Fibulin-1 is required for morphogenesis of neural crest-derived structures

Here we report that mouse embryos homozygous for a gene trap insertion in the fibulin-1 (Fbln1) gene are deficient in Fbln1 and exhibit cardiac ventricular wall thinning and ventricular septal defects with double outlet right ventricle or overriding aorta. Fbln1 nulls also display anomalies of aortic arch arteries, hypoplasia of the thymus and thyroid, underdeveloped skull bones, malformations of cranial nerves and hemorrhagic blood vessels in the head and neck. The spectrum of malformations is consistent with Fbln1 influencing neural crest cell (NCC)-dependent development of these tissues. This is supported by evidence that Fbln1 expression is associated with streams of cranial NCCs migrating adjacent to rhombomeres 2-7 and that Fbln1-deficient embryos display patterning anomalies of NCCs forming cranial nerves IX and X, which derive from rhombomeres 6 and 7. Additionally, Fbln1-deficient embryos show increased apoptosis in areas populated by NCCs derived from rhombomeres 4, 6 and 7. Based on these findings, it is concluded that Fbln1 is required for the directed migration and survival of cranial NCCs contributing to the development of pharyngeal glands, craniofacial skeleton, cranial nerves, aortic arch arteries, cardiac outflow tract and cephalic blood vessels.     

Heparan sulfate expression in the neural crest is essential for mouse cardiogenesis

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1^−/− embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect.     

Importance of genetic predisposition and maternal environment for the occurrence of congenital malformations in offspring of diabetic rats

Previous experimental studies have implicated a genetic component in the induction of malformations in the offspring of diabetic rats. We have compared the outcome of diabetic pregnancy in two outbred (sub)strains of Sprague-Dawley rats (with low incidence [H] and high incidence [U] of skeletal malformations in the offspring) and hybrids between them. The fetuses of diabetic H mothers had no skeletal malformations and the lowest frequency of resorptions (8-9%), regardless of embryo type (H/H or H/U). When the diabetic mother was U or from the hybrid strain (H/U) and the offspring were of the mixed H/U type, we found increased resorption (16-21%) and skeletal malformation (3-5%) rates. If instead the embryos contained a major U genome [either U/U or U/(H/U)], further increased resorptions (23-30%) and skeletal malformations (17-19%) resulted. The H/H and U/U embryonic susceptibility to defined teratogens (3-6 mg/ml D-glucose, 4-8 mM B-hydroxy-butyrate) were compared in whole embryo culture and found to be similar, suggesting that the malformations occurring in vivo may have a different etiology than those found in vitro. In the rat model studied, diabetes in the mother appears to cause a disturbance of early stages of embryogenesis in genetically predisposed embryos. This early disturbance results in skeletal malformations and seems to require inducing factor(s) in addition to increased levels of D-glucose and B-hydroxybutyrate. The findings are in concert with the notion of a mixed genetic-environmental etiology of malformations in (diabetic) pregnancy.     

Fetal cardiac effects of maternal hyperglycemia during pregnancy

Maternal diabetes mellitus is associated with increased teratogenesis, which can occur in pregestational type 1 and type 2 diabetes. Cardiac defects and with neural tube defects are the most common malformations observed in fetuses of pregestational diabetic mothers. The exact mechanism by which diabetes exerts its teratogenic effects and induces embryonic malformations is unclear. Whereas the sequelae of maternal pregestational diabetes, such as modulating insulin levels, altered fat levels, and increased reactive oxygen species, may play a role in fetal damage during diabetic pregnancy, hyperglycemia is thought to be the primary teratogen, causing particularly adverse effects on cardiovascular development. Fetal cardiac defects are associated with raised maternal glycosylated hemoglobin levels and are up to five times more likely in infants of mothers with pregestational diabetes compared with those without diabetes. The resulting anomalies are varied and include transposition of the great arteries, mitral and pulmonary atresia, double outlet of the right ventricle, tetralogy of Fallot, and fetal cardiomyopathy. A wide variety of rodent models have been used to study diabetic teratogenesis. Both genetic and chemically induced models of type 1 and 2 diabetes have been used to examine the effects of hyperglycemia on fetal development. Factors such as genetic background as well as confounding variables such as obesity appear to influence the severity of fetal abnormalities in mice. In this review, we will summarize recent data on fetal cardiac effects from human pregestational diabetic mothers, as well as the most relevant findings in rodent models of diabetic cardiac teratogenesis. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Coronary artery embryogenesis in cardiac defects induced by retinoic acid in mice

