Oral vitamin B1-substitution does not decrease genetically determined cleft rate in mice (A/WySn).

PURPOSE: Cleft lip and palate (CL/P) are one of the most common human birth defects. Animal experiments and clinical investigations show a clear reduction of teratogenic clefts by a high-dose vitamin B supplementation during early pregnancy, especially in families at risk (reduction of recurrence). The aim of this work was to examine the influence of thiamine (vitamin B1) on CL/P appearance in genetically determined A/WySn mice within different supplementation starting points. MATERIALS AND METHODS: A total of 24 A/WySn female mice were orally supplemented with high doses (80 mg/kg) of thiamine at different times of pregnancy (5 groups, n = 90). The influence of thiamine on the abortion rate and CL/P appearance in the offspring was analyzed with respect to the concentration of thiamine in the serum and amniotic fluid (HPLC-chromatography). Immunochemical analyses of the ThTr-1 und ThTr-2 receptor-status were performed in midface sections of A/WySn-fetuses and the corresponding placenta, with and without CL/P. RESULTS: High doses of orally supplemented thiamine did not reduce the CL/P appearance in A/WySn mice. However, the different starting points of vitamin B1 substitution had some influence. Additionally, an obvious decrease in aborted fetuses was noticed in all supplemented groups. The oral substitution caused a clear increase of the serum concentration in all mothers, but showed no increase of the amniotic fluid concentration. Then immunohistochemistry detected an overexpression of ThTr-1 in the midface and an irregular localization of ThTr-2 in the placenta of fetuses with clefts. CONCLUSION: Our results suggest a time-dependent influence of thiamine on CL/P appearance in female mice. The prophylactic/periconceptional, but not the therapeutic supplementation, starting point can be proposed as a crucial step for regular facial and palatal fusion in embryonic development. The absolute rate of CL/P was not reduced, and the concentration of the water-soluble thiamine could not increase in the amniotic fluid. Thus the proposed local effect of thiamine failed in the development of genetically determined mice.

The role of folate metabolism in orofacial development and clefting.

Folate deficiency has been associated with numerous diseases and birth defects including orofacial defects. However, whether folate has a role in the face during early orofacial development has been unclear. The present study reveals that pharmacological and antisense oligonucleotide mediated inhibition of DHFR, an integral enzyme in the folate pathway, results in specific changes in the size and shape of the midface and embryonic mouth. Such defects are accompanied by a severe reduction in the muscle and cartilage jaw elements without significant change in neural crest pattern or global levels of methylation. We propose that the orofacial defects associated with DHFR deficient function are the result of decreased cell proliferation and increased cell death via DNA damage. In particular, localized apoptosis may also be depleting the cells of the face that express crucial genes for the differentiation of the jaw structures. Folate supplementation is widely known to reduce human risk for orofacial clefts. In the present study, we show that activating folate metabolism can reduce median oral clefts in the primary palate by increasing cell survival. Moreover, we demonstrate that a minor decrease in DHFR function exacerbates median facial clefts caused by RAR inhibition. This work suggests that folate deficiencies could be a major contributing factor to multifactorial orofacial defects.

Folic acid rescue of ATRA-induced cleft palate by restoring the TGF-ß signal and inhibiting apoptosis.

BACKGROUND: Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF-ß signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all-trans retinoic acid (ATRA)-induced cleft palate in organ culture were tested. METHODS: C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFß3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis. RESULTS: Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase-9 in MEPM cells were detected in the ATRA group than in the control or the ATRA+FA group. Compared with the control or the ATRA+FA group, ATRA had little effect on TGF-ß3 in MEPM cells but significantly inhibited TGF-ß receptor II. CONCLUSIONS: Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFß signaling in MEPM cells were involved in folic acid rescued ATRA-induced cleft palate.

Biochemical alterations in the palatal processes in fetuses of biotin-deficient mice.

