secalonic-acid and Cleft-Palate

Cyclic AMP is an important second messenger mediating the actions of many hormones and other ligands in a variety of cells. Cells of the developing organism are no exception. Once generated, it releases the catalytic subunit of protein kinase A (PKA) from the inhibitory influence of its regulatory subunit, which then migrates into the nucleus to phosphorylate and enhance the binding of relevant transcription factors to the promoter element CRE of genes involved in above cellular responses. This review summarizes the available data on the essential role of this pathway in embryonic development as well as the functionality, ontogeny and consequences of genetic and chemical disruption of this pathway in the developing orofacial structures, especially the secondary palate as influenced by the mycotoxin, secalonic acid D.

Topics: Animals; Cleft Palate; CREB-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Humans; Mice; Nuclear Proteins; Signal Transduction; Trans-Activators; Xanthones

The mycotoxin, secalonic acid D (SAD), a known animal and potential human cleft palate (CP)-inducing agent, is produced by Pencillium oxalicum in corn. SAD selectively inhibits proliferation of murine embryonic palatal mesenchymal (MEPM) cells leading to a reduction in cell numbers. These effects can explain the reduction in shelf size and the resulting CP seen in the offspring of SAD-exposed mice. Ability of SAD to inhibit proliferation as well as to block the progression of cells from G1- to S-phase of the cell-cycle were also shown in the human embryonic palatal mesenchymal (HEPM) cells suggesting the potential CP-inducing effect of SAD in human beings. Gestation day (GD) 12 mouse embryos and HEPM cells were used to test the hypothesis that the cell-cycle block induced by SAD results from a disruption of stage-specific regulatory components both in vivo and in vitro. The effects of SAD on the activity of various cyclin dependent kinases (CDK) and on the levels of various positive (cyclins and CDK) and negative (CDK inhibitors p15, 16, 18, 19, 21, 27, 57) cell-cycle regulators were assessed by performing kinase assays and immunoblots, respectively.. In the murine embryonic palates, SAD specifically inhibited G1/S-phase-specific CDK2 activity, reduced the level of cyclin E and tended to increase the level of the CIP/kip CDK inhibitor, p21. In the HEPM cell cultures, exposure to IC50 of SAD significantly affected all of the above targets. In addition, a reduction in the levels/activity of CDK 4/6, a reduction in the levels of cyclins D1, D2, D3, E, A, and all INK4 family proteins, and an increase in the level of the CIP/kip CDK inhibitor, p57, were also seen.. These results suggest that the S-phase-specific cell-cycle proteins CDK2, cyclin E and possibly p21 are the common targets of SAD in murine palatal shelves in vivo and in human embryonic palatal mesenchymal cells in vitro and may be relevant to the pathogenesis of SAD-induced CP.

Topics: Animals; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cells, Cultured; Cleft Palate; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Embryonic Development; Humans; Mesoderm; Mice; Palate; Xanthones

The protein kinase C (PKC) family of proteins mediates the action of growth factors and other ligands by activating a network of transcription factors that bind to TRE sequences in the promoters of many genes that regulate cell proliferation, differentiation, extracellular matrix synthesis, apoptosis and others in a cell type-, isozyme- and context-specific manner. The critical role of PKC in embryonic development is indicated by early death of embryos in which one or more of these isozymes are inactivated. Our studies together with others show that palatal PKC signalling is functional and may be essential for normal palate development. Although single gene knockouts have failed to exhibit the cleft palate (CP) phenotype, owing to compensation by other kinases, many chemicals including the mycotoxin, secalonic acid D, disrupt palatal PKC signalling leading to altered palatal mesenchymal gene expression. The potential relevance of such effects to chemical-induced CP is discussed.

Topics: Abnormalities, Drug-Induced; Cleft Palate; Face; Humans; Palate; Protein Kinase C; Response Elements; Signal Transduction; Xanthones

