Search

Dietary glycine inhibited hepatocyte proliferation in response to the carcinogen WY-14,643. Since increased cell replication is associated with hepatic cancer caused by WY-14,643, glycine may have anti-cancer properties. Therefore, these experiments were designed to test the hypothesis that dietary glycine would inhibit the growth of tumors arising from B16 melanoma cells implanted subcutaneously in mice. C57BL/6 mice were fed diet supplemented with 5% glycine and 15% casein or control diet (20% casein) for 3 days prior to subcutaneous implantation of B16 tumor cells. Tumor volume was estimated from tumor diameter for 14 days. Tumors were excised, weighed and sectioned for histology post-mortem. B16 cells and endothelial cells were cultured in vitro to assess effects of glycine on cell growth. Statistical tests were two-sided and a P-value of 0.05 was defined as a significant difference between groups. Weight gain did not differ between mice fed control and glycine-containing diets. B16 tumors grew rapidly in mice fed control diet; however, in mice fed glycine diet, tumor size was 50-75% less. At the time of death, tumors from glycine-fed mice weighed nearly 65% less than tumors from mice fed control diet (P < 0.05). Glycine (0.01-10 nM) did not affect growth rates of B16 cells in vitro. Moreover, tumor volume and mitotic index of B16 tumors in vivo did not differ 2 days after implantation when tumors were small enough to be independent of vascularization. After 14 days, tumors from mice fed dietary glycine had 70% fewer arteries (P < 0.05). Furthermore, glycine (0.01-10 mM) inhibited the growth of endothelial cells in vitro in a dose-dependent manner (P < 0.05; IC50 = 0.05 mM). These data support the hypothesis that dietary glycine prevents tumor growth in vivo by inhibiting angiogenesis through mechanisms involving inhibition of endothelial cell proliferation. [ABSTRACT FROM PUBLISHER]

Increased cell proliferation most likely plays a key role in peroxisome proliferator-induced liver cancer. Recently, Kupffer cells were shown to be responsible for Wy-14,643-induced cell proliferation. However, the mechanism by which peroxisome proliferators activate Kupffer cells is unknown. Since gut-derived endotoxin is a known activator of Kupffer cells, the hypothesis that it is involved was evaluated. Increased cell proliferation and peroxisome induction were unaffected by gut sterilization. Moreover, endotoxin was not detectable in portal blood following treatment with Wy-14,643. Therefore, it is concluded that gut-derived endotoxin is not responsible for Kupffer cell activation. To test the hypothesis that Kupffer cell activation. To test the hypothesis that Kupffer cells are activated by Wy-14,643 directly, Kupffer cell superoxide production was measured following treatment in vitro. Wy-14,643 increased superoxide production in a dose-dependent manner (0.1 and 50 μM) with half-maximal stimulation at 2.5 μM. Treatment with Wy-14,643 for 21 days caused a 2-fold increase in Kupffer cell superoxide production while DEHP did not. Pretreatment of Kupffer cells with staurosporine (0.01-10 pM) completely blocked generation of superoxide demonstrating that protein kinase C is required. Moreover, Wy-14,643 increased Kupffer cell protein kinase C activity 3-fold. Pretreatment of Kupffer cells with the amino acid glycine (0.01-3 mM), which blunts calcium signaling, inhibited Wy-14,643-stimulated superoxide production and increased protein kinase C activity completely. These data are consistent with the hypothesis that potent peroxisome proliferators (Wy-14,643 and MEHP) directly activate Kupffer cell production of oxidants via mechanisms involving protein kinase C. Further, peroxisome proliferator treatments that sustain elevated rates of cell proliferation (e.g. Wy-14,643) activate Kupffer cell superoxide production following long-term dietary treatment supporting the hypothesis that Kupffer cell-derived oxidants are involved in peroxisome proliferator-induced neoplasia. [ABSTRACT FROM PUBLISHER]

Stimulation of cell proliferation caused by peroxisome proliferators was blocked by antibodies against TNFα and agents that inactivate Kupffer cells, a rich source of TNFα, which supports the hypothesis that Kupffer cells play a pivotal role in peroxisome proliferator-induced hyperplasia. Here, the ability of the very potent peroxisome proliferator WY-14 643 to activate the transcription factor NF-κB in rat liver was examined since it is involved in TNFα production. Female Sprague-Dawley rats were treated by gavage with WY-14 643 (100 mg/kg) while control animals were given equivalent doses of vehicle (olive oil). Activation of NF-κB in both whole liver, non-parenchymal cells, Kupffer cells and hepatocytes was assessed for up to 36 h using an electrophoretic mobility shift assay. In whole liver, WY-14 643 transiently increased NF-κB binding maximally 3.5-fold in 2-8 h followed by a steady decline to near control levels at 36 h. As early as 2 h after WY-14 643 treatment, the active form of NF-κB was localized predominantly in Kupffer cells with values 20- to 25-times greater than in hepatocytes. In hepatocytes, a small increase in NF-κB binding was observed but only 8 h after WY-14 643 administration. Pre-treatment with allopurinol, a xanthine oxidase inhibitor and free radical scavenger, suppressed NF-κB activation by WY-14 643 almost completely. It is concluded that NF-κB is activated by reactive oxygen species and plays a central role in the mechanism of action of peroxisome proliferators. Moreover, these findings support the hypothesis that Kupffer cells play a pivotal role in peroxisome proliferator-induced hepatocyte proliferation through rapid NF-κB activation and subsequent induction of TNFα production. TNFα from Kupffer cells stimulates growth in parenchymal cells later via mechanisms that also involve NF-κB. [ABSTRACT FROM PUBLISHER]

