A toxicology study to evaluate the embryotoxicity of metformin compared with the hypoglycemic drugs, the anticancer drug, the anti‐epileptic drug, the antibiotic, and the cyclo‐oxygenase (COX)‐2 inhibitor

Background The safe use of medications in pregnant females, their embryos and in offspring is important. The aim of the present study was to evaluate embryotoxicity of metformin (MET) compared with other hypoglycemic drugs (rosiglitazone [RSG] and glimepiride [GLIM]), the anticancer drug 5-fluorouracil (5-FU), the anti-epileptic drug diphenylhydantoin (DPH), the antibiotic penicillin G (PenG), and the cyclo-oxygenase (COX)-2 inhibitor nimesulide (NIM) in an embryonic stem cell test (EST). Methods Differences in the expression of developmental marker genes following treatment with the test compounds during the course of differentiation (from embryonic stem cell D3 (D3 cells) to myocardial cells) were determined using real-time quantitative polymerase chain reaction. In these studies, 5-FU was used as a positive control and PenG was used as a negative control. The cytotoxicity of these drugs against D3 cells and 3T3 fibroblasts was determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Embryotoxicity was classified according to the prediction model of EST. Results At concentrations >800 μg/mL MET had a greater cytotoxic effect on D3 cells than 3T3 fibroblasts. At the highest concentration of MET (5 mg/mL), the cell viability of D3 cells and 3T3 fibroblasts was 30%, respectively. The size of the embryonic body (EB) differentiation area was almost the same over the concentration range 50–200 μg/mL MET, and there was no significant difference in EB differentiation area until a concentration of 400 μg/mL MET. At a concentration of 800 μg/mL MET, the size of EB outgrowth was significantly reduced. The same assays revealed GLIM, RSG, and NIM to be weakly embryotoxic substances. Conclusions Based on the EST, MET can be classified as a weakly embryotoxic substance, which suggests that it should be prescribed with caution to pregnant women with gestational diabetes. 摘要 背景： 孕期妇女及其胚胎和后代的安全用药十分重要。本研究的目的是利用胚胎干细胞试验方法（embryonic stem cell test，EST）评价二甲双胍（embryotoxicity of metformin，MET）的胚胎毒性，并与其他类的降糖药（罗格列酮和格列美脲），抗癌药（5-氟尿嘧啶），抗癫痫药（苯妥英），抗菌药（青霉素G），以及环氧合酶-2抑制剂（尼美舒利）进行比较。 方法： 采用实时定量聚合酶链式反应测定在细胞分化过程（从胚胎干细胞D3分化为心肌细胞）期间使用受试化合物处理后发育性标志物基因的表达差异。研究中将5-氟尿嘧啶作为阳性对照，将青霉素G作为阴性对照。利用MTT法检测药物对D3细胞和3T3成纤维细胞的细胞毒性。根据EST预测模型对药物的胚胎毒性进行分级。 结果： MET浓度高于800 μg/mL时，与3T3细胞相比MET对D3细胞具有更大的细胞毒性。当MET达到最高浓度（5 mg/mL）时，D3细胞和3T3成纤维细胞的细胞活力分别低于10%和高于30%。MET的浓度范围在50-200 μg/mL时，内胚胎体分化面积大小几乎相同，无显著性差异，直到MET的浓度高于400 μg/mL后才出现明显差异。当MET的浓度为800 μg/mL时，内胚胎体分化集落明显减小。采用相同的试验方法揭示格列美脲、罗格列酮和尼美舒利均为弱胚胎毒性物质。 结论： 基于EST法MET可被分级为弱胚胎毒性物质，提示患妊娠糖尿病的孕期妇女应谨慎使用。