Primary cultures of rabbit renal proximal tubule cells. III. Comparative cytotoxicity of inorganic and organic mercury.

The present study further developed primary cultures of rabbit renal proximal tubule cells (RPTC) as an in vitro model to study chemical-induced toxicity by investigating the comparative cytotoxicity of mercuric chloride (HgCl2) and methyl mercury chloride (CH3HgCl) to RPTC. Confluent monolayer cultures of RPTC exposed to HgCl2 and CH3HgCl for 24 hr exhibited a concentration-dependent loss in cell viability at culture medium concentrations greater than 25 and 2.5 microM, respectively. Vital dye exclusion was a more sensitive indicator of cytotoxicity than the amount of lactate dehydrogenase activity, alkaline phosphatase activity, N-acetylglucosaminidase activity, and protein content remaining on the culture dish. On the basis of vital dye exclusion, HgCl2 was less toxic to proximal tubule cells in culture than CH3HgCl after 24 hr of exposure, whether cytotoxicity was based on LC50 values (34.2 microM HgCl2 vs 6.1 microM CH3HgCl) or total cellular mercury uptake (4.6 nmol Hg2+/10(5) cells vs 1.25 nmol CH3Hg+/10(5) cells). Differences in the extent and rate of metal uptake were also evident. Maximum cellular uptake of Hg2+ occurred within 6-24 hr after exposure and was not concentration-dependent, whereas maximum uptake of CH3Hg+ occurred within 3 hr of exposure and was concentration-dependent. The intracellular distribution of both mercurials between acid-soluble and acid-insoluble binding sites also differed. At noncytotoxic concentrations of HgCl2 (0.04-5 microM), intracellular Hg2+ bound increasingly to acid-soluble binding sites as a function of time, from 15-30% after 6 hr of exposure to 40-60% after 72 hr of exposure. However, at subcytotoxic (25 microM) and cytotoxic (34.2 microM) concentrations, Hg2+ binding to acid-soluble binding sites remained constant at approximately 30-40% for 6, 12, 24, and 72 hr after exposure. In contrast, only 20% of total cellular CH3Hg+ was bound to acid-soluble binding sites after exposure to 0.039 to 6.1 microM CH3HgCl for 6, 12, and 24 hr. Total cellular glutathione content was unaffected after exposure to 0.04-5 microM HgCl2 and 0.039-6.1 microM CH3HgCl, but was depleted 6 hr after exposure to 25 and 34.2 microM HgCl2. These results indicate that CH3HgCl was a more potent cytotoxicant to RPTC in primary culture than HgCl2. Furthermore, compared to Hg2+, the low binding of CH3Hg+ to acid-soluble binding sites and the absence of a redistribution of CH3Hg+ from acid-insoluble to acid-soluble binding sites appeared to contribute to its more potent toxicity to cultured cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the effects of methylmercury on Caenorhabditis elegans

The rising prevalence of methylmercury (MeHg) in seafood and in the global environment provides an impetus for delineating the mechanism of the toxicity of MeHg. Deleterious effects of MeHg have been widely observed in humans and in other mammals, the most striking of which occur in the nervous system. Here we test the model organism, Caenorhabditis elegans (C. elegans), for MeHg toxicity. The simple, well-defined anatomy of the C. elegans nervous system and its ready visualization with green fluorescent protein (GFP) markers facilitated our study of the effects of methylmercuric chloride (MeHgCl) on neural development. Although MeHgCl was lethal to C. elegans, induced a developmental delay, and decreased pharyngeal pumping, other traits including lifespan, brood size, swimming rate, and nervous system morphology were not obviously perturbed in animals that survived MeHgCl exposure. Despite the limited effects of MeHgCl on C. elegans development and behavior, intracellular mercury (Hg) concentrations (≤ 3 ng Hg/mg protein) in MeHgCl-treated nematodes approached levels that are highly toxic to mammals. If MeHgCl reaches these concentrations throughout the animal, this finding indicates that C. elegans cells, particularly neurons, may be less sensitive to MeHgCl toxicity than mammalian cells. We propose, therefore, that C. elegans should be a useful model for discovering intrinsic mechanisms that confer resistance to MeHgCl exposure.     

