Superoxide deficiency attenuates promotion of hepatocarcinogenesis by cytotoxicity in NADPH oxidase knockout mice.

Long-term exposure to carcinogens combined with chronic hepatitis contributes greatly to the worldwide high incidence of hepatocellular carcinoma (HCC). It is still unclear to which extent the release of pro-inflammatory reactive oxygen or nitrogen species contributes to the development of this malignancy. Here, we aim to elucidate the role of superoxide in a model of chemical hepatocarcinogenesis. p47(phox) knockout mice (KO), lacking superoxide formation by phagocytic NADPH oxidase (phox), and wild-type animals (WT) were subjected to two different initiation-promotion protocols: (1) single dose of diethylnitrosamine (DEN) at 6 weeks of age followed by phenobarbital (PB) via diet, or ethanol (EtOH) in drinking water; (2) DEN at neonatal age followed by three cytotoxic doses of DEN at intervals of 6-7 weeks. The appearance of tumors and prestages was quantified. There was no obvious difference in the capacity of DEN to initiate hepatocarcinogenesis in KO and WT mice. PB promoted tumor development in both genotypes without significant difference. EtOH induced steatosis significantly less in KO than in WT liver, but had no effect on tumor formation in either genotype. However, hepatocarcinogenesis by three cytotoxic DEN doses after neonatal initiation was attenuated significantly in KO. Macrophages/monocytes identified by the specific antigen F4/80 were more abundant in KO than in WT liver, possibly reflecting a compensatory response. We conclude that phox-derived superoxide is not essential but is supportive for the promotion of hepatocarcinogenesis by cytotoxic doses of DEN. The production of superoxide may therefore contribute to the promotion of hepatocarcinogenesis by cytotoxic/pro-inflammatory stimuli.

Proteomics reveals acute pro-inflammatory and protective responses in rat Kupffer cells and hepatocytes after chemical initiation of liver cancer and after LPS and IL-6.

Inflammation is a key event in the development of liver cancer. We studied early inflammatory responses of Kupffer cells (KCs) and hepatocyte (HC) after cancer initiation. The chemical carcinogen N-nitrosomorpholine (NNM) was used in a rat model. We applied a comprehensive analytical strategy including metabolic labeling, 2-D PAGE, LC-MS/MS-based spot identification and shotgun proteomics and thus determined the rates of synthesis of individual proteins, compared whole tissue with isolated constituent cells and performed in vivo to in vitro comparisons of NNM effects. NNM increased synthesis of overall and 138 individual proteins identified in HC and/or KC, indicating reprogramming of metabolism favoring protection, repair and replacement of cell constituents in HC and KC. Secretome analysis by 2-D PAGE and shotgun proteomics of HC revealed the induction of acute phase proteins, in case of KC of proteases, cytokines and chemokines, indicating inflammatory effects. All responses were induced rapidly, independently of signals from other cells, and closely mimicked the pro-inflammatory and protective effects of inflammation modulators LPS in KC and IL-6 in HC. In conclusion, the carcinogen NNM exerts pro-inflammatory effects in the liver, partially by direct activation of KC. The acute inflammation and its protective component will enhance formation, survival and proliferation of initiated cells and may therefore act synergistically with the genotoxic action of the carcinogen.

Divide and conquer: rat liver tissue proteomics based on the analysis of purified constituents.

Comparative proteome data of normal and diseased tissue samples are difficult to interpret. Proteins detected in tissues are derived from different cell types and blood constituents. Pathologic or toxicant-induced aberrations may affect the proteome profile of tissues in several ways since different cell types may respond in very different and highly specific manners. The aim of this study was to analyze the proteome profiles of purified rat liver primary cells and of blood plasma in comparison to liver whole tissue. Moreover, we investigated alterations of these profiles induced by the liver toxicant N-nitrosomorpholine (NNM) used as a model compound. Whole liver samples, pure hepatocytes and Kupffer cells as well as blood plasma were obtained from saline- or NNM-treated rats. Proteins were separated by 2-D PAGE and their amounts were estimated by fluorography. Selected proteins were identified by MS analysis of tryptic digests. Among them we identified proteins exclusively expressed in the analysed constituents. Several of these proteins were assigned in the proteome profile of whole-tissue homogenates. Furthermore, we identified several proteins that were modified, up-regulated or down-regulated due to NNM treatment in total liver homogenates. Some of these protein alterations were specifically detected in primary cells isolated from NNM-treated rats. Thus, we demonstrated the successful assignment of NNM-induced proteome alterations in rat liver to the cell type of origin. The currently applied approach may help to better understand pathologic processes at a whole-tissue level.