BACKGROUND: Although normal coronary artery embryogenesis is well described in the literature, little is known about the development of coronary vessels in abnormal hearts. METHODS: We used an animal model of retinoic acid (RA)-evoked outflow tract malformations (e.g., double outlet right ventricle [DORV], transposition of the great arteries [TGA], and common truncus arteriosus [CTA]) to study the embryogenesis of coronary arteries using endothelial cell markers (anti-PECAM-1 antibodies and Griffonia simplicifolia I (GSI) lectin). These markers were applied to serial sections of staged mouse hearts to demonstrate the location of coronary artery primordia. RESULTS: In malformations with a dextropositioned aorta, the shape of the peritruncal plexus, from which the coronary arteries develop, differed from that of control hearts. This difference in the shape of the early capillary plexus in the control and RA-treated hearts depends on the position of the aorta relative to the pulmonary trunk. In both normal and RA-treated hearts, there are several capillary penetrations to each aortic sinus facing the pulmonary trunk, but eventually only 1 coronary artery establishes patency with 1 aortic sinus. CONCLUSIONS: The abnormal location of the vessel primordia induces defective courses of coronary arteries; creates fistulas, a single coronary artery, and dilated vessel lumens; and leaves certain areas of the heart devoid of coronary artery branches. RA-evoked heart malformations may be a useful model for elucidating abnormal patterns of coronary artery development and may shed some light on the angiogenesis of coronary artery formation.     

Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells

Cardiac and cephalic neural crest cells (NCCs) are essential components of the craniofacial and aortic arch mesenchyme. Genetic disruption of the platelet-derived growth factor receptor alpha (PDGFRalpha) results in defects in multiple tissues in the mouse, including neural crest derivatives contributing to the frontonasal process and the aortic arch. Using chimeric analysis, we show that loss of the receptor in NCCs renders them inefficient at contributing to the cranial mesenchyme. Conditional gene ablation in NCCs results in neonatal lethality because of aortic arch defects and a severely cleft palate. The conotruncal defects are first observed at E11.5 and are consistent with aberrant NCC development in the third, fourth and sixth branchial arches, while the bone malformations present in the frontonasal process and skull coincide with defects of NCCs from the first to third branchial arches. Changes in cell proliferation, migration, or survival were not observed in PDGFRalpha NCC conditional embryos, suggesting that the PDGFRalpha may play a role in a later stage of NCC development. Our results demonstrate that the PDGFRalpha plays an essential, cell-autonomous role in the development of cardiac and cephalic NCCs and provides a model for the study of aberrant NCC development.     

Thickness of renal glomerular capillary basement membrane in the offspring of diabetic rats fed a regular or high-sucrose diet

Numerous animal model studies of diabetes mellitus have been reported. Diabetes-induced vascular damage is a common cause of systemic organ damage in humans and animals. Many investigations have been made of human and animal offspring of diabetic mothers. The present report documents the sequential glomerular basement membrane (GBM) thickness in fetuses and infants of diabetic rats. The postnatal increase in GBM thickness was similar in the offspring of control and diabetic rats, and was not related to the sucrose concentration in the diet.     