Prolongation of human neutrophil survival by low-level mercury via inhibition of spontaneous apoptosis

Low levels of organic and inorganic mercury compounds have been reported previously to induce cell death by apoptosis in human peripheral blood mononuclear cells (MNC). but little is known about their potential effects on the viability and death of polymorphonuclear neutrophils (PMN). In contrast to MNC, PMN are known to undergo readily spontaneous apoptosis both in vivo and in vitro. Therefore, it was hypothesized that PMN may differ from MNC in their reactions to low mercury levels. The effects of methylmercuric chloride (MeHgCl) and mercuric chloride (HgCl2) were evaluated in concentration-response and time-course studies on human PMN viability and on their modes of cell death after in vitro incubation at 37 degrees C. Cell death by apoptosis or necrosis was assessed by annexin V-fluorescein isothiocyanate binding to externalized phosphatidylserine in conjunction with propidium iodide, and flow cytometry analysis. Morphologic counting of pyknotic nuclei and the fluorescence properties of the DNA-binding dye Hoechst 33342 in combination with propidium iodide were used to further confirm apoptotic cell death and to characterize the sequence of Hg-induced cell death. Results show that low concentrations of MeHgCl (1-7.5 microM) that were cytotoxic to MNC actually inhibited PMN spontaneous apoptosis. Low-level HgCl, reproduced the anti-apoptotic effects of MeHgCl on PMN, but to a lower extent. Higher concentrations of MeHgCl and HgCl2 were necrogenic to PMN, but MeHgCl was about an order of magnitude more toxic, and discrete differences were observed in the modalities of cell death induced by both species. These data reveal for the first time that (1) low levels of organic and inorganic mercury species protect human PMN from cell death via inhibition of spontaneous apoptosis, and (2) PMN are more resistant than MNC to mercury-induced cytotoxicity. Since delayed apoptosis and increased resistance to toxicant-induced cell death may lead to excessive accumulation of senescent PMN, evidence indicates that findings of this study may have implications for mercury-induced autoimmunity and inflammation.     

Methylmercury increases N-methyl-D-aspartate receptors on human SH-SY 5Y neuroblastoma cells leading to neurotoxicity

Methylmercury (MeHg) is a known neurotoxin, yet the mechanism for low dose chronic toxicity is still not clear. While N-methyl-D-aspartate receptors (NMDARs) were found to be induced after exposure to MeHg in a mink model, its role on neurotoxicity is not known. The aims of this study were to investigate the expression and the functional roles of NMDARs on the induction of cell death in the human SH-SY 5Y neuroblastoma cell line after exposure to MeHg. NMDARs were measured using a radiolabeled phencyclidine receptor ligand [(3)H] (MK801) and cell death was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and lactate dehydrogenase (LDH) release. We found a significant increase in NMDARs followed by increased caspase-3 activity after 4 h of exposure to MeHg (0.25-1 microM). Necrotic cell death was found after 4 and 24 h of exposure to MeHg (0.25-5 microM). The NMDAR antagonists dizocilpine ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate [(+)-MK801]) and Memantine (1-amino-3,5-dimethyl-adamantane) (10 microM) completely attenuated MeHg-mediated cell death by blocking NMDARs, thus demonstrating the importance of NMDARs in mercury neurotoxicity. Intracellular calcium chelator BAPTA-AM (1 microM) partially attenuated the neurotoxicity effect of 1 microM MeHg. These results suggest that MeHg toxicity can be mediated through the binding and increase of NMDARs.     

Induction by mercury compounds of metallothioneins in mouse tissues: inorganic mercury accumulation is not a dominant factor for metallothionein induction in the liver

Among the naturally occurring three mercury species, metallic mercury (Hg(0)), inorganic mercury (Hg(II)) and methylmercury (MeHg), Hg(II) is well documented to induce metallothionein (MT) in tissues of injected animals. Although Hg(0) and MeHg are considered to be inert in terms of directly inducing MT, MT can be induced by them after in vivo conversion to Hg(II) in an animal body. In the present study we examined accumulations of inorganic mercury and MT inductions in mouse tissues (brain, liver and kidney) up to 72 hr after treatment by one of three mercury compounds of sub-lethal doses. Exposure to mercury compounds caused significant mercury accumulations in mouse tissues examined, except for the Hg(II)-treated mouse brain. Although MeHg caused the highest total mercury accumulation in all tissues among mercury compounds, the rates of inorganic mercury were less than 10% through the experimental period. MT inductions that depended on the inorganic mercury accumulation were observed in kidney and brain. However, MT induction in the liver could not be accounted for by the inorganic mercury accumulation, but by plasma IL6 levels, marked elevation of which was observed in Hg(II) or MeHg-treated mouse. The present study demonstrated that MT was induced in mouse tissues after each of three mercury compounds, Hg(0), Hg(II) and MeHg, but the induction processes were different among tissues. The induction would occur directly through accumulation of inorganic mercury in brain and kidney, whereas the hepatic MT might be induced secondarily through mercury-induced elevation in the plasma cytokines, rather than through mercury accumulation in the tissue.     

