Genetic analysis of Asian measles virus strains--new endemic genotype in Nepal.

In many parts of Asia measles virus (MV) continues to be endemic. However, little is known about the genetic characteristics of viruses circulating on this continent. This study reports the molecular epidemiological analysis based on the entire nucleocapsid (N) and hemagglutinin (H) genes of the first isolates from Nepal and Taiwan, as well as of recent MV strains from India, Indonesia, and China. Four isolates collected in various regions in Nepal during 1999 belonged to a new genotype, tentatively called D8. Another Nepalese isolate and one from India belonged to genotype D4. The diversity of the Nepalese strains indicated that measles continues to be endemic in this country. The isolate from Taiwan grouped with D3 viruses and one Chinese strain isolated in The Netherlands was assigned to the previously described clade H, known to be endemic in Mainland China. Molecular characterization emerges as an important tool for monitoring virus endemicity and vaccination efforts.

Monitoring of measles elimination using molecular epidemiology.

The different measles virus genotypes are confined to more or less distinct geographic regions. Molecular characterization of virus isolates has been successfully used to determine epidemiological links between cases and the geographic origin of imported viruses. In Europe, indigenous measles has been eliminated in some countries, but in others the disease is still endemic. Intra-outbreak variability can be used to differentiate between sporadic endemic cases and a 'pseudo-outbreak' of unrelated imported cases. The interruption of virus circulation by mass vaccination campaigns could be demonstrated by comparing the variability of pre-campaign viruses with post-campaign isolates. Simplified tools are being developed that could bring genotyping within reach of laboratories that do not have the possibility of sequencing.

Genetic variability of measles viruses circulating in the Benelux.

In Europe measles incidence remains high and in some parts the disease is likely to be still endemic due to insufficient vaccination. Luxembourg experienced an outbreak with at least 110 cases in 1996, and cases continued to be reported throughout 1997. We used molecular epidemiology to investigate this apparent endemicity. On the basis of their N gene sequences, the isolates were assigned to the typical European C2 and D6 genotypes. Sequence diversity within the outbreak was 0.2%. The nucleotide distance between the C2-viruses of the outbreak and the other C2 isolates was at least three or four times higher, suggesting an independent origin of the latter viruses. Similarly, the four D6 viruses found in Luxembourg were thought to be of at least two or three origins. Thus, we propose here to use intra-outbreak sequence diversity to differentiate between sporadic endemic cases and a "pseudo-outbreak" of multiple unrelated imported cases.

Genotypic and antigenic characterization of hemagglutinin proteins of African measles virus isolates.

A comprehensive phylogenetic study based on the hemagglutinin (H) protein of all known African measles virus (MV) isolates is presented. The study includes 64 new H gene sequences from Ghana. Nigeria and South Africa as well as viruses from Zambia and The Gambia for which only incomplete sequencing data were available and that have previously not been genotyped. The results provide further support to the tentative assignment of the Nigerian and Ghanaian viruses to a new genotype B3 within clade B. A distinct geographic distribution pattern emerged with clade B viruses circulating exclusively in African countries north of the equator. All MV strains from southern Africa grouped in clades A and D with the majority of viruses belonging to genotype D4. The viruses considerably differed by their sensitivity to neutralization by monoclonal antibodies (mAb), but three selected antibodies were sufficient to distinguish between African MVs representing four different genotypes.

A hemagglutinin-derived peptide-vaccine ignored by virus-neutralizing passive antibodies, protects against murine measles encephalitis.

The neutralizing and protective monoclonal antibody BH47 defines the sequential epitope H236-255 of the measles virus hemagglutinin protein (MV-H). The objective of this study was to design peptides combining this B cell epitope (BCE) with different T cell epitopes (TCE) to obtain protective immunity. Most TTB peptides based on the 15mer BCE H236-250 induced MV-crossreactive antibodies, but only certain TCE induced virus neutralizing antibodies. The shortest BCE required for MV-reactivity and -neutralization was the 8mer H243-250 containing residue R243 implicated in CD46 down-regulation. Sera obtained after immunization with the TTB peptide containing the MV-derived TCE F421-435 protected mice against a lethal challenge with a neuro-adapted MV strain. Our results further demonstrate that this TTB peptide is fully immunogenic, even in the presence of protective levels of pre-existing MV-specific antibodies, suggesting that subunit vaccines based on such peptides could potentially be used to immunize infants in the presence of persisting maternal antibodies. It is therefore interesting that neutralizing antibodies were also obtained with a TTB peptide comprising a human promiscuous TCE (tt830). However, our results also emphasize the need to test sera induced with epitope-based vaccines against different virus strains, in particular if the epitope is not fully conserved.

