Oxidative stress-induced mitochondrial dysfunction in a normal colon epithelial cell line

AIM: To determine how a normal human colon cell line reacts to microbial challenge as a way to study oxidative stress-induced responses associated with inflammatory bowel disease. METHODS: or heat killed murine cecal contents (HKC) and examined for several relevant biomarkers associated with inflammation and oxidative stress including cytokine production, mitochondrial autophagy and oxidant status. TNFα, IL-1β and IL-8 protein concentrations were measured within the supernatants. Fluorescent microscopy was performed to quantify the production of reactive oxygen species (ROS) using an oxidation responsive fluorogenic probe. Mitochondrial morphology and mitochondrial membrane potential was assessed by dual staining using COXIV antibody and a dye concentrating in active mitochondria. Mitochondrial ROS scavenger was used to determine the source of ROS in stimulated cells. Autophagy was detected by staining for the presence of autophagic vesicles. Positive controls for autophagy and ROS/RNS experiments were treated with rapamycin and chloroquine. Mitochondrial morphology, ROS production and autophagy microscopy experiments were analyzed using a custom acquisition and analysis microscopy software (ImageJ). Lastly, blocking mitochondrial ROS generation using mitochondrial specific ROS scavenger reversed microbial challenge induced mitochondrial morphologic abnormalities and autophagy. CONCLUSION: The findings from this study suggest that CRL.1790 cells may be a useful alternative to other colon cancer cell lines in studying the mechanisms of oxidative stress events associated with intestinal inflammatory disorders.