Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity.

The sedative drug thalidomide ([+]-alpha-phthalimidoglutarimide), once abandoned for causing birth defects in humans, has found new therapeutic license in leprosy and other diseases, with renewed teratological consequences. Although the mechanism of teratogenesis and determinants of risk remain unclear, related teratogenic xenobiotics are bioactivated by embryonic prostaglandin H synthase (PHS) to a free-radical intermediates that produce reactive oxygen species (ROS), which cause oxidative damage to DNA and other cellular macromolecules. Similarly, thalidomide is bioactivated by horseradish peroxidase, and oxidizes DNA and glutathione, indicating free radical-mediated oxidative stress. Furthermore, thalidomide teratogenicity in rabbits is reduced by the PHS inhibitor acetylsalicylic acid, indicating PHS-catalyzed bioactivation. Here, we show in rabbits that thalidomide initiates embryonic DNA oxidation and teratogenicity, both of which are abolished by pre-treatment with the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). In contrast, in mice, a species resistant to thalidomide teratogenicity, thalidomide does not enhance DNA oxidation, even at a dose 300% higher than that used in rabbits, providing insight into an embryonic determinant of species-dependent susceptibility. In addition to their therapeutic implications, these results constitute direct evidence that the teratogenicity of thalidomide may involve free radical-mediated oxidative damage to embryonic cellular macromolecules.

Structural and histochemical features of the avian blood-brain barrier.

We have investigated the structural and histochemical features of the blood-brain barrier (b-bb) in both adults and embryos of chicken (Gallus domesticus, White Leghorn) and quail (Corturnix coturnix japonica). We found that brain endothelial cells of both species are characterized structurally by tight junctions, a low density of pinocytotic vesicles, and a moderately elevated density of mitochondria. Both alkaline phosphatase and butyryl cholinesterase were found in adult quail brain capillaries, but only alkaline phosphatase was found in adult chick brain capillaries. Aromatic amino acid decarboxylase was not found in brain capillaries of either species. In the chick embryo alkaline phosphatase appeared during the time when b-bb matures functionally; i.e., during the third week of development. However, an elevation in mitochondrial density was not apparent until after hatching. In the quail, alkaline phosphatase and butyryl cholinesterase appeared during the last week of embryonic development. By 2 days posthatching the structural characteristics of the brain capillaries were similar to those in the adult.

Phenytoin covalent binding and embryopathy in mouse embryos co-cultured with maternal hepatocytes from mouse, rat, and rabbit.

The anticonvulsant drug phenytoin is teratogenic in a variety of species including humans. Traditional embryo culture studies have employed the addition of 9000 g supernatant (S-9) or microsomal fractions from induced rat or mouse liver as an exogenous bioactivating system to approximate a maternal contribution. However, cellular fractions, unlike cultured intact hepatocytes, may themselves be embryotoxic, and do not reflect the in vivo balance of bioactivation and detoxification. To evaluate in vitro the known in vivo differential species susceptibility to phenytoin teratogenesis, day 9.5 (day of plug = day 1) mouse embryos either were cultured alone for 24 hr or were co-cultured with hepatocytes from maternal mice, rats or male rabbits, thereby exposing the embryos to the effects of potential species-specific phenytoin metabolism. In the absence of hepatocytes, phenytoin embryotoxicity was concentration dependent (0, 10, 20 and 60 micrograms/mL), with decreases in embryonic growth, reflected by reduced yolk sac diameter and crown rump length, apparent within the maternal therapeutic range (20 micrograms/mL). Covalent binding of the radiolabeled drug to live embryonic tissue was significantly higher than in control embryos previously killed by fixation, suggesting that the embryo can bioactivate phenytoin to a toxic reactive intermediate. Mouse embryos grew equally well with hepatocytes from all three species, indicating interspecies tissue compatibility. The addition of rat and rabbit hepatocytes, but not mouse hepatocytes, significantly enhanced the phenytoin-induced impairment of mouse embryonic development, as demonstrated by reductions in somite number. The phenytoin-induced impairment of mouse embryonic growth was not enhanced by the addition of rat or rabbit hepatocytes, while mouse hepatocytes conferred protection. The covalent binding of phenytoin to extracellular proteins in the culture medium was not enhanced by the addition of mouse hepatocytes. These results suggest that mouse embryos intrinsically can bioactivate phenytoin to a toxic reactive intermediate, with embryopathic consequences. The protection conferred by maternal mouse hepatocytes suggests a species-specific maternal biochemical balance favouring detoxification that is not shared by rat and rabbit hepatocytes, which enhanced phenytoin embryopathy. Thus, while phenytoin teratogenicity likely involves embryonic bioactivation, maternal determinants may contribute variably to teratologic susceptibility in a species-specific manner.