Secalonic acid-D (SAD) is a teratogenic mycotoxin inducing cleft palate (CP) in the offspring of the exposed mice by reducing palatal shelf size secondary to reduced proliferation of the palatal mesenchymal (PM) cells. Co-administration of dimethylsulfoxide (DMSO) reversed the CP-inducing effect of SAD. Although SAD has been shown to affect both protein kinases A (PKA) and C (PKC) pathways, the relevance of each of these pathways to its CP induction is unknown. The present studies were designed to test the hypothesis that the protective effect of DMSO is mediated by its specific reversal of the effect(s) of SAD on one of these two pathways using ELISA-based activity assays, Western blot analysis, electrophoretic mobility shift assays (EMSA), and murine embryonic PM (MEPM) cell growth in culture. Within the PKA pathway, SAD inhibited the activity of the catalytic subunit of PKA and its migration into the nucleus, elevated phosphorylated cyclic AMP (cAMP) response element (CRE)-binding protein (pCREB) level, and reduced the binding of CREB to CRE. In the PKC pathway, SAD reduced the activity of PKC and the binding of transcription factors (TF) to 12-O-tetradecanoate-13 phorbol acetate-response element (TRE). SAD also inhibited MEPM cell growth and the expression of the CRE- and TRE-containing gene, proliferating cell nuclear antigen (PCNA). Reversal, by DMSO, of the effects of SAD on MEPM cell growth, on PCNA expression and on all components of the PKA, but not of PKC, pathway suggests that the perturbation of the PKA pathway by SAD is relevant to its induction of CP in mice.

Topics: Animals; Blotting, Western; Catalysis; Cell Division; Cell Nucleus; Cleft Palate; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Female; Gene Expression; Mice; Palate; Phosphorylation; Pregnancy; Proliferating Cell Nuclear Antigen; Protein Kinase C; Response Elements; Signal Transduction; Teratogens; Transcription Factors; Xanthones

To assess the mechanism(s) of cleft palate induction by secalonic acid D (SAD) in human embryonic palatal mesenchymal (HEPM) cells and compare them with those evaluated in the murine embryonic palate.. Effect of SAD on HEPM cell proliferation was studied by obtaining dose response curves for cell numbers, uptake of 3H-thymidine and the expression of proliferating cell nuclear antigen (PCNA). Effects of SAD on cell cycle were assessed by flowcytometry. Cell-labeling with 3H-glucosamine and immunoblot analysis were conducted to study SAD effects on the synthesis of glycosaminogycans (GAG) and the expression of fibronectin and tenascin, respectively.. SAD induced a concentration-dependent decrease in HEPM cell number and 3H-thymidine uptake beginning at 0.1 microg of SAD/ml. Expression of PCNA and progression of cell cycle from G1 to S phase were inhibited following SAD exposure. Cell viability was significantly reduced only at 7.5 microg/ml of SAD or higher indicating that the reduction in cell numbers by SAD at lower concentrations is likely due to reduced proliferation and at higher concentrations due to both reduced proliferation and cell death. Synthesis of extra cellular matrix components (GAGs, fibronectin or tenascin) by HEPM cells, however, was not inhibited by SAD.. The results of these studies confirmed those of our previous studies with mice and the MEPM cells that SAD may induce cleft palate by reducing numbers of palatal mesenchymal cells by inhibition of their proliferation thereby leading to a reduction in the size of the developing palate shelves.

Topics: Animals; Cell Count; Cell Cycle; Cell Line; Cell Proliferation; Cell Survival; Cleft Palate; Dose-Response Relationship, Drug; Fibronectins; Glycosaminoglycans; Humans; Mesoderm; Mice; Mycotoxins; Palate; Proliferating Cell Nuclear Antigen; Tenascin; Thymidine; Xanthones

Glucocorticoids (GC) induce cleft palate (CP) in the offspring of exposed pregnant mice. Glucocorticoids induce prostaglandin (PG) synthesis inhibition via reduced arachidonic acid (AA) release from membranes, and this results from inhibition of phospholipase A2. This metabolic event is associated with reduced palatal cAMP levels in the prefusion stages (d 13 of gestation). The mycotoxin secalonic acid D (SAD) induces CP in the offspring born to treated mothers, elevates maternal plasma corticosterone levels, and reduces prefusion palatal cAMP levels. In addition, an increase in cAMP was noted in the postfusion period (d 15 of gestation). Since exogenous AA given simultaneously to GC-exposed mothers may protect against GC-induced CP in the offspring, such a possibility was tested for SAD. Pregnant CD1 mice given a teratogenic dose of SAD (30 mg/kg, ip, on gestational d 11) were simultaneously treated with maximal tolerated doses of AA (200 mg/kg, sc, on gestational d 11, 12, and 13). At term, no significant reduction in SAD-induced CP was seen as a result of AA treatment. To evaluate if SAD-induced alterations in palatal cAMP are due to reduced palatal membrane-associated adenylate cyclase (AC) activity during pre- and postfusion periods, SAD-treated mothers were sacrificed at 12-h intervals between gestational d 13.5 and 15.5, palate shelves were collected from the fetuses, and AC activity (cAMP formed/mg protein/min) was assayed in the presence or absence of the enzyme stimulator, sodium fluoride (NaF). Although SAD did not alter unstimulated AC activity, it significantly reduced the NaF-induced stimulation of enzyme activity in the prefusion period. This inhibition could not be reversed by excess GTP in the incubation mixture. Since NaF stimulation of AC indicates post-receptor-site function involving GTP-binding and catalytic units, and since addition of GTP failed to correct SAD-induced alteration of NaF stimulation of the enzyme, it is suggested that SAD may inhibit the AC sensitivity to stimuli by its effect on the catalytic unit in a manner that does not affect enzymic basal activity.