Our previous studies showed that an extract from Camellia sinenesis (green tea), which contains several polyphenols, attenuates nephrotoxicity caused by cyclosporine A (CsA). Since polyphenols are stimulators of mitochondrial biogenesis (MB), this study investigated whether stimulation of MB plays a role in green tea polyphenol protection against CsA renal toxicity. Rats were fed a powdered diet containing green tea polyphenolic extract (0.1%) starting 3 days prior to CsA treatment (25 mg/kg, i.g. daily for 3 weeks). CsA alone decreased renal nuclear DNA-encoded oxidative phosphorylation (OXPHOS) protein ATP synthase-β (AS-β) by 42%, mitochondrial DNA (mtDNA)-encoded OXPHOS protein NADH dehydrogenase-3 (ND3) by 87% and their associated mRNAs. Mitochondrial DNA copy number was also decreased by 78% by CsA. Immunohistochemical analysis showed decreased cytochrome c oxidase subunit IV (COX-IV), an OXPHOS protein, in tubular cells. Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, the master regulator of MB, and mitochondrial transcription factor-A (Tfam), the transcription factor that regulates mtDNA replication and transcription, were 42% and 90% lower, respectively, in the kidneys of CsA-treated than in untreated rats. These results indicate suppression of MB by chronic CsA treatment. Green tea polyphenols alone and following CsA increased AS-β, ND3, COX-IV, mtDNA copy number, PGC-1α mRNA and protein, decreased acetylated PGC-1α, and increased Tfam mRNA and protein. In association with suppressed MB, CsA increased serum creatinine, caused loss of brush border and dilatation of proximal tubules, tubular atrophy, vacuolization, apoptosis, calcification, and increased neutrophil gelatinase-associated lipocalin expression, leukocyte infiltration, and renal fibrosis. Green tea polyphenols markedly attenuated CsA-induced renal injury and improved renal function. Together, these results demonstrate that green tea polyphenols attenuate CsA-induced kidney injury, at least in part, through the stimulation of MB.

It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl(-) influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1-10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor.

TNFalpha, a mediator of hepatotoxicity in several animal models, is elevated in acute and chronic liver diseases. Therefore, we investigated whether hepatic injury and fibrosis due to bile duct ligation (BDL) would be reduced in TNFalpha knockout mice (TNFalpha-/-). Survival after BDL was 60% in wild-type mice (TNFalpha+/+) and 90% in TNFalpha-/- mice. Body weight loss and liver to body weight ratios were reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Following BDL, serum alanine transaminases (ALT) levels were elevated in TNFalpha+/+ mice (268.6+/-28.2U/L) compared to TNFalpha-/- mice (105.9U/L+/-24.4). TNFalpha-/- mice revealed lower hepatic collagen expression and less liver fibrosis in the histology. Further, alpha-smooth muscle actin, an indicator for activated myofibroblasts, and TGF-beta mRNA, a profibrogenic cytokine, were markedly reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Thus, our data indicate that TNFalpha induces hepatotoxicity and promotes fibrogenesis in the BDL model.

Aim: To investigate the effects of (dietary) glycine against oxidant-induced injury caused by bile duct ligation (BDL)., Methods: Either a diet containing 5% glycine or a standard diet was fed to male Sprague-Dawley (SD) rats. Three days later, BDL or sham-operation was performed. Rats were sacrificed 1 to 3 d after BDL. The influence of deoxycholic acid (DCA) in the presence or absence of glycine on liver cells was determined by measurement of calcium and chloride influx in cultivated Kupffer cells and lactate dehydrogenase (LDH) activity was determined in the supernatant of cultivated hepatocytes., Results: Serum alanine transaminase levels increased to about 600 U/L 1 d after BDL. However, enzyme release was blunted by about two third in rats receiving glycine. Release of the alkaline phosphatase and aspartate aminotransferase was also blocked significantly in the group fed glycine. Focal necrosis was observed 2 d after BDL. Glycine partially blocked the histopathological changes. Incubation of Kupffer cells with DCA led to increased intracellular calcium that could be blocked by incubation with glycine. However, systemic blockage of Kupffer cells with gadolinium chloride had no effects on transaminase release. Incubation of isolated hepatocytes with DCA led to a significant release of LDH after 4 h. This release was largely blocked when incubation with glycine was performed., Conclusion: These data indicate that glycine significantly decreased liver injury, most likely by a direct effect on hepatocytes. Kupffer cells do not appear to play an important role in the pathological changes caused by cholestasis.

Aim: To investigate the effects of heme oxygenase-1 (HO-1) against oxidant-induced injury caused by bile duct ligation (BDL)., Methods: Either cobalt protoporphyrin (CoPP), a HO-1 inducer, or saline were injected intraperitoneally in male SD-rats. Three days later, BDL or sham-operations were performed. Rats were sacrificed 3 wk after BDL and livers were harvested for histology. Fibrosis was evaluated by sirius red staining and image analysis. Alpha-smooth muscular actin, which indicates activation of stellate cells, was detected by immunohistochemical staining, and cytokine and collagen-Ialpha (Col-Ialpha) mRNA expression was detected using RNase protection assays., Results: Serum alanine transaminase increased 8-fold above normal levels one day after BDL. Surprisingly, enzyme release was not reduced in rats receiving CoPP. Liver fibrosis was evaluated 3 wk after BDL and the sirius red-positive area was found to be increased to about 7.8%. However, in CoPP pretreated rats sirius red-positive areas were increased to about 11.7% after BDL. Collagen-Ialpha and TGF-beta mRNA increased significantly by BDL. Again, this effect was increased by HO-1 overexpression., Conclusion: Hepatic fibrosis due to BDL is not reduced by the HO-1 inducer CoPP. In contrast, HO-1 overexpression increases liver injury in rats under conditions of experimental chronic cholestasis.

The immunosuppressants ciclosporin (cyclosporin A, CsA) and tacrolimus can cause severe nephrotoxicity. Since CsA increases free radical formation, this study investigated whether an extract from Camellia sinensis, which contains several polyphenolic free radical scavengers, could prevent nephrotoxicity caused by CsA and tacrolimus. Rats were fed powdered diet containing polyphenolic extract (0-0.1%) starting 3 days before CsA or tacrolimus. Free radicals were trapped with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and measured using an electron spin resonance spectrometer. Both CsA and tacrolimus decreased glomerular filtration rates (GFR) and caused tubular atrophy, vacuolization and calcification and arteriolar hyalinosis, effects that were blunted by treatment with dietary polyphenols. Moreover, CsA and tacrolimus increased POBN/radical adducts in urine nearly 3.5 fold. Hydroxyl radicals attack dimethyl sulfoxide (DMSO) to produce a methyl radical fragment. Administration of CsA or tacrolimus with (12)C-DMSO produced a 6-line spectrum, while CsA or tacrolimus given with (13)C-DMSO produced a 12-line ESR spectrum, confirming formation of hydroxyl radicals. 4-Hydroxynonenal (4-HNE), a product of lipid peroxidation, accumulated in proximal and distal tubules after CsA or tacrolimus treatment. ESR changes and 4-HNE formation were largely blocked by polyphenols. Taken together, these results demonstrate that both CsA and tacrolimus stimulate free radical production in the kidney, most likely in tubular cells, and that polyphenols minimize nephrotoxicity by scavenging free radicals.

