Maria Luisa Broccia

Teratogenic effects of sodium valproate in mice and rats at midgestation and at term.

This experiment was carried out with the aims of comparing the embryotoxic potential of valproic acid (VPA) in rats and mice, better defining the malformation pattern in these species, and comparing the embryotoxic effects detectable in mid-pregnancy to those observed in fetuses at term. Pregnant CD:Crl rats were treated subcutaneously (s.c.) at 08:00, 16:00, and 00:00 on day 9 of gestation with 0, 150, or 300 mg/ kg VPA; pregnant NMRI mice were treated s.c. at 00:00 on day 7 of gestation, and at 08:00 and 16:00 on day 8 of gestation with 0,75, 150, or 300 mg/kg VPA. Groups of females were killed on day 9 (mice) or day 11 (rats) of pregnancy and their embryos were carefully examined under a dissecting microscope. The remaining females were killed 1 day before parturition and their fetuses were examined for external, visceral, and skeletal malformations. A very high frequency (84%) of malformed embryos was recorded in the group of mice treated with 300 mg/kg, including open brain folds (73%), somite defects (36%), and heart malformations (20%). The rat embryos were less sensitive: only 43% of them were malformed after treatment with 300 mg/ kg, however, the pattern of malformations was quite similar to that observed in mice. The treatment with 150 mg/kg produced about 32% malformed embryos in mice and only 8.5% in rats. More than 84% of mouse fetuses from mothers treated with the highest dose showed a severe form of exencephaly. The axial skeleton was also severely affected. The postimplantation loss reached 52%. Exencephaly and skeletal malformations were also recorded in mouse fetuses from mothers exposed to 150 mg/kg. The dose of 75 mg/kg was without effects. Exencephaly was not observed in rat fetuses at term. In this species the axial skeleton was the most severely affected region at 300 mg/kg, while the lowest dose produced only sporadic malformations. These results confirm that the mouse is the more sensitive species for the teratogenic effects of VPA. Furthermore, it has been shown that, in both species, the axial skeleton is a system which is very sensitive to the teratogenic effects of VPA. The observed alterations show a possible link between axial specification and VPA and suggest further studies of embryos exposed to VPA for the expression of genes controlling the identity of vertebral segments.

In vitro teratogenic potential of two antifungal triazoles: Triadimefon and triadimenol

SummaryThe teratogenic potential of two antifungal triazoles (Triadimefon and Triadimenol) has been investigated in vitro by the rat postimplantation whole embryo culture method. Rat embryos 9.5 d old were cultured for 48 h in rat serum with Triadimefon (12.5–250 μM) or Triadimenol (6.25–125 μM) and then examined. Some embryos exposed to Triadimenol (6.25–125 μM) were cultured for 12 extra hours in control serum to improve their developmental degree and then immunostain cranial nerves and ganglia. The exposure to the highest doses of triazoles only moderately reduced some morphometrical developmental parameters. By contrast, 25–250 μM Triadimefon and 25–125 μM Triadimenol induced specific concentration-related teratogenic effects at the level of first and second branchial arches. After immunostaining, embryos exposed to 12.5–125 μM Triadimenol showed specific cranial nerve and ganglia abnormalities. The possible implication of neural crest cell alterations on triazole-related abnormalities is discussed.

Glutathione Status in Diabetes-Induced Embryopathies

The mechanism involved in diabetes-induced embryotoxicity is still unclear. Mitochondrial alterations probably produced by oxidative stress have been described in embryos developing in a diabetic environment. Furthermore, oxygen radicals-scavenging enzymes can reduce the embryotoxic effects induced by diabetic conditions. In this work we tried to test if glutathione (GSH), a tripeptide implicated in cellular protection against reactive oxygen species, is involved in diabetes-related embryotoxicity. Rat embryos were explanted on day 11 on gestation from normal and from streptozotocin-diabetic mothers. The embryos were examined morphologically, then protein, DNA and GSH were determined both in embryos and in their visceral yolk sacs. The embryos explanted from diabetic mothers showed signs of developmental retardation and 16% were morphologically abnormal. GSH content was reduced in these embryos in comparison to control, but the GSH/protein in the visceral yolk sacs of conceptuses explanted from diabetic mothers was higher than in control visceral yolk sacs. Our hypothesis is that the reduction of embryonic GSH is a consequence of the alteration in GSH transport across the yolk sac endodermal cells damaged by diabetic conditions. The observed reduction in embryonic GSH could reduce the protection against the oxidative stress condition described in diabetic pathology.

