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Journal of Peking University (Health Sciences) ›› 2024, Vol. 56 ›› Issue (2): 213-222. doi: 10.19723/j.issn.1671-167X.2024.02.003

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

Yuanzhi JIAN1,Fei WANG1,Ning YIN1,Ruoyu ZHOU1,Junbo WANG1,2,*()   

  1. 1. Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing 100191, China
    2. Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing 100191, China
  • Received:2023-04-06 Online:2024-04-18 Published:2024-04-10
  • Contact: Junbo WANG E-mail:bmuwjbxy@bjmu.edu.cn
  • Supported by:
    Science and Technology Innovation 2030-Major Project(2023ZD0406305-04)

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Abstract:

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.

Key words: Cry1Ab protein, Developmental toxicity, Cardiac myocytes, Cell differentiation, Em-bryonic stem cells

CLC Number: 

  • R155.5

Table 1

Primer sequence of myocardial differentiation marker"

PrimerSequences (5′ to 3′)
β-MHCForward: CCTGCGGAAGTCTGAGAAGG
Reverse: CTCGGGACACGATCTTGGC
GATA-4Forward: CACCCCAATCTCGATATGTTTGA
Reverse: GCACAGGTAGTGTCCCGTC
Nkx2.5Forward: GACAAAGCCGAGACGGATGG
Reverse: CTGTCGCTTGCACTTGTAGC
Oct3/4Forward: CGGAAGAGAAAGCGAACTAGC
Reverse: ATTGGCGATGTGAGTGATCTG
GAPDHForward: CGTCCCGTAGACAAAATGGT
Reverse: TTGATGGCAACAATCTCCAC

Figure 1

Morphological observation of ES-D3 and MEF cells The figure shows the co-culture morphology of MEF cells and ES-D3 cells, with ES-D3 cells on the upper layer and MEF cells on the lower layer. The elliptical cell colonies indicated by the black circles in the image represent ES-D3 cells, while the region indicated by the black arrows represents the underlying MEF cells. The observation was made using a 100× optical microscope. MEF, mouse embryo fibroblast; ES-D3, embryonic stem cell line D3."

Figure 2

The myocardial differentiation process of ES-D3 A, cell suspension droplets prepared from ES-D3 cells; B, cell suspension droplets cultured for 3 days; C, embryoid embryos cultured for 3 days under a microscope; D, embryoid embryos cultured for 5-6 days; E, embryoid embryos adherent cultured for 5-6 days; F, embryoid embryos cultured for 16 days, and the arrow shows the myocardial beating area. ES-D3, embryonic stem cell line D3."

Figure 3

Effect of 5-FU on 3T3 cells (A) and ES-D3 (B) proliferation (n=18) 5-FU, 5-fluorouracil; ES-D3, embryonic stem cell line D3. # P < 0.01, vs. control (0 μg/L)."

Figure 4

Effect of Cry1Ab protein on 3T3 cells (A) and ES-D3 (B) proliferation (n=18) ES-D3, embryonic stem cell line D3."

Figure 5

Effect of 5-FU protein on the diameter of EBs on the 3rd (A) and 5th (B) day (n=24) # P < 0.01 vs. control (0 μg/L). 5-FU, 5-fluorouracil; EBs, embryonic bodies."

Figure 6

Effect of Cry1Ab protein on the diameter of EBs on the 3rd (A) and 5th (B) day (n=24) EBs, embryonic bodies."

Figure 7

Effects of 5-FU and Cry1Ab protein at various concentrations on the adhering growth of EBs The figure shows the growth status of EBs in each group on the 16th day of culture, where the central black cell cluster represents the high-density aggregation area of ES-D3, and the outer layer consists of ES-D3 cells forming a multi-layered structure. 5-FU, 5-fluorouracil; EBs, embryonic bodies; ES-D3, embryonic stem cell line D3."

Figure 8

Effect of 5-FU (A) and Cry1Ab protein (B) on ES-D3 myocardial differentiation (n=3) # P < 0.01 vs. control (0 μg/L). 5-FU, 5-fluorouracil; ES-D3, embryonic stem cell line D3."

Figure 9

Effect of 5-FU on the expression of ES-D3 myocardial differentiation markers (n=3) A, β-MHC; B, Nkx2.5; C, GATA-4; D, Oct3/4. *P < 0.05, # P < 0.01 vs. control (0 μg/L). 5-FU, 5-fluorouracil; ES-D3, embryonic stem cell line D3."

Figure 10

Effect of Cry1Ab protein on the expression of ES-D3 myocardial differentiation markers (n=3) A, β-MHC; B, Nkx2.5; C, GATA-4; D, Oct3/4. ES-D3, embryonic stem cell line D3."

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