Limited DNA damage in human endothelial cells after hyperbaric oxygen treatment and protection from subsequent hydrogen peroxide exposure

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Abstract

Background

In vitro studies on hyperbaric oxygen (HBO) therapy suggest that HBO may cause DNA damage, but this has not been evaluated using endothelial cells.

Methods

Human umbilical cord endothelial cells (HUVECs) were exposed either to H2O2 or to HBO for 90 min, with or without subsequent H2O2 exposure. Measurements included the comet assay for DNA damage, and reduced and oxidised glutathione levels.

Results

HUVECs showed sensitivity to H2O2 (EC50 of 0.2 mM for DNA migration). A single 90 min HBO treatment at 2.2 ATA caused a statistically significant (ANOVA, P < 0.05) increase of DNA migration in HUVECs to 6.8 ± 0.3% (mean ± SEM, n = 8), which returned to normal levels (4.9 ± 0.1%, n = 6) after 24 h. Further exposure to 0.2 mM H2O2 after HBO treatment significantly increased the DNA migration in HBO-treated cells immediately post-treatment; but 24 h later the cells showed 22% less DNA damage and higher glutathione than controls.

Conclusion

A single HBO exposure causes limited DNA damage to HUVECs, which repairs quickly. HBO treatment protects against H2O2-induced DNA damage and involves cellular glutathione.

Significance

Endothelial cells are unlikely to be compromised during HBO therapy.

Research Highlights

► Novel use of human umbilical cord endothelial cells, HUVECs, with hyperbaric oxygen. ► HUVECs are a good model for measuring DNA damage and respond to H2O2. ► A single 90 min HBO treatment at 2.2 ATA causes limited DNA damage to HUVECs. ► Cells quickly repaired within 24 h of HBO treatment. ► HBO treatment protects HUVECs from H2O2-induced DNA damage and involves glutathione.

Introduction

Hyperbaric oxygen (HBO) therapy involves breathing 100% oxygen at greater than 1 atmosphere absolute pressure (ATA; i.e., greater than atmospheric pressure at sea level), and has been used successfully to treat patients for wound healing (reviews, [1], [2], [3]). The mechanism of wound healing is not fully understood, but involves angiogenesis into the wound area [4]. We have recently shown that HBO does not damage the aortic endothelium of rodents in vitro [5]. However, some in vitro studies have raised concerns that HBO may cause oxidative DNA damage. Using the comet assay, a single HBO treatment has been shown to induce DNA damage in vitro in whole blood, human lymphocytes, A549 lung cells, and V79 Chinese hamster cells [6], [7], [8], [9].

In vivo studies have shown more variable effects. Although patients and healthy volunteers generally showed no overt HBO-induced oxidative stress in terms of haematology and blood chemistry [10], [11], [12], [13], [14], induction of DNA strand breaks cannot be excluded. Some studies have shown that a single HBO exposure (2.5 ATA, 3 × 20 min) induces DNA strand breaks in leukocytes and lymphocytes from healthy volunteers, and these breaks may arise from oxidative damage of the bases in DNA [10], [11], [15]. However, the effect is transient and HBO-induced DNA damage can be apparently repaired in 2 h [15]. Several studies report no DNA damage 24 h after a first HBO treatment, or following subsequent HBO treatments [7], [10], [11], [15]. However, the situation may be different in patients with illness (i.e., not healthy volunteers). For example, Eken et al. [16] reported persistent DNA damage to lymphocytes from patients with hypoxia-related problems even after the 10th and 20th HBO treatment (2.5 ATA, for 3 × 20 min in each treatment). Furthermore, most of the previous studies on humans have focused on blood cells, but effects of HBO on DNA in the endothelial cells that line blood vessels have yet to be established.

Endothelial cells are central to the success of HBO-dependent angiogenesis [17], and while there are some data suggesting DNA damage in lymphocytes, there are no data on the genotoxicity of HBO to endothelial cells. Even basic experimental information such as the responsiveness of human umbilical vein endothelial cells (HUVECs) to the common oxidising agents used as positive controls in DNA damage experiments (e.g., H2O2, [18]) appears to be lacking. In this study, we aim to test the effects of a single HBO treatment on HUVECs. The experimental approach included establishing positive controls with H2O2 to bench mark the sensitivity of HUVECs to DNA damage, as measured by the comet assay. Then a second series of experiments tested the effect of a single HBO treatment on DNA damage and glutathione pools in HUVECs. In some experiments, cells were also subject to H2O2 exposure after HBO therapy to determine whether HBO protects or exacerbates the damaging effects of H2O2.

Section snippets

Materials and methods

All chemicals were obtained from Sigma-Aldrich (Poole, UK), unless stated otherwise.

Response of HUVECs to H2O2

Exposure of HUVECs to H2O2 caused a concentration-dependent increase in DNA damage (strand breaks) as assessed using the alkaline comet assay (Fig. 1). In control cells (no H2O2 exposure) a typical background level of % of DNA in comet tails (% tail DNA) was 4.2 ± 0.2% (n = 5), and the maximum achieved at the highest H2O2 concentration was 78.5 ± 3.1% (n = 4). The EC50 was 0.19 mM, and so a concentration of 0.2 mM H2O2 was chosen for subsequent experiments involving HBO.

Effect of HBO treatment with or without subsequent H2O2 exposure on levels of DNA damage

In these experiments HUVECs were

Discussion

For the first time, we have successfully applied the comet assay to measure HBO-induced DNA damage and recovery in HUVECs, and have demonstrated that HUVECs are a good model for measuring DNA damage because they respond in the expected way to H2O2, as a well known reference mutagen [23]. Overall, we find that a single HBO exposure causes limited DNA damage to HUVECs, which is quickly repaired within 24 h. In addition, HBO treatment protects the cells against H2O2-induced DNA damage, and the

Acknowledgments

We would like to thank Dr. Jackie Whatmore and Selina McHarg (Peninsula Medical School, Universities of Exeter and Plymouth) for their generous supply of HUVECs, and Dr. Teresa Neuparth for her help with the Comet assay. This work was funded by the Diving Diseases Research Centre (DDRC) and the University of Plymouth.

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