Background & Aims: The mechanisms by which small-for-size liver grafts decrease survival remain unclear. This study investigated the role of free radicals in injury to small-for-size grafts., Methods: Rat liver explants were reduced in size ex vivo and transplanted into recipients of the same or greater body weight, resulting in a graft weight and standard liver weight of approximately 50% and 25%, respectively. A polyphenol extract from Camellia sinenesis (20 microg/mL) or an equivalent concentration of epicatechin was added to the storage solution and the lactated Ringer poststorage rinse solution., Results: Serum alanine aminotransferase release increased from approximately 60 U/L before implantation to 750, 1410, and 2520 U/L after full-size, half-size, and quarter-size transplantation, respectively. Total bilirubin increased slightly after transplantation of full-size and half-size grafts but increased 104-fold in quarter-size grafts. In quarter-size grafts, histological changes included necrosis, leukocyte infiltration, and eosinophilic inclusion body formation. Polyphenol treatment ameliorated these effects by > or =67%. Survival was 30% after transplantation of small-for-size grafts. After polyphenol treatment, survival increased to 70%. Free radicals in bile assessed by spin trapping and 4-hydroxynonenal adducts measured by immunohistochemistry were also greater in reduced-size grafts, an effect ameliorated by polyphenols. Epicatechin, a major polyphenol from Camellia sinenesis, also improved graft function and decreased enzyme release, histopathologic changes, and free radical formation., Conclusions: Increased formation of free radicals occurs after transplantation of reduced-size livers, which contributes to graft dysfunction and failure. Plant polyphenols decrease liver graft injury and increase survival of small-for-size liver grafts, most likely by scavenging free radicals.

Fatty liver caused by ethanol decreases survival after liver transplantation in rats. This study investigated if antioxidant polyphenols from Camellia sinenesis (green tea) prevent failure of fatty grafts from ethanol-treated rats. Donor rats were given ethanol intragastrically (6 g/kg). After 20 h, livers were explanted and stored in University of Wisconsin solution for 24 h. Prior to implantation, the explanted grafts were rinsed with lactated Ringer's solution containing 0 to 60 microg/ml polyphenols. Alanine aminotransferase (ALT) release after liver transplantation was 4.5-fold higher in recipients receiving ethanol-induced fatty grafts than in those receiving normal grafts. Liver grafts from ethanol-treated donors also developed severe focal necrosis. Graft survival was 11% in the ethanol group versus 88% for normal grafts. Polyphenol treatment at 60 microg/ml blunted ALT release by 66%, decreased necrotic areas by 84%, and increased survival to 75%. Ethanol increased alpha-(4-pyridyl-1-oxide)-N-tert.-butylnitrone free radical adducts in bile by 2.5-fold, as measured by electron spin resonance spectroscopy, and caused accumulation of 4-hydroxynonenal in liver sections, effects blunted by polyphenols. Epicatechin gallate, a major polyphenol from C. sinenesis, also decreased enzyme release, minimized pathological changes, and decreased free radical adduct formation. In conclusion, polyphenols scavenged free radicals in ethanol-induced fatty livers and decreased injury after liver transplantation.

Bacterial infections play an important role in the multifactorial etiology of rheumatoid arthritis. The arthropathic properties of Gram-positive bacteria have been associated with peptidoglycan-polysaccharide complexes (PG-PS), which are major structural components of bacterial cell walls. There is little agreement as to the identity of cellular receptors that mediate innate immune responses to PG-PS. A glycosylphosphatidylinositol-linked cell surface protein, CD14, the lipopolysaccharide receptor, has been proposed as a PG-PS receptor, but contradictory data have been reported. Here, we examined the inflammatory and pathogenic responses to PG-PS in CD14 knockout mice in order to examine the role for CD14 in PG-PS-induced signaling. We found that PG-PS-induced responses in vitro, including transient increase in intracellular calcium, activation of nuclear factor-kappaB, and secretion of the cytokines tumor necrosis factor-alpha and interleukin-6, were all strongly inhibited in CD14 knockout macrophages. In vivo, the incidence and severity of PG-PS induced acute polyarthritis were significantly reduced in CD14 knockout mice as compared with their wild-type counterparts. Consistent with these findings, CD14 knockout mice had significantly inhibited inflammatory cell infiltration and synovial hyperplasia, and reduced expression of inflammatory cytokines in PG-PS arthritic joints. These results support an essential role for CD14 in the innate immune responses to PG-PS and indicate an important role for CD14 in PG-PS induced arthropathy.

Accumulation of hydrophobic bile acids during cholestasis leads to generation of oxygen free radicals in the liver. Accordingly, this study investigated whether polyphenols from green tea Camellia sinenesis, which are potent free radical scavengers, decrease hepatic injury caused by experimental cholestasis. Rats were fed a standard chow or a diet containing 0.1% polyphenolic extracts from C. sinenesis starting 3 days before bile duct ligation. After bile duct ligation, serum alanine transaminase increased to 760 U/l after 1 day in rats fed a control diet. Focal necrosis and bile duct proliferation were also observed after 1-2 days, and fibrosis developed 2-3 wk after bile duct ligation. Additionally, procollagen-alpha1(I) mRNA increased 30-fold 3 wk after bile duct ligation, accompanied by increased expression of alpha-smooth muscle actin and transforming growth factor-beta and the accumulation of 4-hydroxynenonal, an end product of lipid peroxidation. Polyphenol feeding blocked or blunted all of these bile duct ligation-dependent changes by 45-73%. Together, the results indicate that cholestasis due to bile duct ligation causes liver injury by mechanisms involving oxidative stress. Polyphenols from C. sinenesis scavenge oxygen radicals and prevent activation of stellate cells, thereby minimizing liver fibrosis.

