Eya1 and Eya2 gene expression is down-regulated during somitic myogenesis in the cadmium-induced omphalocele chick model.

PURPOSE: The molecular mechanisms underlying omphalocele are still largely unknown. Recently, established cadmium (Cd)-induced omphalocele chick model has been used to investigate the pathogenesis of omphalocele. The earliest histologic changes in this model has been observed in somites 4 hours posttreatment, leading us to hypothesize that disruption of migration of somite-derived cells ventrally may cause omphalocele phenotype. Eyes absent (Eya) genes are expressed in the somite (dermomyotome) and play a key role in somitic myogenesis. We designed this study to investigate the hypothesis that Eya1 and Eya2 gene expression is down-regulated during the critical period of early embryogenesis in the Cd-induced omphalocele chick model. METHODS: After 60 hours of incubation, chicks were exposed to either chick saline or Cd and divided into control and Cd (n = 24 for each group). Chicks were then harvested 1 hour, 4 hours, and 8 hours posttreatment. Real-time quantitative polymerase chain reaction was performed to evaluate gene expression levels of Eya1 and Eya2 in the chick embryo, and they were statistically analyzed. Immunofluorescence confocal microscopy was also performed to evaluate protein expression and distribution pattern of Eya1 and Eya2. RESULTS: At 4 hours posttreatment, the relative messenger RNA expression levels of Eya1 and Eya2 were significantly down-regulated in the Cd group compared with controls (P < .05). The intensity of Eya1 and Eya2 immunofluorescence was also markedly diminished at 4 hours in the Cd-treated embryos, whereas in control embryos, strong intensity of immunofluorescence of them was expressed in the dermomyotomal cells. CONCLUSION: Down-regulation of Eya genes during the critical period of early embryogenesis may contribute to omphalocele phenotype in the Cd chick model, interfering with migration of embryonic body wall ventrally.

EphB2/B3 gene expression is down-regulated during early embryogenesis in the cadmium-induced omphalocele chick model.

PURPOSE: In the chick embryo, the administration of cadmium (Cd) induces omphalocele phenotype. The earliest histologic change in this model is observed in the somite 4 hours (H) post treatment, postulating that disruption of somite development in embryogenesis may cause omphalocele phenotype. EphB2 and EphB3 are involved in many embryonic developmental processes, including somitogenesis. EphB2(-/-)EphB3(-/-) double knockouts display omphalocele phenotype. We hypothesized that EphB2/B3 genes are down-regulated in the Cd chick model during the critical period of embryogenesis. METHODS: After 60H incubation, chicks were harvested 1H, 4H, and 8H post treatment with saline or Cd and divided into control and Cd groups. Reverse transcriptase-polymerase chain reaction was performed to evaluate gene expression levels of EphB2/B3. Immunofluorescence confocal microscopy was performed to evaluate protein expression/distribution of EphB2/B3. RESULTS: At 4H post treatment, the messenger RNA expression levels of EphB2/B3 were significantly down-regulated in the Cd group compared with controls (P < .05). The intensity of EphB2/B3 immunofluorescence was markedly diminished at 4H in the Cd-treated embryos, whereas strong immunoreactivity was observed in the somite in controls. CONCLUSION: Downregulation of EphB2/B3 during the narrow window of early embryogenesis may interfere with normal somitogenesis, preventing migration of embryonic body wall ventrally and thus causing omphalocele.

Presenilin genes are downregulated during somitogenesis in the cadmium-induced omphalocele chick model.

