应用胚胎干细胞实验模型体系对异硫氰酸盐发育毒性的评价

Evaluation on developmental toxicity of isothiocyanate by embryonic stem cell test system

目的应用胚胎干细胞实验模型体系探讨苯甲基异硫氰酸盐(BITC)和苯乙基异硫氰酸盐(PEITC)的发育毒性。方法小鼠胚胎干细胞(ESC)经体外悬滴和悬浮培养分化获得心肌细胞及经视黄酸5×10-7mol·L-1诱导培养分化获得神经细胞,利用RT-PCR方法分析BITC和PEITC0,1,2,4和8μmol·L-1对ESC定向分化为特异表达肌球蛋白重链(MHC)基因的心肌细胞以及特异表达巢蛋白(巢蛋白)基因的神经细胞的影响,计算BITC和PEITC对ESC定向分化能力的抑制作用。同时应用MTT法测定BITC和PEITC对ESC129品系小鼠D3和BALB/c品系小鼠3T3细胞活性的抑制作用,结合定向分化抑制作用结果评价BITC和PEITC的发育毒性。结果 BITC和PEITC对ESC体外心肌细胞定向分化半数抑制浓度(ID50)为3.56和3.48mg·L-1,BITC和PEITC对ESC体外定向分化神经细胞的ID50分别为3.87和2.43mg·L-1,依据发育毒性评价公式计算得BITC和PEITC均无心肌发育毒性作用,BITC和PEITC的神经发育毒性作用分别为无和弱。结论 BITC和PEITC均无心肌发育毒性作用,但PEITC具有弱神经发育毒性作用。

OBJECTIVE To investigate effect of developmental toxicity of benzyl isothiocyanate(BITC)and phenethyl isothiocyanate(PEITC) by the embryonic stem cell test system.METHODS Using RT-PCR method,it was studied effect of BITC and PEITC 0,1,2,4 and 8 μmol·L-1 on embryonic stem cells(ESC) differentiating into cardiomyocyte with myosin heavy chain(MHC) expression after hanging and suspending culture and neuron cells with nestin expression after being induced by retinoic acid 5×10-7 mol·L-1.Together with the results of D3 and 3T3 cells viability assessed by MTT,the developmental toxicity characteristics of BITC and PEITC were identified clearly.RESULTS The half-maximal differentiation-inhibitory concentration(ID50) of BITC and PEITC on D3 differentiating into MHC[ ID50(D3,MHC)] was 3.56 mg·L-1 and 3.87 mg·L-1.ID50 of BITC and PEITC on D3 differentiating into nestin[ ID50(D3,nestin)] was 3.48 mg·L-1 and 2.43 mg·L-1.So both BITC and PEITC were identified as no embryotoxicity,but PEITC was judged as weak-developmental neurotoxicity.CONCLUSION BITC and PEITC have no embryotoxicity,but the emphasis that PEITC may cause developmental neurotoxicity should be focused on.