Background: Oxygen-derived free radicals play a central role in ischemia/reperfusion injury after organ transplantation and are degraded by endogenous radical scavengers such as superoxide dismutase (SOD). Overexpression of SOD by delivery of the cytosolic SOD gene with an adenovirus (Ad.SOD1) decreases organ injury and increases survival in a rat model of liver transplantation. However, it is unclear which of the three isoforms of SOD provides the most protective effect. The purpose of this study was to identify the isoform with the highest effectiveness against ischemia/reperfusion injury after transplantation of fatty livers, which are particularly susceptible., Methods: Donor rats were given ethanol by gavage before harvest to induce steatotic livers. Some of the donors were infected with adenoviruses expressing either the gene lacZ encoding bacterial beta-galactosidase (Ad.lacZ), Ad.SOD1, Ad.SOD2 (mitochondrial isoform), or Ad.SOD3 (extracellular isoform). After transplantation, SOD activity in liver, survival, histopathology, transaminases, and activation of nuclear factor (NF)-kappaB, IkappaB kinase, Jun-N-terminal kinase (JNK), and tumor necrosis factor (TNF)-alpha were evaluated., Results: Ad.SOD1 treatment increased survival, blunted transaminase release, and reduced necrosis, whereas Ad.SOD3 had no protective effect. Ad.SOD2 was not as protective as Ad.SOD1. Ad.SOD1 reduced the activation of NF-kappaB, blunted JNK activity, and reduced TNF-alpha activity. Ad.SOD2 treatment resulted in lower kinase, TNF-alpha, and NF-kappaB activities but was not as effective as Ad.SOD1. IkappaB kinase activity was not affected., Conclusion: This study demonstrates that cytosolic SOD represents the most effective isoform of SOD to protect transplanted livers from failure; this may be related to lowered NF-kappaB and JNK activities because of reduced oxygen-derived radical production.

Hemorrhagic shock and resuscitation cause hepatocellular damage by mechanisms involving oxidative stress. However, the sources of free radicals mediating hepatocellular injury remain controversial. Thus, this study tested the hypothesis that NADPH oxidase plays a role in producing hepatocellular injury after hemorrhagic shock and resuscitation. Both wild-type and NADPH oxidase-deficient mice (p47(phox) knockout mice) were subjected to hemorrhagic shock (3 h at 30 mmHg). The mice were resuscitated over 30 min with the shed blood and additional lactated Ringer's solution (50% of the shed blood volume). Serum alanine aminotransferase (ALT) levels increased at 1 and 6 h postresuscitation in wild-type animals to 4735 +/- 1017 IU/L and 1450 +/- 275 IU/L (mean +/- SE), respectively, whereas in knockout mice, this ALT increase was blunted at both time points (732 +/- 241 IU/L and 328 +/- 69 IU/L, P < 0.05). Liver necrosis assessed histologically 6 h after the end of reperfusion was also attenuated in the knockout mice (3.5% +/- 0.95% of area vs. 0.9% +/- 0.26%, P < 0.05). In hemorrhaged wild-type mice, infiltrating neutrophils were twice as numerous compared with hemorrhaged NADPH oxidase-deficient animals 6 h after reperfusion. In knockout animals, hepatic 4-hydroxynonenal content, indicative of lipid peroxidation from reactive oxygen species, was blunted (6.7% +/- 0.6% vs. 26.4% +/- 2.3% of stained area, P < 0.05), as shown by immunohistochemistry. Immunohistochemical staining for 3-nitrotyrosine, indicative of reactive nitrogen species formation, was also blunted in the livers of knockout mice (11.6% +/- 2.8% vs. 37.4% +/- 3.4, P < 0.05). In conclusion, hemorrhagic shock and resuscitation cause hepatocellular damage via NADPH oxidase-mediated oxidative stress. The absence of NADPH oxidase substantially attenuates hepatocellular injury after hemorrhagic shock and resuscitation, blunts neutrophil infiltration, and decreases formation of reactive oxygen and reactive nitrogen species.

Ethanol is known to cause both tolerance and sensitization to endotoxin (lipopolysaccharide). It is also known that ethanol modulates the expression and activity of several intracellular signaling molecules and transcription factors in monocytes and Kupffer cells, the resident hepatic macrophages. Expression of CD14, the endotoxin receptor, is up-regulated following chronic exposure to endotoxin and ethanol. Ethanol-induced oxidative stress is important in the regulation of transcription factor activation and cytokine production by Kupffer cells. Thus, it was hypothesized that acute ethanol increases CD14 expression through a mechanism dependent upon oxidant production. This hypothesis was tested by overexpression of superoxide dismutase via recombinant adenovirus. Mice were infected with adenovirus (3 x 10(9) plaque-forming units, intravenously) containing either Cu,Zn superoxide dismutase (Ad.SOD1) or beta-galactosidase (Ad.lacZ), which caused significant expression of Cu,Zn-SOD in hepatocytes and Kupffer cells. Three days post-infection, mice were given saline or ethanol (5 g/kg, intragastrically). A significant increase in CD14 mRNA was observed 3 h after ethanol, and this increase was almost completely blocked in mice overexpressing Cu,Zn-SOD. Additionally, overexpression of SOD also blunted ethanol-induced activation of redox-sensitive transcription factors NFkappaB and AP-1 and production of cytokines. However, only inhibition of AP-1 with dominant-negative TAK1 but not NFkappaB by dominant-negative IkappaBalpha significantly blunted ethanol-induced increases in CD14, suggesting that AP-1 is important for CD14 transcriptional regulation. It is also shown here that NADPH oxidase is important in the increase in CD14 due to ethanol. Moreover, these data suggest that acute ethanol causes sensitization to endotoxin through mechanisms dependent upon oxidative stress.