PURPOSE: Although the precise pathogenesis of ventral body wall (VBW) defects is not clearly understood, it has recently postulated that disruption of somite development during early embryogenesis may cause failure of proper VBW formation. The administration of cadmium (Cd) after 60 h of incubation induces omphalocele spectrum in the chick embryo. Previous studies have shown that one of the earliest histological changes seen in this model is abnormal cell death in the somite, occurring at 4 h post treatment (4H). However, the molecular mechanism by which Cd acts in this critical period of embryogenesis still remains unclear. Presenilins are expressed in somites and play an important role in vertebrate development, including somitogenesis and thus VBW formation. We designed this study to test the hypothesis that gene expression levels of presenilin 1 (PSEN1) and presenilin 2 (PSEN2) are downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele in the chick model. METHODS: After 60 h of incubation, chick embryos were exposed to either saline or 50 µM cadmium and divided into two groups: control and Cd (n = 8 at each time point for each group). Real-time RT-PCR was performed to evaluate the relative mRNA expression levels of PSEN1 and PSEN2 in the Cd-induced omphalocele chick model. Differences between two groups at each time point were analysed statistically and the significance was accepted at p < 0.05. Immunofluorescence confocal microscopy was performed to evaluate the protein expression/distribution of presenilins in the somite of chick embryo. RESULTS: The relative mRNA expression levels of PSEN1 and PSEN2 were significantly downregulated in the Cd group at 4H compared with controls (p < 0.005) (Table). However, there were no significant differences at the other time points. At 4H, immunofluorescence of presenilins (green) was markedly diminished in the Cd-treated embryos, whereas strong immunofluorescence of them was seen in the somite (dermomyotome) in controls (Fig. 1). 1 Immunofluorescence Confocal Microscopy for PSEN1 and PSEN2 in the dermomyotome of the somite in the trunk level of chick embryo 4H post treatment. Intensity of PSEN1 immunofluorescence (green) was markedly diminished in Cd-treated embryos, whereas strong PSEN1 expression was seen in the dermomyotome in controls. PSEN2 immunofluorescence was also decreased in the Cd-treated embryos, whereas strong PSEN2 immunofluorescence (green) was observed predominantly in the dermomyotome in controls. Immunofluorescence in orange is DNA counter staining by DAPI CONCLUSION: We provide evidence, for the first time, that gene expression of presenilins is downregulated during the narrow window of very early embryogenesis in the Cd chick model. Decreased expression of presenilins may contribute to omphalocele phenotype in Cd chick model, by disrupting somite development.

Alteration of gene expression of IQGAP1 and Rho-family GTPases in the cadmium-induced ventral body wall defects in the chick model.

Cadmium (Cd) induces ventral body wall defects (VBWD) in the chick embryo, with adherens junctions (AJs) breakdown at 4h post treatment (4H). Signalling by which Cd disrupts AJs in this model remains unclear. IQGAP1 regulates AJs via binding to Cdc42 and Rac1, Rho-family GTPases. Activation of IQGAP1-Cdc42 interaction regulates AJs positively, whereas Rac1 activation inhibits AJs. We hypothesised that IQGAP1 and Cdc42 are downregulated and Rac1 is upregulated during embryogenesis in the Cd chick model. Chick embryos were explanted and treated with saline or Cd after 60 h incubation. Chicks were harvested at 1H, 4H and 8H post treatment and RT-PCR and immunohistochemistry were performed. Gene expression levels of IQGAP1 and Cdc42 were significantly downregulated and Rac1 was upregulated in Cd group compared to controls only at 4H. Immunoreactivity of IQGAP1 and Cdc42 was also markedly decreased, whereas Rac1 was increased in Cd group compared to controls at 4H. Alteration of IQGAP and Rho-family GTPases may cause VBWD in Cd chick model by inducing the dissociation of cadherin-mediated AJs.

Epigenetic effect of cadmium on global de novo DNA hypomethylation in the cadmium-induced ventral body wall defect (VBWD) in the chick model.

Administration of the heavy metal cadmium (Cd) induces ventral body wall defects (VBWD) in the chick embryo. In this model, the expression of most genes involved in body wall formation is altered 4h-posttreatment. However, the mechanism by which Cd results in the initiation of altered gene expression remains unclear. Epigenetic mechanisms can change genome function under exogenous influences. Moreover, Cd is one of the environmental factors that can affect epigenomic programming. De novo DNA methylation is essential for normal embryogenesis and is regulated by the DNA methyltransferases (DNMT)3A and DNMT3B. The objective of this study was to investigate the hypothesis that gene expression levels of DNMT3A/3B were altered, resulting in global DNA methylation changes during the critical period of embryogenesis in the Cd chick model. After 60-h incubation, chick embryos (n = 48) were harvested at 1, 4, and 8 h after treatment with saline or Cd, and divided into controls and Cd groups. Quantitative reverse transcription PCR was performed to evaluate the gene expression levels of DNMT3A/3B in the chick embryos and was statistically analyzed using Student's t-test. Immunohistochemistry was performed using a monoclonal antibody against 5-methylcytidine (5'MeC), which labels methyl-rich regions within the nucleus. DNMT3A/3B gene expression levels at 4 h were significantly downregulated in the Cd group compared with controls (p < 0.005/p < 0.00001, respectively). Immunoreactivity of 5'MeC was markedly diminished in the Cd group at 4 h. Our findings demonstrates for the first time that Cd impacts on the expression levels of DNMT3A/3B, which may underlie the pathogenesis of VBWD in the Cd chick model.

