Figure 3.

In vitro effects of dissolved oxygen (DO) on intracellular ATP and reactive oxygen species (ROS). A, Intracellular ATP levels were measured in HSFs incubated for 4 hours in DMEM with (33 mg/L) or without (8 mg/L) elevated levels of DO. The cells were restimulated every hour with the respective treatments. Cells treated with 33 mg/L DO showed significantly higher intracellular ATP levels than control (n = 3, *P < 0.05, independent 2‐tailed t‐test). B, ROS kinetics were measured in HSFs at 15 minutes, 30 minutes, 1 hour, and 4 hours, after incubation in 32 mg/L DO (DMEM w/10% FBS), control (DMEM w/10% FBS, 9 mg/L DO), or positive control (DMEM w/10% FBS, 9 mg/L DO, and 500‐μM H₂O₂). ROS levels are presented as relative fluorescence (RFU, 485/520 nm) × 10³. There were no significant differences between the ROS production in the cells treated with 32 mg/L DO when compared with control. The positive control had significantly higher levels of ROS compared with control at all time points (n = 3, *P < 0.05, ANOVA with Bonferroni post hoc test). C, ROS was measured in HSF incubated for 4 hours in DMEM with 10% FBS with 31 mg/L or 8 mg/L DO. The cells were restimulated every hour with the respective treatments. Cells treated with 31 mg/L had increased ROS levels after 4 hours when compared with control, but the change was not significant. The positive control (DMEM w/10% FBS, 8 mg/L DO, and 500 μM H₂O₂) had significantly higher levels of ROS compared with control (n = 3, *P < 0.05, ANOVA with Bonferroni post hoc test)