Preactivation exposure of RAW 264.7 cells to taurine chloramine attenuates subsequent production of nitric oxide and expression of iNOS mRNA

Abstract

Recent studies demonstrate that taurine chloramine (Tau-Cl) inhibits production of nitric oxide (NO) and other proinflammatory mediators in cultured macrophages when added to the media at the time of activation. Because Tau-Cl may react with various media constituents and it is difficult to measure Tau-Cl in complex solutions, we designed experiments to more carefully control cell exposure to various chloramines and NaOCl. RAW 264.7 cells were exposed to 1 mM of NaOCl, Tau-Cl, or chloramine preparations of the following amino acids: L-alanine (L-Ala-Cl), beta-alanine (beta-Ala-Cl), serine (Ser-Cl), or glycine (Gly-Cl) in Hanks' balanced salt solution (HBSS) for up to 2 h (37 degrees C, 5% CO2). The HBSS solution was then replaced with complete media containing interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) for an additional 24 h before measuring cell viability. The chemical stability of NaOCl and each chloramine was evaluated after various times of preactivation exposure by measuring retention of each solution's UV absorption spectra and ability to oxidize KI. Cytotoxicity of each solution was evaluated by the maintained ability of RAW 264.7 cells to reduce MTT. Whereas Tau-Cl, beta-Ala-Cl, and Gly-Cl were stable chloramines, only Tau-Cl was not cytotoxic. L-Ala-Cl, Ser-Cl, and the highly reactive oxidant NaOCl were unstable and toxic. In further studies RAW 264.7 cells were exposed to Tau-Cl in HBSS for 2 h and the solution was then replaced with complete media containing IFN-gamma and LPS, taxol, lipoarabinomannan, or interleukin-2. Production of NO was measured 24 h later and was inhibited in activated cells that were previously exposed to Tau-Cl. Inhibition of NO production was dependent on Tau-Cl concentration and was accounted for by reduced expression of inducible nitric oxide synthase mRNA, regardless of activator combinations. These results support the idea that Tau-Cl has the potential to function as an inhibitory modulator of inflammations.