BMPR1A-mediated BMP1 signalling is disrupted in the cadmium-induced omphalocele in the chick model.

PURPOSE: In the chick embryo, administration of cadmium (Cd) induces omphalocele phenotype. The earliest histological changes in this model are observed commencing at 4-h post treatment (4H). The molecular mechanisms by which Cd acts during the critical period of embryogenesis remain unclear. Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) regulate many fundamental biological processes and are involved in various embryonic morphogenesis, including ventral body wall (VBW) formation. Homozygous BMP1 mutant mice cause VBW defects. It has been reported that BMPR1A conditional knockouts also exhibit omphalocele phenotype. We designed this study to test the hypothesis that gene expression levels of BMP1 and BMPR1A are downregulated during the critical period of embryogenesis in the Cd chick model. METHODS: After 60-h incubation, chick embryos were exposed to either Cd or saline and then harvested 1H, 4H and 8H. Chicks were divided into control (n = 24) and Cd (n = 24). RT-PCR was performed and differences between the two groups were tested statistically (significance was accepted at p < 0.05). Immunohistochemical study was also performed to evaluate those proteins expression/distribution. RESULTS: The gene expression levels of BMP1 and BMPR1A at 4H were significantly downregulated in Cd group compared to controls. Immunoreactivity of BMP1 and BMPR1A was also markedly decreased in Cd-treated embryos compared to controls. CONCLUSION: Disruption of BMPR1A-mediated BMP1 signalling during the narrow window of early embryogenesis may interfere with normal VBW formation, causing omphalocele phenotype in the Cd chick model.

Altered PITX2 and LEF1 gene expression in the cadmium-induced omphalocele in the chick model.

PURPOSE: Although, recent studies have suggested that disruption of somitogenesis may be involved in ventral body wall (VBW) defects; the molecular mechanisms of VBW defects remain unclear. In the chick embryo, the administration of cadmium (Cd) induces VBW defects similar to the human omphalocele. In this model, the earliest histological change in the somite occurs commencing at 4 h post-Cd treatment (4 h). PITX2 is expressed in somites, and PITX2 mutants have been shown to display VBW defects. PITX2 interacts with lymphoid enhancer factor-1 (LEF1) to regulate somite myogenesis. We designed this study to investigate the hypothesis that PITX2 and LEF1 genes are downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele chick model. MATERIALS AND METHODS: Chick embryos were exposed to Cd or saline after 60 h incubation and harvested at 1, 4, and 8 h posttreatment. Chicks were then divided into two groups: control (n = 24), and Cd (n = 24). RT-PCR was performed and analyzed statistically (significant difference was accepted at p < 0.05). Immunohistochemistry was also performed to evaluate expression/distribution of those proteins. RESULTS: The mRNA expression levels of PITX2 and LEF1 at 4 h were significantly decreased in the Cd group compared with controls, whereas there were no differences at the other time points. Immunoreactivity of those proteins at 4 h was also markedly decreased in somites in the Cd-treated embryos compared with controls. CONCLUSIONS: Downregulation of PITX2 and LEF1 genes may interfere with ventral body wall formation in Cd chick model causing omphalocele by disrupting somite myogenesis.

Pre-treatment with N-acetylcysteine upregulates superoxide dismutase 2 and catalase genes in cadmium-induced oxidative stress in the chick omphalocele model.

