The intracellular distribution of glutathine (GSH) in cultured hepatocytes hasbeen investigated by using the compound monochlorobimane (BmCI) Approximately 10-20% of total cellular GSH in rat liver is sequestered in the mitochondrial matrix (6, 7). The size ofthis pool depends on cytosolic GSH synthesis (8) and the active transport of GSH into mitochondria via a multicomponent system recently described (9).Conventional cell-fractionation studies have not provided evidence for the existence of functionally distinct pools of GSH in hepatocytes other than those in the cytosol and mitochondria. Despite the known functions of GSH in DNA synthesis (10) and protection from oxidative DNA damage (11), little is known about the nuclear localization ofGSH and the factors regulating the nuclear GSH level. Tirmenstein and Reed (12), using fractionation and centrifugation techniques in nonaqueous medium, measured the nuclear GSH content in rat kidney and found values similar to those in the cytosol. Other fractionation techniques (such as selective permeabilization of cell constituents with various detergents) provided equivocal results (13,14). However, the latter investigations have suggested that a nuclear pool of GSH may exist in intact cells.Recent advances in image-analysis technology, together with the development of additional, nontoxic fluorescent indicators that can be used in intact cells, have facilitated an enormous input into the study of various aspects of cell physiology (15,16 ITo whom reprint requests should be addressed. 4412The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

We tested whether rat liver preservation performed by machine perfusion (MP) at 20°C can enhance the functional integrity of steatotic livers versus simple cold storage. We also compared MP at 20°C with hypothermic MP at 8°C, and 4°C. Obese and lean male Zucker rats were used as liver donors. MP was performed for 6 hours with a glucose and N-acetylcysteine-supplemented Krebs-Henseleit solution. Both MP and cold storage preserved livers were reperfused with Krebs-Henseleit solution (2 hours at 37°C). MP at 4°C and 8°C reduced the fatty liver necrosis compared with cold storage but we further protected the organs using MP at 20°C. Necrosis did not differ in livers from lean animals submitted to the different procedures; the enzymes released in steatotic livers preserved by MP at 20°C were similar to those showed in nonsteatotic organs. The adenosine triphosphate/adenosine diphosphate ratio and bile production were higher and the oxidative stress and biliary enzymes were lower in steatotic livers preserved by MP at 20°C as compared with cold storage. In livers from lean rats, the adenosine triphosphate/adenosine diphosphate ratio appears better conserved by MP at 20°C as compared with cold storage. In steatotic livers preserved by cold storage, a 2-fold increase in tumor necrosis factor-alpha levels and caspase-3 activity was observed as compared with organs preserved by MP at 20°C. These data are substantiated by better morphology, higher glycogen content, and lower reactive oxygen species production by sinusoidal cells in steatotic liver submitted to MP at 20°C versus cold storage. MP at 20°C improves cell survival and leads to a marked improvement in hepatic preservation of steatotic livers as compared with cold storage. Liver Transpl 15:20-29, 2009.

Glutathione (GSH), a tripeptide particularly concentrated in the liver, is the most important thiol reducing agent involved in the modulation of redox processes. It has also been demonstrated that GSH cannot be considered only as a mere free radical scavenger but that it takes part in the network governing the choice between survival, necrosis and apoptosis as well as in altering the function of signal transduction and transcription factor molecules. The purpose of the present review is to provide an overview on the molecular biology of the GSH system; therefore, GSH synthesis, metabolism and regulation will be reviewed. The multiple GSH functions will be described, as well as the importance of GSH compartmentalization into distinct subcellular pools and inter-organ transfer. Furthermore, we will highlight the close relationship existing between GSH content and the pathogenesis of liver disease, such as non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), chronic cholestatic injury, ischemia/reperfusion damage, hepatitis C virus (HCV), hepatitis B virus (HBV) and hepatocellular carcinoma. Finally, the potential therapeutic benefits of GSH and GSH-related medications, will be described for each liver disorder taken into account.

