Biliary Excretion Of Methylmercury Research Articles

Impaired biliary excretion and whole body elimination of methylmercury in rats with a 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 μmol/kg of 203Hg-methylmercury chloride. The results for the two doses were similar. Methyhnercury was eliminated by a first order process; however, the biological half-life 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 that 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 (CH 3Hg-SG). This study also provides a direct measure of the contribution of biliary excretion to the whole-body elimination of methylmercury. The biological half-life of the metal was 2.5 times longer in the mutant animals, indicating a critical role for biliary secretion in methyhnercury detoxification.

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.

Effect of lipoic acid on biliary excretion of glutathione and metals

Several metals are excreted in bile as glutathione complexes, and their biliary excretion is facilitated by increased hepatobiliary transport of glutathione. The present study analyzed the effect of lipoic acid (LA; thioctic acid; 37.5–300 μmol/kg, iv), an endogenous disulfide which can be reduced in vivo to a dithiol, on the hepatobiliary disposition of glutathione-related thiols and the biliary excretion of metals (10 μmol/kg, iv) in rats. Administration of LA enhanced the biliary excretion of reduced glutathione in a dose-dependent fashion. Despite increasing glutathione output, LA (150 μmol/kg, iv) did not increase, but rather decreased, the biliary excretion of methylmercury, cadmium, zinc, and copper, which are transported into bile in a glutathione-dependent manner, as indicated by a marked reduction in their biliary excretion after diethyl maleate-induced glutathione depletion. In contrast, biliary excretion of inorganic mercury, which is minimally affected by glutathione depletion, was dramatically enhanced (12- to 37-fold) by LA administration. Following injection of LA, the concentrations of endogenous disulfides in arterial blood plasma (e.g., cystine, glutathione disulfide, cysteine-glutathione, protein-cysteine, and protein-glutathione mixed disulfides) were considerably diminished, while the levels of endogenous thiols (e.g., glutathione and cysteine) were increased. This finding indicates that LA, probably after enzymatic conversion to dihydrolipoic acid, can reduce endogenous disulfides to thiols. It appears that LA induces the transport of glutathione into bile by the temporary formation of dihydrolipoic acid-glutathione mixed disulfide, which after being translocated into bile is cleaved to LA and reduced glutathione. Because the glutathione molecule thus transported into bile cannot complex metals at the thiol group, this might be the mechanism for the observed failure of the LA-induced increase in biliary excretion of glutathione to enhance the hepatobiliary transport of metals that are transported into bile as glutathione complexes (i.e., methylmercury, cadmium, zinc, and copper). The observations also raise the possibility that endogenous dihydrolipoic acid, by forming a stable complex with mercuric ion, may play the role of a carrier molecule in the hepatobiliary transport of inorganic mercury.

Slow biliary elimination of methyl mercury in the marine elasmobranchs, Raja erinacea and Squalus acanthias

The present study examined the ability of two marine elasmobranchs ( Raja erinacea, little skate, and Squalus acanthias, spiny dogfish shark) to excrete methyl mercury into bile, a major excretory route in mammals. 203Hg-labeled methyl mercury chloride was administered via the caudal vein, and bile collected through exteriorized cannulas in the free swimming fish. Skates and dogfish sharks excreted only a small fraction of the 203Hg into bile over a 3-day period: in the skate, the 3-day cumulative excretion (as a % of dose) was 0.44 ± 0.10 ( n = 4, ±SD), 0.71 ± 0.23 ( n = 6), and 1.00 ± 0.34 ( n = 4) for doses of 1, 5, and 20 μmol/kg, respectively, while the shark excreted only 0.15 ± 0.15% ( n = 8) at a dose of 5 μmol/kg. As in mammals, the availability of hepatic and biliary glutathione was a determinant of the biliary excretion of methyl mercury in these species: the administration of sulfobromophthalein, a compound known to inhibit both glutathione and methyl mercury excretion in rats, or of l-buthionine- S,R-sulfoximine, an inhibitor of glutathione biosynthesis, decreased the biliary excretion of both glutathione and mercury in the skate. The slow hepatic excretory process for methyl mercury in the skate and shark was attributed to an inordinately slow rate of bile formation: from 1 to 4 ml/kg·day. An inefficient biliary excretory process in fish may account in part for the long biological half-times for methyl mercury in marine species.