PURPOSE: In the chick embryo, administration of the heavy metal Cadmium (Cd) induces omphalocele phenotype. Cd is a potent inhibitor of antioxidant enzymes and causes accumulation of reactive oxygen species (ROSs) such as hydrogen peroxide. Previous work with the Cd chick model has demonstrated that increased levels of MDA, as a marker for oxidative stress, 24 h post Cd treatment (24H) are identical in chick embryos exposed to Cd. Furthermore, of the several antioxidants assessed, only N-acetylcysteine (NAC) has been shown to reduce MDA levels to control values in the Cd-treated chick embryo. However, the molecular mechanisms by which NAC acts to maintain oxidative stress in the Cd-induced ventral body wall defect chick model remains to be unclear. We designed this study to investigate the hypothesis that gene expression levels of antioxidant enzymes are downregulated in malformed embryos exposed to Cd compared to controls and to determine the effect of pre-treatment with NAC on the expression levels of genes encoding antioxidant enzymes. METHODS: After 60 h incubation, chick embryos were pre-treated with NAC and exposed to either chick saline or Cd. Chicks were then harvested at 24H and divided into five groups: control, Cd group without malformation [Cd(-)], Cd group with malformation [Cd(+)], NAC + Cd(-) and NAC + Cd(+). Real-time PCR was performed to evaluate the relative mRNA expression levels of antioxidant enzymes, including superoxide dismutase (SOD)-1, SOD2, catalase (CAT) and glutathione peroxidase (GPX)-4. Differences between five groups were tested by Tukey-Kramer post-hoc test following one-way ANOVA. Statistical significance was accepted at p < 0.05. Immunohistochemistry was also performed to evaluate protein expression. RESULTS: The mRNA expression levels of SOD2 and CAT were significantly decreased in Cd(+) as compared to controls, whereas there was no significant difference between controls and Cd(-) (p < 0.05 vs. controls). In addition, gene expression levels of SOD2 and CAT were significantly increased in NAC + Cd(-) as compared to Cd(+) and NAC + Cd(+) (p < 0.05 vs. controls). However, there were no significant differences in the expression levels of SOD1 and GPX4 among any groups. Increased immunoreactivity of SOD2 and CAT was also observed in NAC + Cd(-) as compared to Cd(+) and NAC + Cd(+). CONCLUSION: Our results suggest that SOD2 and CAT may play an important role in preventing Cd-induced teratogenesis. Prenatal treatment with drugs which can upregulate SOD2 and CAT transcripts may have a therapeutic potential in preventing omphalocele phenotype.

Disruption of GLI3-ZIC3 interaction in the cadmium-induced omphalocele chick model.

PURPOSE: Administration of heavy metal cadmium (Cd) after 60-h incubation induces omphalocele spectrum in the chick embryo. Although previous studies have shown that the earliest detectable histological changes in the chick Cd model occurs commencing at 4-h post-treatment (4H). However, the molecular mechanism by which Cd acts in the critical period of early embryogenesis still remains unclear. Zic3, a Gli superfamily transcription factor, is expressed in somites and plays an important role in vertebrate development, including somitogenesis and thus ventral body wall formation. Gli3 is also expressed in somites and interacts with Zic3 physically and functionally. It has been reported that Gli3 homozygous double mutants display omphalocele. Zic3 mutant mice have also been known to result in omphalocele phenotype. We designed this study to test the hypothesis that Gli3 and Zic3 gene expression is downregulated during the critical period of very early embryogenesis in the Cd-induced omphalocele in the chick model. METHODS: After 60-h incubation, chick embryos were exposed to either saline or 50 µM cadmium and divided into two groups: control and Cd (n = 24 for each group). Real-time reverse transcription polymerase chain reaction was performed to evaluate the relative mRNA expression levels of Gli3 and Zic3 in the Cd-induced omphalocele chick model. Differences between the two groups at each time point were analyzed statistically and the significance was accepted at p < 0.05. Immunohistochemistry was also performed to evaluate the expression/distribution of those proteins in chick embryo. RESULTS: The relative mRNA expression level of Gli3 and Zic3 was significantly decreased in the Cd group at 4H when compared with controls (p < 0.05). However, there were no significant differences at the other time points. At 4H, immunoreactivity of GLI3 and ZIC3 was also markedly decreased in Cd-treated embryos, whereas strong expression of them was seen in the somite in controls. CONCLUSION: We provide evidence, for the first time, that Gli3 and Zic3 gene expression is downregulated during the narrow window of very early embryogenesis in Cd chick model. Disruption of Gli3-Zic3 interaction in the critical period for ventral body wall formation may contribute to omphalocele phenotype in Cd chick model.

Disruption of noncanonical Wnt/CA2+ pathway in the cadmium-induced omphalocele in the chick model.

