Oxidative stress caused by high concentration ROS can lead to damage to cell structures, including membrane lipids, proteins and DNA, particularly mitochondrial DNA. 8-OHdG, the DNA guanine base oxidation product, is considered to be one of the most abundant DNA lesions resulting from oxidative stress and is a biomarker of the oxidative DNA damage. It is highly mutagenic causing GC to TA transversions.(16, 17) The DNA repair include base excision repair (BER), double strand break repair (DSBR), mismatch repair (MR) and nucleotide excision repair (NER). BER is the main pathway for repair of oxidative DNA damage. Human 8-OHdGuanine DNA glycosylasel (hOGG1), an enzyme involving in BER, can specifically recognize and excise 8-OHdG.(18)
Mitochondria is a major producer of ROS, thus it is susceptible to direct injury of ROS.(19) In this study, we mainly observed the oxidative DNA damage of RPE cells after HBO exposure. Our data showed that in the normal RPE cells, the level of 8-OHdG increased and the proliferation was suppressed after exposure to HBO, which are likely associated with oxidative damage. In addition, the damage was aggravated with increased duration and pressure of HBO. Conversely, HBO therapy could ameliorate oxidative DNA damage of RPE cells in hypoxic statue. Meanwhile, the increased expression of DNA repair enzymes hOGG1 in the normoxic and hypoxic RPE cells demonstrated that HBO could induce the oxidative DNA damage repair.
Previous experiment demonstrated that in rats after inspired hyperoxia, the expression of DNA repair enzymes and the level of 8-OHdG in the rat lens increased.(20) In our study, in normoxic group, HBO could cause oxidative DNA damage to RPE cells, which may related to the generation of ROS. Meanwhile, the increased expression of hOGG1 in RPE cells after HBO therapy reflected the stimulation of oxidative DNA damage. Nevertheless, increased duration and pressure of HBO caused decreased expression of hOGG1, which may in connection with accumulation of ROS and damage in DNA leading to the decline in capability of repair of DNA. There is evidence to support that. Santos JH et al established a feed-forward cascade of ROS production and mtDNA damage, which demonstrated persistence of lesions in the mtDNA involving a drop in mitochondrial membrane potential, secondary ROS generation, and loss of repair capacity. In addition, the feed-forward cascade at last result in the cell apoptosis and death.(21) As in our study, HBO suppressed the proliferation of RPE cells in normoxic status.
In our study, we used 200μmol/L CoCl2 to mimic hypoxic status of RPE cells. CoCl2, a chemical substance, is usually used to mimic hypoxia. On one hand, CoCl2 can inhibit heme oxygenation by replacing Fe2+ in heme with Co2+,thus result in the hypoxic status of cells.(22) On the other hand, CoCl2 can cause the stabilization of hypoxia-inducible factor-α(HIF-α)in cells, which can activate the expression of genes that contain a hypoxia response element.(23) Moreover, previous study detected that, after cells incubated with CoCl2, the production of ROS increased.(24) The increasing expression of 8-OHdG in RPE cells treated with CoCl2 in our study may involve in the generation of ROS.
In hypoxic group, HBO treatment ameliorated oxidative DNA damage of RPE cells. That may be concerned with the increased expression of hOGG1 in RPE cells. Furthermore, a number of experiments have proved that, HBO can upregulate antioxidant gene expression and enhance the activity of antioxidant enzymes in cells.(25-27) There are some studies about preconditioning of HBO on mouse with focal cerebral ischemia, and they discovered upregulated HIF-1α and erythropoietin in rats after HBO,which induced ischemic tolerance and reduced infarct volume.(28, 29)
In conclusion,we have demonstrated that HBO treatment may lead to oxidative DNA damage in normal RPE cells, and there is an increase trend of the damage with the increase of duration and pressure. Meanwhile, HBO can protect from the oxidative damage in hypoxic PRE cells. These findings invite further exploration into the oxidative effect of HBO therapy on retina in vivo and suggest the reasonable and individual treatment applying in clinical work. Furthermore, antioxidant can be used during the HBO therapy to prevent from the oxidative damage.