Nanoparticle-Induced Apoptosis Propagates through Hydrogen-Peroxide-Mediated Bystander Killing: Insights from a Human Intestinal Epithelium In Vitro Model

Abstract

The ability to assess the risks of human exposure to engineered nanomaterials requires fundamental understanding of the fate and potential cytotoxicity of nonbiodegradable nanoparticles, for instance, after oral uptake. In this study, we quantify the impact of nanoparticles with low chemical toxicity on the intestinal membrane in a human intestinal in vitro model. Differentiated human colorectal adenocarcinoma cells, Caco-2, were cultured on a permeable support where they form an epithelial monolayer separating an apical and basal compartment. This model system allows a systematic characterization of the effect of nanoparticles on the cell viability as a function of size, surface chemistry, concentration, and incubation time. We used polystyrene (PS) nanoparticles (20 and 40 nm diameter) with two different surface chemistries (carboxylic acid and amines). The experiments performed show a strong decrease in cell viability as a response to nanoparticle exposure. Incubation times of ≤4 h are sufficient to induce dramatic losses in cell viability after an additional induction period of 4−12 h. Mapping the temporospatial distribution of dead cells in the Caco-2 cell monolayer using optical microscopy reveals that the nanoparticles induce apoptosis in individual cells, which then propagate across the cell monolayer through a “bystander killing effect”. Addition of catalase, which selectively decomposes hydrogen peroxide, leads to a significant decrease in apoptosis levels, indicating that hydrogen peroxide causes the spread of apoptosis across the monolayer. Our findings confirm that ingested nonbiodegradable nanoparticles represent a potential health risk due to their detrimental impact on the intestinal membrane by destroying their barrier protection capability over time.