Relationship between the reduction of tetrazolium salt XTT and DNA strand breakage with aminosugars.
Dihydropyrazine derivatives formed by the self-condensation reaction of D-glucosamine have the DNA breaking activity. To establish the monitoring method of the biological active dihydropyrazines, we investigated the relationship between the XTT (3'-[1-[(phenylamino)-carbonyl]-3, 4-tetrazolium]bis(4-methoxy-6-nitro)benzensulfonic acid hydrate) reducibility and the DNA breaking activity of aminosugars. Aminosugar in 50 mM sodium phosphate buffer (pH 7.4) was incubated at 37 degrees C. At a given time, the XTT reducibility and the DNA breaking activity of the incubated aminosugar were measured. Both XTT reducibility and DNA breaking activity showed a maximum value within 1-4 h after the incubation and then gradually decreased with the incubation time. Superoxide anion was suggested to involve in both of the DNA breaking activity and the XTT reducibility by the addition of the radical scavengers into those assay mixtures. The quantity of remaining covalently closed circular DNA and the XTT reducibility of all aminosugars showed a good correlation (r = 0.825, n = 26). This means that the XTT assay is applicable for the monitoring of those biologically active products derived from aminosugars when the participation of superoxide anion in DNA scission is recognized.
Shimamura
T
T
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku, Kochi 783-8502, Japan.
Takamori
A
A
Ukeda
H
H
Nagata
S
S
Sawamura
M
M
D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D005944:Glucosamine / Q000737:chemistry*; D006595:Hexosamines / Q000737:chemistry*; D007202:Indicators and Reagents; D007700:Kinetics; D015416:Maillard Reaction; D010084:Oxidation-Reduction; D011719:Pyrazines / Q000737:chemistry*; D013778:Tetrazolium Salts
D006595:Hexosamines; D007202:Indicators and Reagents; D011719:Pyrazines; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; D004247:DNA; D005944:Glucosamine