Superoxide generation from Kupffer cells contributes to hepatocarcinogenesis: studies on NADPH oxidase knockout mice.

We hypothesized that superoxide from Kupffer cells (KC) contributes to hepatocarcinogenesis. p47phox(-/-) mice, deficient in phagocyte NADPH oxidase and superoxide generation, received a single dose of the hepatocarcinogen diethylnitrosamine (DEN). The following hepatic effects were observed at time points between 30 min and 35 days. Liver damage after DEN was manifested by loss of body and liver mass and of liver DNA and by an increase in apoptosis, necrosis and signs of inflammation. These effects were massive in wild-type (wt) male mice, but only very mild in p47phox(-/-) mice. Regenerative DNA synthesis subsequent to liver damage was high in wt male mice, but weak in p47phox(-/-) mice. In females the apparent protection by p47phox(-/-) was less pronounced than in males. Therefore, further experiments were performed with males. In KC isolated from wt mice superoxide production was enhanced by DEN pretreatment in vivo. Also, in vitro addition of DEN to KC cultures induced superoxide release, similarly to lipopolysaccharide, a standard KC activator. Thus, DEN directly activates wt KC to produce superoxide. KC from p47phox(-/-) mice did not release superoxide. TNFalpha production by isolated KC was transiently depressed 0.5 h after DEN treatment in vivo, but recovered rapidly. In blood serum TNFalpha levels of wt mice were elevated for the initial 6 h. TNFalpha in KC cultures and in serum of p47phox(-/-) mice was reduced. DEN in vivo induced DNA damage ('comets') in hepatocytes. This damage was extensive in wt mice but much less in p47phox(-/-) mice. These studies suggest two conclusions: (i) superoxide generation by phagocytes during liver damage and inflammation aggravates genotoxic and cytotoxic effects in hepatocytes and may thus contribute to tumor initiation and promotion; (ii) DEN has a direct stimulatory effect on KC to release superoxide and TNFalpha.

Response of isolated hepatocytes from carcinogen sensitive (C3H) and insensitive (C57BL) mice to signals inducing replication or apoptosis.

The mouse strain C3H shows high incidence of liver tumors in carcinogenicity testing, while the strain C57BL exhibits low incidence. The F1 generation hybrids, B6C3F1, which are widely used in long-term carcinogenesis bioassays, are of intermediate sensitivity. We asked whether this strain difference could be due to different susceptibility of the parenchymal cells to signals inducing replication or apoptosis. Hepatocytes were isolated and cultured according to standard protocols. We tested (1) for the induction of DNA synthesis by epidermal growth factor (EGF), (2) for its inhibition by TGF-beta1, and (3) for the induction of apoptosis by TGF-beta1. Basal rates of DNA synthesis in untreated hepatocytes cultured from C3H and B6C3F1 mice were 6.5 and 3.5 times higher, respectively, than in hepatocytes from C57BL on day 3. Moreover, addition of EGF (10 ng/ml) increased DNA synthesis on day 3 in hepatocytes from C3H (4.2-fold) and B6C3F1 (2.7-fold) more strongly than in hepatocytes from C57BL. Treatment with TGF-beta1 inhibited basal and EGF-stimulated DNA synthesis dose-dependently. Inhibition was maximal at 1 ng TGF-beta1/ml in cultures from C57BL mice, and at 0.3 ng/ml in hepatocytes from C3H mice. In untreated hepatocytes from both strains virtually no apoptotic figures (condensed or fragmented nuclei, Hoechst 33285 staining) were found. After treatment with TGF-beta1 the incidence of apoptotic nuclei in hepatocytes from C57BL was higher than in cells from C3H mice (1.7% vs 3% on day 3). Thus it appears that hepatocytes from C57BL mice possess a lower growth potential, as indicated by a low basal rate of DNA synthesis and low inducibility by EGF, but a higher sensitivity to induction of apoptosis by TGF-beta1 than hepatocytes of the C3H strain. These findings may be helpful to explain the different susceptibility to induction of hepatocarcinogenesis in C3H and C57BL mice.