Low oxygen decreases intracellular reactive oxygen species

Abstract

The purpose of this study was to test the hypothesis that intracellular reactive oxygen species (ROS) accumulates in a pattern directly proportional to the oxygen tension in which trophoblast cells are cultured. Basic science experimental study. HTR8 and RS trophoblast cells were grown in monolayer cell culture at either 20%, 5%, or 2% oxygen tension for 48 hours on coverslips. The steady-state amount of intracellular reactive oxygen species was assayed with 0.8 μmol of 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA) to assay for superoxide; 10μmol of 3'-p-Aminophenyl fluorescein (APF) to assay for hydroxyl anion, and 10μmol of 2', 7' dichlorodihydrofluorescein diacetate (DCHFDA) to assay for hydrogen peroxide. Cells were immediately imaged with florescent microscopy at emissions of 465nm, 515nm, and 510nm, respectively. The intracellular levels of hydroxyl anion and hydrogen peroxide were clearly decreased in low oxygen. However, ROS was maximally decreased in 5% oxygen tension; there was no further diminution in 2% oxygen. The greatest decrease was seen with hydrogen peroxide, which was decreased by 50%. Superoxide levels were not clearly affected by the oxygen tension. This study confirmed that there is a reduction in the amount of hydrogen peroxide and hydroxyl anions at 5% and 2% oxygen tension. Because hydroxyl is derived from the metabolism of hydrogen peroxide, it is anticipated that the greatest affect would be on H2O2. The lack of change in superoxide may be explained by its short half-life and the sensitivity of our assay. Since reactive oxygen species (ROS) are involved in both cell signaling and cellular damage, the balance between these two actions is critical. These assays can be further applied to determine whether a threshold of ROS would improve embryo development in vitro and avoid oxidative stress induced damage.