Henry D. Connor

NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease

In North America, liver disease due to alcohol consumption is an important cause of death in adults, although its pathogenesis remains obscure. Despite the fact that resident hepatic macrophages are known to contribute to early alcohol-induced liver injury via oxidative stress, the exact source of free radicals has remained a mystery. To test the hypothesis that NADPH oxidase is the major source of oxidants due to ethanol, we used p47(phox) knockout mice, which lack a critical subunit of this major source of reactive oxygen species in activated phagocytes. Mice were treated with ethanol chronically, using a Tsukamoto-French protocol, for 4 weeks. In wild-type mice, ethanol caused severe liver injury via a mechanism involving gut-derived endotoxin, CD14 receptor, production of electron spin resonance-detectable free radicals, activation of the transcription factor NF-kappaB, and release of cytotoxic TNF-alpha from activated Kupffer cells. In NADPH oxidase-deficient mice, neither an increase in free radical production, activation of NF-kappaB, an increase in TNF-alpha mRNA, nor liver pathology was observed. These data strongly support the hypothesis that free radicals from NADPH oxidase in hepatic Kupffer cells play a predominant role in the pathogenesis of early alcohol-induced hepatitis by activating NF-kappaB, which activates production of cytotoxic TNF-alpha.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

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.

Cyclosporin A increases hypoxia and free radical production in rat kidneys: prevention by dietary glycine

The major side effect of cyclosporin A is severe nephrotoxicity. It is likely that cyclosporin A causes vasoconstriction leading to hypoxia-reperfusion injury; therefore, these experiments were designed to attempt to obtain physical evidence for hypoxia and free radical production in kidney following cyclosporin A. Rats were treated daily with cyclosporin A (25 mg/kg ig) for 5 days, and pimonidazole, a hypoxia marker, was injected 2 h after the last dose of cyclosporin A. A dose of α-(4-pyridyl-1-oxide)- N- tert-butylnitrone (4-POBN) was injected 3 h after cyclosporin A to trap free radicals. Cyclosporin A doubled serum creatinine and decreased glomerular filtration rates by 65% as expected. Pimonidazole adduct binding in the kidney was increased nearly threefold by cyclosporin A, providing physical evidence for tissue hypoxia. Moreover, cyclosporin A increased 4-POBN/radical adducts nearly sixfold in the urine but did not alter levels in the serum. Glycine, which causes vasodilatation and prevents cyclosporin A toxicity, minimized hypoxia and blocked free radical production; however, it did not alter cyclosporin A blood levels. These results demonstrate for the first time that cyclosporin A causes hypoxia and increases production of a new free radical species exclusively in the kidney. Therefore, it is concluded that cyclosporin A causes renal injury by mechanisms involving hypoxia-reoxygenation, effects which can be prevented effectively by dietary glycine.The major side effect of cyclosporin A is severe nephrotoxicity. It is likely that cyclosporin A causes vasoconstriction leading to hypoxia-reperfusion injury; therefore, these experiments were designed to attempt to obtain physical evidence for hypoxia and free radical production in kidney following cyclosporin A. Rats were treated daily with cyclosporin A (25 mg/kg ig) for 5 days, and pimonidazole, a hypoxia marker, was injected 2 h after the last dose of cyclosporin A. A dose of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was injected 3 h after cyclosporin A to trap free radicals. Cyclosporin A doubled serum creatinine and decreased glomerular filtration rates by 65% as expected. Pimonidazole adduct binding in the kidney was increased nearly threefold by cyclosporin A, providing physical evidence for tissue hypoxia. Moreover, cyclosporin A increased 4-POBN/radical adducts nearly sixfold in the urine but did not alter levels in the serum. Glycine, which causes vasodilatation and prevents cyclosporin A toxicity, minimized hypoxia and blocked free radical production; however, it did not alter cyclosporin A blood levels. These results demonstrate for the first time that cyclosporin A causes hypoxia and increases production of a new free radical species exclusively in the kidney. Therefore, it is concluded that cyclosporin A causes renal injury by mechanisms involving hypoxia-reoxygenation, effects which can be prevented effectively by dietary glycine.

