Abstract: BACKGROUND & AIMS: Alcohol consumption exacerbates liver injury in chronic hepatitis C, and enhanced mitochondrial oxidative stress is one possible mechanism. The aim of this study was to determine whether hepatitis C virus core protein and alcohol-inducible cytochrome P450 2E1 contribute to reactive oxygen species production and cytotoxicity in human hepatoma cells. METHODS: Huh-7 cells expressing core protein, cytochrome P450 2E1, or both were exposed to 0.1 mmol/L tertiary butyl hydroperoxide, tumor necrosis factor alpha, and/or 25 mmol/L ethanol. Cytotoxicity, reactive oxygen species production, glutathione content, and mitochondrial membrane potential were measured. RESULTS: Expression of core/cytochrome P450 2E1 synergistically enhanced cell death induced by either tertiary butyl hydroperoxide or tumor necrosis factor alpha. After tertiary butyl hydroperoxide treatment, total reactive oxygen species production was increased more than 3-fold compared with cells that did not express core and cytochrome P450 2E1. Mitochondrial depolarization and reduced glutathione depletion occurred as well, and cell death was prevented by inhibition of mitochondrial permeability transition or caspase activity. Confocal microscopy showed that the mitochondria themselves were the origin of the reactive oxygen species. In the absence of core/cytochrome P450 2E1 expression, mitochondrial changes and cell death did not occur. Ethanol treatment further decreased mitochondrial reduced glutathione content and exacerbated mitochondrial reactive oxygen species production, depolarization, and cell death. All these effects were prevented by the antioxidant N -acetylcysteine. CONCLUSIONS: Mitochondrial reactive oxygen species production is induced by hepatitis C virus core and cytochrome P450 2E1, resulting in a reduction of mitochondrial antioxidant capacity and sensitivity to oxidants and tumor necrosis factor alpha. Alcohol further depletes mitochondrial reduced glutathione, which exacerbates depolarization and cell death. Sensitization of mitochondria to oxidative insults is thus a potential mechanism for alcohol-related exacerbation of liver injury in chronic hepatitis C.

Abstract: OBJECTIVES: Pancreaticobiliary maljunction, an anomalous union of the pancreatic duct with the common bile duct, is a risk factor for biliary carcinoma. We hypothesized that, in patients with pancreaticobiliary maljunction, persistent regurgitation of pancreatic juice into the biliary tract induces oxidative DNA damage. We assessed the expression of an oxidative DNA base-modified product, 8-hydroxy-2'-deoxyguanosine, in gallbladder epithelium. DESIGN: Eleven noncancerous gallbladders from patients with pancreaticobiliary maljunction, 12 gallbladder carcinomas from patients without pancreaticobiliary maljunction and 14 noncancerous gallbladders from patients without pancreaticobiliary maljunction (control) were studied. METHODS: Immunohistochemistry was performed for 4-hydroxy-2-nonenal-modified protein (as a marker for lipid peroxidation), 8-hydroxy-2'-deoxyguanosine and p53 gene product. RESULTS: Stronger cytoplasmic staining of 4-hydroxy-2-nonenal-modified protein was observed in the gallbladder epithelium from patients with pancreaticobiliary maljunction than in epithelium from gallbladder cancer patients or from control subjects with normal gallbladders. Clear, strong nuclear staining of 8-hydroxy-2'-deoxyguanosine was observed in the gallbladder epithelial cells from patients with pancreaticobiliary maljunction. Densitometric quantitation revealed significantly higher expression of 8-hydroxy-2'-deoxyguanosine in gallbladder epithelial cells from patients with pancreaticobiliary maljunction (index 27.3 +/- 3.1) than in cells from patients with gallbladder carcinoma (11.4 +/- 1.5; P < 0.05) or from control subjects with normal gallbladder (6.4 +/- 1.0; P < 0.05). Positivity of p53 was 27% in gallbladder epithelium associated with pancreaticobiliary maljunction, 75% in gallbladder carcinoma epithelium and 0% in control epithelium. CONCLUSIONS: These results suggest that reactive oxygen species are produced in the gallbladder of patients with pancreaticobiliary maljunction and that oxidative DNA injury is related to carcinogenesis in these patients.