Molecular epidemiology of Nigerian and Ghanaian measles virus isolates reveals a genotype circulating widely in western and central Africa.

Sub-Saharan Africa is one of the regions of the globe with the highest measles-related morbidity and mortality. Yet only seven virus isolates from this vast region have been phylogenetically characterized on the basis of their nucleoprotein, the last one in 1991. To characterize the prevalent wild-type viruses and to understand their circulation pattern, a large panel (n = 45) of isolates was collected in Ghana and Nigeria in 1997 and 1998. On the basis of their nucleoprotein sequence, the viruses clearly belong to clade B but a reshuffling of the structure of this clade was proposed, tentatively extending the number of genotypes from two to three on the basis of quantitative criteria. The sequences revealed the co-circulation of at least two distinct viruses in the cities of Lagos and Ibadan, suggesting that the number of susceptible individuals seems to be high enough to support endemic circulation of at least two distinct viruses. The endemic co-circulation of several viruses may well be a characteristic of communities with low vaccination rates. One of these viruses was also found in Accra in 1998 as well as in a 1994 case linked to distant Kenya, suggesting that clade B viruses are prevalent in sub-Saharan Africa while non-B viruses seem to dominate the south of Africa.

Promotion of transition metal-induced reactive oxygen species formation by beta-amyloid.

beta-amyloid protein appears to be involved in the neural degeneration associated with Alzheimer's disease. However, its mechanism of action is poorly understood. The ability of the neurotoxic peptide fragment (25-35) derived from beta-amyloid, to promote the generation of reactive oxygen species (ROS) by a postmitochondrial fraction (P2) derived from rat cerebrocortex, has been examined. The peptide fragment, when incubated together with P2, did not cause excess ROS formation. However, 10 microM FeSO4 or 10 microM CuSO4 were able to enhance ROS production in the P2 fraction and this was increased further in the concurrent presence of the 25-35 fragment. The corresponding inverse sequence non-neurotoxic peptide (35-25) had no parallel ability to augment iron-stimulated ROS production suggesting a degree of specificity for the observed effect. There was no formation of excess ROS when the 25-35 peptide and 0.5 mM Al2(SO4)3 were incubated with the P2 fraction. However in the presence of both aluminum and iron salts together with the 25-35 peptide, ROS production was augmented to a level significantly higher than that in the absence of aluminum. Polyglutamate, a peptide reported to mitigate aluminum toxicity had no effect on iron-related ROS generation but completely prevented its further potentiation by aluminum. The results indicate that beta-amyloid is able to potentiate the free-radical promoting capacity of metal ions such as iron, copper and aluminum. Such potentiation may be a relevant mechanism underlying beta-amyloid-induced degeneration of nerve cells.

An endogenous ATP-sensitive glutathione S-conjugate efflux mechanism in Xenopus laevis oocytes.

