Hepatic apoptosis and proliferation in male and female rats fed alcohol: role of cytokines

Background: The female liver is more sensitive to the toxic effect of chronic alcohol intake than the male liver. The aim of the study was to compare the influence of gender and sex hormonal status on apoptosis and cell proliferation following chronic ethanol intake.
Methods: Male and female rats were pair fed for 8 weeks a liquid diet containing 36% of their total daily calories as ethanol (ETOH group) or sucrose (control group). Liver samples were analyzed for apoptosis and hepatocyte proliferation by immunohistochemistry. The hepatic production of factors able to influence cell death and proliferation, such as tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6) were determined.
Results: In both male and female rats, ethanol intake promoted apoptosis in the liver. This effect of ethanol was more evident in female than male rat livers. Hepatic TNFα levels, which promote apoptosis, are significantly more elevated in female than in male livers. Hepatic IL-6 production, which promotes hepatocyte proliferation, was induced by ethanol only in males, but not female animals.
Conclusion: This observed difference in cytokine responses may contribute to the enhanced sensitivity of female liver to EtOH-induced injury.     

Up-regulation of Transferrin Receptor Expression in Hepatocytes by Habitual Alcohol Drinking Is Implicated in Hepatic Iron Overload in Alcoholic Liver Disease

Background Hepatic iron overload is often seen in alcoholic liver disease (ALD). We previously reported that the expression of 4-hydroxy-2-nonenal-protein adducts, which is a lipid peroxidative product and can be used as a marker of radical-mediated cellular damage, was increased in iron-overloaded hepatocytes with ALD. However, the mechanism of hepatic iron overload in ALD has not been clarified. In this study, to elucidate the mechanism of hepatic iron overload in ALD, we immunohistochemically investigated the expression of transferrin receptor (TfR), which mainly acts for cellular iron uptake.
Methods Hepatic tissues were obtained from 31 patients with ALD and 5 normal livers by percutaneous needle biopsy under laparoscopy or ultrasound guidance. Chemical detection of hepatic iron accumulation was performed by Perls' Prussian blue stain. Immunohistochemical detection of TfR expression was done using human monoclonal anti-TfR antibody (TR104) according to the avidin-biotin complex method with alkaline phosphatase.
Results Excess iron accumulation was found in 22 hepatic tissues with ALD but not in any normal hepatic tissues. TfR expression was increased in hepatocytes of 18 hepatic tissues with ALD but was not detected in any normal hepatic tissues. The mean duration of abstinence of patients who demonstrated positive TfR expression in hepatocytes was significantly shorter than that of patients who demonstrated negative TfR expression (positive: 14 days; negative: 30 days). However, total ethanol consumption, daily ethanol intake, and serum aspartate aminotransferase and γ-glutamyl transpeptidase values on admission were not significantly correlated with TfR expression in hepatocytes.
Conclusions The up-regulation of TfR expression in hepatocytes is implicated in hepatic iron overload in ALD, and habitual alcohol drinking is an important factor for the induction of TfR expression.     

Chlorzoxazone pharmacokinetics as a marker of hepatic cytochrome P4502E1 in humans

Objective: Previous in vitro studies have demonstrated that hepatic P4502E1 metabolizes chlorzoxazone (CZX, a commonly used muscle relaxant) to 6-hydroxychlorzoxazone (6-OH-CZX). We thus assessed whether measurement of the plasma 6-OH-CZX/CZX ratio after a CZX challenge could serve as a marker of hepatic P4502E1 content.
Methods: Three subject groups were included: recently drinking alcoholics (  N = 6  ), abstinent alcoholics (  N = 5  ), and nonalcoholic subjects with liver disease (  N = 5  ) undergoing liver biopsy. Excess tissue was procured for immunochemical determination of hepatic P4502E1 content. Within an hour of the biopsy, 750 mg CZX was administered orally and serial plasma samples were collected for 6 h.
Results: Recently drinking alcoholic subjects had a higher area under the curve for plasma 6-OH-CZX (1.354 ± 0.258 μg · min · ml⁻¹) then abstinent alcoholic subjects (0.296 ± 0.080 μg · min · ml⁻¹, p < 0.005) and subjects with nonalcoholic liver disease (0.428 ± 0.061 μg · min · ml⁻¹,   p < 0.005  ). The use of the plasma 6-OH-CZX/CZX ratio at 90, 120, and 180 min discriminated between recently drinking alcoholic and nondrinking subjects. Hepatic P4502E1 content significantly correlated with the maximal 6-OH-CZX concentration (  r = 0.76  , p = 0.001) and other pharmacokinetic parameters. In the recently drinking group, the area under the curve for plasma 6-OH-CZX significantly decreased after 8 days of abstinence.
Conclusions: Measurement of plasma 6-OH-CZX after administration of a CZX challenge can serve as a marker of hepatic P4502E1 activity and thus help avoid adverse drug reactions secondary to P4502E1 induction, particularly in heavy drinkers.     