PURPOSE: Cadmium (Cd) has been found to cause ventral body wall defects (VBWDs) in the chick embryo similar to human omphalocele. The earliest detectable histologic changes in Cd-induced VBWD chick model have been observed 4 hours posttreatment. The exact mechanism by which Cd acts in the early embryogenesis remains unclear. Wnt proteins play a key role during embryogenesis, and altered Wnt signaling has been linked to developmental defects. Noncanonical Wnt/Ca(2+) pathway has been implicated in regulating embryogenesis by controlling cell movement and adhesion. Wnt11 can activate protein kinase C (PKC) and calcium/calmodulin-dependent kinase II (CaMKII) in the Wnt/Ca(2+) pathway. We hypothesized that the Wnt11, PKCalpha, and CaMKII gene expression is downregulated in the Cd-induced VBWD during early embryogenesis. METHODS: After 60 hours of incubation, chick embryos were harvested 1 hour (1H), 4H, and 8H after treatment of saline or cadmium and divided into 2 groups: control and Cd (n = 8 at each time-point, respectively). Real-time polymerase chain reaction was performed to evaluate the messenger RNA (mRNA) expression of Wnt11, PKCalpha, and CaMKII in the Cd-induced VBWD chick model. RESULTS: The mRNA expression levels of Wnt11, PKCalpha, and CaMKII were significantly decreased at 1H in Cd group compared to controls (P < .05). However, there were no significant differences in the other time-points. CONCLUSION: Downregulation of Wnt11, PKCalpha, and CaMKII gene expression during the narrow window of early embryogenesis may cause VBWD, interfering with cell movement and adhesion, disrupting Wnt/Ca(2+) pathway.

HoxB2, HoxB4 and Alx4 genes are downregulated in the cadmium-induced omphalocele in the chick model.

PURPOSE: In the chick embryo, administration of cadmium (Cd) induces omphalocele phenotype. HoxB2 and HoxB4, expressed in cell types that contribute to ventral body wall (VBW) formation, act together to mediate proper closure of the VBW, involving a key downstream transcription factor, Alx4. HoxB2 and HoxB4 knockout mice display VBW defects with specific downregulation of Alx4 gene expression, while homozygous Alx4 knockouts show omphalocele phenotype. Although the earliest histological changes in the Cd chick model occur commencing at 4H post treatment, the exact timing and molecular mechanism by which Cd acts is still unclear. We hypothesized that HoxB2, HoxB4 and Alx4 genes are downregulated during the critical timing of very early embryogenesis in the Cd-induced omphalocele chick model. METHODS: After 60H incubation, chick embryos were harvested at 1H, 4H and 8H after treatment with saline or Cd and divided into controls and Cd group (n = 24 for each group). RT-PCR was performed to investigate the gene expression of HoxB2, HoxB4 and Alx4 and statistically analyzed (significance was accepted at p < 0.05). Immunohistochemical staining was also performed to evaluate the protein expression/distribution of HoxB2, HoxB4 and Alx4 in the chick embryo. RESULTS: The expression levels of HoxB2, HoxB4 and Alx4 gene at 4H were significantly downregulated in the Cd group as compared to controls, whereas there were no significant differences at the other time points. Immunoreactivity of HoxB2, HoxB4 and Alx4 at 4H is also markedly decreased in the ectoderm and the dermomyotome in the Cd chick model as compared to controls. CONCLUSION: Downregulation of HoxB2, HoxB4 and Alx4 expression during the narrow window of early embryogenesis may cause omphalocele in the Cd chick model by interfering with molecular signaling required for proper VBW formation. Furthermore, these results support the concept that HoxB2, HoxB4 and Alx4 genes work together to mediate proper VBW formation.

Msx1 and Msx2 gene expression is downregulated in the cadmium-induced omphalocele in the chick model.

PURPOSE: The administration of cadmium (Cd) induces an omphalocele phenotype in the chick embryo. The molecular mechanism by which Cd acts still remains unclear. Msx1 and Msx2 are expressed in the developing body wall and regulate cellular proliferation and differentiation. It has been reported that Msx1/Msx2 double-mutant mice display an omphalocele phenotype. We hypothesized that gene expression levels of Msx1 and Msx2 are downregulated in the Cd chick model during the critical period of embryogenesis. METHODS: After 60 hours of incubation, chick embryos were exposed to either Cd or saline and harvested at 1 hour (1H), 4H, and 8H after treatment. Chicks were divided into 2 groups: control and Cd (n = 8 for each group at each time-point). Real-time polymerase chain reaction was performed to evaluate the messenger RNA levels of Msx1 and Msx2 in the Cd-induced omphalocele chick model and analyzed statistically. Immunohistochemistry was also performed to examine protein expression of Msx1 and Msx2 at each time-point. RESULTS: Messenger RNA expression levels of Msx1 and Msx2 at 1H were significantly decreased in the Cd group compared with controls (P < .01), whereas there were no significant differences at the other time-points. Immunoreactivity of Msx1 and Msx2 at 1H was remarkably decreased in Cd group compared with controls. CONCLUSION: Downregulation of Msx1 and Msx2 gene expression during the narrow window of early embryogenesis may cause an omphalocele by disrupting cellular proliferation and differentiation in the developing body wall.