Effect of Selenium on Biliary Excretion of Methylmercury (Proceedings of the 12 th Symposium on Environmental Pollutants and Toxicology) (Regular Presentations)

Effect of Selenium on Biliary Excretion of Methylmercury (Proceedings of the 12 th Symposium on Environmental Pollutants and Toxicology) (Regular Presentations)

Role of glutathione and hepatic glutathione S-transferase in the biliary excretion of methyl mercury, cadmium and zinc: a study with enzyme inducers and glutathione depletors.

The effect of hepatic glutathione (GSH) depletion and enzyme induction on hepatic glutathione S-transferase (GST) activity, biliary excretion of GSH, methyl mercury, cadmium and zinc was studied in rats. The GSH depletors, methyl iodide and diethyl maleate, did not influence hepatic GST activity but, depending on the substrate used, benzo(a)pyrene, phenobarbital, pregnenolone-16 alpha-carbonitrile (PCN) and trans-stilbene oxide (TSO) increased it by 16-33, 44-89, 53-97 and 208-279%, respectively. GSH depletors decreased (-88%), benzo(a)pyrene and TSO did not affect, phenobarbital and PCN increased (+113 and +149%) the transport of GSH into bile. The biliary excretion of methyl mercury, cadmium and zinc was reduced by GSH depletors (-97, -74 and -93%), and enhanced by phenobarbital (+139, +280 and +220%) and PCN (+150, +121 and +160%). Treatment with benzo(a)pyrene and TSO did not affect the excretion of methyl mercury and zinc into bile, but decreased that of cadmium. These results do not provide evidence for the role of hepatic GST but strongly support the importance of biliary GSH excretion in the hepatobiliary transport of methyl mercury, cadmium and zinc. It is assumed that phenobarbital and PCN enhance the biliary excretion of these metals by increasing the transport of GSH, the carrier molecule, from liver to bile.

N-acetylpenicillamine potentiated excretion of methyl mercury in rat bile: influence of S-methylcysteine.

N-acetylpenicillamine, 5 mmol/kg body weight increased biliary excretion of methyl mercury more than three fold. Upon simultaneous administration of the same dose of N-acetylpenicillamine and 2,5 mmol/kg body weight of S-methylcysteine biliary excretion of methyl mercury increased only 1.5 fold. In both cases biliary sulfhydryl concentration increased to the same extent, about 5 fold. Decreased biliary excretion of methyl mercury, as a result of liver depletion of reduced glutathione by cyclohexene oxide, could be restored by N-acetylpenicillamine. This restoration could be depressed by S-methylcysteine. The experiments undertaken indicate that N-acetylpenicillamine potentiated methyl mercury excretion occurs by a glutathione S-transferase dependent mechanism. Bile, collected after successive administration of methyl mercuric chloride, cyclohexene oxide, S-methylcysteine and N-acetylpenicillamine contained the methyl mercuric derivatives of N-acetylpenicillamine and glutathione together with other methyl mercury carrying components not present in control bile. Whether these components play any role in the mechanism of N-acetylpenicillamine potentiated methyl mercury excretion cannot be stated from the present investigation.

N-acetylpenicillamine potentiation of biliary excretion of methyl mercury: influence of glutathione depletors.

