Acetylaminofluorene inhibits nitric oxide production in LPS-stimulated RAW 264.7 cells by blocking NF-κB/Rel activation

Abstract

The mechanism by which 2-acetylaminofluorene (AAF) inhibited nitric oxide (NO) formation, in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was investigated. The decrease in NO, as demonstrated by measurement of nitrite was found to correlate well with a decrease in inducible nitric oxide synthase (iNOS) mRNA. Since the promoter in iNOS gene contains binding motifs for NF-κB/Rel, AP-1, and NF-IL6 which appear to be important for LPS-mediated iNOS induction, the effect of AAF on the activation of these transcription factors was determined. Treatment of AAF to RAW 264.7 cells induced a dose-related inhibition of NF-κB/Rel in chloramphenicol acetyltransferase 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-κB/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 paralleled by the accumulation of cytoplasmic c-rel, p65, and p50. These data suggest that AAF inhibits iNOS gene expression by a mechanism involving a blockade of LPS-induced nuclear translocation of NF-κB/Rel.