Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction.
Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. The midpoint cytotoxicity values for 1- and 24-h exposures were 61.12 +/- 2.46 and 21.22 +/- 2.44 microg/ml, respectively. Chlorhexidine did not induce synthesis or liberation of reactive oxygen/nitrogen intermediates. When macrophages were treated with various sub-toxic doses for 1 h (1, 5, 10, and 20 microg/ml) and 24 h (0.5, 1, and 5 microg/ml) and stimulated with 200 nM phorbol myristate acetate (PMA) solution, the H2O2 production was not altered; however, the NO production induced by 10 microg/ml lipopolysaccharide (LPS) solution varied from 14.47 +/- 1.46 to 22.35 +/- 1.94 micromol/l and 13.50 +/- 1.42 to 20.44 +/- 1.40 micromol/l (N = 5). The results showed that chlorhexidine has no immunostimulating activity and sub-toxic concentrations did not affect the response of macrophages to the soluble stimulus PMA but can interfere with the receptor-dependent stimulus LPS.
Bonacorsi
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Instituto de Química de Araraquara, Universidade Estadual Paulista Júlio de Mesquita Filho, Araraquara, SP, Brazil.
Raddi
M S G
MS
Carlos
I Z
IZ
D000818:Animals; D000890:Anti-Infective Agents / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D002710:Chlorhexidine / Q000031:analogs & derivatives* / Q000633:toxicity*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000187:drug effects*; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000890:Anti-Infective Agents; D008070:Lipopolysaccharides; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; C010882:chlorhexidine gluconate; D013755:Tetradecanoylphorbol Acetate; D002710:Chlorhexidine