The effect of 1-chloro-2,4-dinitrobenzene (DNB), sulfobromophtalein (BSP) and cyclohexene oxide (cho) on N-acetylpenicillamine (NAPA) potentiated biliary excretion of methyl mercury in rats pretreated with cho for liver glutathione (GSH) depletion, has been tested. DNB, BSP and cho depressed NAPA potentiation of methyl mercury excretion in bile, while simultaneously biliary sulfhydryl concentration increased, due mainly to a relatively large proportion of unchanged NAPA, being excreted in bile. The fact that relatively large amounts of unchanged NAPA are excreted in bile, without affecting mercury excretion, indicates that NAPA per se cannot carry methyl mercury from liver to bile. In addition to unchanged NAPA, bile collected after administration of NAPA and DNB or BSP contained relatively large amounts of GSH conjugates of DNB or BSP, respectively, together with smaller amounts of GSH and another methyl mercury carrying component. The data are interpreted as suggesting that NAPA potentiation of methyl mercury excretion in bile can be due to an increased availability of GSH. However, it cannot be excluded that methyl mercury carrying components other than GSH and NAPA appearing in bile upon NAPA administration could play a role in NAPA potentiation of methyl mercury excretion in bile.

Species difference in biliary excretion of methylmercury

Species difference in the biliary excretion of methylmercury was studied in male rats, mice, rabbits and guinea pigs. The rates of mercury excretion (% dose/2 hr) into the bile of the rats, mice, rabbits and guinea pigs during the 2 hr from 2 to 4 hr after the administration of methylmercury were 0.61, 0.091, 0.036 and 0.019, respectively. These results suggest that biliary excretion and enterohepatic circulation of methylmercury in the latter three species may not influence the fate of this compound as significantly as in rats. Most of the methylmercury excreted into the bile of rats was bound to glutathione (GSH). In the mouse bile, 40% of the methylmercury was bound to GSH and the rest was found in a fraction eluted at the void volume of the Sephadex G-15 column. However, in the case of the rabbits and guinea pigs, methylmercury-GSH was scarcely detectable in the bile and almost all of the methylmercury was eluted at the void volume of the column.

The mechanism of biliary excretion of methyl mercury: studies with methylthiols.

The S-methylated derivatives of N-acetylpenicillamine, thiola and cysteine as well as methyl iodide decreased biliary excretion of methyl mercury markedly. Excretion of sulfhydryl in bile was not influenced by S-methyl-cysteine, S-methylthiola, S-methyl-N-acetylpenicillamine or a low dose of methyliodide (0.5 mmol/kg body weight). This seems to indicate that coupling of methyl mercury to glutathione in the liver before biliary excretion is a glutathione S-transferase dependent reaction. It also indicates that the methylthiols tested, or metabolites of these compounds are likely to be inhibitors of S-transferase. The effect of S-methylcysteine and low doses of methyl iodide probably reflects glutathione S-transferase inhibition as both compounds are metabolized to the S-transferase inhibitor S-methylglutathione in the liver. A higher dose of methyl iodide (1 mmol/kg body weight) seems to deplete the liver of reduced glutathione through S-methylation as illustrated by decreased biliary excretion of sulfhydryl. S-methylthiola and S-methyl-N-acetylpenicillamine are metabolized in the liver to unknown components which are excreted in bile. Whether S-methylthiola and S-methyl-N-acetylpenicillamine are inhibitors of S-transferase themselves or cause inhibition through metabolites cannot be stated from the present investigation.

The influence of some thiols on biliary excretion of methyl mercury.

N-Acetylpenicillamine and thiola increased biliary excretion of methyl mercury and sulfhydryl right after administration. Cysteine increased excretion of methyl mercury in bile after a temporary decrease following administration. During the interval of decreased mercury excretion biliary excretion of cysteine passed through a maximum. This indicates the existence of a common factor of the excretory systems for cysteine and methyl mercury and illustrates that cysteine cannot carry methyl mercury from liver to bile. Relatively large proportions of unchanged thiola and N-acetylpenicillamine were excreted in bile. Bile collected after administration of one of these compounds, in addition to thiola or N-acetylpenicillamine, contained other methyl mercury carrying components not present in control bile. From the experiments undertaken it cannot be stated whether these components play any role in the increased excretion of methyl mercury in bile caused by thiola and N-acetylpenicillamine. The mechanisms of increased biliary excretion of methyl mercury following administration of N-acetylpenicillamine, thiola and cysteine are discussed.

