Biotin deficiency affects the proliferation of human embryonic palatal mesenchymal cells in culture.

It has recently been demonstrated that pregnancy in women may cause mild biotin deficiency without any clinical signs. However, the teratogenicity of biotin deficiency in humans has not been well investigated. On the other hand, our previous studies have shown that maternal biotin deficiency induces many kinds of malformations, such as cleft palate, micrognathia, and micromelia, in all animal fetuses. However the mechanism for cleft palate induction under biotin-deficient conditions is unknown. Therefore, to investigate the possible mechanisms for cleft palate induction in embryos, we investigated the effects of biotin deficiency on human embryonic palatal mesenchymal (HEPM) cells in culture in this study. HEPM cells were cultured in biotin-deficient and biotin-physiological (control) media for 5 wk. The proliferative availabilities of HEPM cells in the biotin-deficient state were significantly lower after wk 2 of culture (41.3% of the control). Biotin concentrations in biotin-deficient cells were drastically lower after wk 1 of culture, whereas those in the control cells remained at almost the same level. Biotinidase activities were also lower in biotin-deficient cells. Holocarboxylases in biotin-deficient cells were fewer after the first week of culture and were almost undetectable after wk 2. The amount of biotinylated histones in the nuclei of biotin-deficient cells was lower than in the control cells. This suppressed proliferation of mesenchymal cells may delay or inhibit the growth of palatal processes in embryos and thus it may partially contribute to the mechanisms for cleft palate induction.

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.