The influence of selinium on methyl mercury toxicity in rat hepatoma cells, human embryonic fibroblasts and human lymphocytes in culture

The effect of methyl mercury and two selenium compounds have been studied in cell cultures. Methyl mercury in concentrations above 1 microM had a pronounced inhibiting effect on the growth of rat Morris hepatoma cells. Glucose and lactate uptake in relation to cell protein was appreciably stimulated by the organic mercury compound. Selenite in low concentration (0.5 microM) and seleno-di-N-acetyl glycine in thousandfold higher concentrations offered considerable protection against these effects of methyl mercury. The same selenite concentration (0.5 microM), which did not affect cell growth, caused an appreciable protection against methyl mercury (6 microM), even if it was added 3 days after methyl mercury. The methyl mercury inhibited the growth of human embryonic fibroblasts and the DNA-synthesis in the human lymphocytes. However, no protective effect of selenite were observed in these cell types. These results suggest that selenium compounds exert their protective effect through cell specific processes rather than by a direct chemical reaction between selenite and methyl mercury.     

Hemolytic Effects of Sodium Selenite and Mercuric Chloride in Human Blood

Many works have reported the interaction between selenium and mercury in the mammalian body and that chalcogen seems to have a protective effect against mercury toxicity. The aim of this study was to investigate the hemolytic effects of sodium selenite and/or mercuric chloride in human blood under in vitro conditions. For this, total venous blood from healthy subjects (males and females) was heparinized and incubated at 37°C for 90 min with different concentrations of sodium selenite and/or mercuric chloride. The hemolytic effects of compounds were evaluated by measuring plasma hemoglobin concentration after centrifugation. In addition, 2-thiobarbituric acid reactive substances (TBARS) from plasma and erythrocytes, as well as erythrocyte nonprotein thiols (NPSH), were also evaluated in order to investigate molecular mechanisms related to selenite- or mercury-induced hemolysis. Mercuric chloride and sodium selenite, alone (400 µM), promoted a small in vitro hemolytic effect in human erythrocytes. However, when blood was exposed to both compounds (200 µM of each), there was an extremely high synergistic hemolytic effect. The exposure of blood to sodium selenite (400 µM), mercuric chloride (400 µM), and both compounds (200 µM each) did not alter erythrocyte TBARS levels. Sodium selenite presented a high oxidant effect toward erythrocyte NPSH; however, this effect was inhibited by mercuric chloride. The current results point to a synergistic hemolytic effect of sodium selenite and mercuric chloride in human blood, suggesting new understanding on the selenium–mercury antagonism. Moreover, this observed hemolysis seems to be not related to lipoperoxidation or thiol depletion.     

Effect of Organic and Inorganic Mercury on Human Sperm Motility

Abstract: The effects of mercuric chloride and methyl mercuric chloride on the motility of human spermatozoa in vitro were investigated. Organic as well as inorganic mercury compounds decreased the percentage of motile spermatozoa. After 15 min. incubation with 40 μM mercuric chloride a significant decrease in sperm motility was observed. Less than 5% of spermatozoa were motile after 30 min. of exposure to 20 μM methyl mercuric chloride. These effects could not be attenuated by addition of 5 μM sodium selenite. The ultrastructural localization of mercury was demonstrated by autometallography. Silver-enhanced mercury deposits could be demonstrated only in spermatozoa exposed to inorganic mercury. In these cells mercury grains were most abundant in membranes of midpiece and tail.     

Hemolytic effects of sodium selenite and mercuric chloride in human blood

Many works have reported the interaction between selenium and mercury in the mammalian body and that chalcogen seems to have a protective effect against mercury toxicity. The aim of this study was to investigate the hemolytic effects of sodium selenite and/or mercuric chloride in human blood under in vitro conditions. For this, total venous blood from healthy subjects (males and females) was heparinized and incubated at 37 degrees C for 90 min with different concentrations of sodium selenite and/or mercuric chloride. The hemolytic effects of compounds were evaluated by measuring plasma hemoglobin concentration after centrifugation. In addition, 2-thiobarbituric acid reactive substances (TBARS) from plasma and erythrocytes, as well as erythrocyte nonprotein thiols (NPSH), were also evaluated in order to investigate molecular mechanisms related to selenite- or mercury-induced hemolysis. Mercuric chloride and sodium selenite, alone (400 microM), promoted a small in vitro hemolytic effect in human erythrocytes. However, when blood was exposed to both compounds (200 microM of each), there was an extremely high synergistic hemolytic effect. The exposure of blood to sodium selenite (400 microM), mercuric chloride (400 microM), and both compounds (200 microM each) did not alter erythrocyte TBARS levels. Sodium selenite presented a high oxidant effect toward erythrocyte NPSH; however, this effect was inhibited by mercuric chloride. The current results point to a synergistic hemolytic effect of sodium selenite and mercuric chloride in human blood, suggesting new understanding on the selenium-mercury antagonism. Moreover, this observed hemolysis seems to be not related to lipoperoxidation or thiol depletion.     