Study of Mitochondrial DNA Deletion in Alcoholics

Background: Recently, it has been reported that single or multiple mitochondrial DNA (Mt-DNA) deletions have been observed frequently in liver tissue and white blood cells (WBC) obtained from patients with alcoholic liver disease (ALD). In this study, we investigated the deletion of the Mt-DNA encoding adenosine triphosphatase (ATPase) region in WBC to clarify whether Mt-DNA heteroplasmy caused by alcohol drinking is reversible. Methods: Blood samples were obtained from 4 healthy volunteers, 56 patients with ALD, and 106 nonalcoholic healthy controls. The Mt-DNA encoded ATPase region was amplified by polymerase chain reaction (PCR) by using two primers: forward primer, 5‘-AACCAACACCTCTTTACAGTGA; and reverse primer; 5‘-TTGGTGGGTCATTATGTGTTGT. Results: Heteroplasmy was observed in one volunteer on day 3 and in the remaining persons on day 4 after the start of alcohol consumption. Heteroplasmy was observed for another 6 days after alcohol consumption stopped, but on the 7th day it had disappeared in all volunteers. In WBC Mt-DNA obtained from ALD patients within 3 days of abstinence, heteroplasmy was observed in 38 of the 56 patients (67.9%), whereas heteroplasmy was not detected in any healthy subjects. In 10 of the 18 ALD patients (56%) who had heteroplasmy within 3 days of abstinence, heteroplasmy disappeared after 4 weeks of abstinence. Conclusion: An acquired mutation of Mt-DNA, at least in the encoding ATPase region, may result from alcohol drinking and may be reversed by stopping drinking.     

Effects of Malotilate on Alcoholic Liver Injury in Rats

Malotilate (diisopropyl 1,3-dithio-2-yldenemalonate), a hepatotrophic drug, was administered to rats with alcohol-pyrazole hepatitis, which is considered to be a suitable experimental model for alcoholic liver injury, in order to elucidate the effects of malotilate on alcoholic liver injury. The number of ballooned hepatocytes and necrotic hepatocytes were smaller in the alcohol-pyrazole hepatitis rats treated with malotilate for 12 weeks (Al-Py Mal group) than for those without malotilate treatment (Al-Py group). Immunohistochemically, the retention of transferrin, one of the secretory proteins from the liver, in the ballooned hepatocytes was inhibited by malotilate. Biochemically, transferrin content in the Golgi fraction of the hepatocytes was significantly lower in the Al-Py Mal group than in the Al-Py group. Hepatic acetaldehyde levels in the Al-Py Mal group were significantly lower than those in the Al-Py group, even though ethanol metabolic rates were not different between the two groups. These results indicated that malotilate prevented the development of hepatocytic injury in alcohol-pyrazole hepatitis by decreasing hepatic acetaldehyde levels and preventing the retention of transferrin in the hepatocytes.     

Lipid peroxidation and antioxidant defense systems in rat liver after chronic ethanol feeding

