title: Activation of respiratory complex II by interferon-gamma and its inhibition by pyrimidine derivatives.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors: Zdeněk
authors: Z
authors: Zídek
authors: Petr
authors: P
authors: Jansa
authors: Eva
authors: E
authors: Kmoníčková
mesh_terms: D000818:Animals; D042963:Electron Transport Complex II / Q000187:drug effects*; D005562:Formazans / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010537:Peritoneum / Q000166:cytology; D011743:Pyrimidines / Q000494:pharmacology*; D013154:Spleen / Q000166:cytology
chemical_list: D005562:Formazans; D011743:Pyrimidines; C478189:respiratory complex II; D007371:Interferon-gamma; D042963:Electron Transport Complex II