Abstract: BACKGROUND: Bilirubin oxidative metabolites (BOMs) are generated from bilirubin as a result of its scavenging action against free radicals. During sepsis, excess amounts of free radicals are produced, and they play an important role in the pathophysiological process. We studied whether urinary excretion of BOMs would increase under septic conditions in humans and compared BOM levels with other well-established clinical parameters of inflammation. METHODS: In 19 septic patients and 28 nonseptic control patients, the BOM concentrations in urine were measured by enzyme-linked immunosorbent assay with an anti-bilirubin antibody. RESULTS: Urinary BOM levels in septic patients were much higher than those in control patients (21.6 +/- 2.5 vs 1.4 +/- 0.4 micromol/g creatinine, P < 0.001). Although there was a linear correlation between urinary BOM and serum total bilirubin levels (r = 0.392, P = 0.008), serum bilirubin levels were not significantly higher in the septic group than in the control group (P = 0.072). BOM levels correlated with body temperature (r = 0.801, P < 0.001), white blood cell counts in the peripheral blood (r = 0.590, P < 0.001), serum C-reactive protein (r = 0.653, P < 0.001), and the acute physiological and chronic health evaluation (APACHE II) score (r = 0.467, P = 0.003). CONCLUSIONS: These results demonstrated a urinary increase in BOMs in septic patients. This increase indicates that urinary BOM level is a possible marker for continuous monitoring of sepsis severity in clinical practice.

Abstract: Heme oxygenase (HO)-1, the rate-limiting enzyme in heme degradation, is induced by oxidative stress and its major end product, bilirubin, is a potent physiological antioxidant. We studied the induction of HO-1 and bilirubin production in intestinal mucosa using a rat model of sepsis. E. coli lipopolysaccharide was administered intraperitonealy to male Wistar rats and intestinal mucosa was harvested. Intestinal lipid peroxides increased significantly at 1 hr and peaked at 170% of the control value at 5 hr. GSH significantly decreased at 3 hr, reaching the nadir of 50% of the control value at 5 hr. HO-1 mRNA was maximally induced fivefold at 3 hr and HO-1 protein maximally increased to 10 times the control value at 7.5 hr. Both bilirubin and bilirubin oxidative metabolites were maximally increased at 10 hr, to 4.3 and 3.7 times the control value, respectively. These data suggest that oxidative stress in sepsis quickly induces HO-1 in intestinal mucosa and that subsequent production of bilirubin works as an antioxidant. The small intestinal mucosa is an active participant in the general response to sepsis.

Abstract: BACKGROUND: Hepatic resection and percutaneous transhepatic cholangioscopic lithotomy (PTCSL) are the two main approaches to the treatment of hepatolithiasis, but comparisons of longterm followup results have not been adequately reported. STUDY DESIGN: Of 86 patients with hepatolithiasis admitted to our institution between 1980 and 1996, we reviewed 54 patients: 26 who underwent hepatic resection and 28 who underwent PTCSL. Five patients who underwent postoperative cholangioscopic lithotomy were included in the former group. The remainder of the hepatolithiasis patients were not treated by hepatic resection or PTCSL and, therefore, were excluded from this study. Hepatic resections were mainly indicated for left-sided localized intrahepatic calculi, atrophic liver, and possible presence of cholangiocellular carcinoma. PTCSL was performed for right-sided, bilateral or recurrent stones at an average of 6 treatments (range 1 to 20 treatments) for each patient. There were no differences between the two groups in terms of gender or age. The recurrence rate of stones and longterm prognosis were analyzed using the Kaplan-Meier method, and other clinical factors listed below were statistically compared. RESULTS: The rate of complete removal of stones was similarly high in each group (96.2% in the hepatic resection group versus 96.4% in the PTCSL group). The complication (38.5% versus 21.4%) and 5-year survival (85.6% versus 100%) rates were comparable. Remaining bile duct stricture (18.2% versus 60.9%, p < 0.01) and 5-year recurrence rates (5.6% versus 31.5%, p < 0.05) were statistically lower in the hepatic resection group than in the PTCSL group. CONCLUSIONS: Hepatic resection, when combined with postoperative cholangioscopic lithotomy, is a preferable treatment for left-sided stones with strictures and bilateral stones.