Ágnes Gyurasics

Glutathione-dependent biliary excretion of arsenic

This study aimed to clarify whether glutathione (GSH) plays a role in the hepatobiliary transport of arsenic. For this purpose, the biliary excretion of 74As was measured in urethane-anaesthetized rats for 2 hr after the administration of labelled sodium arsenite (50 mumol/kg, i.v.) or arsenate (150 mumol/kg, i.v.) and under the influence of sulfobromophthalein (BSP), indocyanine green (ICG) or diethyl maleate (DEM) which are known to diminish hepatobiliary transport of GSH. Although the biliary excretion of arsenic was different after arsenite and arsenate administration in terms of quantity (19% vs 6% of dose in 2 hr, respectively) and time course, arsenic excretion responded similarly to BSP (50 mumol/kg, i.v.), ICG (25 mumol/kg, i.v.) or DEM (4 mumol/kg, i.p.) irrespective of the injected arsenical. Initially the biliary excretion of arsenic in rats with either arsenite or arsenate was significantly reduced, but then moderately increased by BSP and, more lastingly, depressed by ICG, whereas it was virtually abolished by DEM. The responses of arsenic excretion to BSP, ICG and DEM were related, both proportionally and temporally, to the effects exerted by these agents on the hepatobiliary transport of GSH, as assessed by the biliary excretion of non-protein thiols. These findings indicate that the biliary excretion of arsenic after the administration of either arsenite or arsenate is dependent on the hepatobiliary transport of GSH. Transport of arsenic as a GSH complex may account for the GSH dependence of biliary arsenic excretion.

Effect of arsenicals on biliary excretion of endogenous glutathione and xenobiotics with glutathione-dependent hepatobiliary transport.

Sodium arsenite (25-100 mumol/kg, i.v.) and arsenate (75-300 mumol/kg, i.v.) injected into anaesthetized rats increased the biliary excretion of endogenous non-protein thiols (NPSH) in a dose-dependent fashion up to 24- and 31-fold, respectively. Simultaneously with NPSH, glutathione (GS) excretion was increased to a similar extent suggesting that the increment in biliary thiol output originated from enhanced hepatobiliary transport of GS. After administration of labelled arsenicals, biliary excretion of 74As and NPSH followed similar time-courses. Biliary excretion of 74As was more efficient after arsenite than arsenate administration corresponding to the greater potency of arsenite compared to arsenate to increase biliary output of NPSH. Coadministered sulfobromophthalein (BSP) inhibited the biliary excretion of 74As and prevented the arsenical-induced increase in biliary NPSH. Thus, hepatobiliary transport of arsenic apparently proceeds coordinately with that of GS. However, excretion of each molecule of arsenic compound generates transport of several molecules of GS. Though mercuric, methylmercuric, cadmium and zinc ions are thought to be excreted into bile as complexes with GS, the marked arsenical-induced increase in GS excretion only doubled the biliary excretion of inorganic mercury and hardly influenced the transport of other metals into bile. This finding suggests that arsenicals markedly enhance biliary excretion of GS with a free thiol group but barely or not at all that of GS with a thiol group blocked by a firmly bound metal ion. Both arsenicals diminished the biliary excretion of BSP-glutathione conjugate after BSP administration presumably because they impaired conjugation of BSP with GSH due to decreased GS availability. It is assumed that arsenite, and arsenate after reduction to arsenite, forms an unstable complex with GS that is efficiently transported into bile resulting in increased biliary output of GS. It is demonstrated that arsenite-induced perturbation of hepatobiliary disposition of endogenous GS differentially affects biliary excretion of xenobiotics with GS-dependent hepatobiliary transport.

Interactions between selenium and group Va-metalloids (arsenic, antimony and bismuth) in the biliary excretion.

The interrelationship between the biliary excretion of exogenous group Va-metalloids (arsenic, antimony and bismuth) and selenium, as well as endogenous glutathione has been studied in rats injected intravenously with sodium selenite and one of the group Va-metalloids. Arsenic, antimony and bismuth appeared in the bile of rats together with large amounts of non-protein thiols (NPSH, representing glutathione and its SH-containing degradation products) and, with the exception of bismuth, they caused choleresis. Significant interactions were observed in the hepatobiliary disposition between selenium and each of the group Va-metalloids, however, their outcomes were not uniform. When coadministered with sodium arsenite or arsenate, selenite enhanced the initial biliary excretion of arsenic 2- and 8-fold, respectively, without further increasing the concomitant excretion of NPSH or the choleretic effect of arsenicals. However, selenite augmented neither the excretion of antimony or bismuth, nor the simultaneous biliary release of NPSH. In turn, arsenite, arsenate and antimony potassium tartrate increased the initial biliary excretion of selenium more than 10-fold and enhanced the accumulation of selenium in blood (exclusively in the erythrocytes). In contrast, administration of bismuth ammonium citrate diminished both the biliary excretion and the erythrocytic accumulation of selenium, while causing retention of selenium in the blood plasma. In rats receiving arsenic or antimony with selenite, the time courses of the biliary excretion of these group Va-metalloids, selenium and NPSH were similar. It is hypothesised that incorporation of selenol metabolites of selenite into the glutathione complexes of arsenic and antimony, resulting in cholephilic ternary complexes, accounts for the arsenic- and antimony-induced augmentation of the hepatobiliary transport of selenium. However, additional chemical and/or dispositional mechanisms are thought to be responsible for the selenite-induced increase in biliary excretion of arsenic.

