7V-Acetylpenicillamine Potentiated Excretion of Methyl Mercury in Rat Bile: Influence of S-Methylcysteine

Abstract: 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.     

The Mechanism of Biliary Excretion of Methyl Mercury: Studies with Methylthiols

Abstract: 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.     

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.     

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.     

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 Effect of N-acetylhomocysteine and its Thiolactone on the Distribution and Excretion of Mercury in Methyl Mercuric Chloride Injected Mice

Abstract: The distribution and excretion of mercury was studied in mice given a single intravenous dose of 5 μmol/kg of methyl mercuric chloride. Intravenous treatment with N-acetylhomocysteine (10 mmol/kg) increased the urinary excretion of mercury. The corresponding thiolactone mixed into the feed of the mice turned out to be more effective in removing mercury from the body. The toxicity of the thiolactone seemed to be remarkably low compared to other sulphur containing agents. Mercury deposited in the brain was mobilized by oral administration of the thiolactone even if the treatment was delayed until 5 days after the injection of methyl mercury. The results indicate that the formation of a N-acetylhomocysteine-methyl-mercuric-complex is responsible for this effect.     

Renal functional correlates of methyl mercury intoxication: interaction with acute mercuric chloride toxicity.

To determine the effect of methyl mercury and its possible interaction with mercuric chloride on renal function, male Sprague-Dawley rats were treated with methyl mercuric chloride (1 mg/kg per day × 20 days ip) and/or mercuric chloride (1 mg/kg ip at Day 20) in a 2 × 2 factorial experimental design. Methyl mercury depressed urine osmolality and N-methylnicotinamide (NMN) uptake by renal cortical slices but did not affect the uptake of p-aminohippurate (PAH), blood urea nitrogen concentration (BUN), urine volume, or body weight. Urinary excretion of the lysosomal enzymes, β-galactosidase and acid phosphatase, appeared to be decreased, but excretion of the brush border enzyme, alkaline phosphatase, was not affected. Mercuric chloride treatment increased enzyme excretion, BUN, and uptake of NMN by renal cortical slices, while it decreased PAH uptake and urine osmolality, BUN concentration was further increased by combined treatment, yielding the only significant treatment interaction between methyl mercury and mercuric chloride. Prostaglandin E₂ synthesis and release by renal medullary tissue in vitro was not depressed by methyl mercuric chloride pretreatment nor was renal ammoniagenesis or gluconeogenesis. The effects of methyl mercury upon lysosomal enzyme excretion and NMN accumulation are suggestive of lysosomal and mitochondrial dysfunction. The failure to detect significant interaction between methyl mercury and mercuric chloride indicates that methyl mercury neither potentiates nor protects against acute mercuric chloride toxicity at this time and dose.     

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 mumol/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 mumol/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 mumol/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.     

Simultaneous administration of sodium selenite and mercuric chloride decreases efficacy of DMSA and DMPS in mercury elimination in rats

Two chelating agents meso-2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercapto-propane-1-sulphonate (DMPS) were tested for their efficiency in mercury removal from the body of rats in the presence and in the absence of selenium. Female Wistar rats were given a single intraperitoneal injection of mercuric chloride or an equimolar mixture of mercuric chloride and sodium selenite (1.5 micromol/kg body weight). The chelating agents were given orally, in excess (500 micromol DMSA/kg body weight; 300 micromol DMPS/kg body weight), 30 min after the administration of mercury and selenium. The animals were euthanized 24 h after the treatment and mercury in the kidney, liver, and 24 h urine was determined using cold vapour atomic absorption spectrometry (CV-AAS). The simultaneous administration of mercuric chloride and sodium selenite led to a redistribution of mercury in the organs, so that accumulation of mercury in the kidneys was decreased and in the liver increased. Selenite also caused decrease in the level of urinary mercury excretion. Both chelating agents were effective in mercury removal from the body, by increasing its urinary excretion. However, when animals were simultaneously treated with mercury and selenite, the rise of mercury excreted in the urine due to the treatment with chelating agents was lower when compared to animals receiving mercury without selenite. It is concluded that sodium selenite decreases the efficiency of DMSA and DMPS in mercury removal from the body of rats.     