To clarify the role of biotin in palatal formation, we investigated the effects of biotin deficiency on the development of palatal processes in mouse fetuses at midgestation. We also investigated protein expressions in the palatal processes. Pregnant mice were given either a biotin-deficient diet or a biotin-supplemented (control) diet from day 0 of gestation (dg 0). Some dams in the biotin-deficient group were changed to a biotin-supplemented diet on dg 12, 13 or 14. On dg 15, the palatal processes were dissected from these fetuses and their peptides were characterized using two-dimensional electrophoresis and liquid chromatography/tandem mass spectrometry (LC-MS/MS) system. Regarding Trasler's stage for the growth of the palatal processes in mouse fetuses on dg 15, the average stage of palatal development was 5.83 +/- 0.39 in the biotin-supplemented group, 5.39 +/- 0.66 in the dg 13-supplemented group, and 4.64 +/- 0.90 in the biotin-deficient group. The development of the palatal processes significantly increased in relation to the earlier day of biotin supplementation. In a protein analysis of palatal processes by isoelectro focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 19-kDa spot was confirmed around position at pI 6-7 in the biotin-supplemented group, but this protein was not present in either the biotin-deficient group or the dg 13-supplemented group. From the MS/MS database of peptides, adenosine diphosphate (ADP)-ribosylation factor 2 (arf2) and alpha-crystallin were detected in the mesenchyme of the palatal processes. It is suggested that the expression of these proteins may be downregulated by biotin deficiency, inducing the inhibited development of palatal processes.

Molecular, clinical and political approaches to the problem of cleft lip and palate.

The oral facial complex in man appears to be exquisitively sensitive to genetic and environmental influences which is why clefts of the palate are the most common congenital birth anomaly. The development of the palate starts at about the 6th week of inter-uterine life and requires development of the palatal shelves from the maxillary processes of the first arch, shelf elevation, medial edge epithelial breakdown and mesenchyme flow with subsequent establishment of osteogenic and myogenic blastemata. This significant level of matrix turnover is partly regulated by the matrix metalloproteinases and potentially this could be affected by abnormalities in gene function. This may represent a common mechanism for a variety of different genes associated with clefting of the palate. The measurement of outcomes for children born with a cleft requires a wide input from a variety of specialities. The development of these outcome measures requires rigorous testing and validation, but it is now possible to use a variety of outcome measures to establish clinical standards and this has been done nationally. The impact of identifying a need for a change in organisation of service delivery was probably underestimated. It is clear that the current organisations in the National Health Service struggle to implement change, even with a detailed study and hard evidence. Reasons for this are outlined and a potential harder hitting strategy for effecting this change is outlined. The move towards primary care trusts within the latest reorganisation of the Health Service is potentially extremely damaging for specialised services for low incidence anomalies.

B group vitamins and cleft lip and cleft palate.

B group vitamins including folic acid supplementation during pregnancy have been shown to be effective in preventing cleft lip and palate (CLP) in humans. The clinical trials for the prevention of malformation have been mostly empirically based. The aim of the present study was directed toward the elucidation of the mechanisms underlying the preventive measures. The teratogenic potency of vitamin deficiency over the whole period of gestation (days 1-18) and of food restriction during the critical period of palatogenesis (days 12 and 13) were investigated in the genetically different strains of NMRI and A/WySn mice. Furthermore the potential benefit of vitamin B supplementation/treatment in the genetically susceptible CLP strain was demonstrated for comparison with former work on a teratogenetically induced cleft palate model. The results illustrate the higher susceptibility of the NMRI strain to the teratogenic action of deficiency (increase of the CP rate from 3.8% to 25%) in contrast to A/WySn mice, which actually have a high spontaneous but relative teratogenic-resistant clefting rate (28-44%). A deficiency of each of the individual B vitamins is teratogenic, however total B group deficiency has the strongest effect in the case of deficiency of all B vitamins. This produces up to 25% cleft palates in the NMRI strain. Alternatively, vitamin B group treatment in pregnant A/WySn mice did not substantially influence the clefting rate in contrast to our former experience in Halle:AB mice. The results may help to elucidate the interplay of genetic conditions and exogenous (nutritional) factors in both the aetiology and prevention of CLP. This may further clarify the role of the B vitamins in empiric preventive clinical trials.

Nonspecific stimulation of the maternal immune system. II. Effects on gene expression in the fetus.