The effects of chronic ethanol feeding on hepatic lipid peroxidation, ascorbic acid, glutathione and vitamin E levels were investigated in rats fed low or adequate amounts of dietary vitamin E. Hepatic lipid peroxidation was significantly increased after chronic ethanol feeding in rats receiving a low-vitamin E diet, indicating that dietary vitamin E is an important determinant of hepatic lipid peroxidation induced by chronic ethanol feeding. No significant change was observed in hepatic non-heme iron content, but hepatic content of ascorbic acid and glutathione was increased by ethanol feeding. Both low dietary vitamin E and ethanol feeding significantly reduced hepatic alpha-tocopherol content, and the lowest hepatic alpha-tocopherol was found in rats receiving a combination of low vitamin E and ethanol. Plasma alpha-tocopherol was elevated after ethanol feeding, probably because of the associated hyperlipemia. Both the ratio of plasma alpha-tocopherol/plasma lipid and the red blood cell alpha-tocopherol were reduced by ethanol feeding. Furthermore, ethanol feeding caused a marked increase of hepatic alpha-tocopheryl quinone, a metabolite of alpha-tocopherol by free radical reactions. Ethanol feeding caused little changes of alpha-tocopherol and alpha-tocopheryl quinone content in mitochondria, whereas a striking increase in alpha-tocopheryl quinone was observed in microsomes. These data suggest that ethanol feeding causes a marked alteration of vitamin E metabolism in the liver and that the combination of ethanol with a low-vitamin E intake results in a decrease of hepatic alpha-tocopherol content which renders the liver more susceptible to free radical attack.     

Attenuation of alcohol-induced apoptosis of hepatocytes in rat livers by polyenylphosphatidylcholine (PPC)

BACKGROUND: Alcohol consumption increases apoptosis of hepatocytes. This effect appears to be mediated by the induction of hepatic cytochrome P-4502E1(CYP2E1) and its generation of free radicals, which results in an enhanced lipid peroxidation that initiates apoptosis. Because polyenylphosphatidylcholine (PPC), a soybean extract rich in polyunsaturated phosphatidylcholines, decreases the induction of ethanol-specific CYP2E1 and opposes oxidative stress, we hypothesized that PPC supplementation may attenuate hepatocyte apoptosis caused by ethanol ingestion. METHODS: Twenty-eight male Sprague Dawley rats were pair-fed Lieber-DeCarli liquid diets containing 36% of energy as alcohol or an isocaloric amount of carbohydrate for 28 days. Half of the rats were given PPC (3 g/liter), whereas the other half received the same amount of linoleate (as safflower oil) and of choline as the bitartrate. An additional dose of alcohol (3 g/kg) was given intragastrically 90 min before the livers were removed. We assessed apoptosis in formalin-fixed, paraffin-embedded liver sections by using the TUNEL (terminal transferase dUTP nick end labeling) assay. Apoptotic hepatocytes were identified by positive TUNEL staining in conjunction with condensation of nucleoplasm or margination of chromatin. In each rat, 20,000 to 60,000 hepatocytes were counted by light microscopy by using Image-Pro Plus computer software, and the incidence of apoptosis was expressed as the percentage of total hepatocytes. RESULTS: Alcohol feeding resulted in a 4.5-fold increase in apoptosis of hepatocytes compared to pair-fed control rats; PPC supplementation decreased the alcohol-induced apoptosis to less than half. No difference in the incidence of apoptosis between the control and PPC-supplemented rats was found in the absence of alcohol. Apoptosis was distributed randomly in the liver lobules of the rats fed the control diet, whereas the alcohol-induced apoptosis was significantly increased in the perivenular area. PPC supplementation strikingly reduced this effect. CONCLUSIONS: PPC attenuates alcohol-induced apoptosis of hepatocytes; this effect may provide a mechanism for PPC's protection against liver injury, possibly in association with its antioxidative action via the down-regulation of ethanol-mediated CYP2E1 induction.     

Prevalence and risk factors of hepatic steatosis and its impact on liver injury in Chinese patients with chronic hepatitis B infection

