利用全胚胎和微团培养模型评价硝酸镧的发育毒性

目的： 利用大鼠植入后全胚胎培养模型和微团培养模型评价硝酸镧潜在的胚胎早期和中、晚期发育毒性。方法： 将大鼠胚胎随机分为5组后培养于含不同剂量硝酸镧（0、0.12、0.23、0.46及1 mmol/L）的即刻离心血清中，48 h后测量胚胎卵黄囊直径（YSD）、顶臀长（CRL）和头长（HL），计数体节，并根据Brown's评分法对胚胎进行总形态学评分（TMS）。同时，对BALB/c 3T3细胞进行细胞毒性测试。根据欧洲替代方法验证中心（ECVAM）的预测模型对硝酸镧的胚胎早期发育毒性进行评价。分离大鼠胚胎肢芽细胞，制成单细胞悬液，进行微团培养。分别用0.03、0.06、0.13、0.25、0.5、1、2及3 mmol/L硝酸镧进行染毒，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）以反映细胞增殖情况，利用阿利新蓝染色法测定50%细胞分化受抑制时的浓度（ID₅₀）以反映细胞分化情况。根据ECVAM预测模型对硝酸镧胚胎中晚期发育毒性进行评价。结果： 硝酸镧对胚胎生长的无可见有害作用水平（NOAEL）为0.12 mmol/L，0.46 mmol/L硝酸镧可明显降低胚胎YSD、CRL和HL（P < 0.05），对胚胎生长有明显的抑制效应。0.46 mmol/L硝酸镧可诱导胚胎产生发育畸形，减少胚胎体节形成数目（P < 0.05）。硝酸镧对肢芽细胞增殖活性的NOAEL为1 mmol/L，对肢芽细胞分化为软骨细胞的NOAEL为0.25 mmol/L，其IC₅₀和ID₅₀分别为1.57 mmol/L（510.25 μg/mL）和0.99 mmol/L（321.75 μg/mL）。结论： 经全胚胎培养预测模型评价硝酸镧为弱胚胎发育毒性化学物；经微团培养模型评价硝酸镧为无胚胎发育毒性化学物。

Developmental toxicity of lanthanum nitrate in post-implantation whole embryo culture and micromass test

OBJECTIVE: To assess the potential developmental toxicity of lanthanum nitrate at the middle and advanced stage of gestation in post-implantation whole embryo culture and micromass test. METHODS: Rat embryos were randomly divided into 5 groups and cultured in immediately centrifuged serum,and were exposed to different doses of lanthanum nitrate (0,0.12,0.23,0.46 and 1 mmol/L). After 48 hours culture,yolk sac diameter (YSD),crown-rump length (CRL) and head length (HL) were measured, somites were counted, and the total morphological score (TMS) was calculated according to the Brown's Method to evaluated growth and functional development of embryos. Cytotoxicity in BALB/c 3T3 cells was tested by MTT. Toxicity of lanthanum nitrate in the early embryonic stage was evaluated using the prediction models of ECVAM. In addition,limb bud cells of embryos were isolated from matured SD rats and cultured in micro-mass culture for 5 days. These cultures were exposed to 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2 and 3 mmol/L lanthanum nitrate. The 50% inhibition of cell viability and growth (IC₅₀) of limb bud cells was determined by neutral red staining, and the 50% inhibition of cells differentiation (ID₅₀) was measured by alcian blue staining. Toxicity of lanthanum nitrate in the middle and advanced stages of embryos was evaluated with the prediction model of ECVAM. RESULTS: The no observed adverse effect level (NOAEL) of lanthanum nitrate on embryo growth was 0.12 mmol/L. At the concentration of 0.46 mmol/L,lanthanum nitrate decreased the YSD,CRL and HL of embryos (P < 0.05),which showed that lanthanum nitrate significantly retarded growth of embryos. Besides, 0.46 mmol/L lanthanum nitrate induced developmental malformations and reduced the number of somites (P < 0.05). The NOAEL of lanthanum nitrate on the proliferation of limb bud was 1 mmol/L, the NOAEL on chondrocyte differentiation from limb bud cells was 0.25 mmol/L,the IC₅₀ and ID₅₀ for limb bud cells were 1.57 mmol/L (510.25 μg/mL) and 0.99 mmol/L (321.75 μg/mL),respectively. CONCLUSION: Lanthanum nitrate was evaluated as a weak embryotoxic chemical by whole embryo culture and as a nonembryotoxic chemical by micromass test.