Investigation of Reactive Oxygen Species production in Human Hepatocytes

1. Aim/Background: Reactive oxygen species (ROS) have been identified as compounds responsible for producing cellular damage. The purpose of this research is to examine if there is production of reactive oxygen species through free radical intermediates within human hepatocytes treated with morphine, bilirubin, or furosemide. The investigation examines the early stages of biotransformation by measuring the levels of reactive oxygen species produced inside of the treated hepatocytes within the first and second hours of treatment. The experiment was designed upon a case of a jaundiced (elevated bilirubin) infant who received morphine and furosemide and later died through unknown mechanisms. The experiment looks to examine if these drug compounds could contribute to cellular damage. This can help to further understand the potential interactions and complications of free radical intermediates produced during the phases of biotransformation. 2. Method: Previously cultured human hepatocytes were washed by centrifugation and re-suspended in 1x supplemental buffer to a concentration of 1x106 cells/mL and seeded in a dark clear bottom 96-well microplate at 100,000 stained cells/well. The cells were treated with either furosemide, morphine, bilirubin, a Tert-Butyl hydro peroxide (TBHP) positive control, or left as a background. Reactive oxygen generated in the presence of these agents were quantified by fluorescence excitation/emission measurement at 495nm/529nm. Fluorescence was measured at one and two hours. ROS generated convert 2',7'-dichlorodihydrofluorescein diacetate to 2',7'-dichlorodihydrofluorescein within the cells, which fluoresces. The fluorescence intensity detected is equivalent to the level of ROS generated. Wells that were untreated were used as blanks and subtracted from background and TBPH. 3. Results: Furosemide and Morphine did not produce statistically significant levels of ROS (p >0.05) above the background in both hours 1 and 2 of biotransformation and ROS measurement (Figure 1). Although Bilirubin did not produce statistically significant (p >0.05) levels of ROS above the background (Figure 2) during the first hour, it did produce statistically significant levels in the second hour of biotransformation. Each compound’s level of ROS was reduced during the second hour, signaling the removal of intermediate ROS metabolites (Figure 2). The production of ROS in each compound signifies that there is biotransformation to an intermediate that produces ROS. 4. Conclusion: The production of ROS above the background by each of the compounds shows there is an intermediate free radical compound that is produced during the biotransformation of each compound [21]. In this study, although furosemide and morphine did not produce statistically significant levels of ROS in both hours of biotransformation, bilirubin did produce significant levels of ROS in the second hour of biotransformation. This finding is in line with previous studies that shows morphine to offer protective effects against ROS production [16, 17]; and bilirubin demonstrating deleterious production of ROS at high doses [18]. Further work must be done to examine the correlation between the levels of ROS and extent of hepatocellular damage.