John Bannigan

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.

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.

Stereology of the placenta in type 1 and type 2 diabetes.

OBJECTIVE To assess by stereology the placental structure in type 1 (T1DM) and type 2 (T2DM) diabetic pregnancies compared to normal non-diabetic (ND) controls. STUDY DESIGN Prospective case control study. Placentae were sampled in a systematic random fashion. Stereological analysis was performed using a computerised stereology programme (Image Pro 6.2, Media Cybernetics, Inc, Silver Spring MD, USA). Participants were matched for gender of infant and mode of delivery. MAIN OUTCOME MEASURES Volume, length and surface area of placental components; clinical outcome. RESULTS Ten ND, eight T2DM and ten T1DM women consented to the study. There was no difference between the groups regarding maternal age, neonatal birth weight, or placental weight. On stereological examination, terminal villous volume was significantly increased in both diabetic groups compared to ND controls. Capillary volume and length was increased in T1DM pregnancies compared to ND and T2DM. Capillary length was increased in both diabetic groups compared to ND. When all diabetic groups were compared based on severity of glycaemia those with poor glycaemic control (HbA1c>7%) had higher placental capillary volume than those with good glycaemic control. CONCLUSIONS This study demonstrates an association between maternal diabetes and increased terminal villous volume. Additionally capillary volume and length is increased in the placentae of normally grown infants of T1DM diabetic mothers compared to non-diabetic controls. Maternal glycaemia appears to influence capillary, but not stromal, development. This suggests that factors other than glycaemia have a role in placental development in pre-gestational diabetes.

The effects of ethanol on CNS development in the chick embryo.

Human and animal studies show that the central nervous system (CNS) is particularly vulnerable to developmental exposure to alcohol across all stages of development. New critical periods of ethanol sensitivity continue to be defined. The aim of this study was to further examine the stage-specific effects of ethanol on CNS development using a relatively simple programme of neuronal migration and differentiation, the chick embryo spinal cord, and treating at the immediate post-neurulation stage. Embryos (HH-stage 10-12) were explanted into shell-less culture and treated with ethanol (20 microl/40%) or saline (20 microl). At 6,12, 24 and 48 h post-treatment specimens were processed for resin histology. In addition, levels of cell death were analysed using Lysotracker Red, neural crest cell migration patterns were examined using HNK-1 staining and effects on DNA synthesis were evaluated on autoradiographs prepared 1h after exposure to 3H-TdR. This treatment protocol produced significant growth retardation in ethanol specimens examined at 48 h post-treatment. This effect was shown to involve increased levels of cell death, perturbation of DNA synthesis and an abnormal translocation and subsequent loss of cells into the neural tube lumen. No gross malformations were observed. We conclude that these results further highlight the stage-specific effects of ethanol on neurodevelopment.

Enhanced expression of inducible nitric oxide synthase without vasodilator effect in chronically infected lungs

We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression. Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10-15 days later. Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs. In isolated lungs from chronically infected rats, NOS inhibition with N ω-nitro-l-arginine methyl ester increased the mean perfusion pressure (14.4 ± 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 ± 1.0 mmHg) or noninoculated (5.3 ± 0.8 mmHg) lungs ( P < 0.01). Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity. Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors. Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection. Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not. Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression. Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10-15 days later. Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs. In isolated lungs from chronically infected rats, NOS inhibition with N(omega)-nitro-L-arginine methyl ester increased the mean perfusion pressure (14.4 +/- 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 +/- 1.0 mmHg) or noninoculated (5.3 +/- 0.8 mmHg) lungs (P < 0.01). Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity. Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors. Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection. Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not. Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.

Visualizing expression patterns of Shh and Foxf1 genes in the foregut and lung buds by optical projection tomography