Liver regeneration after partial hepatectomy (PH) involves several signaling mechanisms including activation of the small GTPases Ras and RhoA in response to mitogens leading to DNA synthesis and cell proliferation. Peroxisome proliferator-activated receptor-alpha (PPARalpha) regulates the expression of several key enzymes in isoprenoid synthesis, which are key events for membrane association of Ras and RhoA. Thus the role of PPARalpha in cell proliferation after PH was tested. After PH, an increase in PPARalpha DNA binding was observed in wild-type mice, correlating with an increase in the PPARalpha-regulated enzyme acyl-CoA oxidase. In addition, the PPARalpha-regulated genes farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase were significantly increased in wild-type mice. However, these increases were not observed in PPARalpha knockout (PPARalpha -/-) mice. The peak in DNA synthesis observed 42 h after PH was reduced by approximately 60% in PPARalpha -/- mice, despite increases in TNF-alpha and IL-1. Also, under these conditions, membrane association of Ras was high in wild-type mice after PH but was impaired in PPARalpha -/- mice. Accordingly, Ras was significantly elevated in the cytosol in PPARalpha -/- mice. This observation correlated with lower levels of active GTP-bound Ras after PH in PPARalpha -/- mice compared with wild-type mice. Similar observations were made for RhoA. Moreover, deletion of PPARalpha blunted the activation of cyclin-dependent kinase (cdk)2/cyclin E and cdk4/cyclin D complexes. Collectively, these results support the hypothesis that PPARalpha is necessary for cell cycle progression in regenerating mouse liver via mechanisms involving prenylation of small GTPases Ras and RhoA.

Kupffer cells, the resident macrophages of the liver, play a major role in the pathogenesis of several diseases. This unit contains an easy-to-follow procedure for effective isolation of liver Kupffer cells from rats and mice. The protocol provides viable Kupffer cells in large amounts that can be used for further investigations.

Background & Aims: Kupffer cells play a major role in the pathogenesis of several diseases. They release physiologically active substances that often lead to localized tissue injury. Therefore, the aim of this study was to establish a model to protect the liver through supplementation of Kupffer cells that have been transduced by recombinant adenovirus., Methods: Optimal conditions for intravenous injection in rats were established using carbon-labeled Kupffer cells. Adenoviral-transduced Kupffer cells encoding the Cu/Zn-SOD gene (Ad.SOD1) or beta-galactosidase reporter gene (Ad.LacZ) were transplanted into recipient rats. Twenty-four hours after transplantation, 70% hepatic ischemia-reperfusion was used to induce hepatic oxidative stress, and liver injury was determined 8 or 24 hours later., Results: In initial experiments, 10%-20% of the injected carbon-labeled cells were localized in the host liver after 24 hours, representing approximately 1% of the total population of Kupffer cells. Pretreatment of the recipient with a single dose of cyclosporin A maximized Kupffer cell reseeding up to 4%-10% of the total Kupffer cell population, suggesting that efficiency is limited by host immune response. Moreover, reseeded Kupffer cells were retained in host livers for up to 14 days after transplant. In livers of animals injected with Kupffer cells transduced with Ad.LacZ, transgene expression was observed, indicating Kupffer cell functional integrity. Injection of Kupffer cells transduced with Ad.SOD1 significantly blunted the increase in serum transaminases and liver injury because of ischemia-reperfusion compared with controls., Conclusions: This novel approach allows delivery of transduced Kupffer cells in rats, which can be used as an investigative tool as well as a therapeutic strategy against inflammatory liver diseases.

Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE(2), which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in hepatic parenchymal cells by agonists released during stress, such as with PGE(2) and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [Ca(2+)](i) in individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PGE(2) caused a dose-dependent increase in [Ca(2+)](i) from basal values of 130 +/- 10 to maximal levels of 434 +/- 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca(2+) dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of Ca(2+) from intracellular stores, also partially blocked the increase in [Ca(2+)](i) caused by PGE(2), suggesting that intracellular Ca(2+) pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca(2+) pools are involved in the increase in [Ca(2+)](i) caused by PGE(2). Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca(2+)](i) due to PGE(2) in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl(-) was omitted, glycine was much less effective in preventing the increase in [Ca(2+)](i) due to PGE(2). Phenylephrine, an alpha(1)-type adrenergic receptor agonist, also increased [Ca(2+)](i), as expected, from 159 +/- 20 to 432 +/- 43 nM. Glycine also blocked the increase in [Ca(2+)](i) due to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [Ca(2+)](i) in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection.

Intratracheal instillation of lipopolysaccharide (LPS) activates alveolar macrophages and infiltration of neutrophils, causing lung injury/acute respiratory distress syndrome. Free radicals are a special focus as the final causative molecules in the pathogenesis of lung injury caused by LPS. Although in vitro investigation has demonstrated radical generation after exposure of cells to LPS, in vivo evidence is lacking. Using electron spin resonance (ESR) and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we investigated in vivo free radical production by rats treated with intratracheal instillation of LPS. ESR spectroscopy of lipid extract from lungs exposed to LPS for 6 h gave a spectrum consistent with that of a POBN/carbon-centered radical adduct (aN=14.94+/-0.07 G and abetaH=2.42+/-0.06 G) tentatively assigned as a product of lipid peroxidation. To further investigate the mechanism of LPS-initiated free radical generation, rats were pretreated with the phagocytic toxicant GdCl3, which significantly decreased the production of radical adducts with a corresponding decrease in neutrophil infiltration. NADPH oxidase knockout mice completely blocked phagocyte-mediated, ESR-detectable radical production in this model of acute lung injury. Rats treated intratracheally with LPS generate lipid-derived free radicals via activation of NADPH oxidase.

These experiments were designed to determine whether green tea extract (GTE), which contains polyphenolic free radical scavengers, prevents ischemia-reperfusion injury to the liver. Rats were fed a powdered diet containing 0-0.3% GTE starting 5 days before hepatic warm ischemia and reperfusion. Free radicals in bile were trapped with the spin-trapping reagent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and measured using electron spin resonance spectroscopy. Hepatic ischemia-reperfusion increased transaminase release and caused pathological changes including focal necrosis and hepatic leukocyte infiltration in the liver. Transaminase release was diminished by over 85% and pathological changes were almost totally blocked by 0.1% dietary GTE. Ischemia-reperfusion increased 4-POBN/radical adducts in bile nearly twofold, an effect largely blocked by GTE. Epicatechin, one of the major green tea polyphenols, gave similar protection as GTE. In addition, hepatic ischemia-reperfusion activated NF-kappa B and increased TNF-alpha mRNA and protein expression. These effects were all blocked by GTE. Taken together, these results demonstrate that GTE scavenges free radicals in the liver after ischemiareoxygenation, thus preventing formation of toxic cytokines. Therefore, GTE could prove to be effective in decreasing hepatic injury in disease states where ischemia-reperfusion occurs.