Exogenous metallothionein and renal toxicity of cadmium and mercury in rats.

The relative tissue distribution and toxicity of cadmium (Cd) and mercury (Hg) in the liver and kidneys of rats when the metals are administered as either inorganic salts or complexed with MT were studied. Male Sprague-Dawley rats were injected (i.v.) with Cd or Hg inorganic salt of chloride or in a complex of MT at a dose of 0.3 mg/kg body weight. The concentration of MT and metals in plasma and urine was monitored for 7 days, at the end of which the rats were killed. Injection of both HgCl2 and Hg-MT induced the synthesis of MT only in the kidney but not in the liver, whereas CdCl2 and Cd-MT injections induced MT synthesis in both liver and kidney, respectively. Plasma MT levels increased 3 days after CdCl2 but not after HgCl2 injection, suggesting that hepatic MT may be an important source of plasma MT under our experimental conditions. Renal toxicity was observed morphologically and by an increase in blood urea nitrogen, plasma creatinine, proteinuria in rats injected with Cd-MT and both forms of Hg. Urinary MT excretion was significantly elevated in Cd-MT injected rats compared with those injected with CdCl2. However, HgCl2 and Hg-MT injected rats showed no significant difference in urinary MT excretion. The magnitude in the renal accumulation of Hg is similar after the administration of Hg-MT or HgCl2, but our findings suggest that the site of epithelial injury may be different. Injury effects of Hg-MT localized mainly in the terminal portions of the proximal convoluted tubule and the initial portions of the proximal straight tubule whereas inorganic Hg caused necrosis in pars recta segments of the proximal tubule.     

Ebselen protects glutamate uptake inhibition caused by methyl mercury but does not by Hg2+

Alterations of the neurotransmitter release systems in CNS have been reported in a variety of neuropathological processes associated with heavy metal toxicity. Neurotoxic effects of mercurials were investigated in vitro in cerebral cortex slices from young rats. The present study indicates that: (i) the environmental contaminants methylmercury (MeHg) and mercuric chloride (Hg2+) (50 microM) inhibited the glutamate net uptake from the cerebral cortex of 17-day-old rats; (ii) ebselen (10 microM) reverted the MeHg-induced inhibition of glutamate net uptake but did not protect the inhibition caused by Hg2+. At same time, we investigated another diorganochalcogenide, diphenyl diselenide (PhSe)2 and it was observed that this compound did not revert the action of MeHg or Hg2+; (iii) in addition, we observed that exposure of slices to 50 microM MeHg and Hg2+ for 30 min followed by Trypan blue exclusion assay resulted in 58.5 and 67.5% of staining cells, respectively, indicating a decrease in cell viability. Ebselen protected slices from the deleterious effects of MeHg, but not of Hg2+ on cell viability. Conversely, ebselen did not modify the reduction of MTT caused by MeHg and Hg2+; (iv) the protective effect of ebselen on MeHg-induced inhibition of glutamate net uptake seems to be related to its ability in maintaining cell viability.     

Enhancement of ovalbumin-induced antibody production and mucosal mast cell response by mercury.

