Cytokine gene expression and activation of NF-κB in aniline-induced splenic toxicity

Exposure to aniline results in selective toxicity to the spleen, leading to a variety of sarcomas on chronic exposure in rats, and fibrosis appears to be an important initiating preneoplastic lesion of the spleen. However, the molecular mechanism(s) by which aniline leads to fibrogenic response is not well understood. Previously, we have shown that aniline exposure leads to iron overload and induction of oxidative stress in the spleen. We hypothesized that aniline-induced oxidative stress in the spleen causes transcriptional up-regulation of fibrogenic cytokines via activation of redox-sensitive transcription factor, nuclear factor-kappa B (NF-κB). To test this hypothesis, male SD rats were treated with 0.5 mmol/kg/day aniline hydrochloride via drinking water for 30 days. Cytokine mRNAs were measured by real-time quantitative PCR, while cytokine release was determined in the supernatants of the cultured splenocytes using specific ELISAs. IL-1α, IL-6, and TNF-α mRNA levels showed 6.9-, 2.9-, and 2.6-fold increases, respectively, in the spleens of aniline-treated rats in comparison to the controls. The increases in mRNA levels were associated with enhanced secretion of these cytokines in the splenocyte culture supernatants. NF-κB p65 level in the nuclear extracts of cultured splenocytes of aniline-treated rats showed a 2-fold increase in comparison to the controls as quantitated by NF-κB p65-specific ELISA. The binding activity of NF-κB, determined by electrophoretic mobility shift assay (EMSA), also showed an increase in NF-κB binding in the nuclear extracts of the splenocytes from aniline-treated rats. The specificity of NF-κB binding was further confirmed by supershift assays. The results indicate that aniline exposure causes enhanced expression of IL-1α, IL-6, and TNF-α, both at mRNA and protein levels, suggesting their role in splenic fibrosis. Also, the increased NF-κB binding activity suggests that up-regulation of these cytokines in the spleen is a redox-dependent mechanism.