Constitutive efflux mechanisms for reduced glutathione (GSH) and the glutathione S-conjugates S-ethylglutathione (ethyl-SG) and S-(2,4-dinitrophenol)-glutathione (DNP-SG) were examined in Xenopus laevis oocytes. Oocytes were loaded by either microinjection with 50 nl of the 3H-labeled compounds or were exposed to unlabeled 1-chloro-2,4-dinitrobenzene and efflux of DNP-SG synthesized within the oocytes measured spectrophotometrically. Efflux of unlabeled DNP-SG (approximately 1.2 mM intracellular concentration) and microinjected 0.5 mM [3H]DNP-SG was a linear function of time, with approximately 20% released in 3 h at 25 degrees C. [3H] ethyl-SG, 0.5 mM, was released at a comparable rate, whereas only 4% of a tracer dose of [3H]GSH (2.5 mM intracellular GSH) was released in 3 h. Efflux of all three compounds was temperature sensitive and inhibited after ATP depletion but unaffected when Na+ in the culture medium was replaced with K+ or when the pH was changed from 7.5 to either 6.8 or 8.0. Efflux was saturable, with apparent Michaelis constant values of 0.91 +/- 0.19, 0.44 +/- 0.25, and 5.3 +/- 2.2 mM for DNP-SG, ethyl-SG, and GSH, respectively. Bilirubin ditaurate, 0.5 mM, cis-inhibited efflux of 0.5 mM [3H]DNP-SG, 0.5 mM [3H]ethyl-SG, and 2.5 mM [3H]GSH. DNP-SG and ethyl-SG efflux was also cis-inhibited by other glutathione S-conjugates, 0.25 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 0.5 mM sulfobromophthalein, and 0.5 mM dibromosulfophthalin, but not by 0.25 mM taurocholate. [3H]GSH release (2.5 mM) was unaffected by these compounds or by 10 mM extracellular GSH or methionine. These findings indicate that Xenopus oocytes have an endogenous ATP-sensitive mechanism for extruding glutathione S-conjugates, with properties comparable to ATP-dependent glutathione S-conjugate/organic anion transport systems described in a variety of cell types. However, in contrast to mammalian cells, GSH and ethyl-SG release from Xenopus oocytes was also inactivated after cellular ATP depletion but was not sensitive to membrane depolarization in high-K+ medium or trans-stimulated by extracellular GSH, indicating that efflux of these organic anions from Xenopus laevis oocytes is also mediated by an ATP-sensitive mechanism.

Mechanisms of hepatic methylmercury uptake.

The mechanism by which methylmercury is cleared from hepatic portal blood was examined in isolated rat livers perfused single-pass with Krebs-Henseleit buffer. [203Hg]Methylmercury (0.24-24 microM) was infused over a 30-min interval, followed by a 30-min washout, as a complex with the endogenous ligands L-cysteine (CH3Hg-L-cys), glutathione (CH3Hg-SG), and serum albumin (CH3Hg-albumin), or as a complex with dithiothreitol (CH3Hg-DTT), chloride (CH3HgCl), and the D-enantiomer of cysteine (CH3Hg-D-cys). The sulfhydryl-containing compounds were added at a 10-fold molar excess. When administered as the albumin complex, only a small fraction of the [203Hg]methylmercury was cleared from perfusate (approximately 8%) and excreted into bile (0.7%). Hepatic uptake and biliary excretion of methylmercury was considerably higher for the other complexes: The percent of the dose recovered in liver tissue and bile was, respectively, CH3Hg-albumin, 6.9 and 0.7; CH3Hg-L-cys, 15.7 and 2.3; CH3Hg-D-cys, 27.1 and 2.8; CH3Hg-SG, 17.7 and 2.1; CH3HgCl, 66.5 and 3.2; and CH3Hg-DTT, 70.1 and 19.8. For the dithiothreitol complex, hepatic extraction of methylmercury was nearly complete during single-pass perfusion. A comparison of hepatic removal of increasing doses of CH3Hg-L-cys and CH3Hg-D-cys revealed little difference in uptake between these two enantiomers. Moreover, the fraction of methylmercury removed was similar when infused at concentrations of 0.24, 2.4, and 24 microM, indicating no saturability of uptake within this dose range. Methylmercury was not hepatotoxic at concentrations up to 24 microM if administered as a mercaptide; however, the chloride complex (CH3HgCl) produced cholestasis and an increase in perfusion pressure at a concentration of only 0.24 microM. These findings indicate that hepatic methylmercury uptake and toxicity are dependent on the chemical form in blood plasma. Uptake was faster when methylmercury was present as a cysteine or glutathione complex, as compared to the albumin complex; however, the lack of stereoselectivity indicates that the uptake process may be relatively unselective.

Impaired biliary excretion and whole body elimination of methylmercury in rats with congenital defect in biliary glutathione excretion.