Recent studies have demonstrated that glycine blunts the response of Kupffer cells to endotoxin. Based on pharmacological evidence, it was hypothesized that Kupffer cells and other macrophages contain a glycine-gated chloride channel similar to the glycine receptor expressed in neuronal tissues. Moreover, glycine stimulates influx of radiolabeled chloride in Kupffer cells in a dose-dependent manner. RT-PCR was used to identify mRNA of both alpha- and beta-subunits of the glycine receptor in rat Kupffer cells, peritoneal neutrophils, and splenic and alveolar macrophages, similar to the sequence generated from rat spinal cord. Importantly, the sequence of the cloned Kupffer cell glycine receptor fragment for the beta-subunit was >95% homologous with the receptor from the spinal cord. Membranes of these cells also contain a protein that is immunoreactive with antibodies against the glycine-gated chloride channel. These data demonstrate that Kupffer cells, as well as other macrophages and leukocytes, express mRNA and protein for a glycine-gated chloride channel with both molecular and pharmacological properties similar to the channel expressed in the central nervous system.

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to determine whether cocoa flavonoid extract, composed mostly of epicatechin and epicatechin oligomers, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g/kg per day) and cocoa extract (400 mg/kg per day) continuously for 4 weeks using an enteral feeding protocol. Mean body weight gains ( approximately 4 g/day) were not significantly different between treatment groups. Cocoa extract did not affect average daily urine ethanol concentrations ( approximately 200mg/dL). After 4 weeks, serum alanine amino transferase levels of the ethanol group were increased nearly fourfold (110+/-16 IU/L) compared to control values (35+/-3 IU/L); this effect of ethanol was blocked by cocoa extract (60+/-6 IU/L). Additionally, enteral ethanol caused severe fat accumulation, mild inflammation, and necrosis in the liver; cocoa extract significantly blunted these changes. Increases in liver TNFalpha protein levels caused by ethanol were completely blocked by cocoa extract. Further, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation serving as an index of oxidative stress; again this was counteracted by the addition of cocoa extract. These results indicate that dietary flavanols such as those found in cocoa can prevent early alcohol-induced liver injury.

Tissue deposition of protein adducts derived from ethanol metabolism and lipid peroxidation, has been suggested to play a role in the initiation of alcoholic liver disease. The mechanisms modulating adduct formation have, however, remained unclear. We used immunohistochemical methods to examine acetaldehyde (AA) and malondialdehyde (MDA) adducts and cytochrome P4502E1 and P4503A2 expression in rats after administration of (i) an ethanol-diet (n = 6), (ii) ethanol-diet plus gadolinium chloride (GdCl(3)), a selective Kupffer cell toxicant (n = 7), or (iii) control diet (n = 6). A 4 week ethanol treatment resulted in liver steatosis, necrosis, and inflammation and deposition of protein adducts with both AA and MDA, which colocalized with areas of fatty change. The intensities (mean +/- SD) of the immunohistochemical reactions for both AA (2.50 +/- 1.23) and MDA (3.00 +/- 1.10) adducts were significantly higher in the ethanol-fed animals than in the controls (0.083 +/- 0.20) (0.16 +/- 0.25) (p <.001). GdCl(3) prevented adduct accumulation, the mean immunohistochemistry scores being 0.86 +/- 1.07 for AA and 1.64 +/- 0.63 for MDA, the former showing a more striking reduction (p <.01). The hepatic cytochrome enzymes were not different in the ethanol-fed groups with or without GdCl(3). The data indicates that Kupffer cells are involved in the generation of protein adducts with both acetaldehyde and ethanol-induced lipid peroxidation products in alcoholic liver disease.

Nicotine influences energy metabolism, yet mechanisms remain unclear. Since the liver is one of the largest organs and performs many metabolic functions, the goal of this study was to determine whether nicotine would affect respiration and other metabolic functions in the isolated perfused liver. Infusion of 85 microM nicotine caused a rapid 10% increase in oxygen uptake over basal values of 105 +/- 5 micromol/g/h in perfused livers from fed rats, and an increase of 27% was observed with 850 microM nicotine. Concomitantly, rates of glycolysis of 105 +/- 8 micromol/g/h were decreased to 52 +/- 9 micromol/g/h with nicotine, whereas ketone body production was unaffected. Nicotine had no effect on oxygen uptake in glycogen-depleted livers from 24-h fasted rats. Furthermore, addition of glucose to perfused livers from fasted rats partially restored the stimulatory effect of nicotine. Infusion of atractyloside, potassium cyanide, or glucagon blocked the nicotine-induced increase in respiration. Intracellular calcium was increased in isolated hepatocytes by nicotine, a phenomenon prevented by incubation of cells with d-tubocurarine, a nicotinic acetylcholine receptor antagonist. Respiration was also increased approximately 30% in hepatocytes isolated from fed rats by nicotine, whereas hepatocytes isolated from fasted rats showed little response. In the presence of N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), an inhibitor of cyclic AMP-dependent protein kinase A, nicotine failed to stimulate respiration. These data support the hypothesis that inhibition of glycolysis by nicotine increases oxygen uptake due to an ADP-dependent increase in mitochondrial respiration.

Cellular responses to endotoxins are enhanced markedly by LPS-binding protein (LBP). Furthermore, it has been demonstrated that endotoxins and proinflammatory cytokines such as TNF-alpha participate in early alcohol-induced liver injury. Therefore, in this study, a long-term intragastric ethanol feeding model was used to test the hypothesis that LBP is involved in alcoholic hepatitis by comparing LBP knockout and wild-type mice. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. There was no difference in mean urine alcohol concentrations between the groups fed ethanol. Dietary alcohol significantly increased liver to body weight ratios and serum alanine aminotransferase levels in wild-type mice (189 +/- 31 U/L) over high-fat controls (24 +/- 7 U/L), effects which were blunted significantly in LBP knockout mice (60 +/- 17 U/L). Although no significant pathological changes were observed in high-fat controls, 4 wk of dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals as expected (pathology score, 5.9 +/- 0.5). These pathological changes were reduced significantly in LBP knockout mice fed ethanol (score, 2.6 +/- 0.5). Endotoxin levels in the portal vein were increased significantly after 4 wk in both groups fed ethanol. Moreover, ethanol increased TNF-alpha mRNA expression in wild-type, but not in LBP knockout mice. These data are consistent with the hypothesis that LBP plays an important role in early alcohol-induced liver injury by enhancing LPS-induced signal transduction, most likely in Kupffer cells.