Bladder exstrophy: penile lengthening procedure.

A review of bladder exstrophy experience at our institution was undertaken with reference to penile adequacy and intersymphyseal distance. Drift of the symphysis apart even with iliac osteotomy was noted to occur within two years of initial closure but remained stable after three years. This drift can be prevented if the sacral tuberous and sacral spinous ligaments are sectioned. Further, the adequacy of the phallus seems to depend directly on the intersymphyseal distance. Thus, we suggest a variation in the staged approach to bladder exstrophy repair with these facts in mind.

In vitro murine embryotoxicity of cyclophosphamide in embryos co-cultured with maternal hepatocytes: development and application of a murine embryo-hepatocyte co-culture model.

The technique of whole embryo culture provides a sensitive model to evaluate both the effects, and their underlying mechanisms, of drugs and environmental chemicals on embryonic development, independent of maternal influences. However, before teratogenic expression, many teratogens must be enzymatically bioactivated to toxic reactive intermediates. To detect such proteratogens, the embryo culture model may need to be coupled with an exogenous bioactivating system if maternal and/or placental metabolism is involved. We developed a similar embryo-hepatocyte co-culture system using embryos and maternal hepatocytes from mice, which often are more sensitive than rats to chemical teratogens, and which may have a balance of phase II drug metabolising enzymes more similar to humans. This murine system was then used to evaluate the relative maternal and embryonic contributions to cyclophosphamide embryopathy. Day 9.5 (morning of plug = day 1) murine embryos were co-cultured for 24 h in vitro with primary cultures of murine maternal hepatocytes (> 85% viability). Murine embryos were exposed to cyclophosphamide concentrations (0, 7.5, 15, 25 micrograms/ml), similar to those used in rat embryo culture studies. Murine embryos co-cultured with murine maternal hepatocytes developed normally, as did embryos exposed to cyclophosphamide in the absence of hepatocytes. Maternal hepatocytes were necessary for the expression of cyclophosphamide embryotoxicity, which was concentration-dependent, as demonstrated by increasing severity of reductions in crown rump length, yolk sac diameter and somite number. These results show that the co-culture of murine maternal hepatocytes and embryos is feasible, and suggest that maternal bioactivation is required for murine cyclophosphamide embryopathy.

The distribution of cell surface glycoconjugates during mouse secondary neurulation.

During secondary neurulation in the mouse, the neural tube develops from the tail bud by caudal extension of the primary neurocoele. The mesenchymal cells of the tail bud become radially arranged around the neurocoele and undergo a mesenchymal to epithelial transformation to form a neuroepithelium. In order to study the expression of glycoconjugates during the morphogenesis of the secondary neural tube, 14 lectins were applied to serial sections of tail buds at various stages of development. In general, binding was fairly homogeneous during the early stages of tail bud development. However, as development progressed, several lectins became localized to specific structures. The changes were observed to parallel the ongoing development of the secondary neuraxis. sWGA, which is N-acetylglucosamine (GlcNAc) specific, bound mainly to the luminal surface of the secondary neurocoele and to a lesser extent, the notochord. WGA, which has both GlcNAc and sialic acid specificities, showed most intense binding at the luminal and abluminal surfaces of the secondary neurocoele. Binding by the lectin PNA was restricted to the extracellular matrix around the developing secondary neural tube. A comparison of the lectin binding patterns in mouse with those previously reported in chick, demonstrates a less elaborate pattern of lectin binding in murine embryos. This may suggest a less complex expression of glycoconjugates in rodents, in keeping with their comparatively simpler mechanism of secondary neurulation.

N-CAM, polysialic acid and chick tail bud development.

