In this study we investigated intracellular and extracellular oxygen radical production by polymorphonuclear leukocytes (PMNs) during the phagocytosis of periodontopathic bacteria. In in vitro assays, bacteria of the species Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum were phagocytosed at 37 degrees C for 4 h by purified peripheral human PMNs from healthy subjects (n = 6). Superoxide production during phagocytosis was determined by flow cytometry and with a fluorescence/luminescence microplate reader. After phagocytosis, oxidative stress was determined by flow cytometry. Both the intracellular and extracellular oxygen radical production by PMNs phagocytosing F. nucleatum was significantly greater than that of PMNs phagocytosing P. gingivalis and A. actinomycetemcomitans ( P < 0.01 by the Mann-Whitney test). Moreover, after 4 h of incubation, the oxidative stress of PMNs phagocytosing F. nucleatum was significantly greater than that of PMNs phagocytosing P. gingivalis and A. actinomycetemcomitans. We conclude that a high level of superoxide production by PMNs may damage not only periodontopathic bacteria but also PMNs themselves, and may be correlated with the destruction of periodontal tissue.

Key words:super reductive water/bactericidal effect/cytotoxicity/alamar blue assay Abstract:The purpose of this study was to investigate the bactericidal effect against several genera of bacteria and fungi of the newly developed super reductive water (SRW) and its cytotoxicity. SRW has -310mV 1 strain of fungi. Each genus of bacteria (initial concentration of 108 cells/ml) was incubated with SRW for one and ten min. at room temperature. Then the survival rate was determined by colony formation. For the genus E. coli, P. aeruginosa, A. actinomycetemcomitans, F. nucleatum, and P. gingivalis, the number of cells that survived less than 10102 cells/ml, but for the genus S. aureus, S. epidermidis, S. mutans , S. salivarius, S.sanguis, E. faecalis, L. acidophilus, B. subutilis and C. albicans, cell survival was more than 106 cells/ml . The bactericidal activity against E. coli was not reduced in the presence of serum or thiourea. The cytotoxicity of SRW was tested by alamar blue assay with L 929 cell lines established from subcutaneous tissue in C 3 H mice. The cytotoxicity (LD 50) of SRW determined by alamar blue assay was 33w/w% . These results suggested that newly developed SRW has an excellent bactericidal effect against only gram-negative bacteria and lower cytotoxicity, and that the application of SRW is restricted to the disinfection of gramnegative bacteria.