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to test the hypothesis that the antioxidant polyphenolic extract of green tea, comprised predominantly of epigallocatechin gallate, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g kg(-1) day(-1)) and green tea (300 mg kg(-1) day(-1)) continuously for 4 weeks using an intragastric enteral feeding protocol. Mean body weight gains (approximately 4 g/day) were not significantly different between treatment groups, and green tea extract did not the affect average concentration or the cycling of urine ethanol concentrations (0-550 mg dl(-1) day(-1)). After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (35+/-3 IU/l) by enteral ethanol (114+/-18); inclusion of green tea extract in the diet significantly blunted this increase (65+/-10). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver. While not affecting fat accumulation or inflammation, green tea extract significantly blunted increases in necrosis caused by ethanol. Furthermore, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation and an index of oxidative stress; green tea extract blocked this effect almost completely. TNFalpha protein levels were increased in liver by alcohol; this phenomenon was also blunted by green tea extract. These results indicate that simple dietary antioxidants, such as those found in green tea, prevent early alcohol-induced liver injury, most likely by preventing oxidative stress.

Although glycine prevents renal tubular cell injury in vitro, its effect in vivo is not clear. The purpose of this study was to investigate whether a bolus injection of glycine given before reperfusion plus continuous dietary supplementation afterward would reduce renal injury caused by ischemia-reperfusion. Female Sprague-Dawley rats received a semisynthetic powdered diet containing 5% glycine and 15% casein (glycine group) or 20% casein (control group). Two days later, renal ischemia was produced by cross-clamping the left renal vessels for 15 min, followed by reperfusion. The right kidney was removed before reperfusion. The postischemic glomerular filtration rate (GFR) showed that renal function was less impaired and recovered more quickly in rats receiving glycine. For example, at day 7, GFR in controls (0.31 +/- 0.03 ml x min(-1) x 100 g(-1)) was about one-half that of glycine-treated rats (0.61 +/- 0.06 ml x min(-1) x 100 g(-1), P < 0.05). Furthermore, tubular injury and cast formation observed in controls was minimized by glycine (pathology score, 3.2 +/- 0.4 vs. 1.0 +/- 0.4, P < 0.05). Urinary lactate dehydrogenase (LDH) concentration was elevated by ischemia-reperfusion in the control group (260 +/- 22 U/l), but values were significantly lower by about fourfold (60 +/- 30 U/l) in glycine-fed rats. Similarly, free radical production in urine was significantly lower in glycine-treated animals. Importantly, on postischemic day 1, binding of pimonidazole, an in vivo hypoxia marker, was increased in the outer medulla in controls; however, this phenomenon was prevented by glycine. Two weeks later, mild leukocyte infiltration and interstitial fibrosis were still observed in controls, but not in kidneys from glycine-treated rats. In conclusion, these results indicate that administration of glycine indeed reduces mild ischemia-reperfusion injury in the kidney in vivo, in part by decreasing initial damage and preventing chronic hypoxia.

Background: Long-term cold storage and reperfusion lead to great liver injury involving major microcirculatory disturbance. This study investigated the effect of storage duration on endothelin-1 production, portal pressure, and microcirculation., Methods: Rat livers were perfused before and after 15 min to 48 h of cold storage in University of Wisconsin (UW) solution., Results: Portal pressure was unchanged for up to 12 h of storage, and increased by 40% and 70% after 24 and 48 h, respectively. Vascular space was increased by a factor of 1.7 following 48 h of storage. Endothelin-1 was released by livers stored in UW solution for 48 h (3.99 +/- 1.85 pg/100 microl). The nonselective endothelin-1 receptor antagonist TAK-044 partially prevented the increase in portal pressure and prevented the increase in vascular space., Conclusions: Cold storage induces a time-dependent elevated portal pressure at reperfusion. The involvement of endothelin-1 in this process offers opportunities to improve liver graft quality by using endothelin-1 inhibitors.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

The purpose of this study was to investigate the effectiveness of superoxide dismutase (SOD) overexpression in an acute model of hepatic oxidative stress. Oxidative stress was established using a warm ischemia-reperfusion model, where nearly 70% of the liver was made hypoxic by clamping the hepatic artery and a branch of the portal vein for 1 hr followed by restoration of blood flow. Animals were infected i.v. with 1 x 10(9) plaque-forming units (PFU) of adenovirus containing the transgene for cytosolic Cu/Zn-SOD (Ad.SOD1), mitochondrial Mn-SOD (Ad.SOD2), extracellular Cu/Zn-SOD (Ad.SOD3), or the bacterial reporter gene for beta-galactosidase (Ad.lacZ) 3 days prior to experiments. Ad.SOD1 and Ad.SOD2 caused a three-fold increase in SOD expression and activity in liver compared to Ad.lacZ-treated control animals. Intravenous administration of Ad.SOD3 increased SOD activity slightly in serum but not in liver. Increases in serum transaminases and pathology due to ischemia-reperfusion were blunted by Ad.SOD1 and Ad.SOD2; however, extracellular SOD had no significant effect. Moreover, lipid-derived free radical adducts (a(N) = 15.65 G and a(H)(beta) = 2.78 G) were increased by ischemia-reperfusion. This effect was blunted by about 60% in Ad.SOD1- and Ad.SOD2-infected animals, but was unaffected by Ad.SOD3. However, when high doses of Ad.SOD3 (3 x 10(10) PFU) were administered. serum SOD activity was elevated three-fold and was protective against hepatic ischemia-reperfusion injury under these conditions. These data demonstrate that adenoviral delivery of superoxide dismutase can effectively reduce hepatic oxidative stress.