Role of glutathione in the biliary excretion of the arsenical drugs trimelarsan and melarsoprol

After administration of the inorganic sodium arsenite or arsenate to rats, the biliary excretion of arsenic is rapid, is accompanied by the biliary output of large amounts of GSH, and is completely arrested by the GSH depletor diethyl maleate (DEM). We studied the biliary excretion of trimelarsan (TMA) and melarsoprol (MAP) in rats in order to determine whether biliary excretion is also significant in the disposition of these trivalent organic arsenicals that are used as therapeutic agents and whether GSH is also involved in their hepatobiliary transport. After injection of either drug (100 micromol/kg, i.v.), arsenic was rapidly excreted in bile (up to 1 micromol/kg. min, approximately 55% of dose/100 min). Concurrently, TMA and MAP increased the biliary output of GSH 3- and 6 fold, and lowered the hepatic GSH content by 24% and 27%, respectively. In TMA-injected rats, pretreatment with DEM or buthionine sulfoximine decreased the initial biliary excretion of arsenic by 75% and 40%, respectively, whereas in MAP-injected rats these GSH depletors diminished arsenic output by 45% and 20%. Both arsenicals reacted with GSH in vitro, giving rise to the same product, which was also shown by HPLC analysis to be a major biliary metabolite of both TMA and MAP. This metabolite was sensitive to gamma-glutamyltranspeptidase in vitro and its biliary excretion was virtually prevented by the GSH depletors, confirming that it is a GSH conjugate (purportedly melarsen-diglutathione). Some TMA was excreted in the bile unchanged, whereas a significant amount of MAP also appeared there as two glucuronides. The biliary excretion of unchanged TMA and MAP glucuronides was increased by experimental depletion of GSH. These studies indicate that the biliary excretion of TMA and MAP (1) is very significant in their disposition, (2) is partially dependent on the hepatic availability of GSH, as these arsenicals are excreted in part as a GSH conjugate, and (3) is concomitant with the increased appearance of GSH in bile, probably originating from dissociation of the unstable GSH conjugate of these arsenicals. Thus, conjugation with GSH is important in the elimination of both TMA and MAP, although glucuronidation is also involved in the fate of MAP.

Increased biliary excretion of glutathione is generated by the glutathione-dependent hepatobiliary transport of antimony and bismuth

We have recently demonstrated that the hepatobiliary transport of arsenic is glutathione-dependent and is associated with a profound increase in biliary excretion of glutathione (GSH), hepatic GSH depletion and diminished GSH conjugation (Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 41: 937-944 and Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 42: 465-468, 1991). The present studies in rats aimed to determine whether antimony and bismuth, other metalloids in group Va of the periodic table, also possess similar properties. Antimony potassium tartrate (25-100 mumol/kg, i.v.) and bismuth ammonium citrate (50-200 mumol/kg, i.v.) increased up to 50- and 4-fold, respectively, the biliary excretion of non-protein thiols (NPSH). This resulted mainly from increased hepatobiliary transport of GSH as suggested by a close parallelism in the biliary excretion of NPSH and GSH after antimony or bismuth administration. Within 2 hr, rats excreted into bile 55 and 3% of the dose of antimony (50 mumol/kg, i.v.) and bismuth (150 mumol/kg, i.v.), respectively. The time courses of the biliary excretion of these metalloids and NPSH or GSH were strikingly similar suggesting co-ordinate hepatobiliary transport of the metalloids and GSH. However, at the peak of their excretion, each molecule of antimony or bismuth resulted in a co-transport of approximately three molecules of GSH. Diethyl maleate, indocyanine green and sulfobromophthalein (BSP), which decreased biliary excretion of GSH, significantly diminished excretion of antimony and bismuth into bile indicating that hepatobiliary transport of these metalloids is GSH-dependent. Administration of antimony, but not bismuth, decreased hepatic GSH level by 30% and reduced the GSH conjugation and biliary excretion of BSP. These studies demonstrate that the hepatobiliary transport of trivalent antimony and bismuth is GSH-dependent similarly to the hepatobiliary transport of trivalent arsenic. Proportionally to their biliary excretion rates, these metalloids generate increased biliary excretion of GSH probably because they are transported from liver to bile as unstable GSH complexes. The significant loss of hepatic GSH into bile as induced by arsenic or antimony may compromise conjugation of xenobiotics with GSH.

