OBJECTIVES: Formation of formazan is a commonly used measure of cytotoxicity of compounds. It is a product of reduction of tetrazolium salts such as 4-[3- (4-iodophenyl)-2- (4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride. The extent of substrates reduction reflects the activity of enzymes succinate dehydrogenase (SDH; respiratory complex II) and lactate dehydrogenase (LDH), respectively. The aim of present study was a) to investigate formazan formation under the conditions of in vitro stimulation of cells with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), and b) to analyse possible interference of pyrimidine analogues with formazan production.
METHODS: Peritoneal cells and splenocytes were obtained from C57BL/6 mice. They were cultured at 37 degrees C, 5% CO2 in humidified incubator. Levels of formazan were determined at the interval of 24 h of culture using the WST-1 and LDH assays. Nitric oxide (NO) was activated by IFN-γ plus LPS and assayed by Griess reagent 24 h afterwards. Pyrimidines were applied concomitantly with immunostimulatory agents.
RESULTS: IFN-γ enhanced concentration of SDH-produced formazan by macrophages (not by splenocytes) by approximately 50%. The activity of LDH remained unaffected. LPS was ineffective in both cases. While pyrimidines with NO-inhibitory properties suppressed the IFN-γ-enhanced levels of SDH-produced formazan, they did not change the LDH-dependent formazan production.
CONCLUSION: IFN-γ augments the SDH-produced formazan by macrophages. It does not change the LDH-dependent formazan formation. The enhancing effect may have a significant impact upon the appropriate interpretation of cytotoxic properties of drugs investigated under the conditions of immune stimulation of cells.