Disturbed morphogenesis of cardiac outflow tract and increased rate of aortic arch anomalies in the offspring of diabetic rats

BACKGROUND: Maternal diabetes (MD) is a risk factor for offspring to develop cardiovascular anomalies; this is of growing clinical concern since the number of women in childbearing age with compromised glucose homeostasis is increasing. Hyperglycemia abrogates cardiovascular development in vitro; however, a link to cardiovascular defects in diabetic offspring remains to be investigated. METHODS: We have studied cardiovascular development in offspring of MD rats by examining serial histological sections of GD 12.0-18.0 offspring. Development of pharyngeal arch artery malformations was analyzed and related to intracardiac anomalies. RESULTS: Pharyngeal arch artery and intracardiac defects were present in 27 of 37 MD GD 13.0-18.0 offspring. Early sixth arch arteries showed abrogated arteriogenesis, whereas fourth arch artery defects developed as a result of abnormal remodeling. Morphometrical analysis showed increased apoptosis in regressing artery segments and reduced apoptosis in persisting artery segments. Double outlet right ventricle with infundibular stenosis (tetralogy of Fallot) was predominantly found in combination with sixth artery defects and pulmonary atresia. As confirmed by morphometric analysis and three-dimensional (3D)-reconstructions, outflow tract defects coincided with endocardial cushion hypoplasia. Cases with teratology of Fallot additionally showed a shorter outflow tract. No relation with apoptosis or disturbed neural crest cell migration was found. CONCLUSIONS: Our data uniquely demonstrate mechanistic differences involved in the development of sixth and fourth artery anomalies. Whereas increased apoptosis induces fourth artery anomalies, pulmonary outflow obstruction abrogates sixth artery differentiation independent of apoptosis. The model presented allows analysis of diabetic conditions on cardiovascular development in vivo, essential for elucidating this teratology.     

Maternal vitamin A supplementation in relation to selected birth defects

High doses of vitamin A cause birth defects in animals. Concern over vitamin A teratogenicity in humans has been prompted by reports of teratogenic effects of the vitamin A analogue, isotretinoin. The pattern of defects observed among isotretinoin- and vitamin A-exposed infants and animals suggests a possible mechanism involving cranial neural crest cell activity. Data from a case-control study were used to assess maternal use of vitamin A supplements alone and vitamin A-containing multivitamin supplements in relation to the occurrence of certain birth defects involving structures derived, at least in part, from cranial neural crest cells. Cases were 2,658 infants with such defects (primarily craniofacial and cardiac malformations). Controls were 2,609 infants with other malformations. Vitamin A supplementation was defined as daily use for at least 7 days of retinol alone or with vitamin D, or of fish oils. Information on vitamin A dose and nutrition was not available. The mothers of six controls used vitamin A supplements in each of the first three lunar months of pregnancy in comparison to the mothers of 15, 14, and 10 cases in lunar months 1, 2, and 3, respectively. Relative risk estimates and 95% confidence intervals were 2.5(1.0-6.2) for lunar month 1, 2.3(0.9-5.8) for lunar month 2, and 1.6(0.6-4.5) for lunar month 3. These findings should be considered tentative because no dose information was available, small numbers of cases and controls were exposed to vitamin A supplements, and relative risk estimates were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperation between the PDGF receptors in cardiac neural crest cell migration

Neural crest cells (NCCs) are essential components of the sympathetic nervous system, skin, craniofacial skeleton, and aortic arch. It has been known for many years that perturbation of migration, proliferation, and/or differentiation of these cells leads to birth defects such as cleft palate and persistent truncus arteriosus (PTA). Previously, we had shown that disruption of the platelet-derived growth factor receptor (PDGFR) alpha in NCCs resulted in defects in craniofacial and aortic arch development, the latter with variable penetrance. Because we observed ventricular septal defects in embryos that are null for the PDGFRbeta, we hypothesized that both PDGF receptors are involved in NCC formation. Here, we show that both receptors are expressed in cardiac NCCs and that the combined loss of the PDGFRalpha and PDGFRbeta in NCCs resulted in NCC-related heart abnormalities, including PTA and a ventricular septal defect (VSD). Using NCC lineage tracing, we observed that loss of PDGF receptor signaling resulted in reduced NCCs in the conotruncus region, leading to defects in aortic arch septation. These results indicate that while PDGFRalpha plays a predominant role in NCC development, the PDGFRbeta is expressed by and functions in cardiac NCCs. Combined PDGF receptor signaling is required for sufficient recruitment of cardiac NCCs into the conotruncal region and for formation of the aortico-pulmonary and ventricular septum.     