We have previously shown that the binding of the lectin wheat germ agglutinin (WGA) to developing tail buds results in a range of caudal axial defects, which were most likely due to the affinity of the lectin for sialic acid residues. In the present study, we examined the distribution and role of a sialic acid-containing glycoprotein, N-CAM, in chick tail bud development. In the early tail bud, anti N-CAM, staining was found in the medullary cord. However, there was no uptake of an antibody specific to N-CAM containing moderate to long chains of polysialic acid (5A5 monoclonal antibody). At later stages, while N-CAM localized throughout the neural tube, staining with the 5A5 antibody was restricted to the floor plate. Sub-blastodermal injection of the anti N-CAM antibody beneath the tail bud region of HH stages 13-14 embryos produced caudal axial malformations. These malformations included the presence of accessory segments of neural tube and/or notochord, and fusion between the neural tube and underlying segment of notochord. Our results suggest that N-CAM is present during the development of the secondary neuraxis from the tail bud, although the highly sialylated form of this molecule could not be visualized until relatively late stages. N-CAM probably plays a role in the normal course of tail bud development, since perturbation of the molecule with an antibody resulted in malformations. Since these malformations were similar to those we have previously reported when we treated similarly staged chick embryos with WGA, there is a possibility that the sialic acid residues recognized and bound by the lectin are those associated with the N-CAM molecule.

The vertebrate tail bud: three germ layers from one tissue.

The tail bud of amniote embryos comprises a mass of apparently undifferentiated mesenchymal cells located at the caudal limit of the embryo, representing the remains of Hensen's node and the primitive streak. These cells have the potential to give rise to a variety of different tissues including the posterior or 'secondary' neural tube, the tail gut, and somites and their derivatives. This seemingly homogeneous accumulation of cells therefore has the capacity to differentiate into tissues which in more cranial regions of the embryo are derived from cells of different germ layers. In this review, the tissue contributions of the tail bud in various vertebrate classes are discussed, with particular attention to the mesenchymal-to-epithelial transformation that characterizes the process of secondary neurulation, and which distinguishes it from the epithelial folding that occurs during primary neurulation in more cranial regions. Recent studies suggest that the transformation is accompanied by extensive changes in the cell surface oligosaccharide complement of the differentiating cells, and that the sialyted form of N-CAM is expressed both temporally and spatially in a manner that suggests a role for it in the process. The pluripotential nature of the tail bud mesenchyme may be revealed experimentally by grafting the tissue ectopically, or by culturing it on different substrata. In the latter case, the mesenchyme can be demonstrated to give rise to myocytes, chondrocytes, neuroepithelium and neural crest derivatives such as melanocytes, depending on the nature of the culture substratum. It is concluded that the tail bud mesenchyme represents a developing system which is readily amenable to experimentation and should provide insights into the general mechanisms of cell differentiation and transformation.

The effects of cytochalasins on the ultrastructure of neurulating hamster embryos in vivo.

Single intraperitoneal injections of cytochalasin B (CB) in dimethylsulfoxide were given to gravid Syrian hamsters on the eighth day of pregnancy at various dose levels. Exencephaly and encephalocele, the only defects which were seen in the term litters, occurred in dose-response patterns reaching peak frequencies of 14.9% and 53.2%, respectively, at the highest dose level, while accompanied by a mortality of 27.7% of implantations. Although these abnormalities were the same as those resulting from cytochalasin D (CD) treatment at this time, the frequencies were lower and the distribution of defects somewhat different. Morphological comparison of embryos fixed at various times after maternal treatment with 7.0 mg/kg CB or 1.5 mg/kg CD demonstrated qualitatively similar changes in response to either teratogen, leading to failure of the cranial neural folds to approximate and close. The principal ultrastructural changes involved alterations in the topography of the apical membranes of neuroectoderm cells. At doses which produced high frequencies of gross defects in the term litters, no changes were seen in the apical bundles of microfilaments in these cells, although much higher dose levels did disrupt these structures. The results support the hypothesis that the cell membrane is the primary target of these teratogens in vivo.

The pathogenesis of retinoic acid-induced vertebral abnormalities in golden Syrian hamster fetuses.

