Redox status impairment in liver and kidney of prematurely senescent mice: effectiveness of DTS phytotherapeutic compound.

T-maze test-selected prematurely senescent mice (PSM) were allocated into two groups: (A) those given DTS (150 mg/kg) orally for 30 days and (B) untreated PSM with age-matched fast T-maze performers as control. After sacrifice, the liver and kidney were analyzed for catalase (CAT) activity, glutathione peroxidase (GPx), superoxide dismutase (SOD), malondyaldehyde (MDA), and plasma thiols. Untreated PSM showed decreased plasma thiols and tissue level of CAT, SOD, GPx, with higher MDA (P < 0.01 vs. fast performers), while DTS (Denshichi-Tochiu-Sen) significantly improved glutathione and cysteine (P < 0.05) and tissue concentration of the above parameters (P < 0.05). Such preliminary data suggest that DTS mitigated oxidative damage in PSM, with likely action on the cytoplasm and mitochondrial matrix.

Liver exposure to xenobiotics: the aging factor and potentials for functional foods.

Hepatocytes isolated from 20- and 4-month Wistar rats and cultured with or without alpha-linolenic acid (LNA) were then added with nutraceutical YHK or sylibin before the test with iron or copper. Overall, YHK proved to be more effective than sylibin in Fe/Cu-induced peroxidative damage on normal and LNA-loaded hepatocytes (p < 0.05). YHK exerted a significant protection against DPPH radical-scavenging activity in the "old" group (p versus sylibin) and against lipophilic generators in both age groups (p < 0.05 versus sylibin). Both compounds were ineffective on age-related increase of surface-charge density. These preliminary data suggest that age per se enhances the vulnerability of hepatocytes to xenobiotics, whereas some safe nutraceuticals seem to exert significant protective effects.

A prospective study of hepatitis B virus markers in patients with chronic HBV infection from Brazilian families of Western and Asian origin.

The purpose of the present study was to determine the frequency of hepatitis B virus (HBV) markers in families of HBsAg-positive patients with chronic liver disease. Serum anti-HBc, HBsAg and anti-HBs were determined by enzyme immunoassay and four subpopulations were considered: genetically related (consanguineous) and non-genetically related (non-consanguineous) Asian subjects and genetically related and non-genetically related Western subjects. A total of 165 and 186 relatives of Asian and Western origin were enrolled, respectively. The occurrence of HBsAg and anti-HBs antibodies was significantly higher (P < 0.0001) in family members of Asian origin (81.8%) than in family members of Western origin (36.5%). HBsAg was also more frequent among brothers (79.6 vs 8.5%; P < 0.0001), children (37.9 vs 3.3%; P < 0.0001) and other family members (33.9 vs 16.7%; P < 0.0007) of Asian than Western origin, respectively. No difference between groups was found for anti-HBs, which was more frequently observed in fathers, spouses and other non-genetic relatives. HBV infection was significantly higher in children of Asian than Western mothers (P < 0.0004). In both ethnic groups, the mothers contributed more to their children's infection than the fathers (P < 0.0001). Furthermore, HBsAg was more frequent among consanguineous members and anti-HBs among non-consanguineous members. These results suggest the occurrence of vertical transmission of HBV among consanguineous members and probably horizontal sexual transmission among non-consanguineous members of a family cluster. Thus, the high occurrence of dissemination of HBV infection characterizes family members as a high-risk group that calls for immunoprophylaxis. Finally, the study showed a high familial aggregation rate for both ethnic groups, 18/19 (94.7%) and 23/26 (88.5%) of the Asian and Western origin, respectively.