Antifungal triazole derivative triadimefon induces ectopic maxillary cartilage by altering the morphogenesis of the first branchial arch

BACKGROUND: The triazole derivative, triadimefon (FON), induces branchial arch abnormalities in post-implantation rat embryos cultured in vitro, and cranio-facial malformations in mouse fetuses. Ectopic maxillary cartilage has been also described as a typical FON-related malformation. This work studies the morphogenesis of the ectopic cartilage in rat embryos and fetuses exposed in vivo to FON during the early postimplantation period. METHODS: Pregnant rats were treated with 0, 250, and 500 mg/kg FON on Day 9.5 of pregnancy (D9.5) and sacrificed at term (D20), during the early fetal period (D17) or at different embryogenetic periods (D10, D11, D12). The skeleton was examined after stain of bone and cartilage or of cartilage alone respectively at term or at D17. The neural crest cell (NCC) migration and compaction was investigated at D10 and D11 and the cranial nerve organization described at D12. RESULTS: Triadimefon is teratogenic in rats under the chosen experimental conditions. The malformations were at the level of the cranio-facial and axial skeleton at term and of the hindbrain nerves in embryos. A NCC abnormal migration and compaction was observed at the level of the first branchial arch: in FON-exposed embryos NCC were detected at the level of both maxillary and mandibular processes, whereas control embryos showed the immunostained tissue only at the level of the mandibular bud. CONCLUSIONS: The pathogenic pathway, proposed to explain the ectopic cartilage, is the displacement of part of the NCC-derived tissues at the maxillary region of the first branchial arch.     

Nutrient intakes in women and risks of anophthalmia and microphthalmia in their offspring

BACKGROUND: There is a paucity of information about risk factors for the human eye anomalies anophthalmia and microphthalmia. In this population-based case-control study we investigated whether periconceptional intakes of supplemental folic acid, dietary folate, vitamin A, and several other nutrients were associated with these eye defects. METHODS: This study included data on deliveries that had estimated due dates from 1997-2002 and were part of the National Birth Defects Prevention Study (the National Birth Defects Prevention Study is a population-based case-control study of a wide spectrum of birth defects, incorporating data from 10 birth defects surveillance systems in the United States [Arkansas, California, Georgia/Centers for Disease Control and Prevention, Iowa, Massachusetts, New Jersey, New York, North Carolina, Texas, and Utah]). Cases were those infants or fetuses born with either anophthalmia or microphthalmia. Liveborn infants without major malformations were eligible as controls. Maternal interviews were conducted, primarily by telephone, in English or Spanish. Participation in the interview was 71% among case mothers and 68% among control mothers. Interviews were completed with 89 case mothers and 4,143 control mothers. A shortened version of the food frequency questionnaire from the Nurse's Health Study was used to assess frequency of intake of 58 food items during the year before pregnancy. RESULTS: Our results did not indicate reduced risks for these eye malformations associated with maternal intake of vitamin supplements containing folic acid. The data did not show an association between malformation risk and higher or lower intakes of vitamin A. We also did not observe strong evidence that an abundance or a lack of dietary intake of any other nutrient was associated with increased risk of the studied eye malformations. CONCLUSIONS: Our observations contribute to a limited body of findings on these rare eye defects.     

