Chandler L Eyre. David A. Eastley (Brigham Young University)
Faculty Advisor: Hancock, Chad (Brigham Young University, NDFS)
Purpose: To determine the effects of H2O2 exposure to human hepatocytes on the iron regulatory proteins such as transferrin receptor (TfR), Ferritin Light-Chain (FLC). Methods: Cells were cultured at an initial density of 35,000 cells/cm2 in 6-well plates, grown until confluent, and then harvested. Iron treatments were done using FeCl3 solution with media to desired concentrations, and then incubating for 24 hours. Groups that received both iron and H2O2 treatments were exposed to 12 hours of iron, followed by incubation with media that included both iron and GOX at 4.8 mU/mL and 0.165 mg/mL of CAT (2-5 KU/mg). BCA protein assay was used to assess protein content, and then normalize each sample to each other for subsequent preparation for western blot. Western blots were carried out were analyzed by fluorescence following densitometry. MTT assays were carried out utilizing the mitochondrial reductase enzyme, and colorimetrically measured to assess cell viability. Results: Iron treatments of 10, 50, and 100 µM for 24-hrs did not result in any significant cell death. Treatment with the same concentrations resulted in a significant decrease (n=12) in TfR for all three groups when compared against control cells that were cultured and harvested simultaneously without any addition the growth media. (10 µM p<0.01), (50 µM p<0.01), and (100 µM p<0.01). FTL in the 10 µM group was significantly decreased (n=11, p<0.01), but not for the 50 µM, or 100 µM groups with p values of 0.9867, and 0.9612, respectively. H2O2 treatments produced concentrations of 5-10 µM, mimicking neutrophil release during inflammatory response. Conclusion: Our assay is sufficient to mimic neutrophil release of H2O2. Iron treatments are able to induce TfR decrease but seem to have a threshold at 10 µM for increasing FTL for storage. We hypothesize that this may be due to a dysregulation at higher concentrations.