The Effect of Selenium on the Biliary Excretion and Organ Distribution of Mercury in the Rat after Exposure to Methyl Mercuric Chloride

Abstract The influence of selenium compounds on the biliary excretion and the organ distribution of mercury after injection of methyl mercuric chloride (4 μmol/kg) have been tested. Selenite, seleno-di-N-acetylglycine and seleno-methionine strongly inhibited the biliary excretion of mercury. Selenite even in a molar dose of 1/40 of the methyl mercury dose inhibited the biliary excretion of mercury. The less toxic seleno-di-N-acetylglycine was needed in larger molar doses and did not act as rapidly as selenite. Biliary excreted methyl mercury is known to be partly reabsorbed in the gut. Subsequently a part of it is deposited in the kidneys since drainage of the bile lowered the kidney content of mercury. Rats given selenium compounds in combination with bile drainage showed further reduction of the kidney mercury content than bile duct drainage alone. Thus the demonstrated lowering effect of selenium compounds on the kidney mercury content cannot be completely explained by an inhibition of biliary excretion of mercury. The mercury concentration in the brain was increased by the selenium compounds; the effect being dependent of the selenium dose reaching a maximum at an equimolar selenite - to methyl mercury dose ratio. The mechanisms by which selenium influences the methyl mercury kinetics are discussed.     

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 mumol/kg, respectively, while the shark excreted only 0.15 +/- 0.15% (n = 8) at a dose of 5 mumol/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 X day. An inefficient biliary excretory process in fish may account in part for the long biological half-times for methyl mercury in marine species.     

The effect of selenium on the biliary excretion and organ distribution of mercury in the rat after exposure to methyl mercuric chloride.

The influence of selenium compounds on the biliary excretion and the organ distribution of mercury after injection of methyl mercuric chloride (4 mumol/kg) have been tested. Selenite, seleno-di-N-acetylglycine and seleno-methionine strongly inhibited the biliary excretion of mercury. Selenite even in a molar dose of 1/40 of the methyl mercury dose inhibited the biliary excretion of mercury. The less toxic seleno-di-N-acetylglycine was needed in larger molar doses and did not act as rapidly as selenite. Biliary excreted methyl mercury is known to be partly reabsorbed in the gut. Subsequently a part of it is deposited in the kidneys since drainage of the bile lowered the kidney content of mercury. Rats given selenium compounds in combination with bile drainage showed further reduction of the kidney mercury content than bile duct drainage alone. Thus the demonstrated lowering effect of selenium compounds on the kidney mercury content cannot be completely explained by an inhibition of biliary excretion of mercury. The mercury concentration in the brain was increased by the selenium compounds; the effect being dependent of the selenium dose reaching a maximum at an equimolar selenite--to methyl mercury dose ratio. The mechanisms by which selenium influences the methyl mercury kinetics are discussed.     

Metabolization of 14C-N-acetylpenicillamine and 14C-cysteine in relation to 1-chloro-2,4-dinitrobenzene and sulphobromophtalein conjugation and biliary excretion in the rat

Upon intraperitoneal administration of N-(1-14C)-acetylpenicillamine (NAPA) to rats, at a dose of 1-2 mmol/kg body weight, a 14C-labelled metabolite of NAPA together with unchanged NAPA were excreted in bile. The NAPA metabolite was characterized by acid hydrolysis and thin-layer chromatography. Rats previously depleted of liver glutathione by intraperitoneal injection of cyclohexene oxide, 2.5 mmol/kg body weight, were given intraperitoneal injections of 14C-cysteine, 0.5 mmol/kg, or 14C-NAPA, 1-2 mmol/kg, together with 1-chloro-2,4-dinitrobenzene (DNB), 1 mmol/kg, or bromosulphophtalein (BSP), 0.5 mmol/kg body weight. Simultaneous administration of 14C-cysteine and DNB or BSP lead to rapid incorporation of 14C-activity in glutathione and glutathione conjugates of DNB and BSP being excreted in bile. Upon simultaneous administration of 14C-NAPA and DNB or BSP, 14C-labelled metabolite of NAPA not conjugated to DNB or BSP, and unchanged NAPA, together with non-labelled glutathione conjugates of DNB or BSP, were excreted in bile. This is interpreted as indicating that neither NAPA nor NAPA metabolite are capable of forming conjugates of DNB or BSP in rat liver.     

The effects of spironolactone on the biliary excretion of mercury,cadmium, zinc,and cerium in rats

The effect of spironolactone (Sp) pretreatment on the biliary excretion of intravenously injected heavy metals (mercury, cadmium, zinc and cerium) was investigated in rats, using five metal compounds (four inorganic metals in chloride form, and methyl mercuric chloride). The oral administration of Sp ($\text{5 mg}\text{100 g}$) 1–3 hr before the bile excretion study increased the biliary recovery of mercury more than ten times over a period of 4 hr in rats injected with mercuric chloride, but did not increase the biliary excretion of the other three metals (Cd, Zn and Ce). Multiple-dose pretreatment (2 doses a day for 3 days) also increased the biliary excretion of mercury but much less than in the acutely treated rats. Cadmium excretion was significantly decreased by multiple dose pretreatment. Sequential nuclear imagings after intravenous injection of ¹⁹⁷HgCl₂, demonstrated clear differences in the tissue distribution of mercury between control and Sp-treated rats.     