Background and Aims:  The clinical significance of hepatic steatosis in chronic hepatitis B infection (CHB) is unclear. The aims of this study were thus to investigate the prevalence and risk factors for hepatic steatosis in patients with CHB and its relationship with liver injury.
Methods:  Consecutive patients with biopsy-proven CHB at Hangzhou Sixth People's Hospital between January 2005 and June 2007 were included. Patients co-infected with other viruses or suffering from liver disease of any other cause were excluded. Liver steatosis, necroinflammation and fibrosis were assessed by both Brunt and Scheuer classifications.
Results:  A total of 1915 patients (1497 men) with a mean age of 31 ± 9.5 years were analyzed. Hepatic steatosis was present in 260 (14%) patients. The steatosis involved < 33% of hepatocytes in 90% of cases, and was more frequent among men than women (15% vs 8%, P  < 0.001). Two-thirds (178 of 260) of patients with steatosis were hepatitis B e antigen (HBeAg)-positive, but there was no correlation with either serum HBeAg status or hepatitis B virus DNA titer. Degree of inflammation and fibrosis were more mild among those with steatosis than those without. Multivariate analysis showed that steatosis was independently associated with body mass index, serum triglyceride, apolipoprotein B, uric acid, and fasting blood glucose. However, fibrosis was only independently associated with age and inflammatory grade, and the latter associated with viral load and fibrosis stage.
Conclusions:  Hepatic steatosis is common in CHB, it is associated with metabolic factors not viral ones, and does not appear to affect the severity of liver disease.     

Ascorbate inhibits apoptosis of Kupffer cells during warm ischemia/reperfusion injury

BACKGROUND/AIMS: This study sought to determine whether ascorbate (Asc), a scavenger of reactive oxygen species, inhibits apoptosis of hepatic cells consisting of hepatocytes, Kupffer cells, and sinusoidal endothelial cells (SECs) in the rat liver after warm ischemia/reperfusion (I/R) injury. METHODOLOGY: Hepatic warm ischemia (69% of the total liver) was induced for 30 min, followed by reperfusion for 60 min. In some animals, ascorbate (at 1 or 10 mg/kg) was infused intravenously immediately before the onset of reperfusion. Hepatic cell apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). Mitochondrial release of cytochrome c into the cytoplasm was assessed by Western blot analysis, and the activation of caspase-3 in liver tissue was determined by colorimetric assays. RESULTS: Assays of cytochrome c release and caspase-3 showed increased levels of these apoptotic related proteins and enzyme activity. While few apoptotic hepatocytes or SECs were detected in the ischemic group by TUNEL staining, the number of TUNEL-positive Kupffer cells was approximately 4.5-fold greater than that seen in the sham-treatment group. Ascorbate treatment reduced this increase in apoptotic Kupffer cells. CONCLUSION: The hepatic cells most vulnerable to oxidative stress in the first hour of reperfusion were Kupffer cells. These may play a key role in hepatic warm I/R injury.     

Augmented regeneration of partial liver allograft induced by nuclear factor-κB decoy oligodeoxynucleotides-modified dendritic cells

AIM:To investigate the effect of NF-κB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs) on regeneration of partial liver allograft.METHODS:Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs, respectively. GMCSF-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A),GM-CSF-propagated DCs (group B), GM-CSF+IL-4-propagated DCs (group C), and GM-CSF+NF-κB decoy ODNs(group D) or scrambled ODNs -propagated DCs (group E) were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation.DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24h, 48h, 72h and 84h,respectively. Liver graft-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ level at the time of the maximal liver allograft regeneration were measured with ^51Cr release assay, semiquantitative RT-PCR and ELISA, respectively.RESULTS: Regeneration of liver allograft was markedly promoted by NF-κB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84h in group C rats and 48h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats.NF-κB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ production.At the time of the maximal regeneration of liver allograft,the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-γ mRNA expression and serum IFN-y production were detected in group D rats. The apoptotic index of hepatocytes, the activityof liver- resident NK cells, the hepatic TFN-γ mRNA expression level and the serum IFN-γ level in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft.CONCLUSION:The data suggest that the augmented regeneration of partial liver allograft induced by NF-κB decoy ODNs-modified Des may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liverresident NK cells and the reduced IFN-γ production.     

Augmented regeneration of partial liver allograft induced by nuclear factor-κB decoy oligodeoxynucleotides-modified dendritic cells