Comparative study of sodium valproate-induced skeletal malformations using single or double staining methods

The teratogenic activity of xenobiotics is usually investigated by examining visceral and skeletal abnormalities of term fetuses. Although the rodent fetal skeleton is only partially ossified, the single stain for bone is the most commonly used method in routine teratology testing, while the double stain for evaluation of both bone and cartilage is often used only in basic research. The present work compares data obtained from rat fetuses using the two methods after exposure to the teratogenic agent sodium valproate at specific embryonic stages of development. Pregnant rats were treated with 400mg/kg sodium valproate and sacrificed at term of pregnancy. Even if both methods were able to identify sodium valproate as a teratogenic molecule, correct and complete interpretation of data was possible only by using the double stain. Our results show the inability of the single stain to correctly discriminate between major and minor abnormalities.

The Inhibition of Embryonic Histone Deacetylases as the Possible Mechanism Accounting for Axial Skeletal Malformations Induced by Sodium Salicylate

In spite of the large use of salicylates, introduced into clinical practice more than 100 years ago, their anti-inflammatory and cancer preventive mechanisms are still under study. Teratogenic effects of salicylates have been reported in experimental animals since 1959 but the pathogenic pathways and the mechanisms of action were never described until now. The aim of this work is to verify if the inhibition of embryonic histone deacetylase (HDAC) enzymes and the consequent tissue hyperacetylation could be the mechanism responsible for axial skeletal defects described after the exposure of pregnant rodents to sodium salicylate (SAL). E8 pregnant CD-1 mice were intraperitoneally treated with SAL 0-150-300-450 mg/kg and sacrificed at 1, 3, 5 h after treatment or at term of gestation (E18). E8 embryos were processed for Western blotting and immunostaining analyses, while skeletons of E18 fetuses were double stained for bone and cartilage. A group of control E8 embryos were used to prepare embryonic nuclear extract for the HDAC enzyme assay. A significant SAL dose-related HDAC inhibition activity, compatible with a mixed-type partial inhibition mechanism, was detected. A clear dose-related hyperacetylation of histones was observed in embryos exposed in utero to SAL, with a peak at 3 h after treatment of dams. The most hyperacetylated organs were somites and the heart. Histone hyperacetylation is suggested to be the mechanism accounting for SAL-related axial skeletal and cardiovascular defects and is proposed as the mechanism responsible for other biological effects of salicylates.

Teratogenesis study of dioxane in rats.

The industrial solvent dioxane (1,4-diethylene dioxide) was evaluated for teratogenic potential in Sprague-Dawley rats. The compound was administered on days 6-15 of gestation by gavage (0, 0.25, 0.5 and 1.0 ml/kg/day). A slight maternal toxicity, as evidenced by reduced weight gain, was observed with 1.0 ml/kg. Animals were killed and subjected to uterine examination on day 21 of pregnancy. There were no differences between control and dioxane-treated groups in implantation numbers, live fetuses, postimplantation loss or major malformations. Embryotoxicity, manifested by reduced fetal weight, occurred only at the highest dose level.

EFFECTS OF EXCESS AND DEPRIVATION OF SEROTONIN ON IN VITRO NEURONAL DIFFERENTIATION

SummaryThe neutrotransmitter serotonin (5HT) possesses developmental functions in vertebrates and invertebrates. Rodent embryos express 5HT receptors even before neural development, but the role of this neurochemical seems to be particularly important during axonal morphogenesis and differentiation and in neural crest cell migration. Moreover, 5HT inhibitors are teratogenic in mammals, inducing brain and heart abnormalities. The aim of this study was to investigate the effects of nonphysiological concentrations of 5HT (5HT excess as well as deprivation) on developing rat neural cells using the micromass method. This simple and rapid micromass method allows the culture of mesencephalic cells capable of achieving and maintaining a significant degree of differentiation. Mesencephalic cells from 13 d post coitum (pc) rat were cultured and exposed to exogenous 5HT (1, 10, 50, or 100 μM) or to the specific 5HT2 receptor inhibitor mianserin (0.5, 5, 25, or 50 μM) during the whole culture period (5 d). The micromass morphology, the cytoskeletal organization, the pathological apoptosis, and the differentiative capability of cultured mesencephalic cells have been analyzed. The results show that 10–100 μM 5HT and 0.5–50 μM mianserin are able to disrupt the normal micromass morphology; 5HT and mianserin are unable to interfere with the cytoskeletal structures; mianserin (but not 5HT) induces pathological apoptosis on micromass cells at concentration levels of 0.5–50 μM; 5HT (but not mianserin) alters the neural differentiation at concentration levels of 10–100 μM. In conclusion, our results demonstrate that an excess of 5HT inhibits the capability of mesencephalic neurons to differentiate as shown by the alterations of the expression of the neuronal differentiative proteins glial-derived neurotrophic factor and Neu-N; on the other hand, the blocking of 5HT2 receptors induces apoptosis in differentiating neurons.

