Chloropicrin induces endoplasmic reticulum stress in human retinal pigment epithelial cells

Chloropicrin is an aliphatic volatile nitrate compound that is mainly used as a pesticide. It has several toxic effects in animals and can cause irritating and other health problems in exposed humans. Since the mode of chloropicrin action is poorly understood, the aim of this study was to investigate molecular responses underlying chloropicrin toxicity. We used human retinal pigment epithelial cells (ARPE-19) as a model cell type because the eyes are one of the main target organs affected by chloropicrin exposure. Transmission electron microscopy images revealed that exposure to a chloropicrin concentration that decreased cell viability by 50%, evoked the formation of numerous electron-lucent, non-autophagy vacuoles in the cytoplasm with dilatation of the endoplasmic reticulum (ER). Lower concentrations led to the appearance of more electron-dense vacuoles, which contained cytoplasmic material and were surrounded by a membrane resembling autophagy vacuoles. According to immunoblotting analyses chloropicrin increased the amount of the ER-stress related proteins, Bip (about 3-fold compared to the controls), IRE1α (2.5-fold) and Gadd 153/Chop (2.5-fold), evidence for accumulation of misfolded proteins in the ER. This property was further confirmed by the increase of reactive oxygen species (ROS) production (2-2.5-fold), induction of heme oxygenase-1 (about 6-fold), and increase in the level of the tumour suppressor protein p53 (2-fold). Thus, the cytotoxicity of chloropicrin in the retinal pigment epithelium is postulated to be associated with oxidative stress and perturbation of the ER functions, which are possibly among the mechanisms involved in oculotoxicity of chloropicrin.