Protective effect of molybdenum on the acute toxicity of mercuric chloride

The effect of molybdenum on the acute toxicity of mercuric chloride was examined. Rats that were given mercuric chloride alone (0.03 mmol/kg, once, sc) all died within 2 days after treatment. However, rats pretreated with sodium molybdate (1.24 mmol/kg, once a day for 3 days, ip) all survived for 6 days after administration of the same dose of mercuric chloride. The pretreatment with molybdenum decreased the mercury content in the liver, kidney, and spleen, but the molybdenum content in the liver, kidney, and large intestine were increased in comparison with those of the rats given molybdenum alone. Moreover, pretreatment with molybdenum caused polyuria in contrast with anuria in rats given mercuric chloride alone. These data suggest that a protective mechanism of molybdenum is the ability of this metal to decrease the mercury content in the liver, kidney, and spleen by enhancement of urinary excretion of mercury.     

Inhibition of biliary excretion of indocyanine green by the thiol-oxidizing agent, diazenedicarboxylic acid bis[N,N'-dimethylamide].

Biliary excretion of Indocyanine green (ICG) in Sprague-Dawley rats during constant intravenous infusion of the dye in vivo was inhibited by intraperitoneally administered diazenedicarboxylic acid bis[N,N'-dimethylamide] (diamide, 0.5 mmol/kg body wt), a glutathione-specific thiol-oxidizing agent. Significant inhibition of ICG excretion was observed also when ICG was injected rapidly 90 min after diamide administration. Disappearance of ICG from the plasma was not affected by diamide. Oxidized glutathione in bile increased transiently following diamide administration but returned to the basal level within 30 min. Hepatic concentrations of reduced and oxidized glutathione were not different from those of controls when determined 90 min after diamide administration. The inhibition of ICG excretion was completely prevented by subsequent administration of dithiothreitol (0.5 mmol/kg) 30 min after that of diamide. The results, therefore, suggest that the biliary excretion of ICG was inhibited by secondary changes in the redox status of thiols in hepatocytes caused by a transient increase in oxidized glutathione.     

Excretion of Methyl Mercury in Rat Bile: The Effect of Thioctic Acid,Thionalide, Hexadecyl- and Octadecylmercaptoacetate

Abstract: 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.     

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.     

Excretion of Zinc in Rat Bile – A Role of Glutathione

Abstract: Fractionation of the bile from rats injected with ⁶⁵ZnCl₂ (5 μmol/kg) showed that zinc was mainly bound to low molecular weight compounds eluted corresponding to the zinc-glutathione complexes. Diethylmaleate (3.9 mmol/kg), cyclohexene oxide (4.9 mmol/kg) and acrylamide (3.5 mmol/kg) administered intraperitoneally to rats caused a rapid decrease in the endogenous excretion of both zinc and reduced glutathione into bile. This depression probably reflects the conjugation of the aforementioned substances to glutathione in the liver cells. These results indicate that zinc is transferred from liver to bile by glutathione dependent process and most likely as zinc-glutathione complexes.     

Effect of zinc pretreatment on mercuric chloride-induced lipid peroxidation in the rat kidney

The effect of zinc on mercuric chloride-induced lipid peroxidation in the rat kidney was investigated. The rats received zinc acetate (2.0 mmol/kg, po) for 2 days before being given mercuric chloride (15 mumol/kg, sc) and were killed 6, 12, and 24 hr after the last injection. Lipid peroxidation occurred in the rat kidney 12 hr after mercury administration, and this mercury-induced lipid peroxidation was significantly reduced by zinc pretreatment. A decrease in vitamin C and E contents in the kidney was observed 12 hr after the administration of mercury, and this decrease was prevented by zinc pretreatment. In the kidney of rats pretreated with zinc, the activities of the protective enzymes, glutathione peroxidase and glucose-6-phosphate dehydrogenase, were increased after mercury injection. Non-protein sulfhydryl content (mostly glutathione) also rose markedly. The results indicate that zinc not only induces metallothionein, but also increases protective enzyme activities and glutathione content, which would tend to inhibit lipid peroxidation and suppress mercury toxicity.