Mesenchyme-derived factors enhance preneoplastic growth by non-genotoxic carcinogens in rat liver.

Many frequently prescribed drugs are non-genotoxic carcinogens (NGC) in rodent liver. Their mode of action and health risks for humans remain to be elucidated. Here, we investigated the impact of two model NGC, the anti-epileptic drug phenobarbital (PB) and the contraceptive cyproterone acetate (CPA), on intrahepatic epithelial-mesenchymal crosstalk and on growth of first stages of hepatocarcinogenesis. Unaltered hepatocytes (HC) and preneoplastic HC (HCPREN) were isolated from rat liver for primary culture. DNA replication of HC and HCPREN was increased by in vitro treatment with 10 µM CPA, but not 1 mM PB. Next, mesenchymal cells (MC) obtained from liver of rats treated with either PB (50 mg/kg bw/day) or CPA (100 mg/kg bw/day), were cultured. Supernatants from both types of MC raised DNA synthesis of HC and HCPREN. This indicates that PB induces replication of HC and HCPREN only indirectly, via growth factors secreted by MC. CPA, however, acts on HC and HCPREN directly as well as indirectly via mesenchymal factors. Transcriptomics and bio-informatics revealed that PB and CPA induce extensive changes in the expression profile of MC affecting many growth factors and pathways. MC from PB-treated rats produced and secreted enhanced levels of HBEGF and GDF15, factors found to suppress apoptosis and/or induce DNA synthesis in cultured HC and HCPREN. MC from CPA-treated animals showed enhanced expression and secretion of HGF, which strongly raised DNA replication of HC and HCPREN. In conclusion, our findings reveal profound effects of two prototypical NGC on the hepatic mesenchyme. The resulting release of factors, which suppress apoptosis and/or enhance cell replication preferentially in cancer prestages, appears to be crucial for tumor promotion by NGC in the liver.

Proinflammatory mesenchymal effects of the non-genotoxic hepatocarcinogen phenobarbital: a novel mechanism of antiapoptosis and tumor promotion.

Many environmental pollutants and drugs, including steroid hormones, hypolipidemics and antiepileptics, are non-genotoxic carcinogens (NGC) in rodent liver. The mechanism of action and the risk for human health are still insufficiently known. Here, we study the effects of phenobarbital (PB), a widely used model NGC, on hepatic epithelial-mesenchymal crosstalk and the impact on hepatic apoptosis. Mesenchymal cells (MC) and hepatocytes (HC) were isolated from control and PB-treated rat livers. PB induced extensive changes in gene expression in MC and much less in HC as shown by transcriptomics with oligoarrays. In MC only, transcript levels of numerous proinflammatory cytokines were elevated. Correspondingly, ELISA on the supernatant of MC from PB-treated rats revealed enhanced release of various cytokines. In cultured HC, this supernatant caused (i) nuclear translocation and activation of nuclear factor-κB (shown by immunoblots of nuclear extracts and reporter gene assays), (ii) elevated expression of proinflammatory genes and (iii) protection from the proapoptotic action of transforming growth factor beta 1 (TGFß1). PB treatment in vivo or in vitro elevated the production and release of tumor necrosis factor alpha from MC, which was identified as mainly responsible for the inhibition of apoptosis in HC. In conclusion, our findings reveal profound proinflammatory effects of PB on hepatic mesenchyme and mesenchymal-epithelial interactions. The resulting release of cytokines acts antiapoptotic in HC, an effect crucial for tumor promotion and carcinogenesis by NGC.