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 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.

Oxidative Stress and Autophagy in the Regulation of Lysosome-Dependent Neuron Death

“…Alternatively, chloroquine-induced oxidative damage to lysosomal membranes and the accumulation of oxidatively modified lipoproteins may result from macroautophagy inhibition combined with its inhibition of lysosomal proteases (4,43,53,62,163) mechanisms that may be responsible for its induction of lipofuscin, as previously described (70). Chloroquine also was shown recently to reduce intracellular levels of glutathione (110), which could lead to an increased production of cytosolic hydrogen peroxide and concomitant extralysosomal damage of macromolecules and organelle membranes.…”

“…The intralysosomal accumulation of chloroquine has been shown to induce profound alterations in lysosome function, including inhibition of both the proteolytic maturation and enzyme activities of CB and CD (4,43,53,62,101,163), which may be secondary to chloroquine-induced increase in intralysosomal pH and disruption of pH optima for these enzymes. In our laboratory, we observed similar results in SH-SY5Y cells, such that a death-inducing concentration of chloroquine markedly decreases maturation of CD, as measured by Western blot (Fig.…”

Comparative Response of Rat and Rabbit Conceptuses In Vitro to Inhibitors of Histiotrophic Nutrition

“…Because the rodent embryo cannot utilize proteins supplied by the dam, the endocytosed protein component of the histiotroph is catabolized to amino acids by lysosomal proteases (Freeman et al., ; Freeman and Lloyd, , ; Ambroso et al., ). Cysteine proteases play a critical role in this process from early postimplantation through late gestation (Freeman and Lloyd, ; Beckman et al., ), and inhibition of protease activity reduces VYS protein catabolism and embryonic protein synthesis resulting in teratogenicity both in vitro and in vivo (Beck and Lowy, ; Freeman and Lloyd, ; Freeman and Brown, ; Daston et al., ; Ambroso and Harris, , ; Sant et al., ). Administration of the cysteine protease inhibitor leupeptin to pregnant rats on GD 8.5 to 9.5 increased the incidence of resorptions and malformations (Freeman and Lloyd, ).…”

Whole embryo culture: a “New” technique that enabled decades of mechanistic discoveries

“…This link between inhibition of lysosomal proteinases and altered fluid balance in the embryo is especially intriguing, as such ''blisters'' are commonly observed in response to toxicant treatment in WEC. More recent WEC research by Ambroso and Harris has suggested that the mechanism by which the anti-malarial drug and in vivo teratogen, chloroquine, acts is via reversible inhibition of cysteine proteinase cathepsin B and L activity (Ambroso and Harris, 1994). These studies on histiotrophic nutrition are critically important for human risk assessment, as there is considerable evidence suggesting that this mechanism occurs only in species that rely on an inverted yolk sac placenta during organogenesis, but not in humans and other primates that utilize a chorioallantoic placenta throughout gestation (Beck, 1976;Carney et al, 2004;Holson et al, 2005).…”