AIM: To investigate the effect of NF-κB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs)on regeneration of partial liver allograft.METHODS: Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL4 to obtain immature DCs or mature DCs, respectively. GMCSF-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A),GM-CSF -propagated DCs (group B), GM-CSF+IL-4 -propagated DCs (group C), and GM-CSF+NF-κB decoy ODNs (group D) or scrambled ODNs -propagated DCs (group E)were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation. DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24 h, 48 h, 72 h and 84 h,respectively. Liver graft-resident NK cell activity, hepatic IFN-y mRNA expression and recipient serum IFN-γ level at the time of the maximal liver allograft regeneration were measured with 51Cr release assay, semiquantitative RT-PCR and ELISA, respectively.RESULTS: Regeneration of liver allograft was markedly promoted by NF-κB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72 h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84 h in group C rats and 48 h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats.NF-κB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-γmRNA expression and recipient serum IFN-γ production. At the time of the maximal regeneration of liver allograft, the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-γ mRNA expression and serum IFN-γ production were detected in group D rats. The apoptotic index of hepatocytes, the activity of liver- resident NK cells, the hepatic IFN-γ mRNA expression level and the serum IFN-γlevel in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft.CONCLUSION: The data suggest that the augmented regeneration of partial liver allograft induced by NF-κB decoy ODNs-modified DCs may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liverresident NK cells and the reduced IFN-γ production.     

Mitochondrial oxidative stress and CD95 ligand: a dual mechanism for hepatocyte apoptosis in chronic alcoholism

Apoptosis plays an important role in the progression of alcohol-induced liver disease to cirrhosis. Oxidative stress is an early event in the development of apoptosis. The major aim of this study was to study the conditions in which oxidative stress occurs in chronic alcoholism and its relationship with apoptosis of hepatocytes. We have found that oxidative stress is associated with chronic ethanol consumption in humans and in rats, in the former independently of the existence of alcohol-induced liver disease. Ethanol or acetaldehyde induces apoptosis in hepatocytes isolated from alcoholic rats, but not in those from control rats. Inhibition of aldehyde dehydrogenase, but not of cytochrome P450 2E1, prevents ethanol-induced cell death. Ethanol-induced apoptosis is caused by increased reactive oxygen species (ROS) driven by increased availability of the reduced form of nicotinamide-adenine dinucleotide (NADH) owing to mitochondrial acetaldehyde metabolism and it is prevented by blocking the opening of mitochondrial permeability transition (MPT) pores with cyclosporine A. Inhibition of nitric oxide (NO) synthase or addition of antioxidant vitamins C and E completely prevented ethanol-induced apoptosis. Mitochondrial oxidative stress, which occurs during chronic alcoholism, renders hepatocytes susceptible to apoptosis. On the other hand, the CD95 ligand expression was up-regulated by acetaldehyde. In conclusion, ethanol induces apoptosis via 2 different pathways: MPT and up-regulation of the expression of CD95-Fas ligand. The overproduction of ROS by mitochondria, driven by acetaldehyde metabolism, is a common trigger of both mechanisms.     

Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase

Because alcoholic liver disease has been linked to oxidative stress, we investigated the effect of a compromised antioxidant defense system, Cu, Zn-superoxide dismutase (Sod1) deficiency, on alcohol-induced liver injury. C57BL/129SV wild-type (Sod1(+/+)) and Sod1 knockout (Sod1(-/-)) mice were fed dextrose or ethanol (10% of total calories) liquid diets for 3 weeks. Histologic evaluation of liver specimens of Sod1(-/-) mice fed ethanol showed the development of liver injury ranging from mild to extensive centrilobular necrosis and inflammation. Sod1(+/+) mice fed ethanol showed mild steatosis; both Sod1(+/+) and Sod1(-/-) mice fed the dextrose diet had normal histology. Alanine transaminase levels were significantly elevated only in Sod1(-/-) mice fed ethanol. Cytochrome P450 2E1 (CYP2e1) activity was elevated about 2-fold by ethanol in Sod1(+/+) and Sod1(-/-) mice. Ethanol consumption increased levels of protein carbonyls and lipid peroxidation aldehydic products in the liver of Sod1(-/-) mice. Hepatic adenosine triphosphate (ATP) content was reduced dramatically in Sod1(-/-) mice fed ethanol in association with a decrease in the mitochondrial reduced glutathione (GSH) level and activity of MnSOD. Immunohistochemical determination of 3-nitrotyrosine (3NT) residues in liver sections of the Sod1 knockout mice treated with ethanol showed a significant increase of 3NT staining in the centrilobular areas. In conclusion, a rather moderate ethanol consumption promoted oxidative stress in Sod1(-/-) mice, with increased formation of peroxynitrite, protein carbonyls, and lipid peroxidation and decreased mitochondrial GSH and MnSOD. We speculate that the increased oxidative stress causes mitochondrial damage and reduction of ATP content, leading to alcoholic liver injury. This model may be useful in further mechanistic studies on alcohol-induced liver injury.     