The contribution of endogenous fluorophores - such as proteins, bound and free NAD(P)H, flavins, vitamin A, arachidonic acid - to the liver autofluorescence was studied on tissue homogenate extracts and on isolated hepatocytes by means of spectrofluorometric analysis. Autofluorescence spectral analysis was then applied to investigate the response of single living hepatocytes to experimental conditions resembling the various phases of the organ transplantation. The following conditions were considered: 1 h after cells isolation (reference condition); cold hypoxia; rewarming-reoxygenation after cold preservation. The main alterations occurred for NAD(P)H and flavins, the coenzymes strictly involved in energetic metabolism. During cold hypoxia NAD(P)H, mainly the bound form, showed an increase followed by a slow decrease, in agreement with the inability of the respiratory chain to reoxidize the coenzyme, and a subsequent NADH reoxidation through alternative anaerobic metabolic pathways. Both bound/free NAD(P)H and total NAD(P)H/flavin ratio values were altered during cold hypoxia, but approached the reference condition values after rewarming-reoxygenation, indicating the cells capability to restore the basal redox equilibrium. A decrease of arachidonic acid and vitamin A contributions occurred after cold hypoxia: in the former case it may depend on the balance between deacylation and reacylation of fatty acids, in the latter it might be related to the vitamin A antioxidant role. An influence of physico-chemical status and microenvironment on the fluorescence efficiency of these fluorophores cannot be excluded. In general, all the changes observed for cell autofluorescence properties were consistent with the complex metabolic pathways providing for energy supply.

Western blot analysis of protein extracts from rat liver revealed the presence of the mGlu5 receptor, one of the G-protein-coupled receptors activated by glutamate (named ''metabotropic glutamate receptors'' or mGlu receptors). mGlu5 expression was particularly high in extracts from isolated hepatocytes, where levels were comparable with those seen in the rat cerebral cortex. The presence of mGlu5 receptors in hepatocytes was confirmed by reversetranscription polymerase chain reaction (RT-PCR) analysis, immunohistochemistry in neonate or adult rat liver, as well as by immunocytochemical analysis in HepG2 hepatoma cells, where the receptor appeared to be preferentially distributed in cell membranes. Interestingly, mGlu1 receptors (which are structurally and functionally homologous to mGlu5 receptors) were never found in rat liver or hepatocytes. In hepatocytes exposed to anoxic conditions for 90 minutes, glutamate, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and quisqualate, which all activate mGlu5 receptors, accelerated the onset and increased the extent of cell damage, while 4-carboxy-3-hydroxyphenylglycine (4C3HPG), an agonist of mGlu2/3 receptors, was inactive. 2-Methyl-6-(2-phenyl-1-ethynyl)-pyridine (MPEP), a novel, noncompetitive, highly selective mGlu5 receptor antagonist, not only abolished the toxic effect of 1S,3R-ACPD, but, unexpectedly, was protective by itself against anoxic damage. This suggests that hepatocytes express mGlu5 receptors and that activation of these receptors by endogenous glutamate facilitates the development of anoxic damage in hepatocytes. (HEPATOLOGY 2000; 31:649-655.)Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS), and evidence for peripheral glutamatergic fibers in mammals is still lacking. However, glutamate receptors have been identified in peripheral organs, including taste buds, 1 myenteric plexus, 2 and pancreatic islet cells. 3 Glutamate receptors may either form membrane ion channels (ionotropic glutamate receptors) or couple to guanosine triphosphate-binding proteins (''metabotropic'' glutamate [mGlu] receptors). mGlu receptors form a family of 8 subtypes, which are subdivided into 3 groups on the basis of their structural homology, pharmacological profile, and transduction pathways. Group I includes mGlu1 and -5 receptors, which are coupled to polyphosphoinositide (PI) hydrolysis and activated by 3,5-dihydroxyphenylglycine (DHPG) and quisqualate; group II (mGlu2 and -3) and -III (mGlu4, -6, -7, and -8) mGlu receptors are instead negatively coupled to adenylyl cyclase activity. 4,5 1S,3R-ACPD behaves as a non-subtype-selective agonist, with a preferential activity on group I and -II mGlu receptors. 4 We were intrigued by the finding that 1S,3R-ACPD and quisqualate stimulate PI hydrolysis in cultured hepatocytes, and this effect is reduced by the mixed mGlu receptor antagonist, ␣-methyl-4-carboxyphenylglycine (MCPG). 6 This suggests that either mGlu1 or -5 (or a related unknown receptor subtype) are expressed and...