Chronic alcohol administration increases gut-derived endotoxin in the portal blood, which activates Kupffer cells through nuclear factor kappaB (NF-kappaB) to produce toxic mediators such as proinflammatory cytokines, leading to liver injury. Therefore, a long-term intragastric ethanol feeding protocol was used here to test the hypothesis that NF-kappaB inhibition would prevent early alcohol-induced liver injury. Adenoviral vectors encoding either the transgene for IkappaB superrepressor (AdIkappaB-SR) or the bacterial beta-galactosidase reporter gene (AdlacZ) were administered intravenously to Wistar rats. Animals were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin (control) for 3 weeks. There was no significant difference in mean urine alcohol concentrations between the groups fed ethanol. IkappaB-SR expression was increased for up to 2 weeks after injection, but was undetectable at 3 weeks. NF-kappaB activation was increased by ethanol and associated with up-regulation of tumor necrosis factor alpha (TNF-alpha). These increases were blunted significantly up to 2 weeks by AdIkappaB-SR. Dietary alcohol significantly increased liver to body weight ratios and serum alanine transaminase (ALT) levels in AdlacZ-treated animals, effects that were blunted significantly in AdIkappaB-SR-treated rats. Ethanol caused severe steatosis, inflammation, and focal necrosis in AdlacZ-treated animals. These pathologic changes were significantly decreased by AdIkappaB-SR. The protective effects of IkappaB-SR were significant 2 weeks after injection, but were lost at 3 weeks when IkappaB-SR was no longer expressed. Ethanol increased 4-hydroxynonenal as a maker of oxidative stress in both AdlacZ and AdIkappaB groups. These data support the hypothesis that NF-kappaB inhibition prevents early alcohol-induced liver injury even in the presence of oxidative stress.

Acute cyclosporin A (CsA) treatment inhibits mitochondrial respiration, yet effects of chronic treatment remain unclear. Accordingly, the effects of chronic CsA on oxygen metabolism in perfused rat liver and isolated mitochondria were investigated. Basal rates of oxygen uptake of around 120 micromol/g/h in isolated perfused livers from vehicle-treated controls were elevated about 1.6-fold by chronic CsA treatment. In the presence of ammonium chloride, a substrate for urea synthesis, oxygen uptake was about 150 micromol/g/h and was increased about 1.7-fold by CsA, indicating that chronic CsA treatment causes a robust hypermetabolic state in the liver. In isolated mitochondria, state 3 rates of oxygen uptake were increased about 1.6-fold by chronic CsA treatment. Since significant increases in oxygen consumption could cause hypoxia, the hypoxia marker pimonidazole was given. Pimonidazole binding in the liver was increased about 3-fold by chronic CsA. Moreover, intracellular calcium in Kupffer cells isolated from vehicle-treated rats was not altered by CsA addition; however, in cells isolated from chronic CsA-treated rats, CsA increased intracellular calcium about 15-fold and prostaglandin E(2) (PGE(2)) production 3.5-fold. Importantly, dietary glycine (5%) largely blocked chronic CsA-induced activation of Kupffer cells, blunted production of PGE(2), prevented the hypermetabolic state, and minimized tissue hypoxia. Taken together, it is concluded that chronic CsA treatment causes a hypermetabolic state leading to hypoxia and injury to the liver. It is hypothesized that CsA activates Kupffer cells and increases production of PGE(2), which alters mitochondria leading to a hypermetabolic state. Glycine inhibits activation of Kupffer cells thus preventing liver injury.

Evidence has accumulated for a role of toxic oxygen radicals in the pathogenesis of ischemia-reperfusion injury in the kidney. The aim of this study was to evaluate the hypothesis that reducing postischemic renal injury is possible by delivery of the gene for the antioxidant enzyme superoxide dismutase (SOD). Female Sprague-Dawley rats received intravenous injections of recombinant adenovirus (1 x 10(9) pfu) containing the transgenes for Escherichia coli beta-galactosidase (Ad-LacZ, as control) or human Cu/Zn-SOD (Ad-SOD). Three days later, renal ischemia was produced by cross-clamping the left renal vessels for 60 min. The right kidney was removed before reperfusion and processed for the transgene. Renal SOD protein and activity in rats given Ad-SOD was 2.5-fold higher than from the animals receiving Ad-LACZ: Urinary lactate dehydrogenase concentrations were elevated by ischemia-reperfusion in the Ad-LacZ group (1403 +/- 112 U/L), yet values were 50% lower in Ad-SOD-treated rats. Free radical production was elevated by ischemia-reperfusion but was significantly lower in SOD-treated animals. Importantly, on postischemic day 1, glomerular filtration rates were reduced to 0.21 ml/min per 100 g in the Ad-LacZ group, whereas values remained significantly higher (0.39) in the Ad-SOD group. Two weeks after ischemia-reperfusion, inflammation, interstitial fibrosis, tubular atrophy and tissue levels of tumor necrosis factor alpha and interleukin-1 were significantly higher in the Ad-LacZ-treated than in Ad-SOD-treated rats. In conclusion, these results indicate that SOD expression can be increased by delivery of the sod gene to the kidney by intravenous injection and that sod gene transduction minimized ischemia-reperfusion-induced acute renal failure.

Tumor necrosis factor alpha (TNF-alpha) and free radicals are produced in early alcohol-induced liver injury. Recently, pathology caused by alcohol was blocked nearly completely in tumor necrosis factor alpha receptor 1 (TNF-R1) knockout mice. With this model, it is now possible to evaluate whether free radicals are directly toxic or act as redox regulators of TNF-alpha production. Specifically, if free radicals were directly toxic, a parallel decrease in free radicals and pathology in TNF-R1 knockout mice would be predicted. If they only affect TNF-alpha production, radicals would be expected to remain high while pathology is diminished. Accordingly, free radical production in TNF-R1 knockout mice was studied here. The enteral alcohol delivery model used mice lacking TNF-R1 (p55) and wild-type control C57Bl/6J mice. Animals received a liquid diet continuously with either ethanol or isocaloric maltose-dextrin as control for 4 weeks. Urine ethanol levels fluctuated from 10 to 500 mg/dL in a cyclic pattern in mice receiving ethanol. Ethanol elevated liver:body weight ratios, serum alanine transaminase (ALT) levels, and pathology scores in wild-type mice. These parameters were blunted nearly completely in TNF-R1 knockout mice. Ethanol treatment increased free radical production in wild-type mice compared with animals fed a high-fat control diet. There were no differences in intensity of free radical signals regardless of the presence or absence of TNF-R1; however, pathology differed markedly between these groups. These findings are consistent with the hypothesis that free radicals act as redox signals for TNF-alpha production and do not directly damage cells in early alcohol-induced hepatic injury.