The role of transforming growth factor-beta 2 and 3 in formation of ventral body wall in the cadmium-induced omphalocele chick model.

PURPOSE: In the chick embryo, the administration of cadmium (Cd) induces ventral body wall defects (VBWDs) similar to the human omphalocele. Transforming growth factors beta (TGFs-beta) are involved in many developmental processes, including ventral body wall formation. The Tgfbeta2(-/-) Tgfbeta3(-/-) double knockout mice and Tgfbeta2(-/-) Tgfbeta3(+/-) mutants show VBWD, whereas Tgfbeta2(+/-) Tgfbeta3(-/-) mutants display normal ventral body wall fusion. We designed this study to investigate the messenger RNA (mRNA) levels of TGF-beta2 and TGF-beta3 in the Cd-induced omphalocele chick model during early embryogenesis. METHODS: Chick embryos were exposed to either Cd or saline, harvested 1 hour (1H), 4H, and 8H after treatment and then divided into 2 groups: control and Cd (n = 8 at each time-point, respectively). Reverse transcription polymerase chain reaction was performed to evaluate the mRNA levels of TGF-beta2 and TGF-beta3 and statistically analyzed. RESULTS: The mRNA expression levels of TGF-beta2 at 1H were significantly decreased in the Cd group compared to controls (P < .05). However, the levels of TGF-beta3 were not altered at all the time-points studied. CONCLUSION: We provide evidence, for the first time, that TGF-beta2 gene expression is downregulated during a narrow window of early embryogenesis in the Cd chick model. Our data show that TGF-beta2 is the key gene involved in the formation of ventral body wall.

Disruption of calreticulin-mediated cellular adhesion signaling in the cadmium-induced omphalocele in the chick model.

PURPOSE: Administration of cadmium (Cd) causes omphalocele in the chick embryo. The earliest histological changes in the chick Cd model are the breakdown of adherens junctions (AJs). Calreticulin (CRT) plays a key role in Ca(2+) signaling and cell adhesion. Ca(2+) signaling in the Cd chick model is known to be altered. The calcium-dependent adhesion molecule, E-cadherin, and its associate, beta-catenin, are key components of AJs regulated by CRT. CRT knockouts display omphalocele. We hypothesized that CRT, E-cadherin and beta-catenin are downregulated during early embryogenesis in the Cd chick model. METHODS: After 60 h (H) incubation, chicks were harvested 1H, 4H, and 8H post treatment with saline or Cd and divided into controls and Cd. RT-PCR was performed to evaluate mRNA levels of CRT, E-cadherin and beta-catenin in the Cd chick model. RESULTS: The mRNA levels of CRT were significantly decreased in the Cd group at 1H compared to controls (p < 0.05). The mRNA levels of E-cadherin and beta-catenin were significantly decreased at 4H in the Cd group compared to controls (p < 0.05). There were no significant differences at 8H. CONCLUSION: Downregulation of CRT, E-cadherin and beta-catenin genes may cause omphalocele in the Cd chick model by disrupting CRT-mediated Ca(2+) signaling and AJs.

Evidence against a direct role for oxidative stress in cadmium-induced axial malformation in the chick embryo.

Cadmium (Cd) is a powerful inducer of oxidative stress. It also causes ventral body wall defects in chick embryos treated at Hamburger-Hamilton stages 16-17. By measuring malondialdehyde levels (TBARS method) and cotreating with antioxidants (tempol, ascorbate, and N-acetylcysteine), we sought to determine if oxidative stress were directly related to teratogenesis. We also investigated the expression of mRNAs for antioxidant enzymes superoxide dismutase (SOD) -1 and -2, catalase (CAT), and glutathione peroxidase (GPx). RT-PCR showed reductions in SOD-1, SOD-2, and CAT 1 hour after treatment with Cd. MDA levels increased 4 hours after Cd, and remained elevated 24 hours after treatment. Of the antioxidants, only N-acetylcysteine reduced MDA levels to control values. Nonetheless, no antioxidant could reduce embryo lethality or malformation rates. Furthermore, MDA levels 24 hours after treatment were identical in malformed and normal embryos exposed to Cd. Hence, we conclude that oxidative stress may not have a direct role in Cd teratogenesis.

