transforming-growth-factor-beta and aniline

Signaling mechanisms in aniline-induced fibrogenic and/or tumorigenic response in the spleen are not known. Previous studies have shown that aniline exposure leads to iron accumulation and oxidative stress in the spleen, which may cause activation of redox-sensitive transcription factors and regulate the transcription of genes involved in fibrosis and/or tumorigenesis. To test this, male SD rats were treated with 0.5 mmol/kg/day aniline via drinking water for 30 days, and activation of transcription factor AP-1 was determined in the splenocyte nuclear extracts (NEs). AP-1 DNA-binding activity in the NEs of freshly isolated splenocytes from aniline-treated rats increased in comparison to the controls, as determined by electrophoretic mobility shift assay (EMSA). AP-1 binding was also determined in the NEs of cultured splenocytes (2 h and 24 h), which showed even a greater increase in binding activity at 2 h. The specificity of AP-1 binding for relevant DNA motifs was confirmed by competition EMSA and by supershift EMSA using antibodies specific to c-Jun and c-Fos. To further explore the signaling mechanisms in the AP-1 activation, phosphorylation patterns of mitogen-activated protein kinases (MAPKs) were pursued. Aniline exposure induced increases in the phosphorylation of the three classes of MAPKs: extracellular-signal-regulated kinase (ERK 1/2), c-Jun N-terminal kinase (JNK 1/2), and p38 MAPKs. Furthermore, TGF-beta1 mRNA expression showed a 3-fold increase in the spleens of aniline-treated rats. These observations suggest a strong association among MAPK phosphorylation, AP-1 activation, and enhanced TGF-beta1 gene expression. The observed sequence of events subsequent to aniline exposure could regulate genes that lead to fibrogenic and/or tumorigenic response in the spleen.

Topics: Aniline Compounds; Animals; Blotting, Northern; Cell Nucleus; Electrophoretic Mobility Shift Assay; Gene Expression; Male; Mitogen-Activated Protein Kinases; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Spleen; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Aniline exposure produces selective toxicity to the spleen, leading to a variety of sarcomas in rats following chronic exposure. Fibrosis appears to be an important preneoplastic lesion of the spleen. However, early molecular events leading to splenic fibrosis are not known. Earlier studies have shown that aniline exposure in rats leads to excessive deposition of iron and increased lipid peroxidation in the spleen, which may produce changes in the expression of fibrogenic cytokines, such as transforming growth factor-beta 1 (TGF-beta 1), leading to splenic fibrosis. Therefore, this study was designed to establish whether aniline exposure leads to induction/overexpression of TGF-beta 1 and association of such induction with lipid peroxidation (oxidative stress) in the spleen. To achieve this, male Sprague-Dawley rats were given 1 mmol/kg/day aniline hydrochloride in water by gavage for 7 days, while controls received water only. Aniline treatment resulted in significant increases in spleen weight (97%), spleen-to-body weight ratios (104%), and splenocyte population (25%). Malondialdehyde-protein adducts, quantitated by a competitive ELISA, showed a 56% increase in the spleen of aniline-treated rats. TGF-beta 1, measured in the supernatants of cultured splenocytes by an ELISA specific for TGF-beta 1, showed a significant increase (60%) in the total TGF-beta 1 from aniline-treated rats. These increases were further confirmed by Western blot analysis, which showed approximately 2.5-fold increase in cell-associated TGF-beta 1 protein expression in aniline-treated rats. Furthermore, determination of TGF-beta 1 mRNA expression showed a 4-fold increase in the spleens of aniline-treated rats. These results suggest an association between formation of MDA-protein adducts and overexpression of TGF-beta 1 as a result of aniline insult, which together could promote splenic injury and fibrogenesis.

Topics: Aniline Compounds; Animals; Carcinogens; Lipid Peroxidation; Male; Malondialdehyde; Rats; Rats, Sprague-Dawley; Spleen; Transforming Growth Factor beta; Up-Regulation