Multiple liver insults synergize to accelerate experimental hepatocellular carcinoma.

The urgent unmet need for hepatocellular carcinoma (HCC) therapies is addressed here by characterising a novel mouse model of HCC in the context of ongoing liver damage and overnutrition. Male C57Bl/6J mice were treated with diethylnitrosamine (DEN) and thioacetamide (TAA), and some were provided with an atherogenic high fat diet (HFD). Inflammation, steatosis, fibrosis, 87 genes, liver lesions and intratumoural leukocyte subsets were quantified up to 24 weeks of age. Adding HFD to DEN/TAA increased fibrosis, steatosis and inflammation, and the incidence of both HCC and non-HCC dysplastic lesions. All lesions contained α-SMA positive fibroblasts. Macrophage marker F4/80 was not significantly different between treatment groups, but the macrophage-associated genes Arg-1 and Cd47 were differentially expressed. Fibrosis, cancer and cell death associated genes were upregulated in DEN/TAA/HFD livers. Fewer Kupffer cells and plasmacytoid dendritic cells were in tumours compared to control liver. In conclusion, combining a hepatotoxin with an atherogenic diet produced more intrahepatic tumours, dysplastic lesions and fibrosis compared to hepatotoxin alone. This new HCC model provides a relatively rapid means of examining primary HCC and potential therapies in the context of multiple hepatotoxins including those derived from overnutrition.

Fibroblast activation protein is dispensable in the anti-influenza immune response in mice.

Fibroblast activation protein alpha (FAP) is a unique dual peptidase of the S9B serine protease family, being capable of both dipeptidyl peptidase and endopeptidase activities. FAP is expressed at low level in healthy adult organs including the pancreas, cervix, uterus, submaxillary gland and the skin, and highly upregulated in embryogenesis, chronic inflammation and tissue remodelling. It is also expressed by cancer-associated stromal fibroblasts in more than 90% of epithelial tumours. FAP has enzymatic and non-enzymatic functions in the growth, immunosuppression, invasion and cell signalling of tumour cells. FAP deficient mice are fertile and viable with no gross abnormality, but little data exist on the role of FAP in the immune system. FAP is upregulated in association with microbial stimulation and chronic inflammation, but its function in infection remains unknown. We showed that major populations of immune cells including CD4+ and CD8+ T cells, B cells, dendritic cells and neutrophils are generated and maintained normally in FAP knockout mice. Upon intranasal challenge with influenza virus, FAP mRNA was increased in the lungs and lung-draining lymph nodes. Nonetheless, FAP deficient mice showed similar pathologic kinetics to wildtype controls, and were capable of supporting normal anti-influenza T and B cell responses. There was no evidence of compensatory upregulation of other DPP4 family members in influenza-infected FAP-deficient mice. FAP appears to be dispensable in anti-influenza adaptive immunity.

Hepatocellular carcinoma: Mouse models and the potential roles of proteases.

Primary liver cancer is the second most common cause of mortality from cancer. The most common models of hepatocellular carcinoma, which use a chemical and/or metabolic insult, xenograft, or genetic manipulation, are discussed in this review. In the tumour microenvironment lymphocytes, fibroblasts, endothelial cells and antigen presenting cells are important determinants of cell fate. These cells make a range of proteases that modify the biological activity of other proteins, particularly extracellular matrix proteins that alter cell migration of tumour cells, fibroblasts and leucocytes, and chemokines that alter leucocyte migration. The DPP4 family of post-proline peptidase enzymes modifies cell movement and the activities of many bioactive molecules including growth factors and chemokines.

The pro-fibrotic role of dipeptidyl peptidase 4 in carbon tetrachloride-induced experimental liver injury.

Liver fibrosis is a progressive pathological process involving inflammation and extracellular matrix deposition. Dipeptidyl peptidase 4 (DPP4), also known as CD26, is a cell surface glycoprotein and serine protease. DPP4 binds to fibronectin, can inactivate specific chemokines, incretin hormone and neuropeptides, and influences cell adhesion and migration. Such properties suggest a pro-fibrotic role for this peptidase but this hypothesis needs in vivo examination. Experimental liver injury was induced with carbon tetrachloride (CCl4) in DPP4 gene knockout (gko) mice. DPP4 gko had less liver fibrosis and inflammation and fewer B cell clusters than wild type mice in the fibrosis model. DPP4 inhibitor-treated mice also developed less liver fibrosis. DNA microarray and PCR showed that many immunoglobulin (Ig) genes and some metabolism-associated transcripts were differentially expressed in the gko strain compared with wild type. CCl4-treated DPP4 gko livers had more IgM+ and IgG+ intrahepatic lymphocytes, and fewer CD4+, IgD+ and CD21+ intrahepatic lymphocytes. These data suggest that DPP4 is pro-fibrotic in CCl4-induced liver fibrosis and that the mechanisms of DPP4 pro-fibrotic action include energy metabolism, B cells, NK cells and CD4+ cells.

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.

Metalloproteins and phytochelatin synthase may confer protection against zinc oxide nanoparticle induced toxicity in Caenorhabditis elegans.

Zinc oxide nanoparticles (ZnONPs) are used in large quantities by the cosmetic, food and textile industries. Here we exposed Caenorhabditis elegans wild-type and a metal sensitive triple knockout mutant (mtl-1;mtl-2;pcs-1) to ZnONPs (0-50mg/L) to study strain and exposure specific effects on transcription, reactive oxygen species generation, the biomolecular phenotype (measured by Raman microspectroscopy) and key endpoints of the nematode life cycle (growth, reproduction and lifespan). A significant dissolution effect was observed, where dissolved ZnO constituted over 50% of total Zn within a two day exposure to the test medium, suggesting that the nominal exposure to pure ZnONPs represents in vivo, at best, a mixture exposure of ionic zinc and nanoparticles. Nevertheless, the analyses provided evidence that the metallothioneins (mtl-1 and mtl-2), the phytochelatin synthase (pcs-1) and an apoptotic marker (cep-1) were transcriptionally activated. In addition, the DCFH-DA assay provided in vitro evidence of the oxidative potential of ZnONPs in the metal exposure sensitive triple mutant. Raman spectroscopy highlighted that the biomolecular phenotype changes significantly in the mtl-1;mtl-2;pcs-1 triple knockout worm upon ZnONP exposure, suggesting that these metalloproteins are instrumental in the protection against cytotoxic damage. Finally, ZnONP exposure was shown to decrease growth and development, reproductive capacity and lifespan, effects which were amplified in the triple knockout. By combining diverse toxicological strategies, we identified that individuals (genotypes) housing mutations in key metalloproteins and phytochelatin synthase are more susceptible to ZnONP exposure, which underlines their importance to minimize ZnONP induced toxicity.