Food contaminants may contribute to the recent increased incidence of food allergies. We have investigated this hypothesis experimentally. It was our objective to determine whether toxicity to the intestinal tissue by orally applied mercury (Hg) could modulate the immune response to food allergens. Effective mechanisms were studied with functional immunological and toxicological parameters. Brown Norway rats were immunized intraperitoneally by ovalbumin (OVA). Before oral challenge with OVA, immunized and non-immunized animals were exposed to HgCl2. Immunological responses were measured by enzyme-linked immunosorbent assays [anti-OVA-IgE and-IgG, rat mast cell protease II (RMCPII), interferon-gamma, interleukin-4, lymphocyte proliferation] and by flow cytometry (lymphocyte subpopulations). Toxicity of Hg to the intestinal barrier was determined by measuring viability, DNA damage and induction of glutathione S-transferase in isolated intestinal epithelial cells and lymph node cells, and by measuring permeability, short-circuit current and tissue conductance of the intact intestinal epithelium. A single high oral dose of HgCl2 enhanced the serum concentrations of anti-OVA-IgE and IgG (P < 0.05) and of RMCPII (P < 0.05) in immunized rats. The treatment resulted in a higher number of CD4/CD25+ T cells in the lymph nodes (P < 0.05). The multiple application of low HgCl2 doses (5 x 0.2 mg/kg body weight) only resulted in an elevated RMCPII serum concentration (P < 0.05). Neither treatment schedules impaired proliferation and cytokine production of lymphocytes. In non-immunized rats only minor immunological changes were observed. Oral HgCl2 induced genotoxic damage in lymph node cells and in jejunal epithelial cells (P < 0.05). Moreover, HgCl2 increased the permeability of intestinal epithelial tissue and of Caco-2 monolayers and was genotoxic and cytotoxic to isolated intestinal epithelial cells in vitro. In conclusion, these studies indicate that the food contaminant Hg can stimulate the immune response to OVA in immunized rats. One possible mechanism could be the toxicity of Hg to the intestinal epithelial and the lymph node cells. Whether humans with allergies respond to high oral doses of Hg in a similar way needs to be investigated in further studies.     

The effects of dimercaptosuccinic acid on the excretion and distribution of mercury in rats and mice treated with mercuric chloride and methylmercury chloride.

1 All five rats in a group survived if dimercaptosuccinic acid (DMSA), a water soluble derivative of 2,3-dimercaptopropanol (BAL), was given in doses of 10-40 mg/kg intraperitoneally 30 min, 4 and 24 h after administration of 2.4 mg/kg Hg as HgCl2, whereas three out of a group of five died if DMSA was not given. DMSA 20 mg/kg increased urinary excretion and decreased the body burden significantly more than 10 mg/kg DMSA, but further doubling of the dose had only marginal effects. 2 DMSA was able to reduce body burden and increase urinary excretion of Hg when intraperitoneal treatment started eight days after the subcutaneous administration of HgCl2. 3 DMSA was effective in decreasing body burden and the brain concentration of Hg in rats dosed orally with methylmercury (MeHgCl) when intraperitoneal treatment started with 40 mg/kg DMSA 24 h after Hg. Increase in the urinary excretion of mercury was responsible for the decrease in body burden. 4 DMSA was effective when given in the drinking water of rats or mice both against inorganic Hg and MeHgCl. In mice treated intraperitoneally with MeHgCl, DMSA 19.5 mug/ml in the drinking water caused a significant decrease in the body burden and increase in the excretion of Hg. 5 DMSA was about four times more efficient than D-penicillamine in decreasing the body burden of Hg. As their toxicity is in the same range, the higher efficiency of DMSA offers a larger margin of safety for the mobilization of Hg.     

Chloride secretion induced by mercury and cadmium: action sites and mechanisms.

The actions of two mercury compounds, HgCl2 and methyl mercury chloride (MeHg), and of CdCl2 on the epithelium of the rat colon were studied with the whole-cell patch-clamp technique and the Ussing chamber. MeHg (50 microM) induced an increase of membrane outward current (I(out)) in enterocytes of isolated crypts patched from the basolateral side. This action was inhibited by a Cl- channel blocker and a K+ channel blocker, indicating an increase of both the Cl- and the K+ conductance. In contrast, HgCl2 (50 microM) did not affect I(out), whereas CdCl2 (50 microM) decreased it slightly. In mucosal preparations all three compounds induced a concentration-dependent increase in short-circuit current (Isc) when administered to the serosal, i.e., contraluminal side. Sensitivity to chloride transport blockers and anion replacement experiments revealed that the increase in Isc represented Cl- secretion. In contrast to the actions of luminally applied mercury compounds, the increase of tissue conductance (Gt) was only small. Tetrodotoxin and indomethacin suppressed the effect of the metal compounds on Isc and Gt, while atropine diminished it only partly. This indicates that the secretory action of these heavy metals has not only a direct effect on epithelial cells but is also mediated by prostaglandins and cholinergic and noncholinergic neurons.     