Administration of single doses of retinoic acid to hamsters on days 7, 8, and 9 of pregnancy resulted in missing, irregular, and abnormally fused centers of ossification in the vertebrae of fetuses recovered near term. A study of early events in embryonic tissues following maternal treatment with 60 mg/kg of the teratogen on day 8 revealed a variety of changes which could be linked to the development of the bony defects. By 12 hours following treatment, the mean number of somites in teratogen-exposed embryos was significantly reduced in comparison to controls. Within 24 hours of maternal treatment, lesions were observed in the aortae of the retinoic acid-exposed embryos. The vessels were consistently damaged caudally with dissection of aortic contents into the adjacent unsegmented mesoderm. Kinking of the neural tube, notochordal irregularities, and a loss of intercellular relationships in the paraxial mesoderm accompanied the vascular lesions. By 36 hours following treatment, abnormalities were evident in the appearance of the caudal somites, and at later stages these appeared to translate into defects in the sclerotomes and subsequently, the vertebrae. The observations suggest that vascular damage plays a significant role in the induction of the vertebral defects by disrupting somitogenesis. Moreover, the results support the hypothesis that retinoic acid produces abnormalities in the vertebral skeleton by a mechanism different from that which has been suggested to operate in the induction of defects in the limb skeleton.

Studies on the silk glands of Calpodes ethlius stoll, (Lepidoptera, hesperiidae).

Each silk gland of Calpodes ethlius consists of five distinct regions: the duct, the green, anterior, middle and posterior regions. Although the gland increases approximately tenfold in length during the larval life, the number of cells remains constant with a concomitant increase in ploidy which is not constant either throughout larval life or in the different regions of the gland. Histochemistry on the glands of the mid-fifth instar larva shows that progressively more mucosubstances are deposited in the lumen, so that while in the distal regions there is only one weakly acidic deposit, this is increased to three more acidic bands in the proximal regions. These bands can be correlated with materials of different electron density. All five regions have characteristic secretory ultrastructure, with prominent secretory vesicles or granules and microvilli. However, the posterior and middle regions have electron-translucent vesicles and relatively short microvilli, while the other three regions have electron dense granules and a more complex, microvillate apical surface. This complexity is greatest in the duct which suggests that it may function in water reabsorption.

Early stages of development in the caudal neural tube of the golden Syrian hamster (Mesocricetus auratus).

Secondary neurulation is the morphogenetic process whereby the caudal segments of the neural tube are derived from cells in the embryonic tail bud. Comparative studies have demonstrated similar characteristics in the mechanism of secondary neurulation among tailless species, which are thought to be due to the evolutionary reduction in tail length (Hughes and Freeman, 1974). In order to explore this hypothesis further, light and scanning electron microscopy was used to study early stages of neurulation in the tail buds of hamster embryos. The golden Syrian hamster is a relatively common laboratory rodent with a reduced tail. In this species, secondary neurulation first became apparent in embryos with approximately 17 pairs of somites. This was well before closure of the posterior neuropore which occurred at the 21-somite stage. The lumen of the neural tube appeared to extend into the tail bud in an even and progressive fashion accompanied by reorientation and rearrangement of tail-bud cells. The mechanism appeared to be similar to that reported in long-tailed rodents.

A comparative study of the effects of retinoic acid given during the critical period for inducing spina bifida in mice and hamsters.

Spina bifida occurred in the offspring of golden Syrian hamsters treated on day 8 of gestation, and CD-1 mice treated on day 9, with 80 mg/kg of retinoic acid. Light microscopic examination of term fetuses demonstrated that myeloschisis was the characteristic form of the defect in mice, whereas myelocystocele was the predominant type of spina bifida induced in the hamster. To investigate the pathogenesis of these defects, a comparative study was undertaken by light microscopy and scanning electron microscopy of the changes occurring in caudal embryonic tissues during the initial 48 hr following maternal treatment. Within 18 hr of exposure, similar effects were observed in both species. These included distortion of the neural folds at the level of the posterior neuropore, vascular damage and hematoma formation, malformation of the notochord, and abnormalities of secondary neurulation. No differences were observed that we thought could account for the apparent variation in the pattern of malformations seen in the term litters. Rather, the dissimilarity may reflect species differences in the position of the posterior neuropore in the neuraxis and in the extent to which secondary neurulation contributes to the development of the lumbosacral cord segments.

Prenatal hamster development following maternal administration of PGE2 at midterm.