Developmental changes in the biliary excretion of methylmercury and glutathione.

The long half-time for methylmercury in the neonatal rats is explained by the neonatal liver's inability to secrete the toxin into bile, which in adults is the main route of elimination. The ability to secrete mercury into bile develops between 2 and 4 weeks of age and is correlated with the increasing ability of the developing liver to secrete glutathione into bile.

Excretion of methyl mercury in rat bile: the effect of thioctic acid, thionalide, hexadecyl- and octadecylmercaptoacetate.

Thioctic acid markedly increases the sulfhydryl and sulphide content of bile. This probably reflects the reduction of thioctic acid in the liver, followed by biliary excretion of a reduced derivative. The total biliary excretion of methyl mercury was not increased. Thionalide markedly inhibits biliary excretion of methyl mercury. Simultaneously, the sulfhydryl and sulphide content of bile decreases. This is probably caused by the conjugation of thionalide to glutathione in the liver, thereby blocking the biliary excretion of methyl mercury. Hexadecylmercaptoacetate increases the biliary content of methyl mercury moderately after a temporary decrease, whereas biliary sulfhydryl and sulphide concentrations were unchanged. Octadecylmercaptoacetate does not change the biliary content of methyl mercury, sulfhydryl and sulphides significantly. Smaller parts of hexadecylmercaptoacetate, octadecylmercaptoacetate and thionalide seemed to be excreted as such in bile. These results indicate that methyl mercury cannot be transported from liver to bile as complexed to the sulphides thioctic acid, thionalide, hexa- and octadecylmercaptoacetate.

Organ distribution and cellular uptake of methyl mercury in the rat as influenced by the intra- and extracellular glutathione concentration

Intravenous administration of CH 3HgCl (4 μmole/kg) premixed with glutathione or cysteine (8 μmole/kg) to female rats caused a rapid uptake of mercury in the kidney and a depressed content in the liver and blood as compared to CH 3HgCl given alone. GSH depletion in the tissues, produced by injection of diethylmaleate, DEM (3.9 mmole/kg) did not influence the kidney uptake of mercury from administered CH 3Hg +-GSH, whereas the uptake of injected CH 3HgCl was depressed. Both GSH and cysteine (8 μmole/kg) promoted the biliary excretion of methyl mercury. In suspensions of rat erythrocytes and isolated hepatocytes, additions of GSH reduced the cellular uptake of CH 3Hg + from the medium, whereas this was increased in the hepatocytes by adding cysteine or methionine. Cysteine addition slightly reduced the uptake of CH 3Hg + in the erythrocytes. GSH-depletion as obtained by DEM pretreatment of the cells, reduced the CH 3Hg + uptake into hepatocytes by 40%, in contrast to only a negligible effect on the erythrocytes. Our results support previous reports that a physiological CH 3Hg +-GSH-complexation takes place intracellularly, at least in liver cells. Our results are furthermore consistent with the assumption that biliary excreted CH 3Hg +-GSH, which can be reabsorbed, only to a limited extent is taken up by the liver, whereas this GSH-complexation and reabsorption is of importance for the CH 3Hg +-uptake in the kidneys.

Excretion of methyl mercury in rat bile: the effect of diethylmaleate, cyclohexene oxide and acrylamide.

