Regulation by Ascorbic Acid and HOO• Radicals of Extracellular DNA Network Formation and Internalization Activity of...

The aim of the study was to identify the regulatory potential of ascorbic acid and HOO• radicals in the formation of extracellular DNA networks and the internalization activity of mononuclear cells from rat blood and spleen.

The isolated mononuclear cells from rat blood and spleen were exposed to ascorbic acid (10 and 50 μM), hydroperoxyl radicals (HOO•), and their combination. The following parameters were assessed: membrane toxicity (using the trypan blue exclusion test), internalization activity with latex particles (endocytosis), extracellular DNA networks formation, intracellular reactive oxygen species production kinetics (flow cytometry with the intracellular fluorescent probe dihydrorhodamine 123, DHR123), tryptophan fluorescence, glycated protein levels, and cellular redox balance (NAD+ and NADH).

Ascorbic acid at the concentrations of 10 and 50 μM suppressed the formation of extracellular DNA networks. At a concentration of 50 μM, it exerted a membrane-toxic effect, reduced tryptophan fluorescence, increased glycated protein levels, and shifted the redox balance toward oxidation (increased NAD+ and decreased NADH). HOO• radicals at concentrations ≥200 μM demonstrated concentration-dependent effects: stimulation of endocytosis, increased formation of extracellular DNA networks, and elevated intracellular production of reactive oxygen species. At a concentration of 600 μM, HOO• radicals reduced tryptophan fluorescence and depleted both NAD+ and NADH levels. The stimulatory effects on endocytosis and extracellular DNA network formation in response to HOO• radicals were the most pronounced in splenic mononuclear cells.The analysis of the combined action of HOO• radicals and ascorbic acid showed that ascorbic acid at a concentration of 10 μM protected cells from the membrane-toxic effects of HOO• radicals and reduced the formation of extracellular DNA networks induced by HOO• radicals. The ascorbic acid at a concentration of 50 μM enhanced the membrane-toxic effect of HOO• radicals and reduced the internalization activity of mononuclear cells.

HOO• radicals at concentrations ≥200 μM stimulated the internalization activity of mononuclear cells and the intracellular generation of reactive oxygen species, whereas HOO• radicals at 600 μM caused a decrease in both oxidized and reduced coenzyme levels and increased the formation of extracellular DNA networks. Ascorbic acid at a concentration of 10 μM exhibited antioxidant properties and protected cells from the damaging effects of HOO• radicals, while at 50 μM it acted as a synergistic pro-oxidant, enhancing the membrane-toxic effect of the radicals and reducing the internalization activity of cells.