing most normal cells. I discuss the potential (and limits) of teratogens in cancer therapy, linking diverse topics from morning sickness of pregnancy, embryonic pathways and poisonous plants to the mechanism of action of anticancer teratogens and their combinations with less selective cytotoxic agents.     

Deletion induction in bacteria. I. The role of mutagens and cellular error-prone repair

The amber mutation trpD28 of Salmonella typhimurium shows a complex reversion pattern on anthranilate (AA)-supplemented minimal medium. Under such conditions it is possible to recover revertants of two phenotypes, prototrophs (MM+) and anthranilate utilizers (AA+), each phenotype brought about by several mutational events. Since one class of AA+ revertants is caused by deletion of the trpD28 mutation, this constitutes a useful system for quantitative studies of the effects of mutagenic agents and cellular factors on the production of deletions. In the present study we have tried to assess the relative contribution of chemical mutagens vs. cellular mutator factors in causing this class of mutations. Strains of S. typhimurium in which the spontaneous reversion rate of trpD28 was modified by pKM101, (strain SO1007), mutL (strain SO1018) and both (strain SO1008), as well as the wild type (strain SO939) were treated with nitrous acid (HNO2) and mitomycin C (MC), mutagens reported to induce deletions in bacteria. The results showed that while the absolute frequency of deletions increased exponentially with dose of mutagen in parallel with the total reversion frequency, the relative frequency (percent) of these mutations was characteristic for each strain and for the most part unaffected by the dose of mutagen. It appears that deletions, spontaneous or induced, occur as a fixed percentage of total mutations and are brought about by the cells' own repair capacity and characteristic DNA metabolism. Perhaps these mutations are the result of untargeted events during SOS misrepair.     

Predictive value of minor anomalies: II. Use in cohort studies to identify teratogens

Cohort studies of putative human teratogens can identify the full spectrum of phenotypic effects, including both major malformations and minor anomalies. Cohort studies which include the much more common minor anomalies make it possible to use a relatively small number of exposed and unexposed infants to identify an increase in the frequency of malformations. We evaluated this use of minor anomalies in a cohort study of newborn infants who had been exposed in utero to three putative teratogens: insulin-dependent diabetes mellitus in the mother and the use of the anticonvulsant phenytoin and exogenous sex hormones by the mother. In addition, the reproducibility of identifying minor anomalies was tested by comparing the results of examinations by two independent observers of 444 unexposed infants. The frequency of minor anomalies was increased among infants of diabetic mothers. However, the reproducibility of identifying minor anomalies was poor. We conclude that the examination of teratogen-exposed infants for minor anomalies cannot be used in epidemiologic studies of putative teratogens unless special efforts are made to maximize consistency in the identification of these features.     

4-Phenylbutyrate restores the functionality of a misfolded mutant low-density lipoprotein receptor

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low-density lipoprotein receptor. To date, more than 900 different mutations have been described. Transport-defective mutations (class 2) causing partial or complete retention of the receptor in the endoplasmic reticulum are the predominant class of mutations. In a cell culture system (Chinese hamster ovary cells), we show that chemical chaperones are able to mediate rescue of a transport-defective mutant (G544V), and that the ability to obtain rescue is mutation dependent. In particular, the low molecular mass fatty acid derivative 4-phenylbutyrate mediated a marked increase in the transport of G544V-mutant low-density lipoprotein receptor to the plasma membrane. Thirty per cent of the mutant receptor was able to escape from the endoplasmic reticulum and reach the cell surface. The rescued receptor had reduced stability, but was found to be as efficient as the wild-type low-density lipoprotein receptor in binding and internalizing low-density lipoprotein. In addition to 4-phenylbutyrate, we also studied 3-phenylpropionate and 5-phenylvalerate, and compared their effect on rescue of the G544V-mutant low-density lipoprotein receptor with their ability to increase overall gene expression caused by their histone deacetylase inhibitor activity. No correlation was found. Our results indicate that the effect of these agents was not solely mediated by their ability to induce gene expression of proteins involved in intracellular transport, but rather could be due to a direct chemical chaperone activity. These data suggest that rescue of mutant low-density lipoprotein receptor is possible and that it might be feasible to develop pharmacologic chaperones to treat familial hypercholesterolemia patients with class 2 mutations.     

Induction of postaxial forelimb ectrodactyly with anticonvulsant agents in A/J mice

Exposure of A/J mice on day 9.5 of gestation to the derivatives of three acidic anticonvulsant agents, namely dimethadione, sodium valproate, and sodium diphenylhydantoin, each induced postaxial forelimb ectrodactyly predominantly of the right side. This specific malformation has previously been associated with the administration of acetazolamide to rodents; however, several agents can induce this same defect including other carbonic anhydrase inhibitors, carbon dioxide, cadmium, ethanol, ammonium chloride, and 13-cis retinoic acid. The relative potency of the three agents indicates no direct relationship to the pKa of the acid. Other than ectrodactyly, each of the anticonvulsant agents induced a compound-specific spectrum of malformations despite the uniform administration time. This finding suggests that these agents are capable of acting via different mechanisms or by the differential spatial and temporal dynamics of a common mechanism.     