Biliary excretion of methylmercury, a major route of elimination of this toxic compound, was less than 2% of control in Eisai hyperbilirubinemic (EHBR) rats, a mutant Sprague-Dawley strain with a defect in biliary excretion of a variety of organic anions, including glutathione S-conjugates and reduced glutathione (GSH). Biliary GSH excretion in EHBR rats was also < 2% of controls, confirming previous findings. Impaired biliary methylmercury and GSH excretion was not explained by decreased hepatic content of these compounds. Indeed, hepatic methylmercury and GSH concentrations in EHBR rats were actually double those of controls. To assess the significance of the impaired biliary excretion in the whole body elimination of the toxicant, 203Hg excretion was measured over a 17-day period after intraperitoneal administration of either 0.5 or 5 mumol/kg of 203Hg-methylmercury chloride. The results for the two doses were similar. Methylmercury was eliminated by a first order process; however, the biological half-line was significantly longer in the EHBR rats, 46 to 54 days versus 18 to 22 days. Fecal excretion was the main route of elimination in both control and mutant animals. At necropsy (17 days), 16% to 25% of the 203Hg dose was recovered in the liver of the EHBR rats, whereas livers of control animals contained less than 2%of the administered dose. These findings demonstrate the biliary excretion of methylmercury is markedly impaired in EHBR rats and is associated with a low biliary GSH excretion, providing support for the hypothesis that methylmercury is normally transported across the canalicular membrane by a GSH-dependent mechanism, and presumably as a GSH mercaptide (CH3Hg-SG).(ABSTRACT TRUNCATED AT 250 WORDS)

ATP depletion and inactivation of an ATP-sensitive taurine channel by classic ion channel blockers.

Cell volume regulation in different cell types is mediated in part by plasma membrane channel(s) that allow taurine and other important intracellular organic osmolytes to efflux from the cell. Previous studies have demonstrated that intracellular ATP is required for activation of a volume-sensitive taurine-permeable channel. The present study examined the relation between cellular ATP and ADP concentrations and swelling-induced [14C]taurine efflux and anion current (whole-cell patch-clamp) after exposure of isolated skate (Raja erinacea) hepatocytes to metabolic poisons and a series of ion channel blockers. When intracellular ATP content was lowered with gradually increasing concentrations of 2,4-dinitrophenol, a sigmoidal relation between ATP content and volume-activated [14C]taurine efflux was observed. Taurine efflux was progressively inhibited over a relatively narrow range of intracellular ATP levels, indicating that physiologic alterations in cellular nucleotides may modulate the opening of the channel. Surprisingly, the inhibition of [14C]taurine efflux by a number of ion channel blockers [glibenclamide, 5-nitro-2-(3-phenylpropylamino)benzoate, diphenylamine-2-carboxylate, ketoconazole, gossypol, niflumic acid, and quinine] was related to a decrease in cellular ATP concentrations and ATP/ADP ratios, rather than to a direct interaction with the channel. In contrast, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and pyridoxal-5-phosphate inhibited volume-activated anion channels but had no effect on cellular ATP levels. These findings suggest multiple sites for regulation of volume-sensitive osmolyte channels and indicate that some putative ion channel blockers may actually alter the activity of ATP-regulated transporters by depleting cellular ATP.

Multiple canalicular transport mechanisms for glutathione S-conjugates. Transport on both ATP- and voltage-dependent carriers.

A large number of structurally distinct electrophiles are conjugated to glutathione within hepatocytes, and the resulting glutathione S-conjugates are selectively transported across the canalicular membrane into bile. To test the hypothesis that a single multi-specific, ATP-dependent carrier mediates biliary secretion of glutathione S-conjugates, the present study compared the driving forces and substrate specificity for canalicular transport of S-ethylglutathione (ethyl-SG), a low molecular weight and relatively hydrophilic thioether, and S-(2,4-dinitrophenyl)-glutathione (DNP-SG), a larger and more hydrophobic anion, using isolated rat liver canalicular membrane vesicles. In agreement with previous findings, DNP-SG transport was stimulated by ATP, although there was considerable transport in the absence of ATP. ATP-independent DNP-SG transport was unaffected by a Na+ gradient, was enhanced by a valinomycin-induced K+ diffusion potential, and was saturable, with both high affinity (Km = 8 +/- 2 microM) and low affinity (Km = 0.5 +/- 0.1 mM) components. High affinity ATP-independent DNP-SG uptake was cis-inhibited by GSH, GSH monoethyl ester, glutathione S-conjugates, other gamma-glutamyl compounds, sulfobromophthalein, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In contrast, ATP-dependent DNP-SG uptake was unaffected by GSH, GSH ester, S-methyl glutathione, or S-carbamidomethyl glutathione, but was strongly inhibited by sulfobromophthalein, DIDS, and by high molecular weight and relatively hydrophobic glutathione S-conjugates. Transport of the low molecular weight ethyl-SG conjugate was only minimally stimulated by ATP (10-20%). ATP-independent ethyl-SG uptake was electrogenic, saturable (Km = 10 +/- 1 microM) and was inhibited by GSH and all glutathione S-conjugates tested. These findings indicate the presence of multiple canalicular transport mechanisms for glutathione S-conjugates and demonstrate that the physicochemical properties of the S moiety are major determinants of transport. Relatively high molecular weight hydrophobic conjugates are substrates for both ATP-dependent and -independent mechanisms, whereas low molecular weight glutathione S-conjugates are transported largely by electrogenic carriers.

