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.

There have been numerous reports that chemicals which induce peroxisomes in rodent liver increase DNA synthesis in isolated hepatic parenchymal cells, but not as well in vitro as in vivo. It is also known that tumour necrosis factor α (TNFα) is mitogenic in isolated hepatocytes. Since Kupffer cells are a major source of TNFα in the liver and have recently been shown to be activated by peroxisome proliferators, the possibility exists that the effect of peroxisome proliferators on DNA synthesis in parenchymal cells is via Kupffer cell contamination of isolated hepatocyte preparations. The purpose of this study was to evaluate this hypothesis by studying the effect of model peroxisome proliferators on purified hepatocyte preparations. Hepatocytes were prepared from rat liver by standard calciumfree and collagenase perfusion. Subsequently, cells were centrifuged through Percoll to remove contaminating nonparenchymal cells. Cells were at least 99.9% pure as assessed by cell counting using specific markers for hepatocytes (resorufin O-glucoside) and Kupffer cells (FITClabelled latex beads). Hepatocytes were cultured in Williams medium ϩ 10% fetal bovine serum for 24 h followed by culture for 48 h in Williams medium plus or minus drug or mitogen additions. Under these conditions epidermal growth factor stimulated DNA synthesis assessed by incorporation of [ 3 H]thymidine~5-fold over control levels. The peroxisome proliferators WY,14-643 and nafenopin, however, had no effect on DNA synthesis, although they did increase acyl-CoA oxidase as expected. In contrast, TNFα increased cell proliferation nearly 10-fold in purified hepatocytes, an effect nearly doubled by WY-14,643. Further, when conditioned medium from purified Kupffer cells incubated with WY-14,643 was added to pure hepatocytes, DNA synthesis was increased over 2-fold in a time-dependent manner. Collectively, these data support the hypothesis that peroxisome proliferators do not influence DNA synthesis in isolated hepatocytes per se. Rather, they stimulate cytokine production by Kupffer cells which in turn increases DNA synthesis in parenchymal cells. An increase in mitogenic cytokine production by Kupffer cells is necessary for stimulation of DNA synthesis in purified rat parenchymal cells.

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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.

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.