Methylmercury,but not inorganic mercury,causes abnormality of centrosome integrity (multiple foci of gamma-tubulin), multipolar spindles and multinucleated cells without microtubule disruption in cultured Chinese hamster V79 cells

Abnormalities of centrosome integrity and spindle organization in the cultured Chinese hamster fibroblast cell line V79 exposed to methylmercury (MeHgCl) and inorganic mercury (Hg(2+)) were investigated in conjunction with inductions of mitotic arrest and multinucleated cells. The centrosome integrity and spindle organization were investigated by immunofluorescence of centrosome proteins, gamma-tubulin, and beta-tubulin, respectively. MeHgCl at subtoxic concentrations caused an increase in mitotic index 6 h after exposure. Ameboid cells with multiple pseudopodia were also induced and chromosomes were distributed even in the pseudopodia. After the increase in mitotic index caused by MeHgCl, multinucleated cells with multiple micronuclei appeared. MeHgCl caused abnormality of centrosome integrity (multiple foci of gamma-tubulin) colocalized with aberrant spindles in a concentration-dependent manner, while it did not cause disruption of centrosome integrity and microtubule organization in interphase cells. In addition, MeHgCl led to the appearance of monoastral cells with a one-dot signal of gamma-tubulin. By contrast, Hg(2+) did not cause any of the changes induced by MeHgCl. Thus, MeHgCl caused centrosome abnormality and the related changes without microtubule disruption, suggesting that the mitotic centrosome is a critical target for the cytotoxic effects of MeHgCl.     

Direct and indirect actions of HgCl2 and methyl mercury chloride on permeability and chloride secretion across the rat colonic mucosa.

The actions of two mercury compounds, the inorganic HgCl2 and the organic methyl mercury chloride (MeHg), and of CdCl2 on ion transport across the rat colon were studied with the Ussing chamber and the everted sac method. The mercury compounds (5-50 microM), but not CdCl2, administered to the luminal side, induced a large, concentration-dependent increase of tissue conductance (Gt). The transepithelial movement of the extracellular marker, mannitol, was enhanced in the presence of the mercury compounds, indicating that they cause an increase in the permeability of the epithelium. Morphological studies revealed that at least for HgCl2 this increase of the permeability was associated with a loosening of the tight junctions, severe alterations of the enterocytes, and a loss of the continuity of the epithelium. After washing out HgCl2, cells neighboring the altered enterocytes developed lateral processes and in this way restored the continuity of the epithelial layer. In parallel, the mercury compounds induced an increase of short-circuit current (Isc), which is indicative of an induction of Cl- secretion. The increase of Isc, but not that of Gt, was suppressed by indomethacin and, in the case of MeHg, also by tetrodotoxin. These results suggest that MeHg and HgCl2 induce Cl- secretion by an indirect effect on the epithelium, which is mediated by prostaglandins.     

Stimulation of erythrocyte phosphatidylserine exposure by mercury ions

The sequelae of mercury intoxication include induction of apoptosis. In nucleated cells, Hg2+-induced apoptosis involves mitochondrial damage. The present study has been performed to elucidate effects of Hg2+ in erythrocytes which lack mitochondria but are able to undergo apoptosis-like alterations of the cell membrane. Previous studies have documented that activation of a Ca2+-sensitive erythrocyte scramblase leads to exposure of phosphatidylserine at the erythrocyte surface, a typical feature of apoptotic cells. The erythrocyte scramblase is activated by osmotic shock, oxidative stress and/or energy depletion which increase cytosolic Ca2+ activity and/or activate a sphingomyelinase leading to formation of ceramide. Ceramide sensitizes the scramblase to Ca2+. The present experiments explored the effect of Hg2+ ions on erythrocytes. Phosphatidylserine exposure after mercury treatment was estimated from annexin binding as determined in FACS analysis. Exposure to Hg2+ (1 microM) indeed significantly increased annexin binding from 2.3+/-0.5% (control condition) to 23+/-6% (n=6). This effect was paralleled by activation of a clotrimazole-sensitive K+-selective conductance as measured by patch-clamp recordings and by transient cell shrinkage. Further experiments revealed also an increase of ceramide formation by approximately 66% (n=7) after challenge with mercury (1 microM). In conclusion, mercury ions activate a clotrimazole-sensitive K+-selective conductance leading to transient cell shrinkage. Moreover, Hg2+ increases ceramide formation. The observed mechanisms could similarly participate in the triggering of apoptosis in nucleated cells by Hg2+.     

