Jian Yuanzhi, Wang Fei, Yin Ning, Zhou Ruoyu, Wang Junbo
Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing 100191, China.
Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing 100191, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2024 Apr 18;56(2):213-222. doi: 10.19723/j.issn.1671-167X.2024.02.003.
To evaluate the developmental toxicity of Cry1Ab protein by studying its effects on cell proliferation and differentiation ability using a developmental toxicity assessment model based on embryonic stem-cell.
Cry1Ab protein was tested in seven dose groups (31.25, 62.50, 125.00, 250.00, 320.00, 1 000.00, and 2 000.00 μg/L) on mouse embryonic stem cells D3 (ES-D3) and 3T3 mouse fibroblast cells, with 5-fluorouracil (5-FU) used as the positive control and phosphate buffer saline (PBS) as the solvent control. Cell viability was detected by CCK-8 assay to calculate the 50% inhibitory concentration (IC) of the test substance for different cells. Additionally, Cry1Ab protein was tested in five dose groups (125.00, 250.00, 320.00, 1 000.00, and 2 000.00 μg/L) on ES-D3 cells, with PBS as the solvent control and 5-FU used for model validation. After cell treatment, cardiac differentiation was induced using the embryonic bodies (EBs) culture method. The growth of EBs was observed under a microscope, and their diameters on the third and fifth days were measured. The proportion of EBs differentiating into beating cardiomyocytes was recorded, and the 50% inhibition concentration of differentiation (ID) was calculated. Based on a developmental toxicity discrimination function, the developmental toxicity of the test substances was classified. Furthermore, at the end of the culture period, mRNA expression levels of cardiac differentiation-related markers (Oct3/4, GATA-4, Nkx2.5, and β-MHC) were quantitatively detected using real-time quantitative polymerase chain reaction (qPCR) in the collected EBs samples.
The IC of 5-FU was determined as 46.37 μg/L in 3T3 cells and 32.67 μg/L in ES-D3 cells, while the ID in ES-D3 cells was 21.28 μg/L. According to the discrimination function results, 5-FU was classified as a strong embryotoxic substance. There were no statistically significant differences in cell viability between different concentrations of Cry1Ab protein treatment groups and the control group in both 3T3 cells and ES-D3 cells (>0.05). Moreover, there were no statistically significant differences in the diameter of EBs on the third and fifth days, as well as their morphology, between the Cry1Ab protein treatment groups and the control group (>0.05). The cardiac differentiation rate showed no statistically significant differences between different concentrations of Cry1Ab protein treatment groups and the control group (>0.05). 5-FU significantly reduced the mRNA expression levels of β-MHC, Nkx2.5, and GATA-4 ( < 0.05), showing a dose-dependent trend ( < 0.05), while the mRNA expression levels of the pluripotency-associated marker Oct3/4 exhibited an increasing trend ( < 0.05). However, there were no statistically significant differences in the mRNA expression levels of mature cardiac marker β-MHC, early cardiac differentiation marker Nkx2.5 and GATA-4, and pluripotency-associated marker Oct3/4 between the Cry1Ab protein treatment groups and the control group (>0.05).
No developmental toxicity of Cry1Ab protein at concentrations ranging from 31.25 to 2 000.00 μg/L was observed in this experimental model.
通过使用基于胚胎干细胞的发育毒性评估模型，研究Cry1Ab蛋白对细胞增殖和分化能力的影响，以评估其发育毒性。
在七个剂量组（31.25、62.50、125.00、250.00、320.00、1000.00和2000.00μg/L）中对小鼠胚胎干细胞D3（ES-D3）和3T3小鼠成纤维细胞进行Cry1Ab蛋白测试，以5-氟尿嘧啶（5-FU）作为阳性对照，磷酸盐缓冲盐水（PBS）作为溶剂对照。通过CCK-8法检测细胞活力，以计算受试物质对不同细胞的50%抑制浓度（IC）。此外，在五个剂量组（125.00、250.00、320.00、1000.00和2000.00μg/L）中对ES-D3细胞进行Cry1Ab蛋白测试，以PBS作为溶剂对照，5-FU用于模型验证。细胞处理后，采用胚体（EBs）培养法诱导心脏分化。在显微镜下观察EBs的生长情况，并测量其在第三天和第五天的直径。记录分化为跳动心肌细胞的EBs比例，并计算50%分化抑制浓度（ID）。根据发育毒性判别函数对受试物质的发育毒性进行分类。此外，在培养期结束时，使用实时定量聚合酶链反应（qPCR）对收集的EBs样本中与心脏分化相关的标志物（Oct3/4、GATA-4、Nkx2.5和β-MHC）的mRNA表达水平进行定量检测。
5-FU在3T3细胞中的IC为46.37μg/L，在ES-D3细胞中的IC为32.67μg/L，而ES-D3细胞中的ID为21.28μg/L。根据判别函数结果，5-FU被分类为强胚胎毒性物质。在3T3细胞和ES-D3细胞中，不同浓度的Cry1Ab蛋白处理组与对照组之间的细胞活力无统计学显著差异（>0.05）。此外，Cry1Ab蛋白处理组与对照组之间在第三天和第五天的EBs直径及其形态方面无统计学显著差异（>0.05）。不同浓度的Cry1Ab蛋白处理组与对照组之间的心脏分化率无统计学显著差异（>0.05）。5-FU显著降低了β-MHC、Nkx2.5和GATA-4的mRNA表达水平（<0.05），呈剂量依赖性趋势（<0.05），而多能性相关标志物Oct3/4的mRNA表达水平呈上升趋势（<0.05）。然而，Cry1Ab蛋白处理组与对照组之间在成熟心脏标志物β-MHC、早期心脏分化标志物Nkx2.5和GATA-4以及多能性相关标志物Oct3/4的mRNA表达水平方面无统计学显著差异（>0.05）。
在该实验模型中，未观察到浓度范围为31.25至2000.00μg/L的Cry1Ab蛋白具有发育毒性。