Diethylmaleate, cyclohexene oxide and acrylamide administered intraperitoneally to rats, have been shown markedly to inhibit biliary excretion of methyl mercury. Simultaneously the sulphhydryl and sulphide content of the bile decreases. These results probably reflect the conjugation of acrylamide, diethylmaleate and cyclohexene oxide to glutathione in the liver, thereby blocking the biliary excretion of methyl mercury. A high concentration of liver glutathione seems to be a prerequisite for the normal translocation of methyl mercury from liver to bile. These results indicate that methyl mercury is transported from liver to bile as a glutathione complex.

The interrelationship between non-protein bound thiols and the biliary excretion of methylmercury

The biliary excretion of methyl mercury was investigated in male rats injected with various thiol compounds, methionine and 3'-methyl-4-dimethyl-azobenzene (3'-MeDAB) after the i.v. administration of 1.0 mg kg of Hg as Me 20HgCl. All the thiol compounds (2 m-moles of glutathione (GSH) and 4 m-moles kg of cysteine, d-penicillamine or dimercaptosuccinic acid (DMSA) reduced the blood concentration of Me 203Hg +, but only cysteine, GSH and d-penicillamine increased the liver content of mercury and its biliary excretion. No significant changes were seen in the volume of bile flow after treatment. There was no correlation between the increase in non-protein thiol concentration in blood and either the liver content or the biliary excretion of Me 203Hg +. Although those thiol compounds that stimulated the biliary excretion of methylmercury also increased the liver concentration of non-protein thiol groups, there was no proportionality between either the extent or time course of these effects. An initial inhibition of excretion was caused by 4 m-moles kg of cysteine though the concentration of non-protein thiols in the liver was at that time the highest. After the administration of cysteine and GSH, excretion increased from 30 to 120 min while the concentration of non-protein thiols in the liver declined. 3'-Methyl-4-dimethylazobenzene (3'-MeDAB) and methionine caused a larger increase in liver non-protein thiol concentration than GSH but their effect on the biliary excretion of Me 203Hg + was smaller. On the assumption that d-penicillamine spares GSH from other metabolic processes, these results are consistent with the role of GSH in the biliary excretion of methylmercury. They do not support a direct relationship between free GSH and the biliary excretion of methylmercury.

ENHANCEMENT BY PHENOBARBITAL OF THE BILIARY EXCRETION OF METHYL MERCURY AND CADMIUM IN RATS

The effect of phenobarbital on the biliary excretion of metals in rats was com-pared among methyl mercury, inorganic mercury and cadmium compounds from which phenobarbital prevented animals.In rats given an i.v. administration of 1mg/kg Cd or Hg, large amounts of cadmium (15.1% of the dose) were found in the bile within 5 hr after the dose, but small amounts of mercury were excreted with bile (1.4 and 3.3% in inorganic mercury and methyl mercury groups, respectively). Phenobarbital significantly increased the biliary excretion of methyl mercury and cadmium (154 and 140% of controls, respectively), but not that of inorganic mercury. Concentrations of methyl mercury in the bile also increased in the treated rats, but those of cadmium did not change when compared with controls. Distribution of those metals in organs was not affected significantly by phenobarbital except for an increase in mercury content in blood obtained from rats given methyl mercury. These different effects of phenobarbital may reflect individual feature of in metabolism of three metals in the liver and those affinity to tissues.

Effect of phenobarbitone on the biliary excretion of methylmercury in rats and mice.

REABSORPTION of methylmercury excreted with bile1 is one of the major factors responsible for its long biological half-life. Without preventing reabsorption there is no point in increasing the biliary excretion of this highly toxic compound. The discovery that polythiolated resins given in food can trap methylmercury in the intestine and can increase its faecal excretion2 has, however, changed the situation and prompted research to increase biliary excretion with penicillamine of BAL (ref. 3). It is known that phenobarbitone enhances bile flow4 and the biliary excretion of a variety of compounds5, so we investigated its effect on the biliary excretion of methylmercury in rats and on the faecal excretion of methylmercury in mice given polythiolated resin in their food.

Canalicular bile production in dogs.

Canalicular bile production in dogs.