Hepatology highlights
Authors: Neil Kaplowitz
Journal: Hepatology (2005)

Vesicular stomatitis virus (VSV) is a nonpathogenic RNA virus that is sensitive to interferon suppression in normal but not cancer cells because the latter have defective antiviral pathways. Because of rapid viral replication in cancer cells, a lytic response precedes the development of an antiviral neutralizing immune response which otherwise would undermine the desired effect. Shinozaki et al. tested the efficacy of such an approach in therapy of multifocal HCC in rats. Rats were infused with rat HCC cells via portal vein to establish multifocal HCC. They were then fit with an indwelling hepatic artery cannula and infused with VSV at 0, 2 and 4 days. In serum and normal liver the virus disappeared very rapidly but was sustained for at least 4 days in the tumor tissue. The lytic response after repeated doses was more rapid than the neutralizing immune response (7 days). There was no increase in serum transaminases or histological changes in normal tissue. Repeated low doses of virus were much more effective than a single large dose — completely necrotic tumors in 29% of all nodules at day 5 and overall survival was prolonged with 18% “cure” (long term survival) (see Fig.) Thus, optimization of virotherapy with rapid acting oncolytic agents which are delivered in repeated doses directly to the liver cancer via hepatic artery has considerable promise in prolonging survival and even curing HCC. (See HEPATOLOGY 2005;41:196-203) Considerable evidence has accumulated in recent years to support a major role for the innate immune system in modulating the severity of acetaminophen (APAP) hepatotoxicity downstream of hepatocellular metabolism, covalent binding and GSH depletion. A key question is what triggers the participation of the innate immune system. Su et al. address this question by examining the toxicity of APAP in lipopolysaccharide binding protein lipopolysaccharide (LBP) null mice. LPS binds to LBP which then activates Kupffer cells by interacting with CD14 coupled to toll-like receptor-4. The null mice exhibited less necrosis and improved survival. This was accompanied by marked suppression of the upregulation of TNF, IL-10 and IL-6 mRNA. Interestingly, although protection against APAP injury was partial, cytokine expression was near-completely suppressed. No differences in expression of CYP was observed in null mice and GSH depletion and covalent binding were the same as in wild type. Thus, APAP metabolism was not changed in null mice. Finally, after pretreatment of null mice with adenovirus expressing LBP, plasma levels of LBP were restored and APAP-induced liver injury was significantly increased compared to adenovirus expressing β-galactosidase. These studies suggest that LPS, via LBP, plays a role in the activation of the innate immune response which contributes to APAP toxicity. However, since LPS levels in the portal vein did not change, it is not clear if LPS plays a “permissive” role in activating Kupffer cells (possibly sensitized by other effects of APAP) or if LBP exerts unknown effects independent of LPS. (See HEPATOLOGY 2005;41:187-195.) HCC complicates cirrhosis which itself is a proliferative premalignant state. An important strategy in management of cirrhosis would be to prevent the development of HCC. TGF-α activation of EGFR is one potential pathway in promoting mitogenesis in the cirrhotic liver and HCC. Schiffer et al. addressed this issue by examining the effect of gefitinib, an EGFR-tyrosine kinase inhibitor, in a rat model of cirrhosis and HCC. Rats received daily ip diethylnitrosamine (DEN); cirrhosis developed in 12 weeks and multifocal HCC in 16 weeks of treatment (see Fig.) Therefore rats with cirrhosis were randomized to receive gefitinib versus vehicle from 12-18 weeks. TGF-α expression and hepatocyte proliferation were increased in the cirrhotic liver and even further increased in HCC nodules. Gefitinib treatment exerted a significant chemopreventive effect (3.7 vs 18.1 HCC nodules per liver). EGFR and downstream ERK phosphorylation were blocked by the treatment. The residual tumors showed persistent ERK activation along with Akt and IGF-2 which are thus mitogenic pathways independent of EGFR. In conclusion, this is an important study demonstrating that chemoprevention is a feasible strategy in situations where there is established cirrhosis and a stimulus to HCC formation. Clinical applications of this strategy are eagerly awaited. However, one concern is the effect of antiproliferative strategies on the maintenance of the cirrhotic hepatocyte functional mass over longer time intervals. (See HEPATOLOGY 2005;41:307-314.) Trovafloxacin, a quinolone antibiotic, is an idiosyncratic hepatotoxin. Postmarketing experience in more than 2 million patients uncovered reports of 150 cases of hepatotoxicity including 14 cases of acute liver failure. To address the mechanism of this adverse effect, Liguori et al. compared trovafloxacin to four other quinolones. None caused toxicity in animal models or in cultured human hepatocytes at very high doses. Microarray analysis of human hepatocytes, however, revealed changes which distinguished trovafloxacin from the other drugs in this class. A set of genes indicative of oxidative stress was