The effects of moderate ethanol consumption on the liver of the monkey, Macaca fascicularis

BACKGROUND: Although evidence has accumulated for the cardioprotective effects of moderate ethanol consumption, little is known about the effects on the liver of consuming the equivalent of two drinks per day. The objective of this study was to determine the effects of moderate ethanol administration on the hepatic content of enzymes involved in ethanol oxidation, on hepatic lipid accumulation, and on serum markers of liver function/damage in the monkey, Macaca fascicularis. METHODS: Ovariectomized, adult monkeys were maintained for 34 months on an atherogenic diet containing cholesterol 1.21 mg/kJ. They were trained to drink ethanol plus vehicle at a dose of 0.5 g/kg body weight, which was administered 5 days a week for 2 years. Blood was collected for ethanol concentrations (1 hr after ethanol administration) and was also assayed for gamma-glutamyltransferase, alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities. Liver obtained at necropsy was analyzed for triglyceride and cholesterol contents and for alcohol dehydrogenase, cytochrome P450 2E1, and cytochrome P450 3A4 by Western blots. RESULTS: The blood ethanol concentrations measured 1 hr after ethanol administration were relatively constant over the 2-year dosing period. Hepatic levels of alcohol dehydrogenase and the cytochrome P450s were not significantly different between ethanol-consuming animals and control animals. Ethanol-associated increases in liver triglyceride were not significant due to high variability in hepatic lipid content in both the controls and ethanol consumers. However, covariance analyses using pretreatment concentrations of plasma cholesterol and apolipoprotein A-I suggested that the ethanol-related increase in hepatic free cholesterol was significant. Relative to controls, alcohol consumers had higher levels of serum ALT and a transient increase in ALP at 5 months. CONCLUSIONS: The observations made in this study on primates administered an atherogenic diet suggest that moderate ethanol ingestion has modest effects on the liver, including slightly increased ALT and ALP values. However, additional studies will be required to verify that this level of consumption is hepatotoxic when ingested over extended periods. This is still a concern because some human studies suggest that levels of ethanol considered to be cardioprotective cause liver injury when consumed over a lifetime.     

An insight into the protection of rat liver against ischemia/reperfusion injury by 2-selenium-bridged β-cyclodextrin

Aim:  The reperfusion following liver ischemia results in the damage and apoptosis of hepatocytes. The aim of this study was to investigate the possible effects and mechanism of a new synthesized glutathione peroxidase (GPX) mimic, 2-selenium-bridged β-cyclodextrin (2-SeCD), on rat liver ischemia-reperfusion (I/R) injury.
Methods:  Male Wistar rats ( n  = 32) were randomly divided into four groups: I. sham-operated group, II. I/R group, III. I/R +2-SeCD group, IV. I/R + Ebselen group. Hepatic I/R was administered by 90 min of ischemia and 12 h of reperfusion. Liver tissues were collected at the end of reperfusion period for measurement of various biochemical parameters.
Results:  The serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) activity and tissue malondialdehyde, myeloperoxidase levels were increased in I/R group, while the increase was significantly reduced by 2-SeCD treatment. The glutathione level, depressed by I/R, was elevated back to normal levels by treatment with 2-SeCD. Severe hepatic damage were observed by light and transmission electron microscopy whilst pretreatment with 2-SeCD resulted in tissue and cellular preservation. Furthermore, 2-SeCD reduced cytochrome c release from mitochondria and subsequent DNA fragmentation by regulating Bcl-2/Bax expression ratio. Results suggested that 2-SeCD was more effective than ebselen in the reversal of the alteration in tissue structural and biochemical parameters caused by I/R injury.
Conclusion:  2-selenium-bridged β-cyclodextrin playes an important role in the protection of liver against I/R injury and this treatment may be a novel pharmacological agent for liver surgery.     