Cadmium teratogenesis in the chick: period of vulnerability using the early chick culture method, and prevention by divalent cations.

Cadmium (Cd) is teratogenic in chick embryos following treatment in ovo or in shell-less culture. We investigated the ability of other divalent cations (Mn, Ni, Se, Mg and Ca) to influence the effects of Cd. As the proposed mechanism of protection of these ions is prevention of Cd influx by blocking or competing for Ca channels, we also assessed verapamil, a Ca-channel blocker. We used a new, completely ex ovo method, explanting the embryos onto an agar-albumen substrate (0.6% agar diluted 1:1 with thin albumen) to which test substances were added. Following 48-96 h incubation, chicks were explanted onto medium containing 7.5 microM Cd acetate or equimolar sodium acetate. Morphology and somite numbers were assessed at explantation, and again following 24h incubation on the culture media. In addition, 60-h embryos were explanted onto media containing various concentrations of the aforementioned agents, alone or in combination with 7.5 microM Cd. Chicks were vulnerable to Cd teratogenesis between Hamburger-Hamilton stages 13 and 18. Co-treatment with Se, Mn and Ni prevented malformation at 2x, 50 x and 100 x the molar dose of Cd, respectively. Ca, Mg and verapamil failed to protect. These results indicate that some, but not all, divalent cations protect against Cd malformation, but the mechanism of rescue remains unresolved.

Downregulation of ROCK-I and ROCK-II gene expression in the cadmium-induced ventral body wall defect chick model.

PURPOSE: In the chick embryo, administration of the heavy metal cadmium (Cd) after 60 h incubation induces the ventral body wall defect (VBW) with similarities to the human omphalocele. Rho-associated coiled-coil-containing protein kinase (ROCK) I and ROCK-II mediate signalling from Rho to the actin cytoskeleton in the Wnt non-canonical pathway. ROCK-I knockout (KO), ROCK-II KO, and ROCK-I/ROCK-II double heterozygous mice have been shown to cause failure of closure of the VBW. The exact mechanism by which Cd acts in the Wnt signalling pathway still remains unclear. We designed this study to test the hypothesis, that the gene expression levels of ROCK-I and ROCK-II are downregulated during the critical period of embryogenesis in the Cd-induced VBW defect chick model. METHODS: Chick embryos were harvested 1 h (1H), 4 h (4H), and 8 h (8H) after treatment of cadmium and divided into two groups: control (n = 8 at each time point), and Cd (n = 8 at each time point). Real-time RT-PCR was performed to evaluate the relative mRNA levels of ROCK-I and ROCK-II expression in the Cd-induced VBW defect chick model. Differences between the two groups at each time point were tested by using Mann-Whitney's U test and statistical significance was accepted at P < 0.05. RESULTS: The relative mRNA levels of ROCK-I and ROCK-II at 4H were significantly decreased in Cd group compared to controls (P < 0.01 and P < 0.001, respectively). The expression levels of ROCK-I and ROCK-II at 1H and 8H were not significantly different between Cd group and controls. CONCLUSIONS: Our results provide evidence, for the first time, that the gene expression levels of ROCK-I and ROCK-II are significantly downregulated at 4 h after treatment of Cd in the VBW defect model of chick embryo. We speculate that the downregulation of ROCK-I and ROCK-II gene expressions during this narrow window of embryogenesis may cause VBW defect by disrupting Wnt non-canonical pathway.

Adriamycin produces a reproducible teratogenic model of gastrointestinal atresia in the mouse.