Evidence that free radicals are involved in graft failure following orthotopic liver transplantation in the rat : an electron paramagnetic resonance spin trapping study

&NA; The purpose of these studies was to determine whether free radicals were formed as a consequence of reperfusion during orthotopic liver transplantation and whether their formation was related to graft failure. Grafts were stored for 18 hr in Euro-Collins solution or for 48 hr in University of Wisconsin solution (nonsurvival conditions) and reperfused with blood containing the spin trap α-phenyl N-tert-butylnitrone (PBN). Venous blood samples (4–5 ml) were collected, and serum was extracted with chloroform and methanol (2:1) and analyzed for radical adducts by electron paramagnetic resonance (EPR) spectroscopy. In samples from livers stored under nonsurvival conditions, EPR spectra were detected indicating the presence of PBN radical adducts. In contrast, radical adduct formation was 3− to 4-fold lower in similar experiments performed with untransplanted livers or with livers stored under survival conditions (1 hr in Ringers solution or 24 hr in UW solution). Oxygen radicals are most likely involved in the production of radical adducts because formation was nearly completely prevented by superoxide dismutase plus catalase or Carolina rinse, which contains glutathione, desferrioxamine mesylate, and allopurinol. Radical adduct formation was much greater in a blood-free perfusion system where oxygen delivery was high, suggesting that blood elements are not necessary for radical adduct formation. An inverse correlation between survival of livers stored in UW solution and radical adduct signal was observed in this study. Thus, it is concluded that free radicals formed during reperfusion are involved in the mechanism of graft failure following liver transplantation in the rat.

The role of gut-derived bacterial toxins and free radicals in alcohol-induced liver injury.

Previous research from this laboratory using a continuous enteral ethanol (EtOH) administration model demonstrated that Kupffer cells are pivotal in the development of EtOH‐induced liver injury. When Kupffer cells were destroyed using gadolinium chloride (GdCl3) or the gut was sterilized with polymyxin B and neomycin, early inflammation due to EtOH was blocked. Anti‐tumour necrosis factor (TNF)‐α antibody markedly decreased EtOH‐induced liver injury and increased TNF‐mRNA. These findings led to the hypothesis that EtOH‐induced liver injury involves increases in circulating endotoxin leading to activation of Kupffer cells. Pimonidazole, a nitro‐imidazole marker, was used to detect hypoxia in downstream pericentral regions of the lobule. Following one large dose of EtOH or chronic enteral EtOH for 1 month, pimonidazole binding was increased significantly in pericentral regions of the liver lobule, which was diminished with GdCl3. Enteral EtOH increased free radical generation detected with electron spin resonance (ESR). These radical species had coupling constants matching α‐hydroxyethyl radical and were shown conclusively to arise from EtOH based on a doubling of the ESR lines when 13C‐EtOH was given. α‐Hydroxyethyl radical production was also blocked by the destruction of Kupffer cells with GdCl3. It is known that females develop more severe EtOH‐induced liver injury more rapidly and with less EtOH than males. Female rats on the enteral protocol exhibited more rapid injury and more widespread fatty changes over a larger portion of the liver lobule than males. Plasma endotoxin, ICAM‐1, free radical adducts, infiltrating neutrophils and transcription factor NFκB were approximately two‐fold greater in livers from females than males after 4 weeks of enteral EtOH treatment. Furthermore, oestrogen treatment increased the sensitivity of Kupffer cells to endotoxin. These data are consistent with the hypothesis that Kupffer cells participate in important gender differences in liver injury caused by ethanol.

[17O]Oxygen hyperfine structure for the hydroxyl and superoxide radical adducts of the spin traps DMPO, PBN and 4-POBN

[17O]oxygen hyperfine coupling constants are reported for the superoxide and hydroxyl radical adducts with the spin traps 5,5-dimethyl-1-pyrroline N-oxide, N-t-butyl-alpha-phenylnitrone and alpha-(4-pyridyl 1-oxide)-N-t-butylnitrone. These couplings provide spectroscopic evidence that the spin adducts have been correctly identified.

Primary nonfunction of fatty livers produced by alcohol is associated with a new, antioxidant-insensitive free radical species

The formation of free radicals after orthotopic liver transplantation in the rat correlates with graft failure. Fatty livers from alcoholics transplant poorly, so these studies were designed to examine the effect of alcohol on free radical formation in a rearterialized rat liver transplantation model. Treatment of rats for 3-5 weeks with either a high-fat or an ethanol-containing liquid diet caused characteristic pericentral lipid accumulation. After storage in University of Wisconsin cold storage solution (UW) and transplantation, a reperfusion injury characterized by increased postoperative AST levels (greater than 1500 U/l in about 3 hours) was observed in rats fed high-fat or alcohol-containing diets, whereas parenchymal cell injury was seen much less in low-fat controls. Survival was around 63% in the low-fat group but decreased to 12 and 18% in the high-fat and alcohol groups, respectively. Furthermore, intracellular lipid content correlated inversely with survival. In untransplanted livers, the spin trap alpha-phenyl N-tert-butylnitrone (PBN) was infused, and blood samples were collected and extracted with chloroform:methanol. Signals indicative of carbon-centered PBN radical adducts were barely detectable in all untransplanted groups studied by electron paramagnetic resonance. In contrast, a robust 6-line complex spectrum was obtained from all groups studied immediately after 48 hours of cold storage in UW solution and transplantation. A mixture of 3 radical species was identified. Two had coupling constants similar to lipid-derived free radicals, whereas the third is a new species with unique coupling constants and is most likely oxygen derived. In low-fat controls, the signal was reduced significantly by superoxide dismutase (SOD)/catalase; however, SOD/catalase had no effect on free radicals in lipid-loaded livers. Thus, both dietary high fat and alcohol exposure produce a unique SOD/catalase-insensitive free radical species that may be involved in the mechanism of failure of fatty livers after orthotopic liver transplantation.