Developmental Sensitivity to the Induction of Great Vessel Malformations by N‐(2‐Aminoethyl)ethanolamine

N‐(2‐Aminoethyl)ethanolamine (AEEA) induced malformations of the great vessels in the offspring of rats treated during gestation and early lactation (Schneider et al., 2012. Birth Defects Res B Dev Reprod Toxicol [in press]). The aim of this study was to determine if in utero exposure alone was sufficient to induce these malformations or whether a peri‐postnatal exposure or physiological component was required. Three groups of five time‐mated female Wistar Han rats were administered AEEA (250 mg/kg/day) by gavage from gestation day (GD) 6 to GD 19 (groups 1 and 2) or from GD 6 to postnatal day 3 (group 3). Animals were euthanized on GD 21 (group 1) or postnatal day 4 (groups 2 and 3), and the hearts of the offspring were examined for changes to the great vessels. The incidence of malformations in group 1 was 91.1%, and primarily consisted of high aortic arch and abnormal carotid course. One fetus had an aortic aneurysm. All fetuses in groups 2 and 3 were malformed, primarily exhibiting abnormal carotid course and aneurysms, which mainly affected the aorta, ductus arteriosus, and pulmonary trunk. The incidence of high aortic arch was lower relative to group 1. Aneurysms were more prevalent in group 3 compared to group 2. These findings indicate that exposure to AEEA during gestation alone was sufficient to induce malformations of the great vessels and aneurysms, which may be triggered by physiological changes that occur during or after birth, but that the critical period of susceptibility to AEEA‐induced aneurysms in the rat extends beyond gestation into the early postnatal period.     

The Potential of Cr3 [Triaqua‐μ3‐Oxo‐Hexa‐μ‐Propionatotrichromium(III) Chloride] to Reduce Birth Defects in the Offspring of Diabetic CD‐1 Mice

Diabetes mellitus is a growing concern worldwide and leads to multiple complications during pregnancy. Pharmacologic doses of chromium (Cr) have been linked with improving insulin sensitivity and other positive benefits in the treatment of diabetes in animal models. By using streptozotocin induced hyperglycemia in female CD‐1 mice, reproductive outcomes of diabetic and chromium‐dosed diabetic females were examined. After dosing 10 mg/kg Cr in the form of triaqua‐μ₃‐oxo‐hexa‐μ‐propionatotrichromium(III) chloride or Cr3 during gestation days 8–16 (GD8–GD16), all females were sacrificed on gestation day 17 (GD17) and examined for maternal weight gain. The fetuses were examined for gross malformations and for skeletal malformations. The offspring of Cr3‐dosed females tended to have a reduction in the incidence of supernumerary ribs. While hyperglycemia still had negative impacts on the health of dams and their offspring, administration of Cr led to an apparent trend in the reduction in the number of malformations and incidence of supernumerary ribs compared to those of untreated diabetic mothers     

Combined supplementation of folic acid and vitamin E diminishes diabetes-induced embryotoxicity in rats

BACKGROUND: Oxidative stress and enhanced apoptosis may be involved in the induction of embryonic dysmorphogenesis in diabetic pregnancy. Administration of folic acid or vitamin E diminishes embryonic dysmorphogenesis. We aimed to evaluate the effect of combined treatment with folic acid and vitamin E on the disturbed development in embryos of diabetic rats. METHODS: Pregnant nondiabetic and diabetic rats were treated with daily injections of 15 mg/kg folic acid or with 5% vitamin E in the diet. A third group received combined treatment. Day 10 and day 11 embryos were evaluated for development and apoptotic profile. RESULTS: We found increased malformations, resorptions, and profound growth retardation in embryos of diabetic rats compared to control embryos. Vitamin E or folic acid alone, or the 2 compounds combined, normalized embryonic demise. Maternal diabetes caused decreased nuclear factor-kappaB (NF-kappaB) activity and B-cell lymphoma 2 (Bcl-2) protein level, and increased Bcl-2-associated x proteins (Bax) in embryos. Supplementation of vitamin E alone normalized the Bax protein level in a diabetic environment. Administration of folic acid to diabetic rats increased NF-kappaB activity and Bcl-2 protein level. Combined treatment normalized Bcl-2 and Bax protein level in a diabetic environment. CONCLUSIONS: Combined supplementation of folic acid and vitamin E to pregnant diabetic rats diminished diabetes-induced malformations and resorptions, concomitant with normalization of apoptotic protein levels. No treatment completely abolished the embryonic demise; therefore, other mechanisms than oxidative stress and apoptosis are likely to be involved in diabetic embryopathy.     

