TGF-beta 1 and AhR are involved in the mechanism of action of Hexachlorobenzene in the generation of preneoplastic foci in rat liver

ANA CLARA DEL TOMASO PORTAZ; FLORENCIA CHIAPPINI; CAROLINA PONTILLO; MARÍA JESÚS OBREGÓN; ANDREA RANDI; MARCELA SUSANA SÁNCHEZ; DIANA KLEIMAN DE PISAREV; LAURA ALVAREZ

Lisboa

Congreso; International Congress on Environmental Health; 2012

Hexachlorobenzene (HCB) is a toxic polyhalogenated aromatic compound, widely distributed in the environment. HCB exposure is associated with a wide variety of toxic effects in humans and experimental animals. Serious hepatotoxic, neurotoxic, developmental and endocrine dysfunctions have been reported. HCB is also known to be a promoter of liver foci growth and rat mammary tumors. HCB is a weak agonist of the arylhydrocarbon receptor (AhR), which is a cytosolic ligand-dependent transcription factor. It has been proposed that upon ligand binding, and translocation to the nucleus, the AhR dimerizes with the AhR nuclear translocator and binds to xenobiotic-responsive elements in the enhancer region of target genes. HCB also stimulates growth factor receptor signaling pathways and c-Src kinase activity in rat liver. We have previously demonstrated that HCB increases TGF-β1 expression and induces loss of the homeostatic balance between cell growth and cell death in rat liver. TGF-β1 in normal epithelial cells has an antiproliferative and pro-apoptotic action, through different pathways. In tumor cells this response is lost or reduced, and TGF-β1 may become a stimulator of proliferation, invasion, angiogenesis, and metastasis. The molecular interplay between TGF- β1 signaling and the AhR pathway has been reported. Our aim was to study: 1) the effect of HCB on cell proliferation, TGF-β1, AhR, c-Myc and p53 expression, in the liver of female Wistar rats, treated with dietilnitrosamina (DEN) (50 mg/kg), to initiate carcinogenesis, followed by HCB (100 mg /kg p. c.), administered by gavage, for 8 weeks (DEN+HCB). 2) the expression of GST-P, TGF-β1 and AhR in the preneoplastic lesions under the initiation/promotion protocol. 3) the role of AhR on HCB (5, 50, 500 and 5000 nM)-induced cell proliferation, ERK1/2 activation, and TGF-β1 expression in the human transformed liver cell line Hep-G2. Our results, in vivo, showed that HCB significantly increased the protein levels of PCNA, 60%, c-Myc, 36 %, and p53, 45%, evaluated by inmunoblot, in the liver of DEN+HCB group compared to DEN. Protein levels of TGF-β1 and AhR were also increased 50 % and 40 %, (p ≤ 0.01), respectively, in the DEN+HCB group compared to DEN. HCB significantly increased TGF-β1 (45 %), mRNA levels, evaluated by RT-PCR, in livers from DEN+HCB group compared to DEN.Protein levels of TGF-β1, and AhR, determined by immunohistochemistry, increased 62 and 51%, (p ≤ 0.001, p ≤ 0.01, respectively), in focal areas compared to non focal areas. HCB increased PCNA, TGF-β1 and phosphorylated ERK1/2 protein levels in a dose- and time-dependent manner in HEP-G2 cells. When cells were preincubated with the AhR antagonist, 4,7 orthophenanthroline, HCB (5 M) stimulatory effect on the former parameters were blocked. Conclusion: Overexpression of TGF-β1 and AhR in the focal areas of rat liver, suggest that these proteins may be involved in HCB-stimulatory effect on cell proliferation. AhR is a mediator of HCB-inducing effect on cell proliferation, TGF-β1 expression, and ERK1/2 activation as demonstrated in HEP-G2 cell line.