A prospective study of hepatitis B virus markers in patients with chronic HBV infection from Brazilian families of Western and Asian origin

The purpose of the present study was to determine the frequency of hepatitis B virus (HBV) markers in families of HBsAg-positive patients with chronic liver disease. Serum anti-HBc, HBsAg and anti-HBs were determined by enzyme immunoassay and four subpopulations were considered: genetically related (consanguineous) and non-genetically related (non-consanguineous) Asian subjects and genetically related and non-genetically related Western subjects. A total of 165 and 186 relatives of Asian and Western origin were enrolled, respectively. The occurrence of HBsAg and anti-HBs antibodies was significantly higher (P < 0.0001) in family members of Asian origin (81.8 percent) than in family members of Western origin (36.5 percent). HBsAg was also more frequent among brothers (79.6 vs 8.5 percent; P < 0.0001), children (37.9 vs 3.3 percent; P < 0.0001) and other family members (33.9 vs 16.7 percent; P < 0.0007) of Asian than Western origin, respectivelly. No difference between groups was found for anti-HBs, which was more frequently observed in fathers, spouses and other non-genetic relatives. HBV infection was significantly higher in children of Asian than Western mothers (P < 0.0004). In both ethnic groups, the mothers contributed more to their children's infection than the fathers (P < 0.0001). Furthermore, HBsAg was more frequent among consanguineous members and anti-HBs among non-consanguineous members. These results suggest the occurrence of vertical transmission of HBV among consanguineous members and probably horizontal sexual transmission among non-consanguineous members of a family cluster. Thus, the high occurrence of dissemination of HBV infection characterizes family members as a high-risk group that calls for immunoprophylaxis. Finally, the study showed a high familial aggregation rate for both ethnic groups, 18/19 (94.7 percent) and 23/26 (88.5 percent) of the Asian and Western origin, respectively.

Bcl-X(L) antisense sensitizes human colon cancer cell line to 5-fluorouracil.

Resistance to 5-fluorouracil (5-FU) has been frequently found in the treatment of digestive tract cancer patients. Our previous study suggested that high expression of endogenous Bcl-X(L), might be associated with resistance to 5-FU in colorectal cancer. The aim of this study is to analyze the role of Bcl-X(L) in 5-FU resistance and to explore a new therapeutic strategy using Bcl-X(L) antisense. First, western blot analysis shows that Bcl-X(L) rather than Bcl-2 is overexpressed in primary adenocarcinoma of colon. Second, when Colo320 cells, with undetectable endogenous Bcl-XL expression, were transfected with Bcl-XL gene, they acquired high resistance to 5-FU. Finally, antisense oligodeoxynucleotides (ODNs) that targeted the start codon of Bcl-X(L) mRNA (AS1) prove to be the most effective in DLD1 cells with high endogenous Bcl-X(L) expression. Bcl-X(L) protein expression was decreased in a dose-dependent manner when the cells were treated with AS1 ODNs, while non-sense and sense controls and 5-FU had no effect on Bcl-X(L) protein. 5-FU treatment induced a level of apoptosis 10-fold higher in DLD1 cells than in untreated control cells, while the same dose of 5-FU induced a 55-fold higher level of apoptosis in DLD1 cells treated with Bcl-XL antisense oligodeoxynucleotides (P = 0.0003). Moreover, AS1 ODNs coupled with 5-FU decreased viable colon cancer cells 40% more than did 5-FU alone (P < 0.05). These results suggest that Bcl-X(L) is an important factor for 5-FU resistance and the suppression of Bcl-X(L) expression by the specific antisense ODNs can increase the sensitivity of colon cancer cells to 5-FU.

Large isoform of hepatitis delta antigen activates serum response factor-associated transcription.

Hepatitis delta virus infection sometimes causes severe and fulminant hepatitis as a coinfection or superinfection along with the hepatitis B virus. To elucidate the underlying mechanism of injury caused by hepatitis delta virus, we examined whether two isoforms of the hepatitis delta antigen (HDAg) had any effect on five well defined intracellular signal transduction pathways: serum response factor (SRF)-, serum response element (SRE)-, nuclear factor kappaB-, activator protein 1-, and cyclic AMP response element-dependent pathways. Reporter assays revealed that large HDAg (LHDAg) activated the SRF- and SRE-dependent pathways. In contrast, small HDAg (SHDAg) did not activate any of five pathways. LHDAg enhanced the transcriptional ability of SRF without changing its DNA binding affinity in an electrophoretic mobility shift assay. In addition, LHDAg activated a rat SM22alpha promoter containing SRF binding site and a human c-fos promoter containing SRE. In conclusion, LHDAg, but not SHDAg, enhances SRF-associated transcriptions. Despite structural similarities between the two HDAgs, there are significant differences in their effects on intracellular signal transduction pathways. These results may provide clues that will aid in the clarification of functional differences between LHDAg and SHDAg and the pathogenesis of delta hepatitis.

