Ambroso J L, Harris C
Department of Environmental and Industrial Health, University of Michigan, Ann Arbor 48109-2029.
Biochem Pharmacol. 1994 Feb 11;47(4):679-88. doi: 10.1016/0006-2952(94)90131-7.
The teratogenicity of chloroquine (CQ) has been hypothesized to result from its effects on lysosomal function, specifically the ability of the visceral yolk sac (VYS) to capture and degrade external macromolecules. Using the rat whole embryo culture system, we evaluated the ability of CQ to accumulate in conceptual tissues and its effects on aspects of VYS function known to be important in the uptake and processing of nutrients. When CQ was added directly to the culture medium, it was found to accumulate rapidly in conceptual tissues, particularly the VYS. Tissue concentrations of CQ in the embryo proper reached approximately 10-fold those in the medium, whereas concentrations in the VYS exceeded by 100-fold the medium concentration within a 4-hr exposure on gestational day (GD) 10. Embryotoxic concentrations of CQ (10-30 microM) enhanced the activity of lysosomal cysteine proteinases measured in vitro under optimum pH conditions in both embryonic and VYS homogenates after a 26-hr treatment from GD 10-11. A different pattern of response in enzyme activity was observed between embryos and VYSs that could be attributed to the preferential accumulation of CQ in the VYS. Nonembryotoxic concentrations of CQ (1-7.5 microM) induced a concentration-dependent increase in VYS enzyme activity that peaked in conceptuses exposed to 20 microM CQ (an intermediate embryotoxic concentration). The enhanced cysteine proteinase activity was time dependent and appeared to increase gradually in conceptuses exposed to 10-20 microM CQ during the 26-hr culture period. This was in contrast to the rapid accumulation of CQ in conceptual tissues seen on gestational day 10. Protein content in the VYS was increased significantly after a 9-hr exposure of whole conceptuses to CQ (20 microM), indicating an inhibition of VYS proteolytic activity in situ. After 24 hr of exposure to 20 microM CQ, VYS protein content was not significantly different from control, but embryonic protein was reduced significantly by 20%. These observations are consistent with a model of reversible inhibition of VYS proteolysis by CQ followed by a compensatory increase in lysosomal proteinase activity. VYS fluid-phase pinocytosis was also assessed after CQ exposure and found to be inhibited only in the highest CQ concentration tested (30 microM). Lower concentrations of CQ that were still embryotoxic (10-20 microM) did not affect VYS fluid-phase pinocytosis, suggesting that inhibition of this activity is not primarily responsible for CQ embryotoxicity.
氯喹（CQ）的致畸性被认为是其对溶酶体功能产生影响的结果，特别是内脏卵黄囊（VYS）捕获和降解外部大分子的能力。我们使用大鼠全胚胎培养系统，评估了CQ在胚胎组织中的蓄积能力及其对VYS功能中已知在营养物质摄取和加工方面很重要的各个方面的影响。当将CQ直接添加到培养基中时，发现它在胚胎组织中迅速蓄积，尤其是在VYS中。胚胎本身的CQ组织浓度达到培养基中的约10倍，而在妊娠第10天暴露4小时后，VYS中的浓度超过培养基浓度100倍。在妊娠第10天至11天进行26小时处理后，胚胎毒性浓度的CQ（10 - 30 microM）增强了在最佳pH条件下体外测定的胚胎和VYS匀浆中溶酶体半胱氨酸蛋白酶的活性。胚胎和VYS之间观察到不同的酶活性反应模式，这可能归因于CQ在VYS中的优先蓄积。非胚胎毒性浓度的CQ（1 - 7.5 microM）诱导VYS酶活性呈浓度依赖性增加，在暴露于20 microM CQ（中等胚胎毒性浓度）的胚胎中达到峰值。增强的半胱氨酸蛋白酶活性是时间依赖性的，并且在26小时培养期内暴露于10 - 20 microM CQ的胚胎中似乎逐渐增加。这与妊娠第10天在胚胎组织中观察到的CQ快速蓄积形成对比。将整个胚胎暴露于CQ（20 microM）9小时后，VYS中的蛋白质含量显著增加，表明原位VYS蛋白水解活性受到抑制。暴露于20 microM CQ 24小时后，VYS蛋白质含量与对照无显著差异，但胚胎蛋白质显著减少20％。这些观察结果与CQ对VYS蛋白水解的可逆抑制模型一致，随后溶酶体蛋白酶活性出现代偿性增加。在CQ暴露后还评估了VYS液相胞饮作用，发现仅在测试的最高CQ浓度（30 microM）下受到抑制。仍然具有胚胎毒性的较低浓度CQ（10 - 20 microM）不影响VYS液相胞饮作用，这表明该活性的抑制不是CQ胚胎毒性的主要原因。