Gastrointestinal atresia is a major cause of bowel obstruction in the newborn. Experimental models and clinical observations have demonstrated the heterogeneous nature of its pathogenesis. A proportion is due to late intra-uterine vascular insults and some are genetic in nature. Epidemiological studies have found gastrointestinal atresia to occur with other birth defects, in particular VACTERL anomalies, suggesting that a subset of cases may result from an early disturbance to intestinal morphogenesis. Adriamycin is teratogenic in rats, producing gastrointestinal atresia and VACTERL anomalies. The mouse is the foremost mammal studied by developmental biologists, offering an expanding wealth of knowledge and scientific research techniques. The aim of this study was to create an Adriamycin mouse model for investigating the development of gastrointestinal atresia. CBA/Ca mice were accurately time-mated (n = 30). Four different doses of Adriamycin (0-saline control, 4, 5 and 6 mg/kg) at three different timings of injections were compared (12 groups). Dams received two intraperitoneal injections, 24 h apart, commencing on day 7, 7.5 or 8. Foetuses were harvested on day 18. Gastrointestinal atresia and VACTERL anomalies were examined using a dissecting microscope. Adriamycin produced type IIIa gastrointestinal atresia in six treatment groups. The effect of Adriamycin depended on the timing and dose of the injections. VACTERL anomalies were only found in four treatment groups, proposing overlapping critical embryological windows for these malformations. Gastrointestinal atresia can be induced by the teratogen Adriamycin, occurring with and without VACTERL anomalies. This produces a reproducible mouse model in which the molecular pathogenesis of gastrointestinal atresia may be studied.

Prenatal treatment with retinoic acid promotes pulmonary alveologenesis in the nitrofen model of congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Severe pulmonary hypoplasia remains the main cause of the high mortality in newborn infants with congenital diaphragmatic hernia (CDH). Retinoids are a family of molecules derived from vitamin A, which play an important role in lung development. We hypothesized that retinoids promote alveologenesis at the end of gestation and therefore designed this study to investigate the effects of retinoid acid on nitrofen-induced hypoplastic lungs in CDH. METHODS: Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9 of gestation. Retinoic acid 5 mg/kg was given intraperitoneally on days 18, 19, and 20 of gestation and fetuses were recovered on day 21. We had 4 study groups: control (n = 24), control + retinoic acid (n = 22), CDH (n = 24), and CDH + retinoic acid (n = 19). Lungs from the 4 study groups were fixed, and the following stereological measurements were performed on vertical random sections: total lung volume, volume density of airspaces, volume density of air walls, gas exchange surface area, alveolar volume, and total number of alveoli per lung. Total DNA content of each lung was measured using a spectrophotometer. RESULTS: Total lung volume increased in CDH lungs after the addition of retinoic acid but remained the same in the control group. Gas exchange surface area was larger in CDH lungs after the addition of retinoic acid but remained unchanged in the control group. The total number of alveoli per lung was higher after the addition of retinoic acid. Total DNA content as well as total DNA content-lung weight ratio of the left lung increased significantly in the CDH group after the addition of retinoic acid compared with CDH without retinoic acid. CONCLUSIONS: Our results demonstrate that prenatal treatment with retinoic acid stimulates alveologenesis in hypoplastic lungs in CDH.

Cadmium: toxic effects on the reproductive system and the embryo.

The heavy metal cadmium (Cd) is a pollutant associated with several modern industrial processes. Cd is absorbed in significant quantities from cigarette smoke, and is known to have numerous undesirable effects on health in both experimental animals and humans, targeting the kidneys, liver and vascular systems in particular. However, a wide spectrum of deleterious effects on the reproductive tissues and the developing embryo has also been described. In the testis, changes due to disruption of the blood-testis barrier and oxidative stress have been noted, with onset of widespread necrosis at higher dosage exposures. Incorporation of Cd into the chromatin of the developing gamete has also been demonstrated. Ovarian Cd concentration increases with age, and has been associated with failure of progression of oocyte development from primary to secondary stage, and failure to ovulate. A further mechanism by which ovulation could be rendered ineffective is by failure of pick-up of the oocyte by the tubal cilia due to suboptimal expansion of the oocyte-cumulus complex and mis-expression of cell adhesion molecules. Retardation of trophoblastic outgrowth and development, placental necrosis and suppression of steroid biosynthesis, and altered handling of nutrient metals by the placenta all contribute to implantation delay and possible early pregnancy loss. Cd has been shown to accumulate in embryos from the four-cell stage onwards, and higher dosage exposure inhibits progression to the blastocyst stage, and can cause degeneration and decompaction in blastocysts following formation, with apoptosis and breakdown in cell adhesion. Following implantation, exposure of experimental animals to oral or parenteral Cd causes a wide range of abnormalities in the embryo, depending on the stage of exposure and dose given. Craniofacial, neurological, cardiovascular, gastrointestinal, genitourinary, and limb anomalies have all been described in placentates, with axial abnormalities and defects in somite structure noted in fish and ventral body wall defect and vertebral malformation occurring in the chick. In this paper, we examine the mechanisms by which Cd can affect reproductive health, and consider the use of micronutrients in prevention of these problems.