Subacute hepatic failure due to hepatitis E

Background and Aim:  The data available on subacute hepatic failure due to hepatitis E virus is scarce. The aim of this study is to analyze the clinical spectrum and outcome of this condition.
Methods:  This is a retrospective hospital-based study of patients with acute hepatitis E and subacute hepatic failure from January 2001 to June 2006.
Results:  We encountered 12 patients with this condition during the study period. There were four females and eight males (age 39 ± 16). Jaundice and ascites were present in all. The model for end stage liver disease (MELD) score was 25 ± 8. All of them had normal-sized liver on ultrasonogram. Transjugular liver biopsies were done in nine patients and revealed extensive bridging, submassive necrosis and cholestasis. Complications included spontaneous bacterial peritonitis (four) and urinary tract infections (two), renal failure (three) and encephalopathy (three). The in-hospital mortality was 25% (3/12). The remaining nine patients left the hospital alive with normalization of liver functions in eight of them over the next few months.
Conclusion:  Subacute hepatic failure caused by hepatitis E is a distinct entity with a better prognosis compared with the previously published series of subacute hepatic failure. Liver biopsy is useful to differentiate from hepatitis E virus superinfection on underlying chronic disease. Poor prognostic factors were female sex, younger age, encephalopathy and persistent renal failure. These patients should be considered for liver transplantation.     

Vascular endothelial growth factor reduces Fas-mediated acute liver injury in mice

Background and Aim:  Fulminant hepatitis is still a fatal liver disease, and no specific treatment for it has been available. Vascular endothelial growth factor (VEGF) is the focus of attention because of its various actions. We investigated the effect of vascular endothelial growth factor (VEGF) on Fas-induced fulminant hepatic failure (FHF).
Method:  Male Balb/c mice were treated with an intraperitoneal injection of an anti-Fas antibody (Jo-2 Ab) with or without premedication with intraperitoneally administered human recombinant VEGF.
Results:  The serum level of alanine aminotransferase (ALT) was up to 300 times higher that of normal mice following the Jo-2 Ab injection, and histological analysis revealed hepatic injury and massive hepatocyte apoptosis. The VEGF significantly suppressed an elevation in serum ALT levels and hepatocyte apoptosis. Immunohistochemically, VEGF-treated mice showed that Bcl-xL in hepatocytes was strongly expressed.
Conclusions:  Since hepatocytes do not express VEGF receptors, we speculated that VEGF acts on sinusoidal endothelial cells (SECs) and promotes production of cytokines such as hepatocyte growth factor in SECs, resulting in reducing apoptosis through an increase expression of Bcl-xL in hepatocytes. We suggest that VEGF has a potent antiapoptotic effect on hepatocytes through cell–cell interaction between SECs and hepatocytes.     

Antifibrotic effects of ambrisentan,an endothelin-A receptor antagonist,in a non-alcoholic steatohepatitis mouse model

AIM: To examine the effects of the endothelin type A receptor antagonist ambrisentan on hepatic steatosis and fibrosis in a steatohepatitis mouse model.METHODS: Fatty liver shionogi(FLS) FLS-ob/ob mice(male, 12 wk old) received ambrisentan(2.5 mg/kg orally per day; n = 8) or water as a control(n = 5) for 4 wk. Factors were compared between the two groups, including steatosis, fibrosis, inflammation, and endothelin-related gene expression in the liver.RESULTS: In the ambrisentan group, hepatic hydroxyproline content was significantly lower than in the control group(18.0 μg/g ± 6.1 μg/g vs 33.9 μg/g ± 13.5 μg/g liver, respectively, P = 0.014). Hepatic fibrosis estimated by Sirius red staining and areas positive for α-smooth muscle actin, indicative of activated hepatic stellate cells, were also significantly lower in the ambrisentan group(0.46% ± 0.18% vs 1.11% ± 0.28%, respectively, P = 0.0003; and 0.12% ± 0.08% vs 0.25% ± 0.11%, respectively, P = 0.047). Moreover, hepatic RNA expression levels of procollagen-1 and tissue inhibitor of metalloproteinase-1(TIMP-1) were significantly lower by 60% and 45%, respectively, in the ambrisentan group. Inflammation, steatosis, and endothelin-related m RNA expression in the liver were not significantly different between the groups.CONCLUSION: Ambrisentan attenuated the progression of hepatic fibrosis by inhibiting hepatic stellate cell activation and reducing procollagen-1 and TIMP-1 gene expression. Ambrisentan did not affect inflammation or steatosis.