Inhibition by (±)-indenestrol A of interferon gamma-stimulated nitric oxide formation in murine macrophage RAW 264.7 cells

We examined the effects of (±)-indenestrol A (IA), an antioxidative and superoxide-producing metabolite of diethylstilbestrol (DES), on the activation of murine macrophages (RAW 264.7 cells) in vitro, particularly with regard to interferon (IFN)-γ-induced nitric oxide (NO) production. (±)-IA inhibited NO production more strongly than DES as assessed by a nitrite assay. The inhibitory effect of (±)-IA on IFN-γ-induced intracellular NO production was confirmed by direct staining of intracellular NO with diaminofluorescein-2 diacetyl. Inhibition of NO production was confirmed by Western blot analysis of IFN-γ-induced NO synthase. Under IFN-γ-stimulated conditions, the IFN-γ activation site (GAS), which was the most important transcription factor, was significantly inhibited by (±)-IA. (±)-IA also promoted the activation of NF-κB. (±)-IA at 1 and 3μM delayed the onset of apoptosis. Our results suggest that (±)-IA inhibited the activation of macrophages, resulting in the suppression of NO-mediated apoptosis. These results suggest a novel mechanism for the carcinogenic promoting activity of DES via its metabolite, (±)-IA.