Enhancement of selenium excretion in bile by sulfobromophthalein: elucidation of the mechanism

This work was intended to explore the mechanism whereby sulfobromophthalein (BSP), an electrophilic and cholephilic organic acid, increases the biliary excretion of selenium in rats injected with sodium [75Se]selenite. In such animals, neither BSP-glutathione conjugate nor dibromosulfophthalein, nonelectrophilic congeners of BSP, enhanced the hepatobiliary transport of selenium, suggesting that reaction of nucleophilic selenite metabolites formed in vivo with the injected BSP may be involved. Indeed, HPLC analysis of bile from rats receiving [75Se]selenite and BSP revealed two peaks (X and Y) that were simultaneously detected both by absorbance as BSP metabolites and by radioactivity as [75Se] metabolites, indicating that these represent selenium-containing BSP metabolites. Pretreatment of rats with inhibitors of selenium methylation, such as periodate-oxidized adenosine (PAD) and ethionine, drastically diminished the size of peak X, while increasing (PAD) or not influencing (ethionine) the size of peak Y. This finding indicates that production of metabolite X, but not Y, is dependent on formation of methylated selenium metabolites. A compound chromatographically indistinguishable from that in peak X was formed in vitro during incubation of BSP with methylselenol, suggesting that biliary metabolite X is identical to the reaction product of BSP and selenite-derived methylselenol. Incubation of BSP with selenite in the presence of a thiol, namely glutathione, cysteine or N-acetylcysteine (which convert selenite into nucleophilic products, i.e. the respective selenopersulfides and hydrogen selenide) resulted in product(s) chromatographically identical to the biliary selenium-containing BSP metabolite(s) of peak Y, irrespective of the nature of the thiol used. Thus, biliary metabolite(s) Y may be reaction products of BSP and hydrogen selenide. Finally, BSP significantly diminished exhalation of dimethyl selenide in selenite-injected rats, purportedly because it reacted with precursors of dimethyl selenide, that include hydrogen selenide and methylselenol. In summary, BSP increases biliary excretion of selenium in rats receiving selenite because it forms selenium-containing BSP metabolites that are readily transported into bile. It is suggested that the in vivo reaction of nucleophilic selenite metabolites with electrophilic compounds may influence the fate of selenium and may contribute to some of the effects of this essential and anticarcinogenic metalloid.

Role of glutathione and methylation in the biliary excretion of selenium. the paradoxical effect of sulfobromophthalein

Biotransformation of selenite involves both reactions with GSH and methylations. Therefore, the role of GSH, methylation, and the hepatobiliary GSH transporter was investigated in the biliary excretion of selenium in rats injected with sodium [75Se]selenite (1-10 micromol/kg, i.v.). Biliary output of selenium exhibited an apparent capacity limitation with an approximately 3 nmol/kg x min maximal rate and a dose-related decline in the fractional excretion. HPLC analysis of bile indicated absence of selenite and presence of selenodiglutathione (GS-Se-SG) and/or its hydrolysis products as the major biliary selenite metabolites. Depletion of hepatic glutathione by D,L-buthionine-[S,R]-sulfoximine or diethyl maleate decreased selenium excretion into bile by 60 and 80%, respectively. In contrast, inhibitors of methylation, i.e. periodate-oxidised adenosine or ethionine doubled the rate of biliary selenium excretion. While indocyanine green--an inhibitor of hepatobiliary GSH transport--failed to influence biliary selenium output, sulfobromophthalein (BSP)--another inhibitor of this sort--dramatically enhanced it. This effect was found to be a function of the dose of both selenite and BSP. The degree of BSP-induced enhancement of the selenium excretion rate gradually increased with elevation of the selenite dose, approaching 20-fold at 10 micromol/kg selenite. In contrast, the stimulatory effect of BSP on biliary selenium output was maximal at 50-100 micromol/kg and gradually lessened with elevation of the BSP dose above 100 micromol/kg. In summary, this study revealed that the biliary excretion of selenium depended on availability of hepatic GSH, probably for formation of GS-Se-SG, the putative cholephilic selenite metabolite. Methylation counteracted selenium excretion into bile and thus may contribute to the apparent capacity limitation of biliary selenium excretion. Finally, selenium output into bile was insensitive to inhibitors of the hepatobiliary GSH transporter, and was enhanced, paradoxically, by BSP several-fold. The mechanism of this unexpected effect is explored in the adjoining article.