Topics: Adenylyl Cyclases; Animals; Arachidonic Acid; Cleft Palate; Cyclic AMP; Dose-Response Relationship, Drug; Female; Mice; Palate; Pregnancy; Sodium Fluoride; Xanthenes; Xanthones

Secalonic acid D (SAD) is a teratogenic mycotoxin that causes cleft palate in the offspring of treated pregnant mice. To investigate the role of maternal corticosterone in the teratogenicity of SAD, pregnant CD-1 mice were treated with 30 mg/kg of SAD i.p. on day 11 of pregnancy in either 5% (w/v) NaHCO3 or 20% (v/v) dimethyl sulfoxide (DMSO) in NaHCO3. Radioimmunoassay (RIA) was performed to determine plasma corticosterone at 24, 48, 72, and 96 hr after dosing. No interference by EDTA, SAD, DMSO, or pentobarbital was noticed on the RIA. Significant (P less than .01) elevations in plasma corticosterone concentrations were seen 24 and 48 hr following dosing of SAD in NaHCO3 with concentrations reaching a peak just prior to the onset of shelf elevation and fusion. Simultaneous treatment with DMSO, an agent known to antagonize the teratogenic effect of SAD, completely abolished the SAD-induced corticosterone elevation at the 24 hr time point and significantly (P less than .01) reduced it at the 48 hr time point. To evaluate the specificity of the role of corticosterone in the teratogenicity of SAD, plasma samples from mature males similarly treated with either single or multiple doses of SAD ranging from 15 to 45 mg/kg were assayed for corticosterone. A dose of SAD comparable to that used in the pregnant females failed to significantly change plasma corticosterone concentrations in the males. An elevation corresponding only to 75% of that in the females was seen in males receiving multiple doses of SAD totaling three times the dose used in the females. As with females, DMSO completely abolished plasma corticosterone elevation by SAD in the males. These results demonstrate, for the first time, the effect of SAD on a mammalian endocrine system and provide evidence for a specific involvement of elevated maternal plasma corticosterone concentrations in SAD teratogenicity.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Corticosterone; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Female; Male; Maternal-Fetal Exchange; Mice; Pregnancy; Radioimmunoassay; Xanthenes; Xanthones

Secalonic acid D (SAD) induces cleft palate in the developing mouse by an unknown mechanism. To investigate possible roles of cyclic nucleotides (cAMP and cGMP) in the teratogenesis of SAD, cAMP and cGMP levels were measured in the extracts of fetal palates of gestational age 13(0) through 16(12) (days(hours]. Fetuses were obtained from pregnant CD-1 mice treated on day 11(0) of gestation, with either 5% (wt./vol.) sodium bicarbonate (NaHCO3, control) or 30 mg/kg of SAD in 5% NaHCO3, intraperitoneally (i.p.). Radioimmunoassay was used to quantitate cyclic nucleotide levels. Cyclic AMP levels peaked on day 14(12) of gestation in controls. In the SAD group there was a significant decrease in cAMP levels on days 13(12) and 14(0) of gestation and a decrease of 30% in total cAMP on days 13(0) through 14(0) of gestation, with little or no change at the peak on day 14(12). On day 15(12), however, the SAD group had a 68% increase in palatal cAMP level over the control. Control levels of cGMP appeared to follow a diurnal pattern reaching maximal levels at the end of the dark cycle. In contrast, SAD decreased the cGMP level by 31% on day 13(12) (P less than 0.05) and increased it 100% above that of the control level on day 15(0) (P less than 0.01). Total cGMP during days 15(0) through 16(12) of gestation was 33% higher than control (P less than 0.01). The number of clefts in the SAD group was significantly higher at all points following palate closure in the control fetuses (14(0) through 16(12] with values ranging from 20% to 34% versus 0% in the control (P less than 0.01-0.005). Morphological changes on days 13(0) through 15(0) indicated a failure of shelf elevation in middle and posterior palatal regions of SAD-treated fetuses. These results suggest that the induction of cleft palate by SAD is associated with dynamic changes (initial decrease followed by later increase), in vivo, in established cyclic nucleotide patterns and support a mechanistic role for cyclic nucleotide-mediated cellular processes in normal as well as abnormal palate development.

