利用全胚胎和微团培养模型评价硝酸铈的发育毒性

doi:10.3969/j.issn.1004-616x.2021.03.004

摘要/Abstract

摘要： 目的：利用大鼠植入后全胚胎培养模型和微团培养模型模拟胚胎发育早期和中、晚期过程，评价硝酸铈的发育毒性，以期为进一步开展体内实验研究及合理制定人群稀土接触的健康指导值提供科学依据。方法：取孕9.5 d的SD大鼠胚胎，分别在含0.00、0.50、0.75、1.00 mmol/L硝酸铈的大鼠即刻离心血清中37℃旋转培养，48 h后根据Brown's评分法对胚胎的生长发育及形态功能进行评分，结合BALB/c 3T3细胞毒性结果，利用欧洲替代方法验证中心（ECVAM）全胚胎预测模型评价硝酸铈胚胎早期发育毒性。分离孕13 d的SD大鼠胚胎肢芽原代细胞进行微团培养，分别用0.03、0.06、0.13、0.25、0.50、1.00、2.00和3.00 mmol/L硝酸铈进行染毒，培养5 d后利用中性红活细胞摄取染色法测定50%细胞增殖受抑制时的浓度（IC50），利用阿利新蓝染色法测定50%细胞分化受抑制时的浓度（ID50），根据ECVAM微团培养预测模型评价硝酸铈胚胎中、晚期发育毒性。结果：全胚胎培养模型中硝酸铈对胚胎生长的无可见有害作用水平（NOAEL）为0.50 mmol/L，0.75 mmol/L以上浓度硝酸铈可显著降低胚胎卵黄囊直径和顶臀长（P < 0.05），抑制胚胎生长，并可致胚胎体节数目减少（P < 0.05），胚胎发育畸形。微团培养模型中硝酸铈对肢芽细胞增殖活性的NOAEL为0.25 mmol/L，对肢芽细胞分化为软骨细胞的NOAEL为0.12 mmol/L，其IC₅₀和ID₅₀分别为1.23和0.76 mmol/L。结论：经全胚胎培养预测模型和微团培养模型评价硝酸铈为弱发育毒性化学物。

关键词: 硝酸铈, 全胚胎培养, 微团培养, 发育毒性

Abstract: OBJECTIVE： To evaluate developmental toxicity of cerium nitrate with in vitro postimplantation whole embryo cultures (representing early，middle and late embryonic developments) using a micromass test. METHODS： Embryos of GD 9.5 SD rats were cultured at 37 ℃ for 48 h in immediately centrifuged serum which contained different doses of cerium nitrate (0.00， 0.50， 0.75 and 1.00 mmol/L). Growth，development and total morphological scores (TMS) of the embryos were documented according to the Brown's Method. Results of BALB/c 3T3 cytotoxicity and of the early embryonic development toxicity of cerium nitrate were evaluated using the whole embryo prediction model by ECVAM. In addition， limb bud cells of embryos were isolated from GD13 SD rats and cultured in micro-mass culture for 5 days with 0.03，0.06， 0.13， 0.25， 0.50， 1.00， 2.00 and 3.00 mmol/L cerium nitrate. The 50% inhibitions of cell viability and growth (IC₅₀) of limb bud cells were determined by neutral red staining and the 50% inhibition of cells differentiation (ID₅₀) was measured by alcian blue staining. The embryonic toxicities of cerium nitrate in the middle and advanced stages of embryos were evaluated with the prediction model of the micro-mass test (ECVAM). RESULTS： In the in vitro post-implantation whole embryo culture test，the no-observed-adverseeffect level (NOAEL) of cerium nitrate on embryo growth was 0.50 mmol/L. Cerium nitrate above 0.75 mmol/L inhibited embryonic growth， and reduced the yolk sac diameter (YSD) and crown-rump length (CRL) significantly (P < 0.05). Cerium nitrate at 0.75 mmol/L also reduced the number of somites (P < 0.05) which indicated developmental malformation of embryos. In the micro- mass test， the NOAEL of cerium nitrate on proliferation of limb buds was 0.25 mmol/L，the NOAEL on differentiation from limb bud cells to chondrocytes was 0.12 mmol/L， the IC50 and ID50 for limb bud cells were 1.23 and 0.76 mmol/L， respectively. CONCLUSION： Based on the prediction model of whole embryo cultures and the micro- mass test， our results indicate that cerium nitrate was a weak embryotoxic chemical.

Key words: cerium nitrate, whole embryo culture, micromass test, developmental toxicity

中图分类号:

994.6

刘青云, 康陈萍, 肖倩倩, 郝卫东. 利用全胚胎和微团培养模型评价硝酸铈的发育毒性[J]. 癌变·畸变·突变, 2021, 33(3): 178-186,207.

LIU Qingyun, KANG Chenping, XIAO Qianqian, HAO Weidong. Developmental toxicity of cerium nitrate in post-implantation whole embryo cultures using a micro-mass test[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2021, 33(3): 178-186,207.

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