Isolation of the missing 5'-end of the encoding region of the bovine leukemia virus cell receptor gene.

The missing 5'-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5') was isolated from a lambda gt11 cDNA library using the 32P-labeled EcoRI-SamI fragment corresponding to the 5'-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5' cDNA revealed that the 1058 bp EcoRI fragment at its 5'-end contained a new 114 amino acid long sequence, and at its 3'-end contained a completely identical 88 amino acid overlapping region with the 5'-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venter's tag sequences of an unidentified gene from the human brain library.

Hepatic uptake of intact glutathione S-conjugate, inhibition by organic anions, and sinusoidal catabolism.

To identify potential mechanisms for hepatic removal of circulating glutathione (GSH) conjugates, uptake and metabolism of S-2,4-dinitrophenylglutathione (DNP-SG) were examined in isolated perfused livers from rat and guinea pig. Guinea pig livers perfused with 5 mumol of DNP-SG in a recirculating system (50 microM initial concn) rapidly cleared the conjugate from the perfusate (half time 3.7 min), whereas clearance was considerably slower in rat liver (half time 35 min). Disappearance of DNP-SG from the perfusate was accompanied by a simultaneous appearance of DNP-SG and its metabolites in bile. Addition of acivicin, an inhibitor of gamma-glutamyltransferase (gamma-GT), to the perfusate resulted in a marked decrease in DNP-SG clearance by guinea pig liver but had no effect in rat liver, suggesting that in the guinea pig this process is largely dependent on sinusoidal gamma-GT activity. However, even in the presence of acivicin, rat and guinea pig livers removed nearly one-half of the administered DNP-SG from the recirculating perfusate over 30 min. High concentrations of DNP-SG were found in bile (up to 3.7 mM), indicating that the liver is capable of transporting the intact conjugate from the circulation. When rat livers were perfused with higher concentrations of DNP-SG (100 and 250 microM), biliary excretion of DNP-SG increased dose dependently, with concentrations in bile reaching 10 mM at the higher dose. This was accompanied by a dose-dependent choleresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione as a primary osmotic driving force in hepatic bile formation.

Indirect evidence suggests that transport of glutathione (GSH) across the canalicular plasma membrane into bile contributes to the formation of the bile acid-independent fraction of bile flow. To directly test this hypothesis, the present study measured bile flow in isolated perfused rat livers whose biliary GSH excretion rate was selectively modulated by administration of GSH monoethyl ester (50, 100, and 200 mumol infused over a 20-min interval), a high dose of GSH itself (550 mumol over 20 min), and the three amino acid components of GSH (70 mumol each) with and without methionine (35 mumol). Animals were starved overnight to decrease hepatic GSH levels, and livers were pretreated with acivicin to inhibit gamma-glutamyl transferase. Livers perfused single pass with Krebs-Henseleit buffer excreted bile acids at a relatively low rate of 1-3 nmol.min-1 x g-1, and this rate was unaffected by agents used to alter biliary GSH efflux. In comparison, basal biliary GSH efflux rates were 8-13 nmol.min-1 x g-1. Administration of the GSH ester produced a dramatic dose-dependent choleresis, a stimulation of biliary GSH excretion, and resulted in the biliary excretion of intact GSH ester. Changes in total biliary GSH excretion and bile flow were temporally and quantitatively related. Infusion of GSH and amino acid supplementation also resulted in higher rates of bile flow and biliary GSH excretion, but their effects were more modest.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholestasis, altered junctional permeability, and inverse changes in sinusoidal and biliary glutathione release by vasopressin and epinephrine.

