A. Roberts1, S. Papillon1, A. Dossa1, M. Frey3,4,5, H. Ford1,3,5, C. Gayer1,3 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 3University Of Southern California,Keck School Of Medicine,Los Angeles, CA, USA 4University Of Southern California,Pediatrics,Los Angeles, CA, USA 5Children’s Hospital Los Angeles,Developmental Biology And Regenerative Medicine,Los Angeles, CA, USA
Introduction: Necrotizing enterocolitis (NEC) is a severe neonatal intestinal disorder of premature infants. Animal models have shown how gavage of secondary bile acids can induce histologic changes in the intestinal epithelium that are identical to the damage seen in NEC. Intestinal bacteria convert primary bile acids (CA, CDCA) into more toxic secondary forms (DCA, LCA). Additional metabolism yields ursodeoxycholic acid (UDCA). The exact mechanism of cellular injury among different bile acids has not been elucidated. Apoptosis, necrosis, and necroptosis, a form of organized cell necrosis, are all potential mechanisms of cell death. We hypothesized that different bile acid metabolites will exert toxicity by separate mechanisms.
Methods: IEC-6 and YAMC cell survival were assessed using an MTS-based assay. Z-VAD-FMK, a pan-caspase inhibitor, was used to measure apoptosis, along with measuring caspase-3 cleavage by western blot. Cell necrosis was quantified with an LDH-release assay. The role of necroptosis was assessed using necrostatin-1, a RIP1-kinase inhibitor, and confirmed with western blot of non-cleaved RIP3 protein.
Results: Preliminary data in our lab shows that DCA and LCA are toxic to IEC-6 intestinal epithelial cells at lower doses than UDCA or primary bile acids. These results were also seen in a colon cell line (YAMC). Pre-treatment with Z-VAD increased survival in intestinal cells treated with DCA and LCA by 43% and 31% respectively, but did not change survival for primary bile acids at LD50 doses, indicating secondary bile acid-induced apoptosis. Western blot demonstrated increased caspase-3 cleavage with secondary bile acid treatment, confirming this mechanism. Bile acids conjugated with either glycine or taurine caused death by necrosis, with up to a 3-fold increase in LDH release. Unconjugated bile acids did not release a significant amount of LDH. Necrostatin-1 increased cell survival after treatment with UDCA, and western blot showed a 5-fold increase in RIP3 expression in these cells, both suggesting that UDCA toxicity occurs via necroptosis.
Conclusion: Our results show that bile acids lead to intestinal cell death via different mechanisms. Conjugated bile acids exert toxicity primarily by cell necrosis. Secondary bile acids are the most toxic, and induce apoptosis. UDCA in contrast, seems to cause necroptosis. The primary mechanism of toxicity for primary bile acids is not yet clear. Additionally, bile acids appear to have similar toxicity in the small intestine and colon. Interestingly, the colon normally has higher levels of secondary bile acids compared to the small intestine. This is the first report describing UDCA-associated necroptosis, and the first to compare intestinal epithelial cell toxicity from bile acids in different portions of bowel. Understanding the differential mechanisms of toxicity among specific bile acid metabolites may provide novel therapeutic targets in NEC, where secondary bile acids are more abundant.