selectively upregulated. Trovafloxacin depleted GSH in HepG2 cells at a much lower concentration than the other quinolones. This was preceded by increased peroxide exposure (DCF fluorescence). Further analysis revealed unique regulation of 142 genes, including some involved in RNA transcription. Since trovafloxacin inhibits bacterial topoisomerase and mammalian topoisomerase II is essential for transcription by RNA polymerase II, gene expression changes caused by trovafloxacin were compared to that caused by the topoisomerase II inhibitor, etoposide. Similar downregulation of genes involved in RNA transcription was observed with both drugs. Interestingly, the gene expression changes were very different in rat hepatocytes, although a few were the same as in human cells. These studies use a toxicogenomic strategy to identify changes in gene expression in human hepatocytes which may provide a unique signature and clues to pathogenesis, even in the absence of toxicity in the experimental system. Although this is a logical and meritorious approach in providing plausible candidate mechanisms contributing to toxicity or to susceptibililty, the evidence is circumstantial, there were many alterations (which are important?), and there is a presumption that gene expression changes are involved in toxicity. Thus, the mechanism of trovafloxacin induced hepatotoxicity in rare individuals remains unknown. (See HEPATOLOGY 2005;41:177-186.) Previous work has strongly implicated oxidative stress in experimental alcohol-induced liver injury. Of the sources of oxidative stress in studies using null mice, NADPH oxidase in nonparenchymal cells was implicated as more important in liver injury while Cyp2e1 did not seem to play a role (although this is controversial). Another consequence of oxidative stress, particularly relevant to HCC development, is DNA damage. Bradford et al. assessed oxidative DNA damage induced by ethanol in the rodent intragastric feeding model and the contribution of the sources of oxidative stress. Feeding ethanol to rats or mice for 4 weeks resulted in increased oxidative DNA adducts, mutagenic apurinic/apyrimidinic sites, and expression of base excision DNA repair genes that are known to remove oxidative DNA lesions. These markers were unaltered in p47 phox-null mice (decreased NADPH oxidase) but were completely absent in Cyp2e1-null mice. Furthermore, treatment with the p450 inhibitor, 1-aminobenzotriazole (ABT), blocked oxidative DNA damage. Interestingly, liver injury still occurred in Cyp2e1-null and ABT-treated mice. The findings suggest that ethanol-induced steatohepatitis and oxidative DNA damage are caused by unrelated mechanisms. Thus, oxidative DNA damage is uniquely dependent on CYP2e1 mediated reactive oxygen generation which may play an important role in the development of cancer in the livers of alcoholic and perhaps nonalcoholic fatty liver cirrhosis. It will be of interest to see if antioxidant treatment is effective in preventing DNA damage which would support a chemoprevention strategy for patients. (See HEPATOLOGY 2005;41:336-344.) Recent evidence suggests that EGFR plays a critical role in Fas (CD95) apoptosis. This involves JNK dependent EGFR/CD95 association and EGFR catalyzed tyrosine phosphorylation of CD95 leading to CD95-membrane translocation and DISC (Death-inducing signaling complex)-formation. Eberle et al. used fluorescence resonance energy transfer (FRET) technique to study the interaction between CD95 and EGFR in Huh-7 hepatoma cells which have low endogenous CD95. EGFR and CD95 coupled to different dyes were expressed in the cells allowing their close association to be viewed as a FRET-signal. Hyperosmolarity or CD95L induced EGFR/CD95 association (FRET-signal), CD95 phosphorylation and DISC formation (see Fig. which demonstrates the association of intracellular EGFR and CD95 and mobilization to the plasma membrane and membrane blebs induced by FasL). No intracellular FRET signal was observed in the presence of a JNK inhibitor demonstrating that association of EGFR and CD95 was blocked. On the other hand an inhibitor of EGFR-tyrosine kinase did not block this association but prevented translocation of the complex to the plasma membrane. Microtubule inhibitors also blocked translocation. CD95 mutants (tyrosine phosphorylation incompetent) did not translocate to the plasma membrane even though they associated with EGFR. Thus, EGFR must both associate with and phosphorylate CD95 in order for the complex to undergo microtubule-dependent translocation. Inhibition of any of these steps blocked CD95L induced apoptosis. DISC formation required plasma membrane targeting of the complex and did not occur in the cytosol, thus explaining the mechanism of the previously described protective effect of colchicine. This is an elegant study which elucidates the role of EGFR in promoting the Fas death pathway and offers a number of therapeutic targets to protect the liver. It is of interest that the DISC formation associated with hyperosmolarity was transient and did not propagate the apoptosis cascade, whereas it was sustained and did so in response to CD95L. One aspect requiring further exploration is how CD95L signals to activate membrane trasnslocation of the CD95/EGFR complex. (See HEPATOLOGY 2005;41:315-326.)