Acetylaminofluorene inhibits nitric oxide production in LPS-stimulated RAW264.7 cells by blocking NF-kappa B/Rel activation

The mechanism by which 2-acetylaminofluorene (AAF) inhibited nitric oxide ( NO) formation, in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was i nvestigated. The decrease in NO, as demonstrated by measurement of nitrite was found to correlate well with a decrease in inducible nitric oxide synth ase (iNOS) mRNA. Since the promoter in iNOS gene contains binding motifs fo r NF-kappa B/Rel, AP-1, and NF-IL6 which appear to be important for LPS-med iated iNOS induction, the effect of AAF on the activation of these transcri ption factors was determined. Treatment of AAF to RAW 264.7 cells induced a dose-related inhibition of NF-kappa B/IRel in chloramphenicol acetyltransf erase activity, while either AP-1 or NF-IL6 activation was not affected by AAF. Treatment of RAW 264.7 cells with AAF inhibited protein/DNA binding of NF-kappa B/Rel to its cognate site as measured by electrophoretic mobility shift assay. In addition, AAF treatment caused a significant reduction of nuclear c-rel, p65, and p50 protein levels, and this decrease was parallele d by the accumulation of cytoplasmic c-rel, p65, and p50. These data sugges t that AAF inhibits iNOS gene expression by a mechanism involving a blockad e of LPS-induced nuclear translocation of NF-kappa B/Rel. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.