BACKGROUND: Maternal immune stimulation reduces malformations caused by chemical teratogens. Mechanisms for this effect are not known. Altered expression of regulatory molecules (e.g., transforming growth factor [TGF-beta], tumor necrosis factor-alpha [TNF-alpha]) has been reported in fetuses from immunostimulated mice, which may affect gene expression. Expression of selected genes that function to control proliferation, differentiation, or apoptosis was evaluated in chemical-exposed fetuses, with or without maternal immunostimulation. METHODS: Ethyl carbamate (urethane) was given to pregnant ICR mice on day 10 of gestation to induce cleft palate. Before teratogen administration, the immune system of the female mice was stimulated by footpad injection with Freund's complete adjuvant (FCA) or by intraperitoneal injection with interferon-gamma (IFN-gamma). RESULTS: Maternal immunostimulation with interferon-gamma (IFN-gamma) decreased severity of the cleft palate lesion caused by urethane, while FCA decreased both incidence and severity of cleft palate. Gestation day 14 fetuses from urethane-exposed mothers displayed decreased expression of cell cycle/apoptotic genes bcl2alpha, bcl2beta, pkCalpha, and p53 in fetal heads. Immune stimulation with IFN-gamma-normalized expression of bcl2alpha, bcl2beta, and pkCalpha to control levels. Urethane also decreased the ratio of expression of bclalpha/p53, bclbeta/p53, and pkCalpha/p53, while maternal injection with IFN-gamma restored these expression ratios to control levels. Maternal immunization with FCA also significantly increased bcl2alpha/p53, bcl2beta/p53, and pkCalpha/p53 gene expression ratios. CONCLUSIONS: These results suggest that (1) the maternal immune system may possess heretofore unrecognized regulatory activity in fetal development, and (2) protection against urethane-induced cleft palate may be mediated through maternal immune regulation of fetal gene expression.

Further studies on protective effects of vitamins in cyclophosphamide-induced cleft palate.

The effects of vitamins B1, B6, and E administered 1 day before and simultaneously with an embryotoxic dose of cyclophosphamide (CPM) were studied using random-bred mice ICR (VELAZ). A single dose of 20 mg/kg CPM administered i.p. or i.m. on day 12 of pregnancy induced in the foetuses cleft palate and malformations of the upper and lower limbs. Both vitamins B1 and B6 decreased nonsignificantly the overall proportion of malformed foetuses and changed the spectrum of malformations, the decrease in foetuses with malformed upper limbs being statistically significant. In the groups supplemented with vitamins the mean foetal weight was higher in litters without malformed upper limbs than in litters with malformed upper limbs and those of both types in the control group. Similar effects of vitamin E were statistically nonsignificant. It is most likely that vitamins influence the pharmacokinetics of cyclophosphamide or its metabolites. The data provided offer no explanation for the claimed beneficial effects of vitamins for preventing the occurrence of orofacial clefts in patients at risk.

Epigenetically regulated genomic expressions for shortened stature and cleft palate are regionally specific in the 11-day mouse embryo.

Chronokinetic synergism, a holistic and extremely sensitive experimental design, has shown in the mouse embryo that site-specific epigenetic forces differentially regulate genesis of the palate (cleft palate) and limb bud organogenesis (shortened stature). Acute exposures of 11-day pregnant mice to minimally effective doses of thymidine or ethanol followed 5 or 8 hr later by minimal exposure to retinoic acid have enabled quantitative and qualitative assay for genomic-epigenetic interactions. These site-specific morphogenetic regulations occurred during palatal genesis from the maxillary prominence of the first pharyngeal arch and during limb bud prechondrogenesis. Thymidine is presumed to induce its response by inhibition of DNA polymerase and hence by transitory cytostatic block. (Embryo size was not detectably changed). Ethanol is interpreted, guilt by associated response, indirectly to interfere with histone regulation of transcription. Two central findings have demonstrated the coordinated regulation of genomic and epigenetic positional information. First, thymidine or ethanol as epigenetic probes for limb prechondrogenesis and palatal precursor cells have activated distinctive site-specific responses. Second, responses to chronokinetic synergisms have indicated that epigenetic regulators for limb and palate dysmorphogenesis may affect distinctly different phases of the cell division cycle and hence induce differential DNA expressions. Although each of palate and limb is concurrently susceptible to epigenetic regulation, their differential intrinsic genomic capabilities appear to have been uncoupled. The putative homeostatic balance of genomic expressions in the palate precursor and the prechondrogenic limb bud cells of the 11-day mouse embryo has been characterized as epigenetically regulated, alternatively expressed, and positionally restricted. We propose that the chronokinetic synergisms have disclosed the existence of distinctive palate-determining genes and stature-determining genes.