Propanil inhibits tumor necrosis factor-alpha production by reducing nuclear levels of the transcription factor nuclear factor-kappa B in the macrophage cell line IC-21

Tumor necrosis factor-alpha (TNF-alpha) is an essential proinflammatory cyt okine whose production is normally stimulated by bacterial cell wall compon ents, such as lipopolysaccharide (LPS), during an infection. Macrophages st imulated with LPS in vitro produce several cytokines, including TNF-alpha. LPS-stimulated primary mouse macrophages produced less TNF-alpha protein an d message after treatment with the herbicide propanil (Xie et al,, Toxicol. Appl. Pharmacol. 145, 184-191, 1997). Nuclear factor-kappaB (NF-kappaB) ti ghtly regulates TNF-alpha transcription. Therefore, as a step toward unders tanding the mechanism of the effect of propanil on TNF-alpha transcription, IC-21 cells were transfected with a TNF-alpha promoter-luciferase construc t, and the effect of propanil on luciferase activity was measured, Cells tr ansfected with promoter constructs containing a kappaB Site showed decrease d luciferase activity relative to controls after propanil treatment, These observations implicated NF-kappaB binding as an intracellular target of pro panil. Further studies demonstrated a marked reduction in the nuclear level s of the stimulatory p65 subunit of NF-kappaB after propanil treatment, as measured by fluorescence confocal microscopy and Western blot analysis. The p50 subunit of NF-kappaB was not found to be reduced after propanil exposu re by Western blot. Electrophoretic mobility gel shift assays showed decrea sed DNA binding of both p65/p50 heterodimers and p50/p50 homodimers to the kappa B3 site of the TNF-alpha promoter of propanil-treated cells. The mark ed reduction in nuclear p65/p50 NF-kappaB levels and diminished binding to the TNF-alpha promoter in propanil-treated cells are consistent with reduce d TNF-alpha levels induced by LPS. (C) 2001 Academic Press.