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.     

Effect of selenium on the uptake of methyl mercury across perfused gills of rainbow trout Oncorhynchus mykiss

The uptake of methyl mercury was measured across the perfused gills of rainbow trout Oncorhynchus mykiss. The effect of selenium, either in the blood (perfusion medium), or in the water was investigated. Methyl mercury was effectively taken up from the water across the gills into the perfusate. The uptake rate reached a stable level after 30 min perfusion. When the gills were placed in mercury free water after exposure to mercury in the water for 1 h, they continued to liberate significant amounts of accumulated mercury into the perfusate. Exposure to selenite (SeIV) or selenate (VI) (0.075–0.75 μM) in the external medium did not affect the uptake of methyl mercury across the gills or the liberation of the metal from the gills. Internal selenite or selenate (7.5 μM) augmented the uptake of methyl mercury across the gills and internal selenite also increased the amounts of liberated methyl mercury from the gills in the unload period. Internal selenium, increased the mercury accumulation in the gills, whereas, external selenium did not alter the mercury accumulation in the gills. Uptake of selenium from the water across the gills occurred very slowly.     

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.     

Effect of vitamin E on selenium cytotoxicity in chick ganglia cultures

Short term exposure of cultured chick ganglia nerve fibers and neuroglia to selenite produced half maximal inhibitory growth effects in concentrations of 6.2 (2.9--13.0) X 10(-5) M and 1.1 (0.7--1.7) X 10(-4) M, respectively. Complete inhibition of growth for both cell types was apparent at 1 X 10(-3) M, while slight stimulation of nerve fiber outgrowth occurred at 1 X 10(-6) M. The addition of vitamin E to these mixed nerve elements produced effects not distinguishable from controls, while on the other hand affording significant protection to the cytotoxic effects of selenite (1 X 10(-4) M). Nerve fibers were more sensitive to selenite than were glial cells. Furthermore, selenite with and without vitamin E exerted a darkening effect on the explant cultures at 1 X 10(-4) M, which became absent or reduced above or below this concentration. The high affinity and binding properties of selenium for the non-polar hydrophobic bi-lipid layer and SH functions of membranes may be implicated in this metal-membrane interaction. The cytotoxic effects of selenite in vitro even though non-specific are protected by the simultaneous presence of vitamin E.     

Effect of methylated forms of selenium on cell viability and the induction of DNA strand breakage.

Selenobetaine (SB) and selenobetaine methyl ester (SBME) are methylated selenonium derivatives that undergo metabolism to release methyl selenide and dimethylselenide, respectively, as primary metabolites. Since methylation of selenium is considered to be detoxifying, the toxicologic activity of SB or SBME may differ from that of inorganic forms of selenium, such as selenite, that undergo reduction and can induce cell damage. In this study, the effects of SB, SBME and selenite on the viability and long-term growth potential of a mouse leukemia cell line (L1210) were compared. Treatment with 20 microM selenite reduced the rate of cell doubling and the long-term growth potential of cells as measured by colony-forming ability. These effects of selenite were accompanied by a reduction in DNA integrity, assessed by alkaline elution analysis for single-strand breaks. Exposure to 500 microM SB or SBME for 24 hr reduced the colony-forming ability of cells in the absence of any effect on dye exclusion or induction of single-strand breaks in DNA. Exposure of cells to 500 microM SB or SBME resulted in levels of intracellular selenium similar to those after exposure to 20 microM selenite. These observations indicate that it is possible to maintain high intracellular levels of selenium, by exposure to methylated selenocompounds, without affecting DNA integrity. These findings also suggest that DNA fragmentation resulting from exposure to selenite occurs during its reductive metabolism and not from the accumulation of a methylated metabolite of selenium. The fact that SB or SBME reduced the ability of L1210 cells to form colonies in agar in the absence of either DNA fragmentation or any effect on the ability of treated cells to exclude a vital dye suggests that both methylated compounds alter the long-term proliferative potential of cells via a mechanism(s) distinct from that associated with cell injury and death by necrosis. Efforts are underway to determine the origin of these effects.     