Glycine improves survival after hemorrhagic shock in the rat.

This study investigated the effect of glycine on hemorrhagic shock in the rat. Rats were bled to maintain mean arterial pressure at 30-35 mm Hg for 1 h and subsequently resuscitated with 60% shed blood and lactated Ringers solution. Only 20% of rats receiving saline just prior to resuscitation survived 72 h after shock. Survival was increased by glycine (11.2-90.0 mg/kg, i.v.) in a dose-dependent manner (half-maximal effect = 25 mg/kg) and reached maximal values of 78% at 45 mg/kg. Eighteen hours after resuscitation, creatinine phosphokinase increased 23-fold, transaminases increased 33-fold, and creatinine was elevated 2.4-fold, indicating injury to the heart, liver, and kidney, respectively. Pulmonary edema, leukocyte infiltration, and hemorrhage were also observed. In the kidney, proximal tubular necrosis, leukocyte infiltration, and severe hemorrhage in the outer medullary area occurred in rats receiving saline. Glycine reduced these pathological alterations significantly. It has been reported that oxidative stress and tumor necrosis factor(TNF)-alpha-production are involved in the pathophysiology of multiple-organ injury after shock. In this study, free radical production was increased 4-fold during shock, an effect blocked largely by glycine. Increases in intracellular calcium and production of TNF-alpha by isolated Kupffer cells stimulated by endotoxin were elevated significantly by hemorrhagic shock, alterations which were totally prevented by glycine. Taken together, it is concluded that glycine reduces organ injury and mortality caused by hemorrhagic shock by preventing free radical production and TNF-alpha formation.

A search for oxygen-centered free radicals in the lipoxygenase/linoleic acid system.

Studies of the oxygenation of linoleic acid by soybean lipoxygenase utilizing electron spin resonance spectroscopy and oxygen uptake have been undertaken. The spin trap, alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) was included in the lipoxygenase system to capture short-lived free radicals. Correlation of radical adduct formation rates with oxygen uptake studies indicated that the major portion of radical adduct formation occurred when the system was nearly anaerobic. Incubations containing [17O]oxygen with nuclear spin of 5/2 did not have additional ESR lines as would be expected if an oxygen-centered 4-POBN-lipid peroxyl radical adduct were formed indicating that the trapped radical must be reassigned as a carbon-centered species. To establish the presence of [17O2]oxygen in our incubations, a portion of the gas from the lipoxygenase/linoleate experiments was used to prepare the 4-POBN-superoxide radical adduct utilizing a superoxide producing microsomal/paraquat/NADPH system.

Ischemic preconditioning prevents free radical production and mitochondrial depolarization in small-for-size rat liver grafts.

Background. Ischemic preconditioning (IP) renders tissues more tolerant to subsequent longer episodes of ischemia. This study tested whether IP attenuates injury of small-for-size liver grafts by preventing free radical production and mitochondrial dysfunction. Methods. IP was induced by clamping the portal vein and hepatic artery for 9 min. Livers were harvested 5 min after releasing the clamp. Mitochondrial polarization and cell death were assessed by intravital confocal/multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide. Free radicals were trapped with &agr;-(4-pyridyl 1-oxide)-N-tert-butylnitrone and measured using electron spin resonance. Results. After quarter-size liver transplantation, alanine aminotransferase, serum bilirubin, necrosis, and apoptosis all increased. IP blocked these increases by more than 58%. 5-Bromo-2′-deoxyuridine labeling and increases of graft weight were only ∼3% and 0.2% in quarter-size grafts without IP, respectively, but increased to 32% and 60% in ischemic-preconditioned grafts, indicating better liver regeneration. Eighteen hours after implantation, viable cells with depolarized mitochondria in quarter-size grafts were 15 per high power field, and dead cells were less than 1 per high power field, indicating that depolarization preceded necrosis. A free radical adduct signal was detected in bile from quarter-size grafts. IP decreased this free radical formation and prevented mitochondrial depolarization. IP did not increase heat shock proteins 10, 27, 32, 60, 70, 72, 75 and Cu/Zn-superoxide dismutase (SOD) but increased heat shock protein-90, a chaperone that facilitates protein import into mitochondria, and mitochondrial Mn-SOD. Conclusion. Taken together, IP decreases injury and improves regeneration of small-for-size liver grafts, possibly by increasing mitochondrial Mn-SOD, thus protecting against free radical production and mitochondrial dysfunction.