Effects of Hyperbaric Oxygen on Proliferative and Apoptotic Activities and Reactive Oxygen Species Generation in Mouse Fibroblast 3T3/J2 Cell Line

Abstract

Background Hyperbaric oxygen (HBO) therapy is widely used to treat problem wounds associated with pathologic conditions compromising blood supply and tissue oxygenation because increased tissue oxygen levels enhance collagen synthesis, cell proliferation, and angiogenesis. However, little is known about the dose of hyperoxia needed to achieve optimal therapeutic effects. Moreover, HBO, by enhancing the production of reactive oxygen species (ROS), may also exert cytotoxic effects. In vitro models are simplified systems that may aid the development of treatment protocols with HBO. Hence, we have investigated the effects of HBO on the growth and ROS production of the 3T3/J2 fibroblast cell line in relation to the pressure and the duration of exposure. Methods 3T3/J2 fibroblasts were plated (5 x 103 cells/cm2) on six-well microtiter plates in phosphate buffered saline (PBS), put in a compression chamber, and exposed to 100% oxygen at a pressure of 1.0 or 2.5 atmosphere absolute (ATA) for 15, 30, 60, or 120 minutes. Then the cells were incubated in Dulbecco's modified minimum essential medium (DMEM) for 24, 48, or 72 hours, and at the end of the post-HBO incubation period, their number was determined. In other experiments, cells were detached just after HBO exposure, seeded on 60 mm Petri dishes, and cultured for 10 days in DMEM, and the colony forming units were counted. The effects of HBO exposure (2.5 ATA) on the apoptotic rate of cultured cells were investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and enzyme-linked immunosorbent assays. To measure ROS production, 60 minutes before HBO exposure, 2',7'-dichlorofluorescin (DCF) diacetate (200 nmol/mL) was added to PBS, and after HBO exposure (2.5 ATA), cells were lysated, and fluorescence-emission intensity was measured and converted to μmol DCF/μg protein. Results At 1.0 ATA, all HBO exposures increased the proliferation rate of cultured fibroblasts and their clonal growth efficiency. At 2.5 ATA, 15-minute exposure to HBO was ineffective, whereas 30- and 60-minute exposures raised the proliferation rate and clonal growth efficiency. Conversely, a 120-minute exposure significantly decreased these parameters compared with control cultures. The exposure of cells to HBO at 2.5 ATA for 120 minutes raised the apoptotic rate of cultured fibroblasts, whereas shorter exposure times were ineffective. All exposure periods to HBO at 2.5 ATA enhanced ROS production from cultured fibroblasts. Conclusions Collectively, our findings allow us to conclude that (1) all of the exposure periods to HBO at 1.0 ATA or 30- and 60-minute periods at 2.5 ATA enhance cell growth, (2) 120-minute exposure to HBO at 2.5 ATA exerts a marked proapoptotic effect, and (3) no evident relationships occur between the effects of HBO on cell growth and ROS production.