Gastric anti-ulcer and cytoprotective effect of selenium in rats

Selenium, a trace element, in the form of sodium selenite has been studied for its ability to protect the gastric mucosa against the injuries caused by hypothermic restraint stress, aspirin, indomethacin, reserpine, dimaprit, and various other gastric mucosal-damaging (necrotizing) agents in rats. The results demonstrate that oral administration of sodium selenite produces a significant inhibition of the gastric mucosal damage induced by all the procedures used in this study. Selenium, in a nonantisecretory dose, produced a marked cytoprotective effect against all the necrotizing agents. The cytoprotective effect of selenium against the effects of 80% ethanol and 0.6 M HCl was significantly reversed by prior treatment with a dose of indomethacin that inhibits prostaglandin biosynthesis. These data indicate that sodium selenite inhibits the formation of these lesions by the mucosal generation of prostaglandins. The concentrations of nonprotein sulfhydryls (NP-SH) were significantly decreased in the gastric mucosa following the administration of necrotizing agents--80% ethanol and 0.6 M HCl. Treatment with sodium selenite, which significantly reduced the intensity of gastric lesions, did not replenish the reduced levels of gastric mucosal NP-SH, thus ruling out the mediation of its protective effect through sulfhydryls. The antisecretory effect of sodium selenite, which becomes evident only in the high dose of 20 mumol/kg, may be responsible for the inhibition of gastric lesions induced by aspirin, indomethacin, reserpine, and dimaprit. Our findings show that selenium possesses significant anti-ulcer and adaptive cytoprotective effects. However, further detailed studies are required to confirm these effects, to establish its mechanism(s) of action, and to determine its role in the prophylaxis and treatment of peptic ulcer disease.     

Effects of selenium on toxicity and ultrastructural localization of mercury in cultured murine macrophages

The effects of selenium on cellular toxicity and histochemical distribution of mercury were examined in a cell culture system of mouse peritoneal macrophages. Selenium protected against the toxicity of mercury in cultures exposed to 4 microM of mercuric chloride. Selenomethionine caused a significant increase in cell survival throughout the experiments, while sodium selenite delayed the toxicity of mercury for a while, after which selenite itself had a toxic effect. The amount of mercury visualized by autometallography was increased in macrophage cultures pre-exposed to sodium selenite or selenomethionine. The additional mercury made visible by this histochemical demonstration was located in the cytoplasm as well as in the lysosomes.     

Maternal selenium deficiency enhances the fetolethal toxicity of methyl mercury

The effect of maternal selenium deficiency on methyl mercury fetotoxicity was examined in the ICR strain of mice. Pregnant mice were fed either selenium-deficient diets based on torula yeast or selenium-supplemented diets which were identical to the former except that 0.1, 0.2, or 0.4 mg of selenium per kilogram of diet was added as sodium selenite. Fetolethality of methyl mercury was exacerbated by maternal selenium deficiency when mothers were administered sc 15, 25, or 35 mumol/kg/day of methylmercuric chloride (MMC) on the 13, 14, and 15th days of pregnancy. One-tenth part per million of selenium in the diet was sufficient to protect the fetuses against MMC fetolethality when dams were administered 25 mumol/kg/day of MMC. Mercury concentrations in maternal and fetal tissues were independent of the dietary selenium level. Selenium concentration and glutathione peroxidase (GSH-Px) activity in maternal tissues were unaffected by MMC administration. In fetal liver, on the other hand, selenium concentration was increased and GSH-Px activity was decreased concurrently by maternal MMC administration in the selenium-supplemented groups. Therefore, as far as GSH-Px activity was concerned, the bioavailability of selenium was markedly decreased in fetal liver by maternal injection of MMC. The increase in selenium content in fetal liver, which was observed only in the selenium-supplemented groups, may play an important role in protection against fetolethal toxicity of MMC.     