The mechanism for the vasopressin- and epinephrine-induced decrease in bile formation and increase in sinusoidal efflux of glutathione was investigated in rat livers perfused with recirculating fluorocarbon emulsion. Vasopressin and epinephrine transiently decreased bile flow and excretion of endogenous bile acids and glutathione and increased the bile/perfusate ratio of [14C]sucrose, suggesting an increase in junctional permeability, but had no effect on the bile/perfusate ratio of [3H]polyethylene glycol-900. The decreased biliary glutathione was balanced by an increase in sinusoidal efflux, such that total hepatic release remained unchanged. The adrenergic antagonist dihydroergotamine blocked the effects of epinephrine. To examine whether an increase in junctional permeability per se could account for the changes in glutathione efflux, biliary permeability was increased by either bile duct ligation, lowering of perfusate Ca2+ concentration with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or addition of taurolithocholate, a cholestatic bile acid. All three maneuvers produced a decrease in biliary glutathione excretion and a concomitant increase in sinusoidal glutathione efflux, whereas total glutathione release was largely unaffected. The effects of EGTA were partially reversed if CaCl2 was reintroduced into the perfusate. Because the GSH/GSSG ratio in perfusate could not be measured in this experimental system due to the spontaneous oxidation of GSH to GSSG, additional experiments in the nonrecirculating mode examined the effects of vasopressin and bile duct ligation on sinusoidal release of GSH and GSSG. In control livers there was no detectable GSSG in perfusate (less than 0.5 nmol.min-1.g-1). After vasopressin administration, the additional sinusoidal glutathione was mainly as GSH, although there was also a significant amount of GSSG (1-2 nmol.min-1.g-1). The additional glutathione released into perfusate after bile duct ligation was 47% as GSSG. When vasopressin was administered to livers whose bile duct had been ligated, its ability to enhance sinusoidal glutathione release was diminished, suggesting that the effects of vasopressin and bile duct ligation are not additive. These observations support previous findings that vasopressin and epinephrine can modulate hepatocyte tight junctional permeability and demonstrate that these hormones produce cholestasis and inverse changes in sinusoidal and biliary glutathione efflux. Other maneuvers that increased biliary permeability to [14C]sucrose also produced cholestasis and a redistribution of glutathione efflux from bile to perfusate, suggesting that an increase in junctional permeability may allow biliary glutathione to reflux from bile to plasma.

Altered hepatic junctional permeability, bile acid excretion and glutathione efflux during oxidant challenge.

Hepatobiliary functions during administration of the thiol-oxidizing agents t-butyl hydroperoxide, hydrogen peroxide, diamide and menadione were monitored in the isolated rat liver perfused with a recirculating fluorocarbon emulsion. Because these agents are detoxified largely by glutathione-dependent mechanisms, efflux of reduced glutathione and glutathione disulfide into bile and perfusate was measured, along with bile flow, excretion of endogenous bile acids, lactate dehydrogenase release and the bile/perfusate ratios of [14C]sucrose (a marker of junctional permeability) and [3H]polyethylene glycol-900 (a substance concentrated in bile, presumably by a vesicular transport pathway). All agents enhanced the rate of glutathione efflux from the liver, reflecting increased formation of glutathione disulfide. At low doses of the thiol-oxidants, most of the additional glutathione released by the liver appeared in bile, whereas at higher doses the fraction appearing in the sinusoidal circulation increased. This enhanced glutathione disulfide release was associated with a decrease in endogenous bile acid excretion and in the bile/perfusate ratio of [3H]polyethylene glycol, and an increase in the bile/perfusate ratio of [14C]sucrose. These deleterious changes were reversible, and were noted at doses that had no effect on lactate dehydrogenase release, and only small effects on perfusion pressure. Bile flow was decreased by t-butyl hydroperoxide and H2O2, and increased by diamide (100 mumol) and menadione (20 mumol). These results indicate that alterations in hepatic thiol-redox status lead to an impairment of bile acid excretion, junctional integrity and bile formation. These functional parameters appear sensitive to the effects of oxidizing agents, and probably contribute to the pathogenesis of hepatobiliary dysfunction during the metabolism of certain drugs and xenobiotics.

Determinants of glutathione efflux and biliary GSH/GSSG ratio in perfused rat liver.

