Chloroquine accumulation and alterations of proteolysis and pinocytosis in the rat conceptus in vitro

Abstract

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 embroyo culture system, we evaluated the ability of CQ to accumulate in conceptal 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 conceptal tissues, particuarly 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 μM) 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 μM) induced a concentration-dependent increase in VYS enzyme activity that peaked in conceptuses exposed to 20 μM CQ (an intermediate embryotoxic concentration). The enhanced cysteine proteinase activity was time dependent and appeared to increase gradually in conceptuses exposed to 10–20 μM CQ during the 26-hr culture period. This was in contrast to the rapid accumulation of CQ in conceptal 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 μM), indicating an inhibition of VYS proteolytic activity in situ. After 24 hr of exposure to 20μM 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 μM). Lower concentrations of CQ that were still embryotoxic (10–20 μM) did not affect VYS fluid-phase pinocytosis, suggesting that inhibition of this activity is not primarily responsible for CQ embryotoxicity.