Protective Effects of Ca2+ Channel Blockers against Methyl Mercury Toxicity

Abstract: The protective effects of Ca²⁺ channel blockers against the toxicity of methyl mercury were examined by both in vivo and in vitro experiments. In the in vivo study we first examined the effects of the Ca²⁺ channel blockers (20 mg/ kg/day), flunarizine, nifedipine, nicardipine, and verapamil against the toxic level of methyl mercury treatment (5 mg/kg/ day of methyl mercuric chloride for 12 consecutive days). However, there was a difference in potency of the effects among the reagents. All the Ca²⁺ channel blockers prevented a decrease in body weight and/or the appearance of the symptoms of neurological disorders in the rats treated with methyl mercury. In the next experiment, we examined flunarizine at different levels of supplementation (1,25 and 50 mg/kg/day). Flunarizine in a dose-dependent manner prevented a decrease in body weight, appearance of the symptoms of neurological disorder and mortality in the rats treated with methyl mercury. Flunarizine treatment (25 mg/kg/day) for the first 5 days did not affect mercury distribution among the tissues, suggesting that the mechanism of protection against methyl mercury-induced toxicity may be attributed to its own pharmacological effect. In the in vitro study we examined the effect of flunarizine (0, 0.5, 5 and 50 μM) using primary cultures of cerebellar granular cells in 96-well culture plates. Viable cell numbers were estimated 1 and 3 days after treatment with methyl mercury. The estimated 50% lethal concentration (LC50) of methyl mercury was higher in plates treated with 5 and 50 uM of flunarizine both on days 1 and 3, indicating that flunarizine protected the primary cultured cerebellar granular cells against the toxicity of methyl mercury. As such, Ca²⁺ channel blockers protected against the toxicity of methyl mercury both in vivo and in vitro, suggesting that Ca²⁺ plays an important role in the mechanisms of methyl mercury toxicity.     

In vitro enhancement of erythrocyte merucry uptake by spironolactone.

Four groups of male Wistar rats were fed the following regimen for 40 days: (1) 20 ppm methylmercury chloride (MMC); (2) 20 ppm MMC + 3 ppm sodium selenite; (3) 3 ppm sodium selenite; (4) basal diet. The basal diet which contained 0.4 ppm “organic selenium” originating mainly from fish meal and wheat was resumed on day 41. Protective effect of selenite over toxicity of methylmercury was observed in terms of both growth rate and morbidity.Concentrations of total mercury, methylmercury and selenium were determined on Days 0, 20, 41, 47, 54, and 61 in the brain, liver, kidney, and blood. It was noted that methylmercury increased accumulation of selenium in all the organs analyzed while selenium retention varied according to the type of selenium and the organs. Modification by selenite, despite its protective effect, remained equivocal in regard to the organ accumulation of mercury and its retention therein.     

Development of reflexes in neonatal mice prenatally exposed to methylmercury and selenite

The development of reflexes in neonates exposed prenatally to methylmercury and selenite was investigated. Pregnant mice were assigned to one of 4 treatments; methylmercury (MeHg), selenite(Se), combination of 2 compounds (MeHg X Se) and saline control (NaCl). Mice were injected subcutaneously (s.c.) on day 9 of gestation. The dose of each compound was 30 mumol/kg. Mercury (Hg) concentrations in the neonatal brain and liver of the MeHg X Se group were slightly lower than in the MeHg group. The results of behavioral examination revealed that the MeHg X Se group showed significantly improved development compared with the MeHg group. These facts suggest the possibility that selenium compounds have protective effects against methylmercury neurotoxicity in fetuses and neonates.     

Interactions between mercuric chloride and sodium selenite on cultured rat cerebrum

Explants of rat cerebrum in culture were treated with toxic concentration of HgCl₂ of 1.10^?4 M and with varying concentrations of sodium selenite. Treatment with sodium selenite resulted in a reduced neurotoxicity of HgCl₂, a maximal effect being attained at a selenite concentration of 1.10^?5 M. However, 1.10^?5 M sodium selenite was itself toxic. In in vitro cell systems, the toxicity of either mercury or selenium is decreased in the presence of the other element.     

Effect of dietary selenium on the metabolism of aflatoxin B1 in turkeys

To investigate the biochemical mechanism of the previously reported protective effect of dietary selenium against aflatoxin toxicity, the hepatic metabolism of aflatoxin B1 in turkey poults was examined at various dietary selenium concentrations. Diets were supplemented with 0.2, 2.0 or 4.0 ppm selenium (as sodium selenite) and 500 ng aflatoxin B1/g diet in an 18-day trial. Free and conjugated aflatoxin and metabolites were quantified using high-performance liquid chromatography. The proportion of liver aflatoxins in conjugated forms increased and the ratio of free aflatoxin B1/M1 decreased with increasing dietary selenium concentrations. These in vivo results provide evidence of selenium-induced enhancement of aflatoxin detoxification processes. In a similar experiment using 2.0 ppm selenium and 750 ng aflatoxin B1/g diet, the concentration of hepatic reduced glutathione, cytochrome P-450 and the activity of enzymes involved in the metabolism of aflatoxin B1 and glutathione were determined. Although the selenium supplement increased glutathione peroxidase activity, dietary selenium had no effect on reduced glutathione or cytochrome P-450 concentrations or on the activities of glutathione transferase E, glucuronyl transferase and cytochrome c reductase. These data indicate that the protective action of selenium is not mediated by an increase in glutathione availability for aflatoxin conjugation or by effects on the activities of these enzymes as measured in vitro.     

