[Developmental toxicity of Cry1Ab protein in the embryonic stem-cell model].

OBJECTIVE: 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.

METHODS: 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 (IC50 ) 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 (ID50 ) 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.

RESULTS: The IC50 of 5-FU was determined as 46.37 μg/L in 3T3 cells and 32.67 μg/L in ES-D3 cells, while the ID50 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 ( P >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 ( P >0.05). The cardiac differentiation rate showed no statistically significant differences between different concentrations of Cry1Ab protein treatment groups and the control group ( P >0.05). 5-FU significantly reduced the mRNA expression levels of β-MHC, Nkx2.5, and GATA-4 ( P < 0.05), showing a dose-dependent trend ( P < 0.05), while the mRNA expression levels of the pluripotency-associated marker Oct3/4 exhibited an increasing trend ( P < 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 ( P >0.05).

CONCLUSION: No developmental toxicity of Cry1Ab protein at concentrations ranging from 31.25 to 2 000.00 μg/L was observed in this experimental model.