Cytoskeletal abnormalities occurring during oxidative stress generated by the metabolism of the redox cycling compound 2-methyl-1,4-naphtoquinone (menadione) have been investigated in different mammalian cells in culture. Extraction of the whole cytoskeleton as well as the intermediate filament- and the microtubule-enriched fractions from menadione-treated cells revealed a marked depletion of protein sulfhydryl groups. The analysis of the whole cytoskeletal fraction by PAGE showed a menadione-dependent and thiol-sensitive oxidation of actin, leading to the formation of high-molecular-weight aggregates. In addition, the extraction of this fraction with high concentrations of KCl entailed only a partial solubilization of actin. The comparative cytochemical analysis performed on treated cells showed a menadione-dependent clustering of actin microfilaments. The metabolism of menadione induced microtubule depolymerization and inhibition of GTP-induced microtubule assembly from soluble cytosolic components. The latter phenomenon was prevented by previously treating the cytosolic fraction with thiol reductants such as dithiothreitol. Menadione increased the protein content of the intermediate-size filament fraction, partially purified by one or more cycles of disassembly/assembly, and particularly enriched in polypeptides reacting with antikeratin antibodies. Furthermore, a reversible and oxidation-dependent change of the electrophoretic mobility of some polypeptides in this fraction was detected. The immunocytochemical investigation of intermediate-size filament distribution in menadione-treated cells, however, revealed only minor modifications mainly consisting of perinuclear condensation of cytokeratin structures. These findings suggest that cytoskeletal structures (actin microfilaments, microtubules, and intermediate-size filaments) are actually significant targets in quinone-induced oxidative stress.

Autofluorescence emission of liver tissue depends on the presence of endogenous biomolecules able to fluoresce under suitable light excitation. Overall autofluorescence emission contains much information of diagnostic value because it is the sum of individual autofluorescence contributions from fluorophores involved in metabolism, for example, NAD(P)H, flavins, lipofuscins, retinoids, porphyrins, bilirubin and lipids, or in structural architecture, for example, fibrous proteins, in close relationship with normal, altered or diseased conditions of the liver. Since the 1950s, hepatocytes and liver have been historical models to study NAD(P)H and flavins as in situ, real-time autofluorescence biomarkers of energy metabolism and redox state. Later investigations designed to monitor organ responses to ischaemia/reperfusion were able to predict the risk of dysfunction in surgery and transplantation or support the development of procedures to ameliorate the liver outcome. Subsequently, fluorescent fatty acids, lipofuscin-like lipopigments and collagen were characterized as optical biomarkers of liver steatosis, oxidative stress damage, fibrosis and disease progression. Currently, serum AF is being investigated to improve non-invasive optical diagnosis of liver disease. Validation of endogenous fluorophores and in situ discrimination of cancerous from non-cancerous tissue belong to the few studies on liver in human subjects. These reports along with other optical techniques and the huge work performed on animal models suggest many optically based applications in hepatology. Optical diagnosis is currently offering beneficial outcomes in clinical fields ranging from the respiratory and gastrointestinal tracts, to dermatology and ophthalmology. Accordingly, this review aims to promote an effective bench to bedside transfer in hepatology.

Excitation at 366-465 nm of bilirubin in aqueous solution with solubilizing agents results in emission spectra composed by two main bands. The variation of their relative contributions as shown by changes in the spectral shape are consistent with the bilirubin bichromophore nature. This latter accounts for an exciton-coupling phenomenon, intramolecular interchromophore energy transfer efficiency being affected by microenvironment. Excitation at 366 nm, despite the poor absorption of bilirubin, gives rise to appreciable emission signals from both pure compounds and bile - collected from functionally altered rat livers - favouring the spectral shape response to environment and molecular conformation changes. As compared to the merely bile flow estimation, real-time detection of fluorescence, revealing composition variations, improves near-UV optical-biopsy diagnostic potential in hepatology.