Hypochlorous acid-induced oxidative stress in Chinese hamster B14 cells: viability, DNA and protein damage and the protective action of melatonin.

This study provides further evidence for the toxicity of hypochlorous acid (HOCl) in mammalian cells. Using the Chinese hamster B14 cell line, a significant decrease in cell viability was demonstrated after exposure to 100-200 microM HOCl for 1 h. Loss of viability was accompanied by a slight increase in DNA damage as shown by the Comet assay and by oxidation of cellular thiols. Exposure of B14 cells, erythrocyte membranes and human serum albumin to HOCl resulted in an extensive protein carbonyl accumulation. Thus, the cytotoxicity of HOCl may be due to both protein damage (carbonyl formation and oxidation of protein thiol groups) and DNA damage. The well-known antioxidant melatonin interacted with the oxidant and significantly protected cells during HOCl exposure, diminishing its cytotoxic effects and reducing protein carbonyl generation.

Hypochlorous acid-induced membrane pore formation in red blood cells.

The hyperproduction of hypochlorous acid (HOCl), an extremely toxic biological oxidant generated by neutrophils and monocytes, is involved in the pathogenesis of many diseases. In these studies, we attempted to determine the membrane and cellular events associated with HOCl-induced erythrocyte impairment and haemolysis. In vitro human erythrocyte exposure to HOCl (0.1-1.0 mM) resulted in rapid oxidation of reduced glutathione, an increase in cell osmotic fragility and the formation of transient membrane pores. The process of glutathione oxidation depended on the [oxidant]/[cell number] ratio. The HOCl-induced haemolysis observed was apparently mediated by pore formation and altered membrane electrolyte permeability. The estimated pore radius was approximately 0.7 nm and the average number per cell was 0.01. The rate constant of HOCl-produced haemolysis depended on pH. There were significant differences in haemolysis of HOCl-treated erythrocytes which had maximal stability at pH 7.2-7.3.

The mechanism of Zn-phthalocyanine photosensitized lysis of human erythrocytes.

The phthalocyanines have recently been suggested as one of most effective possible sensitizers for photodynamic therapy and the blood viral inactivation. The further characterisation of the mechanism of human red blood cell lysis and membrane alterations upon photodynamic treatment in the presence of Zn-phthalocyanine was the aim of this study. It was found that there were (2.7+/-0.4).10(7) dye binding sites per red blood cell with the association constant equal to (1.4+/-0.3).10(4) M(-1). Two types of the photosensitized haemolysis: haemolysis during irradiation ("light" haemolysis) and post-irradiation haemolysis ("dark" haemolysis) were studied. The erythrocyte membrane hyperpolarisation, membrane fluidisation and cell swelling preceded the "light" haemolysis. The modification of the erythrocyte membrane band 3 protein by DIDS (an inhibitor of anion exchange) increased the rate of the "light" haemolysis. The rate of "dark" haemolysis was higher and that of "light" haemolysis was lower in potassium media in comparison to sodium ones. The rates of photohaemolysis depended on the erythrocyte membrane potential: a decrease of membrane potential inhibited both types of haemolysis. The cell shrinkage in the presence of sucrose (up to 15 mM) inhibited the "dark" haemolysis but significantly increased the "light" haemolysis. Oxidation of intracellular oxyHb to metHb by nitrite, which drastically decreases intracellular oxygen concentration, as well as GSH concentration, inhibited the rate of the "light" haemolysis. The results allow for the conclusion that the mechanism of photochemical ("light") haemolysis is not of a colloid-osmotical type, in contrast to the post-irradiation ("dark") haemolysis. The photochemical oxidation or denaturation of band 3 protein plays a significant role in the formation of haemolytic holes. The membrane lipid peroxidation, as well as glutathione oxidation, does not participate in the process of photosensitized haemolysis. From the inhibition of "dark" haemolysis by sucrose the apparent pore radius was estimated to be about 1.1 nm. The pores appear to be transient short-lived ones, the average pore number per cell was 0.02.