The effects of PGE2 on embryonic and fetal development were studied in the golden Syrian hamster. Pregnant hamsters were treated on day 8 of gestation with either 0.2, 0.5, 0.75, or 1.0 mg/kg PGE2 delivered I.P. and the near term litters were compared to those of untreated and vehicle treated controls. Fetuses from the treated litters showed significantly higher frequencies of mortality, lower weights, malformations, and missing ossification centres in comparison to control litters. The results demonstrate that PGE2 is teratogenic in the hamster and the developing neural tube and the fetal skeleton are particularly susceptible to the effects of this teratogen.

Sialoconjugates and development of the tail bud.

Using lectin histochemistry, we have previously shown that there are alterations in the distribution of glycoconjugates in the tail bud of chick embryos that parallel the developmental sequence of the caudal axis. If glycoconjugates or the cells bearing them play a role in caudal axial development, then, restriction of their availability by binding with lectins would be expected to produce abnormalities of caudal development. In the present study, we treated embryos at various stages of tail bud development by microinjection with a variety of lectins. Administration of WGA by sub-blastodermal injection resulted in high incidences of secondary neural tube and notochordal abnormalities in lectin-treated embryos. The incidence of malformations was dependent upon both the dose of WGA received and the stage of development at the time of treatment. Using an anti-WGA antibody, we have also shown binding of the lectin in regions where defects were found. The lectin WGA binds to the sialic acid residues of glycoconjugates and to N-acetylglucosamine. Treatment of embryos with Limulus polyphemus lectin (LPL), which also binds to sialic acid, produced results similar to those of WGA. Treatments using lectins with other sugar-binding specificities, including succinylated WGA (with N-acetylglucosamine specificity only) produced defects that differed from those produced by WGA and LPL, and only with the administration of much higher doses. The results suggest that glycoconjugates in general and sialoconjugates in particular, or the cells carrying them, may have a role in caudal axial development.

Distribution of cell surface glycoconjugates during secondary neurulation in the chick embryo.

Lectin histochemistry was used to examine the expression of cell surface glycoconjugates during secondary neurulation in chick embryos. Fourteen lectins were applied to serial sections of the caudal region of embryos at the various stages of tail bud development. The lectins Bandeiraea simplicifolia, Dolichos biflorus agglutinin, Phaseolus vulgaris leukoagglutinin, soybean agglutinin, Sophora japonica agglutinin, Ulex europaeus agglutinin and succinylated wheat germ agglutinin (sWGA) showed very light or no binding to the developing medullary cord of the tail bud. With the other lectins, staining occurred throughout the early tail bud and solid medullary cord. During cavitation, however, differential expression of cell surface glycoconjugates by different cell populations was observed. The lectins concanavalin A, Lens culinaris agglutinin, Pisum sativum agglutinin, Phaseolus vulgaris erythroagglutinin, Ricinus communis agglutinin and WGA showed basic similarities in the distribution of lectin binding. Of these, the binding pattern of WGA was the most striking. As the medullary cord cells were separating into central mesenchymal and peripheral epithelial populations, WGA bound preferentially to the epithelial cells and the notochord. The lectin PNA, however, became preferentially bound to the mesenchymal cells. Heavy staining by WGA (specific for N-acetylglucosamine and sialic acid) where sWGA staining (specific for N-acetylglucosamine only) was faint suggested that WGA binding was due to the presence of sialic acid containing glycoconjugates.

Effects of retinoic acid on the distribution of glycoconjugates during mouse tail bud development.

Retinoic acid (RA), a potent teratogen of caudal axial development in rodents, has been shown to alter glycoconjugates in a variety of embryonic tissues and teratocarcinomas. In this study, we examined its effects on the expression of cell surface and extracellular matrix glycoconjugates during tail bud development in mouse embryos by using lectin histochemistry. The lectins WGA, sWGA, and PNA showed striking differences in binding between RA-exposed and control embryos. Computer-assisted densitometry revealed a significant increase in binding of all three lectins to the extracellular material of the luminal and abluminal borders of the secondary neural tube and surrounding the notochord in RA-exposed embryos. RA-treated embryos also showed an increased binding affinity for the lectins sWGA and PNA to the cells of the notochord, while WGA showed increased binding to the neuroepithelial cells of the secondary neural tube. The results suggest that RA affects the expression of lectin binding sites during the early development of RA-induced caudal axial defects.