Activation of intracellular signaling by hepatitis B and C viruses: C-viral core is the most potent signal inducer.

To clarify the effects of hepatitis C virus (HCV) infection on hepatocytes, we analyzed and compared the induction of intracellular signals by HCV and hepatitis B virus (HBV) proteins. We examined the influence of 7 HCV (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and 4 HBV (precore, core, polymerase, and X) proteins on 5 well-defined intracellular signaling pathways associated with cell proliferation, differentiation, and apoptosis by use of a reporter assay. Viral protein-expression vectors were cotransfected into mammalian cells with reporter vectors having a luciferase gene driven by the following inducible cis-enhancer elements: the cyclic adenosine monophosphate response element, the serum response element (SRE), and the binding sites for nuclear factor kappaB (NF-kappaB), activator protein 1 (AP-1), and serum response factor (SRF). In addition, the activation of signals by HCV proteins was examined in a reporter plasmid having a natural interleukin-8 (IL-8) promoter upstream of a luciferase gene. Of 11 HCV and HBV proteins, HCV core had the strongest influence on intracellular signals, especially NF-kappaB-, AP-1-, and SRE-associated pathways. HCV core's activation level exceeded that of HBV X protein, a well-characterized transactivator of these signals. Moreover, HCV core activated the IL-8 promoter through NF-kappaB and AP-1. For the other proteins, HCV NS4B showed signal activation, but signals were activated at a lesser extent. The luciferase reporter assay, a recently introduced technique, helped in the elucidation of molecular events underlying the inflammatory and proliferation process in the liver induced by HCV.

Poor association of TT virus viremia with hepatocellular carcinoma.

AIM: The aim of this study was to clarify the relationship between TT virus (TTV) infection and the development of hepatocellular carcinoma. METHODS: TTV from serum was examined in 224 patients with hepatocellular carcinoma (HCC) and 106 patients with chronic liver disease (CLD) but without HCC who were admitted to our hospital between 1995-1997. As controls, 48 patients without liver disease were also examined. TTV DNA was detected using nested PCR method after extraction of DNA from serum. RESULTS: TTV DNA was detected in 29/224 (13%) of patients with HCC; in 14% (4/28) of HCC patients negative for both hepatitis B virus surface antigen (HBsAg) and anti-hepatitis C virus antibody (anti-HCV), in 9% (2/22) of HCC patients positive for HBsAg, and in 12% (21/170) of HCC patients positive for anti-HCV. The prevalence of TTV DNA in HCC patients (13%) was not significantly higher than in CLD patients (22%). There were no significant differences in age, gender, liver function, tumor biology (size, TNM classification), other viral markers, or amount of alcohol intake between TTV-positive and -negative HCC patients. Only a history of blood transfusion was significantly more frequent in TTV-positive HCC patients than in TTV-negative cases (p= 0.02). Coinfection with TTV did not correlate with the severity of HCV-positive liver disease. There was no significant difference in prognosis between TTV-positive and -negative HCC patients. CONCLUSIONS: TTV does not seem to contribute to the development of HCC from chronic liver disease and is not correlated with severity of liver disease.

Susceptibility of lamivudine-resistant hepatitis B virus to other reverse transcriptase inhibitors.

The emergence of resistant hepatitis B virus (HBV), with mutations in the YMDD motif of the polymerase gene after treatment with lamivudine, is becoming an important clinical problem. In this study, susceptibility of wild-type and lamivudine-resistant HBV M552I, M552V, and L528M/M552V mutants to other reverse transcriptase inhibitors was investigated by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, which showed the presence of a single-stranded band (representative of the HBV replicative intermediates) in the drug-free, wild-type HBV-transfected cells. This band was diminished in the samples of wild-type HBV DNA treated with either lamivudine, adefovir, or lobucavir. The band intensities from the lamivudine-resistant mutants were not decreased by treatment with lamivudine, but were decreased by the treatments with adefovir or lobucavir. In contrast, penciclovir and nevirapine did not diminish the intensity of the single-stranded band of wild-type HBV or the lamivudine-resistant mutants. These results demonstrate that lamivudine-resistant HBV is susceptible to adefovir and lobucavir. Lamivudine-resistant HBV should be treated with adefovir or lobucavir, and combination therapy with lamivudine and adefovir/lobucavir may prevent the emergence of lamivudine-resistant HBV.

YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: A study by in vitro full-length viral DNA transfection.

Recently, lamivudine used to treat patients with hepatitis B virus (HBV) infection was revealed to have potent antiviral activity. However, HBV resistance to lamivudine has been reported and shown to have amino acid substitutions in the methionine residue of the conserved tyrosine (Y), methionine (M), aspartate (D), aspartate (D) motif of RNA-dependent DNA polymerase. To explore the consequences of substitutions in this motif (YMDD), we made 7 variants by substituting the methionine of the YMDD motif with isoleucine (I), valine (V), alanine (A), leucine (L), lysine (K), arginine (R), and threonine (T). Replication ability of these variants was evaluated by transfection into human hepatoma cells. Sensitivity to lamivudine was tested for replication-competent variants. Four variants with hydrophobic substitutions (I, V, A, and L) remained replication-competent, whereas 3 others with hydrophilic substitutions (K, R, and T) exhibited impaired replication. Of the 4 replication-competent variants, 2 (I and V) were resistant, and 2 (A and L) were sensitive to lamivudine. Because the polymerase and the surface gene overlap, the introduction of these mutations affected the secretion of hepatitis B surface antigen (HBsAg), namely 4 variants (I, V, L, and R) secreted HBsAg, whereas 3 variants (A, K, and T) did not. Our study elucidated that only one amino acid substitution in the YMDD motif was sufficient to cause lamivudine resistance in vitro. As a result of replication competence and lamivudine sensitivity, only viruses having YIDD or YVDD sequences may appear during treatment with lamivudine. This in vitro system could be used to study HBV mutations, replication competence, and their susceptibility to antivirals.

Does dual infection by hepatitis B and C viruses play an important role in the pathogenesis of hepatocellular carcinoma in Japan?

BACKGROUND: There are contradictory data concerning the synergistic effect of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection on the progression from chronic hepatitis to hepatocellular carcinoma (HCC). METHODS: To clarify the role of coinfection with HBV and HCV in the progression and pathogenesis of HCC, viral and clinicopathologic features were studied in 368 consecutive HCC patients at the University of Tokyo from 1991-1995. RESULTS: Approximately 83% of patients (305 patients) were seropositive for the HCV antibody ("C-viral") and approximately 10% (37 patients) were positive for the hepatitis B surface antigen ("B-viral"). Positivity for both (dual infection) was found in only 2% of patients, and negativity for both in 5%. The incidence of dual infection in HCC patients was Similar to that in 549 patients with chronic hepatitis (1%) and 119 patients with cirrhosis (1%). Of the six HCC patients with dual infection, five patients were positive for the HBV early antigen and HBV DNA was less than measurable, whereas HCV RNA was detected and ranged from 10(3)-10(6) copies/50 microL of serum by competitive reverse transcriptase-polymerase chain reaction, and the clinical features resembled those of "C-viral" HCC. The remaining patient was early antigen positive and had HBV DNA by slot blot analysis, but the serum HCV RNA level was less than measurable. These data indicate that mutually exclusive viral replication occurred in patients with persistent coinfection. To further clarify further the possible involvement of HBV infection in "C-viral" HCC, HBV core antibody (HBcAb) was tested in 192 patients and was found to be positive in 111 and negative in 81. The serum HCV RNA level and clinicopathologic features (such as age and the severity of liver disease) were similar among the "C-viral" HCC patients irrespective of the presence or absence of HBcAb. CONCLUSIONS: Based on these results, coinfection was found to be much less prevalent than generally is claimed, and even in a few HCC patients with the coinfection the mutually exclusive viral replication was noted, suggesting that coinfection plays little if any role in the development of HCC.

Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator.

The hepatitis C virus (HCV) nonstructural region 5A (NS5A) protein, without its 146 amino-terminal amino acids and fused to the DNA-binding domain of GAL4, strongly activates transcription in yeast and human hepatoma cells. Transcriptional activation by the HCV NS5A protein may play a role in viral replication and hepatocarcinogenesis.