Microarray analysis of murine limb bud ectoderm and mesoderm after exposure to cadmium or acetazolamide

BACKGROUND: A variety of drugs, environmental chemicals, and physical agents induce a common limb malformation in the offspring of pregnant mice exposed on day 9 of gestation. This malformation, postaxial, right‐sided forelimb ectrodactyly, is thought to arise via an alteration of hedgehog signaling. METHODS: We have studied two of these teratogens, acetazolamide and cadmium, using the technique of microarray analysis of limb bud ectoderm and mesoderm to search for changes in gene expression that could indicate a common pathway to postaxial limb reduction. RESULTS: Results indicated a generalized up‐regulation of gene expression after exposure to acetazolamide but a generalized down‐regulation due to cadmium exposure. An intriguing observation was a cadmium‐induced reduction of Mt1 and Mt2 expression in the limb bud mesoderm indicating a lowering of embryonic zinc. CONCLUSIONS: We propose that these two teratogens and others (valproic acid and ethanol) lower sonic hedgehog signaling by perturbation of zinc function in the sonic hedgehog protein. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Environmental mutagenesis and use of biomarkers in cancer risk prediction

The field of environmental mutagenesis or toxicology genetics aims to study the genetic damage that leads to mutations produced by physical, chemical and biological agents, to identify these agents and analyze their interactions and ways of action. There are enough experimental and epidemiological evidences implicating mutations in oncogenes, tumor suppressor genes and DNA repair genes as determinants in the onset and progression of the neoplastic process. A valuable tool in public and occupational health is the monitoring of populations exposed to potentially hazardous agents. The objective is to protect the health and quality of life of high risk groups on account of the nature of the agents of exposure. Monitoring of genotoxic effects in exposed populations as well as the analysis of susceptibility polymorphism are visualized as key tools in the realm of future public and occupational health in order to prevent the occurrence of environmental and specially occupational origin of tumors. This paper reviews the main concepts concerning this issue and refers to studies on the subject in Costa Rica.     

DEVELOPMENTAL BIOLOGY AND THE STUDY OF MALFORMATIONS

1. The scientific approach to malformations began with experimental work on abnormal conditions of incubation in the chick. 2. More precise experiments on causing abnormalities had a common origin with experimental embryology. 3. Progress in experimental teratology during the last fifty years is reviewed in a commentary on the four principles formulated by Stockard in 1921. 4. Some relationships are traced between the results of cytogenetical studies in man and in other organisms. 5. An account is given of present knowledge concerning malformations of the neural tube originating either experimentally, spontaneously, or phenotypically. 6. The teratological implications of some recent theories on the expression of the genotype are discussed, particularly in relation to problems of hormones as teratogens. 7. Some account is given of the implication of carbohydrate metabolism and terato-genesis. 8. Teratogenesis is possibly related to cationic balance in early development. 9. It is suggested that one factor retarding progress in the understanding of malformations is the tendency towards the development of teratology in an insufficiently close relationship with other branches of cell biology.     

Oxidative stress as a mechanism of teratogenesis

Emerging evidence shows that redox-sensitive signal transduction pathways are critical for developmental processes, including proliferation, differentiation, and apoptosis. As a consequence, teratogens that induce oxidative stress (OS) may induce teratogenesis via the misregulation of these same pathways. Many of these pathways are regulated by cellular thiol redox couples, namely glutathione/glutathione disulfide, thioredoxinred/thioredoinox, and cysteine/cystine. This review outlines oxidative stress as a mechanism of teratogenesis through the disruption of thiol-mediated redox signaling. Due to the ability of many known and suspected teratogens to induce oxidative stress and the many signaling pathways that have redox-sensitive components, further research is warranted to fully understand these mechanisms.     

Maternal toxicity: a possible etiological factor in embryo-fetal deaths and fetal malformations of rodent-rabbit species

Data from animal teratology studies were surveyed to determine whether embryo-fetal mortality and fetal malformations result from a primary action of the agent on the conceptus or if they are secondary to maternal toxicity--a consequence of administration with high dose levels of test chemicals. A fairly strong association between embryo-fetal mortality and maternal toxicity was revealed by analysis of data from hamsters, mice, rats, and rabbits in 234 studies of chemical and physical agents, of which 83 were conducted at both maternotoxic and nonmaternotoxic doses, 94 only at maternotoxic doses, and 49 at nonmaternotoxic doses. In the above studies, only nine chemicals (four each in hamsters and rabbits and one in rats) were reported to induce embryo-fetal deaths at apparently nonmaternotoxic doses. These findings tend to suggest a contributory role for maternal toxicity in the induction of embryo-fetal deaths. The previously reported hypothesis that certain fetal defects in mice may perhaps be caused by maternal toxicity was also found to be true in a review of data on hamsters, rats, and rabbits. Salient maternal toxicity-associated fetal malformations were exencephaly, encephalocele, micro- or anophalmia, and fused ribs in hamsters and defective (fused, missing, or extra) ribs, vertebrae, and sternebrae, ex-, an-, or microphthalmia, and cleft palate in rats and rabbits. These malformations occurred at low frequencies, generally with no readily apparent dose-response relationship. Presumptive evidence indicates that embryo-fetal deaths, and the above-mentioned fetal malformations in experimental animals, which in published literature are presently attributed to chemical induction for a large number of chemicals, may be a consequence of maternal toxicity per se.