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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.

Discussion

In this study, HEPM cells were cultured in control and biotin-deficient media for 5 wk and the cell proliferation rates were measured every week. The proliferation of biotin-deficient cells was significantly lower after wk 2 of culture than that of the control cells. Crisp et al. (18) have shown decreased cell proliferation rates of human choriocarcinoma JAr cells under biotin-deficient cell culture conditions and this result is consistent with our results. In contrast, the cell proliferation rates of small cell lung cancer NCI-H69 cells (19) and Jurkat cells (20) did not decrease under biotin-deficient culture conditions. This suggests that the susceptibilities differ among the cell lines and that cell proliferative availability may be specifically affected by biotin deficiency.

On the other hand, some studies have revealed that biotin is associated positively with cell proliferation. Zempleni and Mock (21,22) have shown that mitogen-stimulated proliferating peripheral blood mononuclear cells (PBMC) and human lymphocytes accumulated biotin significantly faster than unstimulated controls. For example, when proliferation was stimulated by incubation with pokeweed mitogen for 3 d, biotin uptake increased in a dose-dependent fashion from 481 to 722% of the control value. Other evidence has been provided by a basic study reporting the significant stimulation of BHK cell growth in culture by free biotin, which is not protein bound and is thus more bioavailable (23). This study has also shown that free biotin was found in relatively large amounts in fetal sera and only a very small percentage of free biotin was contained in adult sera and was characterized by lower growth-stimulating activity. In addition, Mantagos et al. (10) reported that human fetal plasma contained more biotin [784 ± 327 ng/L (3.21 ± 1.34 nmol/L)] than maternal plasma [131 ± 102 ng/L (0.54 ± 0.42 nmol/L)]. These studies suggest that biotin utilization is significantly greater in the fetus, which may be consistent with our previous studies. We showed a high incidence of fetal malformations caused by biotin deficiency, whereas the pregnant dams showed no specific signs of biotin deficiency (6,8,24-27).

In conclusion, the proliferative availability of HEPM cells in this study was lower in biotin-deficient cells than in control cells. The biotinylation of biotin-dependent carboxylases and histones was also drastically lower in biotin-deficient cells than in control cells, which resulted from limited access to intracellular biotin. Biotin deficiency may cause cleft palate via delayed or arrested growth of the embryonic palate by the suppressed cell proliferation of HEPM cells. Therefore, this study has revealed a possible mechanism for cleft palate induction by biotin deficiency.