Maternal diabetes and cardiovascular malformations: predominance of double outlet right ventricle and truncus arteriosus

Most studies on the relationship of maternal diabetes to cardiovascular malformations (CVM) have been prospective investigations of pregnancy outcome and therefore could not identify associations with rare cardiac lesions. The results of a retrospective study shed new light on the risks of specific cardiac defects in diabetic pregnancies. The Baltimore-Washington Infant Study, a population-based case-control investigation of CVM, provides information on maternal diabetes reported in personal interviews. Among 2259 mothers of cases, 35 (1.5%) reported diabetes present before pregnancy (called "overt") and 95 (4.2%) reported diabetes only during pregnancy (called "gestational"). Among 2,801 mothers of controls, 14 (0.5%) had overt diabetes and 83 (3.0%) had gestational diabetes. Malformation-specific risks were expressed as odds ratios (OR) with 99.5% confidence intervals (CI). The strongest associations with overt maternal diabetes were found with double outlet right ventricle (OR 21.33; 99.5% CI 3.34, 136.26), and truncus arteriosus (OR 12.81; 99.5% CI 1.43, 114.64). No significant diagnosis-specific associations were found with gestational diabetes. Non-cardiac malformations were present in 23% of infants with CVM whose mothers had overt diabetes and in 26% of infants with CVM whose mother had gestational diabetes, in 32% of infants with CVM whose mothers did not have diabetes, and in 4% of controls. Double outlet right ventricle and truncus arteriosus are malformations dependent upon neural-crest-cell-derived ectomesenchymal tissues; these are precisely the conotruncal abnormalities that result from experimental ablation of the neural crest in chick embryos. The association with diabetes suggests a further etiologic link between these two lesions.     

Delayed effects of experimental maternal diabetes on plasma cholesterol level and vascular prostacyclin synthesis in the offspring

The effect of streptozotocin-induced diabetes during pregnancy in rats on prostacyclin synthesis in aorta and heart of offsprings was investigated. Although the aortic and heart syntheses of 6-keto-PGF1 alpha in the offsprings of diabetic rats were not altered at birth, a significant rise in aorta and a decrease in the heart were evident at weaning. At weaning, offsprings of diabetic rats also show a significant rise in plasma cholesterol. These studies show that maternal diabetes might cause effects in the offspring which might become evident in later life.     

Malformaciones esqueléticas y alteraciones del crecimiento en fetos de ratas con diabetes moderada

Introduction:

Currently, diabetes mellitus represents one of the medical conditions that more frequently complicates pregnancy affecting the fetus''s growth and development.

Objective:

To determine the skeletal malformations and growth alterations in fetuses of diabetic Wistar rats.

Materials and methods:

We used a neonatally streptozotocin-induced mild diabetes model (STZ 100 mg/kg body weight - subcutaneously) in Wistar rats. In adulthood, healthy and diabetic rats were mated with healthy males of the same age and strain. On day 20 of gestation, a cesarean was performed under anesthesia. Fetuses were removed, weighed, and classified as small (SPA), adequate (APA), and large (LPA) for the gestational age. Selected fetuses were processed for skeletal anomaly and ossification sites analysis.