Topics: Animals; Cleft Palate; Cyclic AMP; Cyclic GMP; Embryonic and Fetal Development; Mice; Mice, Inbred Strains; Mycotoxins; Palate; Xanthenes; Xanthones

Dimethylsulfoxide (DMSO) is known to antagonize the teratogenic effects of secalonic acid D (SAD) in mice. To establish the optimum protective dose of DMSO, pregnant CD-1 mice were treated, i.p., with 30 mg/kg of SAD in 5% (w/v) NaHCO3, containing 0, 10, 20, or 30% (v/v) DMSO on day 11 of gestation. Data indicate that at 10% and 20% levels, DMSO affords an apparent dose-related protection against SAD-induced cleft palate, whereas 30% DMSO enhanced fetal resorption with no reduction in the incidence of cleft palate. Ultraviolet spectra and TLC mobility indicated that DMSO at 20% did not directly interact with SAD. Distribution and elimination of 14C-SAD was studied in fetal and maternal tissues from pregnant mice at 24 and 48 hr after exposure to 30 mg/kg of 14C-SAD, i.p., in NaHCO3 (control) or in 20% DMSO. Compared with those not receiving DMSO, maternal exposure to DMSO: 1) significantly reduced (42-75%) radioactivity in fetal heads and bodies, placenta, and maternal tissues other than liver; 2) significantly increased (up to 222%) the radioactivity in maternal liver; and 3) significantly reduced (44-58%) fecal and urinary elimination of SAD-derived radioactivity. These results suggest that the antiteratogenic effect of DMSO against SAD may be at least partly mediated by increased SAD (or its metabolites) retention by maternal liver leading to reduced SAD uptake by the fetus.

Topics: Animals; Body Weight; Cleft Palate; Dimethyl Sulfoxide; Female; Fetal Resorption; Fetus; Mice; Mycotoxins; Pregnancy; Reference Values; Teratogens; Xanthenes; Xanthones

Incidence of cleft palate (CP) in full-term mouse fetuses was evaluated following administration of 25 mg/kg of the mycotoxin, secalonic acid D (SAD), to groups of female mice on each of Days 10, 11, 12, 13, 14, or 15 of pregnancy. Although the highest numerical incidence (45.3%) of cleft palate resulted following SAD exposure on Day 12 of pregnancy, and the response tapered off to 16.9% on Day 10 and 0% on Day 15 of pregnancy, similar responses were produced also following exposures on Days 11 (38.4%) and 13 (39.9%) of pregnancy. Maternal exposure to doses of 0, 15, 20, 25, or 30 mg/kg of SAD, given on Day 12 of pregnancy indicated that although fetuses in the 30-mg/kg group had the highest incidence (51.9%) of CP, the effect was associated with increased resorptions and decreased fetal weights. The 25-mg/kg dose was optimally teratogenic (45.3% cleft palate) and maximally tolerable with neither an increase in resorptions nor a decrease in fetal body weights. Cytotoxicity of the optimally teratogenic dose of SAD (25 mg/kg given ip) on Day 12 of pregnancy was evaluated as a possible mechanism of SAD teratogenicity using indices such as mesenchymal cell density, mitotic index, and the uptake of [3H]thymidine in the developing palatal shelves. No evidence of SAD cytotoxicity was obtained in palatal shelves indicating a possible role for nonlethal cellular effects of SAD in the pathogenesis of CP. These studies also suggest the suitability of the maternal 25-mg/kg dose of SAD to study cellular biochemical effects in the developing embryo without the complicating influence of cytotoxic effects.

Topics: Animals; Birth Weight; Cleft Palate; Embryonic and Fetal Development; Female; Fetal Resorption; Injections, Intraperitoneal; Maternal-Fetal Exchange; Mice; Mitotic Index; Pregnancy; Xanthenes; Xanthones