Utilizing the isolated perfused rat liver, we examined several factors influencing efflux of glutathione [reduced glutathione (GSH) and glutathione disulfide (GSSG)] into perfusate and bile, including the effects of perfusate composition, oxygen delivery to the liver, and acivicin (AT-125), an inhibitor of gamma-glutamyl transferase activity. When livers were perfused with a recirculating Krebs-Ringer bicarbonate buffer only 7-26% of released glutathione was excreted into bile, mainly in its oxidized form (71-90% as GSSG). In contrast, when 20% bovine red blood cells or 20% fluorocarbon emulsion were utilized as perfusates, biliary glutathione accounted for a larger fraction of total hepatic efflux (16-41%), and only 39-65% was excreted as GSSG. To determine whether O2 delivery to the liver could explain some of these differences, biliary and sinusoidal efflux of glutathione were measured as O2 delivery was varied by 1) increasing the perfusion flow rate, 2) altering the concentration of fluorocarbon emulsion (5, 10, and 20%), and 3) changing the PO2 (95% O2-5% CO2 vs. 50% O2-5% CO2-45% N2). Under all experimental conditions, an increase in O2 delivery was accompanied by an increase in bile flow and in the concentration and rate of glutathione efflux into bile but no significant change in sinusoidal efflux of glutathione. Hepatic tissue GSH and GSSG levels were not affected by the various treatments. When gamma-glutamyl transferase activity was inhibited with AT-125, biliary glutathione increased to levels of approximately 50% of total hepatic efflux in fluorocarbon-perfused livers, and only 24-29% of the glutathione was excreted as GSSG.(ABSTRACT TRUNCATED AT 250 WORDS)

Relation between biliary glutathione excretion and bile acid-independent bile flow.

Glutathione efflux into bile of the fluorocarbon-perfused isolated rat liver was altered with eight different agents (L-buthionine-[S,R]-sulfoximine, cefamandole, sodium arsenite, phenobarbital, furosemide, nitrofurantoin, aminopyrine, and benzylamine), and correlations were established between bile flow and biliary excretion of 1) glutathione, 2) endogenous bile acids, and 3) glutathione plus bile acids. Biliary efflux of endogenous bile acids was relatively low (0.5-5 nmol.min-1.g liver-1) and was minimally affected by these agents. Biliary glutathione excretion in control livers was between 4 and 9 nmol.min-1.g-1 and in treated livers ranged from 1 to 21 nmol.min-1.g-1. For each of the various interventions, an increase or decrease in glutathione excretion was always accompanied by a change in bile flow in the same direction; however, these changes were not always directly proportional when comparisons were made between treatment groups. Nevertheless, when bile flow (microliter.min-1.g-1; ordinate) was plotted against glutathione excretion into bile for the pooled data, a significant correlation was observed that was adequately described by a straight line: y = 0.071 chi + 0.72 (r2 = 0.62, P less than 0.001). A similar function described the relation between bile flow and the sum of bile acids and glutathione in bile: y = 0.077 chi + 0.55 (r2 = 0.62, P less than 0.001). In contrast, the taurocholate- or glycocholate-induced choleresis had only minimal effects on glutathione efflux. These findings support the hypothesis that glutathione is one of the osmotic driving forces in bile acid-independent bile formation.(ABSTRACT TRUNCATED AT 250 WORDS)

[Regulation of prolactin and growth hormone gene expression in pituitary cell culture]. / Régulations de l'expression des gènes de la prolactine et de l'hormone de croissance dans des cellules hypophysaires en culture.

The rat pituitary tumor derived cell lines of the "GH" family offer a fruitful model for studying the expressions of the prolactin (rPRL) and growth hormone (rGH) genes in basic and regulated states. In order to assess the potential role of DNA methylation in the basic expressions of rPRL and rGH genes we have used different cell strains which produce either high level of rPRL (GH3B6 cells) or of rGH (GC cells) and minute amounts of both hormones (GH3CDL cells). The cleavage patterns generated by the methylation sensitive enzymes Hpa II and Msp I indicated an inverse correlation between the extent of gene methylation and the level of expression. However the use of 5-azacytidine which decreases DNA methylation suggested a variable importance of gene methylation in the respective control of rPRL and rGH genes depending on the cell lines. In an other hand we attempted to elucidate some of the mechanisms by which thyroliberin (TRH) enhances rPRL gene transcription in GH3B6 cells. Preliminary results indicated that the persistent occupancy of the TRH receptors was required to sustain at least for the first 5 hours the increased rate of rPRL gene transcription. In addition the possible relationship between the TRH-induced acute rPRL release and the stimulation of rPRL gene transcription was investigated. The results suggested that the activators of the C kinase-mediated pathway which are actually involved in the stimulation of the acute release were not sufficient alone for eliciting the maximum TRH response at the gene level.