Results:

In the offspring of diabetic rats, there was a higher percentage of fetuses classified as small or large and a lower percentage of fetuses with adequate weight; the fetal weight mean was lower and there were fewer sites of ossification.Alterations were observed in the ossification of the skull, sternum, spine, ribs and fore and hind limbs; and also, there was a direct correlation between fetal weight and ossification degree There were congenital malformations associated with fusion and bifurcation of the ribs, as well as changes indicative of hydrocephaly, such as the dome shape of the skull, a wide distance between parietals, and the width of the anterior and posterior fontanels.

Conclusion:

Moderate diabetes during pregnancy alters fetal growth and development with macrosomia and intrauterine growth restriction, as well as skeletal malformations.     

Teratogenicity of zinc deficiency in the rat: study of the fetal skeleton

Zinc deficiency (ZD) is teratogenic in rats, and fetal skeletal defects are prominent. This study identifies fetal skeletal malformations that affect calcified and non-calcified bone tissue as a result of gestational zinc deficiency in rats, and it assesses the effect of maternal ZD in fetal bone calcification. Pregnant Sprague-Dawley rats (180-250 g) were fed 1) a control diet (76.4 micrograms Zn/g diet) ad libitum (group C), 2) a zinc-deficient diet (0 microgram/g) ad libitum (group ZD), or 3) the control diet pair-fed to the ZD rats (group PF). On day 21 of gestation, laparotomies were performed. Fetuses were weighed, examined for external malformations, and stained in toto with a double-staining technique for the study of skeletal malformations. Maternal and fetal tissues were used for Zn, Mg, Ca, and P determinations. Gross external malformations were present in 97% of the ZD fetuses. No external malformations were found in fetuses from groups C and PF. Ninety-one percent of cleared ZD fetuses had multiple skeletal malformations, whereas only 3% of the fetuses of group PF had skeletal defects; no skeletal malformations were found in fetuses from group C. Some of the skeletal malformations described in the ZD fetuses, mainly affecting non-calcified bone, were not mentioned in previous reports, thus stressing the importance of using double-staining techniques. Examination of stained fetuses and counting of ossification centers revealed important calcification defects in ZD fetuses. These effects were confirmed by lower Ca and P concentrations in fetal bone with alteration of the Ca:P ratio.     

Oxidative damage in pregnant diabetic rats and their embryos

Free radical mechanisms may be involved in the teratogenesis of diabetes. The contribution of oxidative stress in diabetic complications was investigated from the standpoint of oxidative damage to DNA, lipids, and proteins in the livers and embryos of pregnant diabetic rats. Diabetes was induced prior to pregnancy by the administration of streptozotocin (45 mg/kg). Two groups of diabetic rats were studied, one without any supplementation (D) and another treated during pregnancy with vitamin E (150 mg/d by gavage) (D + E). A control group was also included (C). The percentage of malformations in D rats were 44%, higher than the values observed in C (7%) and D + E (12%) animals. D Group rats showed a higher concentration of thiobarbituric acid reactive substances in the mother's liver, however, treatment with vitamin E decreased this by 58%. The levels of protein carbonyls in the liver of C, D, and D + E groups were similar. The "total levels" of the DNA adducts measured, both in liver and embryos C groups were similar to the D groups. Treatment of D groups with vitamin E reduced the levels by 17% in the liver and by 25% in the embryos. In terms of the "total levels" of DNA adducts, the embryos in diabetic pregnancy appear to be under less oxidative stress when compared with the livers of their mothers. Graziewicz et al. (Free Radical Biology & Medicine, 28:75-83, 1999) suggested "that Fapyadenine is a toxic lesion that moderately arrests DNA synthesis depending on the neighboring nucleotide sequence and interactions with the active site of DNA polymerase." Thus the increased levels of Fapyadenine in the diabetic livers and embryos may similarly arrest DNA polymerase, and in the case of this occurring in the embryos, contribute to the congenital malformations. It is now critical to probe the molecular mechanisms of the oxidative stress-associated development of diabetic congenital malformations.