Congenital malformations of the foregut are common in humans. The respiratory and digestive tubes are both derived by division of the foregut primordium. Sonic hedgehog (Shh) and Fork head box F1 (Foxf1) genes encode regulatory molecules that play a pivotal role in gut and lung morphogenesis and are therefore important candidate genes to be examined in models of foregut developmental disruption. Optical projection tomography (OPT) is a new, rapid and non-invasive technique for three-dimensional (3D) imaging of small biological tissue specimens that allows visualization of the tissue distribution of RNA in developing organs while also recording morphology. To explore the application of OPT in this context, we visualized Shh and Foxf1 gene expression patterns in the mouse foregut and lung buds at several stages of development. Time-mated CBA/Ca mice were harvested on embryonic days 9–12. The embryos were stained following whole mount in situ hybridization with labelled RNA probes to detect Shh and Foxf1 transcripts at each stage. The embryos were scanned by OPT to obtain 3D representations of gene expression domains in the context of the changing morphology of the embryo. OPT analysis of Shh and Foxf1 expression in the foregut and lung buds revealed extra details of the patterns not previously reported, particularly in the case of Foxf1 where gene expression was revealed in a changing pattern in the mesenchyme around the developing lung. Shh expression was also revealed in the epithelium of the lung bud itself. Both genes were detected in complementary patterns in the developing bronchi as late as E12, showing successful penetration of molecular probes and imaging at later stages. OPT is a valuable tool for revealing gene expression in an anatomical context even in internal tissues like the foregut and lung buds across stages of development, at least until E12. This provides the possibility of visualizing altered gene expression in an in vivo context in genetic or teratogenic models of congenital malformations.

Type 2 nitric oxide synthase and protein nitration in chronic lung infection.

Inflammation in the lung can lead to increased expression of inducible nitric oxide synthase (iNOS) and enhanced NO production. It has been postulated that the resultant highly reactive NO metabolites may have an important role in host defence, although they might also contribute to tissue damage. However, in a number of inflammatory lung diseases, including bronchiectasis, iNOS expression is increased but no elevation of airway NO can be detected. A potential explanation for this finding is that NO is rapidly scavenged by reaction with superoxide radicals, forming peroxynitrite, which is preferentially metabolized via nitration and nitrosation reactions. To test this hypothesis, anaesthetized, specific pathogen‐free rats were inoculated with Pseudomonas aeruginosa incorporated into agar beads (chronically infected group) or sterile agar beads (control group). Ten to 15 days later, the lungs were isolated and fixed. Pseudomonas organisms were isolated from the lungs of the chronically infected group. These lungs showed extensive inflammatory cell infiltration and tissue damage, which were not observed in control lungs. Expression of iNOS was increased in the chronically infected group when compared with the control group. However, the mean number of cells staining for nitrotyrosine in the chronically infected group was not significantly different from that in the controls, nor was there an excess of nitrotyrosine, nitrate, nitrite or nitrosothiol concentrations in the infected lungs. Thus, no evidence was found of increased NO metabolites in chronically infected lungs, including products of the peroxynitrite pathway. These findings suggest that chronic infection does not cause increased iNOS activity in the lung, despite increased expression of iNOS. Copyright

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.

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.

The uptake of 5-bromodeoxyuridine by the chicken embryo and its effects upon growth

SummaryWhen embryonic cells in vitro are exposed to bromodeoxyuridine (BUdR) the duration of exposure can be made to last for several cell generation times. Such exposure is known to prevent embryonic cells undergoing terminal differentiation while leaving cell division and basic cell function unaffected. When BUdR is injected into pregnant mammals, it remains available for incorporation in the DNA for only a fraction of one S phase and causes foetal anomalies that are apparently the result of cell death and a transient slowing of the cell generation time but not of failure of cell lines to differentiate.The objectives of our experiments were to ascertain the availability time of BUdR in the chicken embryo in ovo, to assess its teratogenicity and to examine its effects on the growth of the embryo.When 3H-BUdR (0.02 mg) was injected into the albumen space on day 3 of incubation, subsequent scintillation spectrometry and autoradiography showed that the drug was incorporated into the DNA of the embryo for more than 8 h or more than one cell generation time at this stage of development. On the other hand, a trace amount of tritiated thymidine (3H-TdR) was available for only one hour, the difference being probably due to an expansion of the nucleotide precursor pool in the case of BUdR.The injection of 0.02 mg BUdR on day 3 caused growth retardation as manifested by differences in weight and in DNA content between BUdR and saline treated embryos. The difference in DNA content was evident 24 h after treatment and was probably due in part of the cell necrosis in the developing CNS that began 10 h after injection. Differences in weight did not become apparent until 4 days after treatment and were thus thought to be due to factors other than cell necrosis.On day 11 of incubation, the mortality of BUdR treated embryos became significantly greater than that of controls and many survivors after this time had ventral body wall defects. When treatment was delayed until days 4 or 6, the subsequent development of BUdR and saline treated embryos was indistinguishable. The sensitivity of day 3 was thought to be due to the fact that embryo DNA content quadrupled between days 3 and 4 whereas it only doubled per 24 h period thereafter.