Mercury but not organochlorines inhibits muscarinic cholinergic receptor binding in the cerebrum of ringed seals (Phoca hispida)

Elevated concentrations of organochlorines and mercury (Hg) have been reported in marine mammals on a global scale. While risk assessments are generally based on quantifying body burdens of toxicants, much less is known about associated adverse health effects and their underlying mechanisms. The purpose of this study was to characterize the inhibitory effects of methylmercury (MeHg+), mercuric chloride (Hg2+), p,p'-DDT, Arochlor 1254, chlordane,dieldrin, lindane, and toxaphene on [3H]quinuclidinyl benzilate ([3H]-QNB) binding to the muscarinic cholinergic (mACh) receptor in cellular membranes isolated from the cerebrum of ringed seals (Phoca hispida). [3H]-QNB binding to the mACh receptor was saturable with a mean receptor density (B(max)) of 826.9 +/- 68.4 fmol/mg and ligand affinity (K(d)) of 0.31 +/- 0.04 nM. MeHg+ and Hg2+ were the only neurotoxicants that inhibited radioligand binding by greater than 50%. Hg2+ was significantly more potent at inhibiting mACh receptor binding than MeHg+ when the IC50 data were compared (IC50 = 1.92 +/- 0.06 microM versus 2.75 +/- 0.22 microM), but when the data were normalized to derive inhibition constants (K(i)) there was no statistical difference in inhibition (Hg2+ = 1.38 +/- 0.07 mM; MeHg+ = 1.26 +/- 0.12 microM). Toxaphene also inhibited mACh receptor binding by 22.4%, but this was only significant at the highest concentration tested (320 microM). Overall, these data suggest that Hg, and not organochlorines,inhibits ligand binding to the mACh receptor. These mechanistic findings may be used to support and develop specific biomarkers of Hg exposure and neurotoxicity in sensitive ecological species.     

Induction of anti-metallothionein antibody and mercury treatment decreases bone mineral density in mice

Mercuric chloride (HgCl2) is an industrial agent with toxic effects on the immune system, kidney, lung, and nervous tissue, but little is known about its effect on bone. Metallothionein (MT) is a cysteine-rich metal-binding protein that exerts cytoprotective effects against heavy metal toxins. It has been reported that the susceptibility of renal and pulmonary toxicity of mercury was markedly enhanced in MT-null mice compared to control mice. However, there is no report about the effects of anti-metallothionein (anti-MT) Ab induction on mercury toxicity. We investigated the effect of anti-MT Ab induction on mercury-induced bone injury. BALB/c mice were injected with MT (10 microg/mouse ic) five times to induce anti-MT Ab and then treated with HgCl2 (1 mg/kg sc) three times per week for 3 weeks. MT immunization plus HgCl2 treatment dramatically decreased bone mineral density (BMD), and the humoral bone formation indices, alkaline phosphatase (ALP) activity and osteocalcin. MT immunization or HgCl2 treatment alone did not affect either BMD or serum ALP activity and osteocalcin levels. MT immunization impeded HgCl2-induced increase of MT expression in the liver and led to an increase of mercury in serum and the liver but a decrease in the kidney. Furthermore, serum titers of IgE and IgG1 were significantly elevated in the MT-immunized plus HgCl2 treatment group compared with those in the HgCl2 treatment group. Similar results were also observed in splenic secretions of IL-4 and IL-10 based on anti-CD3 Ab stimulation. Taken together, our results indicate that anti-MT Ab induction causes mercury-induced bone injury in BALB/c mice and also enhances mercury-related immune disorders.     

