DNA Cleavage and Trypanosomes Death by a Combination of Alamar Blue and Au(III)

Abstract

Alamar Blue test indicates toxicity of Au(III), but Au(III) ion itself is not toxic in vitro and does not cleave DNA. However, combination of Alamar Blue and Au(III) cleaved DNA through formation of O2-• and H2O2 and showed high toxicity on Trypanosoma brucei brucei that causes African Sleeping Sickness (Trypanosomiasis) in vitro and in vivo tests. The DNA cleavage study was performed by gel electrophoresis and circular dichroism (CD) techniques. In vitro study was carried out with tissue cultured adapted T. b. brucei 427 strain blood-stream form parasites which were grown in HMI-9 medium with and without Au(III) ion and incubated at 37 and#176;C in 5% CO2. Toxicity of the mixture of Alamar Blue and Au(III) ion was also tested in vivo with mice which were infected intraperitoneally with 4.0 x 104 trypanosomes. Mechanism of DNA cleavage and of toxicity in vitro test is discussed for the mixture of Alamar Blue and Au(III) ion. Alamar Blue has long been used for cell proliferation. Recently it has been found that Alamar Blue induces cleavage of DNA in the presence of Au(III) through the formation of O2-• and H2O2. On the other hand, Alamar Blue alone or Au(III) ion is incapable of DNA cleavage. It has also been observed that in the presence of certain metal ions such as Pd(II), Zn(II), Cd(II) and Pb(II), Alamar Blue is incapable of DNA cleavage. The cleavage study was carried by gel electrophoresis and circular dichroism (CD) techniques. It was observed that Form I and Form II of the pBluescript plasmid DNA were converted into Form III and DNA fragment by gel electrophoresis. The intensity of Form II was gradually decreased with increasing concentration of Au(III) with the completed disappearance at its higher concentration. The inhibitory effect by the superoxide dismutase (SOD) and catalase confirmed the formation of O2-• and H2O2 respectively in the presence of Alamar Blue and Au(III) ion. The drastic change in CD spectra from positive ellipticity to negative ellipticity at 270 nm is also suggested the cleavage of DNA with increasing concentration of Au(III) in the presence of Alamar Blue. The toxic effect of the mixture of Alamar Blue and Au(III) on Trypanosoma brucei brucei [that causes African Sleeping Sickness (Trypanosomiasis)] was investigated and found the IC50 value for aqueous Au(III) was 3.8 x 10-7 M while that for Pd(II) was 1.3 and#215; 10-5 M. However, Alamar Blue alone or Au(III) ion did not show any significant toxic effect on the parasite. The toxic effect of the mixture of Alamar Blue and Au(III) ion was also tested in vivo with mice which were infected intraperitoneally with 4.0 x 104 trypanosomes. Mechanism of DNA cleavage and of toxicity in vitro test is discussed for the mixture of Alamar Blue and Au(III) ion.