Organoselenium compounds prevent hyperphosphorylation of cytoskeletal proteins induced by the neurotoxic agent diphenyl ditelluride in cerebral cortex of young rats

In this work we investigated the protective ability of the selenium compounds ebselen and diphenyl diselenide against the effect of diphenyl ditelluride on the in vitro incorporation of ³²P into intermediate filament (IF) proteins from slices of cerebral cortex of 17-day-old rats. We observed that ditelluride in the concentrations of 1, 15 and 50 μM induced hyperphosphorylation of the high-salt Triton insoluble neurofilament subunits (NF-M and NF-L), glial fibrillary acidic protein (GFAP) and vimentin, without altering the immunocontent of these proteins. Concerning the selenium compounds, diselenide (1, 15 and 50 μM) did not induce alteration of the in vitro phosphorylation of the IF proteins. Otherwise, ebselen induced an altered in vitro phosphorylation of the cytoskeletal proteins in a dose-dependent manner. At intermediate concentrations (15 and 30 μM) it increased the in vitro phosphorylation even though, at low (5 μM) or high (50 and 100 μM) concentrations this compound was ineffective in altering the activity of the cytoskeletal-associated phosphorylating system. In addition, 15 μM diselenide and 5 μM ebselen, presented a protective effect against the action of ditelluride, on the phosphorylation of the proteins studied. Considering that hyperphosphorylation of cytoskeletal proteins is associated with neuronal dysfunction and neurodegeneration, it is probable that the effects of ditelluride could be related to the remarkable neurotoxicity of this organic form of tellurium. Furthermore the neuroprotective action of selenium compounds against tellurium effects could be a promising route to be exploited for a possible treatment of organic tellurium poisoning.     

Comparison of the protection given by selenite,selenomethionine and biological selenium against the renotoxicity of mercury

The protective effect of selenite, seleno-dl-methionine and biological selenium against the renotoxicity of mercury was tested in rats. As the source of biological selenium, the liver soluble fraction of rats given 60 moles/kg selenite 3 days before sacrifice was used. The aim of the experiments was to test whether protective efficiency follows the reported order of ability to form HgSe. Mercury was given subcutaneously in doses of 2.5, 5.0 and 7.5 moles/kg HgCl₂ and selenium was given in equimolar doses at the same time as Hg²⁺. Liver soluble fraction, biological selenium or liver soluble fraction supplemented with selenite or seleno-dl-methionine were given orally, while in experiments without liver soluble fraction the two selenium compounds were given subcutaneously. Biological selenium was tested only at the two lower dose levels. Both biological selenium and seleno-dl-methionine decreased the urinary excretion of mercury in the first 48 h, but less so than selenite and only selenite decreased the renal content of mercury at the end of this period. Urinary alkaline phosphatase activity and plasma urea nitrogen at the 2.5 and 5.0 moles/kg dose levels decreased in the order of no selenium > biological selenium > seleno-dl-methionine > selenite. As the reported HgSe formation increases in the same order, the experiments support the role of HgSe formation in the protective effect. The degree of necrotic damage in the P₂ and P₃ regions of the proximal tubular cells increased in the same order as the biochemical indicators at the 5.0 and 7.5 moles/kg dose levels. Necrotic damage at the lower dose level of mercury was slight and differences between groups could be explained by random distribution.     

胰岛素与硒联合应用治疗糖尿病大鼠的可行性分析

目的确定胰岛素和硒联合应用治疗糖尿病大鼠的可行性。方法建立糖尿病模型,胰岛素皮下注射给药,亚硒酸钠口服给药。胰岛素和亚硒酸钠分别设计2个剂量(胰岛素:1和0.5u.kg-1;亚硒酸钠:180和60μg.kg-1)。按2因素2水平的L4(22),运用正交实验设计确定胰岛素和硒联合应用的合理配比剂量。结果胰岛素与亚硒酸钠对糖尿病大鼠血糖的降低作用存在相互影响,胰岛素和硒联合应用的合理配比剂量是1u.kg-1∶180μg.kg-1。结论胰岛素和硒联合应用在降低血糖方面明显优于单用相同剂量的胰岛素或亚硒酸钠,联合用药(胰岛素+硒)发挥了协同作用。