Degradation of methyl and ethyl mercury into inorganic mercury by hydroxyl radical produced from rat liver microsomes

Liver microsomes were prepared from Wistar rat by the Ca²⁺ aggregation method. Under various conditions, ethyl mercury chloride (EtHgCl) or methyl mercury chloride (MeHgCl) was incubated with the microsomal preparations. After the incubation, the amounts of inorganic Hg and hydroxyl radical (·OH) in the preparations were determined. Although the preparations alone produced a small amount of inorganic Hg and ·OH, the addition of NADPH to the preparations increased both inorganic Hg and ·OH production, which were further accelerated by the addition of KCN. The addition of Fe(III)EDTA, a ·OH formation promoter, to the microsome-NADPH-KCN system increased inorganic Hg production, whereas the addition of diethylenetriamine pentaacetic acid, a ·OH formation inhibitor, decreased inorganic Hg production. When ·OH scavengers such as mannitol and dimethyl sulfoxide were added to this system, the inorganic Hg production decreased. These results suggested that the ·OH produced from liver microsomes was responsible for the degradation of MeHg and EtHg. Since both ·OH and inorganic Hg production decreased with a concomitant decrease in NADPH-cytochrome P-450 reductase activities, it is suggested that this enzyme may be involved in the microsomal degradation of MeHg and EtHg.     

Renal and hepatic ALA-D activity and selected oxidative stress parameters of rats exposed to inorganic mercury and organoselenium compounds.

This paper evaluates the ability of organoselenium compounds [ebselen, selenocystine N-ethyl-carbamate (SeCis), bis-4-isopropyl-2-oxazolinyl phenyl diselenide (AASe)] to prevent HgCl(2) toxicity. Rats were injected with HgCl(2) (0 or 17 micromol/kg, sc) 6 h after organoselenium compounds had been injected (0 or 50 micromol/kg, sc). In vivo, HgCl(2) inhibited renal ALA-D activity ( approximately 48%), increased TBARS level in kidney ( approximately 52%) and reduced the hepatic content of non-protein thiol groups ( approximately 40%), but organoselenium compounds did not prevent such effects. SeCis, per se, increased renal TBARS level ( approximately 42%), while AASe increased hepatic content of ascorbic acid ( approximately 38%). In vitro, renal and hepatic ALA-D activity was inhibited by HgCl(2) (>or=25 microM), ebselen (>or=12 microM) and SeCis (>or=4 microM). HgCl(2) (400 microM) significantly increased TBARS production in renal and hepatic tissue preparations in vitro, and this effect was completely or partially prevented by organoselenium compounds. Ebselen exhibited thiol peroxidase activity in our assay conditions, while SeCis exhibited thiol-oxidizing properties regardless of the presence of peroxide. AASe had no effect on thiol oxidation. Results suggest that organoselenium compounds could not prevent mercury toxicity in vivo. The protective effect of these compounds against mercury-induced increase of TBARS production in vitro is probably related to an antioxidant action rather than to mercury binding.     

Increased availability of mercury in rat hepatocytes by complex formation with diethyldithiocarbamate

The availability of different chemical forms of mercury (Hg) was studied in primary cultures of rat hepatocytes incubated with mercuric acetate (HgAc), mercuric diethyldithiocarbamate (Hg(DTC)2) or methyl mercuric chloride (MeHgCl), labelled with 203Hg. The uptake of Hg was linearly related to the concentration in the medium and increased in the order Hg(DTC)2 greater than MeHgCl greater than HgAc when similar concentrations of Hg were used. A maximum concentration of Hg was reached after 4 h incubation with Hg(DTC)2 while incubation with HgAc and MeHgCl resulted in a slow continuous accumulation of Hg for up to 24 h. Hg added as HgAc was bound to proteins in the incubation medium to a greater extent than Hg added as Hg(DTC)2 or MeHgCl. Differences in affinity to the medium, as well as in lipophilicity, may partly explain the observed differential uptake of these Hg compounds. The enzyme alcohol dehydrogenase was inhibited by HgAc and Hg(DTC)2 to a similar extent at comparable cellular concentrations of Hg. On the other hand, glutathione reductase was inhibited to a higher degree by HgAc than by Hg(DTC)2, indicating that Hg(DTC)2 remains at least temporarily in the complexed form and that the enzyme is less susceptible to Hg in this form. Both enzymes were much less susceptible to MeHgCl than to HgAc or Hg(DTC)2. The results from the present study indicate that diethyldithiocarbamate can increase the transport of Hg across the cellular membrane by complex formation with Hg and thereby increase the toxicity of Hg.