VPA-related axial skeletal defects and apoptosis: A proposed event cascade

VPA axial malformations are related to embryonic somitic histone hyperacetylation. In cancer, histone hyperacetylation activates apoptosis. To verify if apoptosis is involved in somitic abnormalities, VPA-exposed embryos were evaluated for DNA fragmentation and for pro- (p53, acetylated p53, caspase 3) and anti-apoptotic (Sirt 1) protein expression. Pregnant mice were i.p. dosed on day 8 with VPA 400mg/kg or TSA (16 mg/kg). Embryos, collected 3, 5, 9 or 24h after treatment, were examined and processed for apoptosis or protein analysis. An event cascade has been observed at the level of somites and proposed as related to VPA-induced axial skeletal defects: increased p53 (3h), DNA fragmentation (9h), abnormalities (24h). TSA, used as alternative HDAC inhibitor, induced apoptosis and somitic abnormalities, strengthening our hypothesized link between HDAC inhibition and axial defects.

Diet composition modifies embryotoxic effects induced by experimental diabetes in rats

Despite improvements in prenatal care, the incidence of congenital malformations in diabetic pregnancies is still 3-4 times higher than in normal pregnancies. These defects could be attributed to alterations of intrauterine environment due to disorder of the maternal metabolism. If this were true, the quality of food could play a role in diabetes-induced embryotoxicity. To check this hypothesis, female CD rats were made diabetic by injecting intravenously 50 mg/kg of streptozotocin 2 weeks before mating. From the first day of pregnancy they were divided into three groups and maintained on the following diets: (1) standard diet (Italiana Mangimi); (2) purified high protein diet (protein 55%, carbohydrates 25.5%, fat 7.5%, fiber 4.5%, ash 7.5%); (3) purified normoprotein diet (protein 19%, carbohydrates 62.5%, fat 7.5%, fiber 4%, ash 7%). Nondiabetic pregnant females fed with standard diet served as negative control. No significant differences were observed in blood glucose levels among the groups (range 410-500 mg/dl). The group fed on normoprotein diet showed at term of pregnancy: (1) higher rate of resorptions; (2) lower fetal weight; (3) higher frequency of major malformations than the groups fed standard and hyperproteic diets. Although we are not able at this time to discriminate between a protective effect of a diet with a high protein content and a disruptive effect of a diet containing high quantity of carbohydrates, the results of this trial support the hypothesis of a fuel-mediated teratogenesis in diabetic pregnancy.

Stage-dependent abnormalities induced by the fungicide triadimefon in the mouse ☆

Aim of this work is the study of abnormalities induced by the triazole triadimefon (FON) administered to pregnant mice at E8, E9, E10, E11 or E12. Pregnant CD-1 mouse were gavaged with FON 500 mg/kg at the selected stages and sacrificed at term and fetuses morphologically examined and processed for visceral and skeletal analysis. Administration of FON on E8, E10-E12 resulted in fetuses with cleft palate (E8 39% and E12 24% representing the peak of sensitivity, in E8 fetuses associated to severe skull basis abnormalities). Other cranial malformations (fusions abnormalities or agenesis of bones) were observed in E8-E10 groups (E8 the most sensitive with 96% of malformed fetuses). Cardiovascular abnormalities were observed in a stage dependent manner at E8-E10 (22.2, 3.8, 7.8%). As far as craniofacial malformation is concerned, we propose that FON acts on two different stages, involved in early and late craniofacial formation.