Chronic mercury exposure induces oxidative stress in female rats by endothelial nitric oxide synthase uncoupling and cyclooxygenase-2 activation, without affecting oestrogen receptor function.

Mercury has been shown to be a significant health risk factor and is positively associated with cardiovascular diseases. Evidence reveals that men are more likely to develop cardiovascular diseases than women during reproductive age. However, the effects of mercury in females remain poorly investigated, despite the finding that female hormones demonstrate a cardioprotective role. In the present study, we evaluated whether chronic mercury chloride exposure could alter blood pressure and vascular function of the female rat aorta. Ten-week-old female Wistar rats were divided into two groups: control (vehicle) and mercury treated (first dose of 4.6 µg/kg, subsequent daily doses of 0.07 µg/kg), im. Mercury treatment did not modify systolic blood pressure (SBP) but increased vascular reactivity due to the reduction of nitric oxide bioavailability associated with the increase in reactive oxygen species from endothelial nitric oxide synthase (eNOS) uncoupling. Furthermore, increased participation of the cyclooxygenase-2 pathway occurred through an imbalance in thromboxane 2 and prostacyclin 2. However, the oestrogen signalling pathway was not altered in either group. These results demonstrated that chronic exposure to mercury in females induced endothelial dysfunction and, consequently, increased aortic vascular reactivity, causing vascular damage to the female rat aorta and representing a risk of cardiovascular diseases.

Renal toxicity of heavy metals (cadmium and mercury) and their amelioration with ascorbic acid in rabbits.

Cadmium and mercury are among the most toxic and dangerous environmental pollutants that may cause fatal implications. Vitamin C is an important chain-breaking antioxidant and enzyme co-factor against heavy metals. The objective of the present study was to evaluate the toxicological effects of cadmium chloride, mercuric chloride, and their co-administration on biochemical parameters of blood serum and metal bioaccumulation in kidneys and also to elucidate the protective effect of vitamin C in rabbits against these metals. In the current research, cadmium chloride (1.5 mg/kg), mercuric chloride(1.2 mg/kg), and vitamin C (150 mg/kg of body weight) were orally administered to eight treatment groups of the rabbits (1, control; 2, vitamin; 3, CdCl2; 4, HgCl2; 5, vitamin + CdCl2; 6, vitamin + HgCl2; 7, CdCl2 + HgCl2, and 8, vitamin + CdCl2 + HgCl2). After the biometric measurements of all experimental rabbits, biochemical parameters viz. creatinine, cystatin C, uric acid, and alkaline phosphatase (ALP) and metal bioaccumulation were determined using commercially available kits and atomic absorption spectrophotometer, respectively. The levels of creatinine (28.3 ± 1.1 µmol/l), cystatin C (1932.5 ± 38.5 ηg/ml), uric acid (4.8 ± 0.1 mg/day), and ALP (51.6 ± 1.1 IU/l) were significantly (P < 0.05) increased due to administration of mercuric chloride but in the presence of vitamin C, the effects of mercuric chloride on creatinine (21.9 ± 1.4 µmol/l), cystatin C (1676.2 ± 42.2 ηg/ml), uric acid (3.9 ± 0.1 mg/day), and ALP (43.3 ± 0.8 IU/l) were less as compared to metal-exposed specimens. Similar results were found in rabbits treated with cadmium chloride and vitamin C and also with co-administration of both metals and vitamin C. Because of the bio-accumulative nature of cadmium chloride and mercuric chloride, these metals were accumulated in kidneys of rabbits, which might lead to deleterious effects. The results of the present study provide an insight into the toxicity of the cadmium chloride, mercuric chloride, and/or their combination on biochemical parameters as well as kidneys of the rabbits and the ameliorating potential of vitamin C against these metals is also evaluated.

Comparison of mercury sulfides with mercury chloride and methylmercury on hepatic P450, phase-2 and transporter gene expression in mice.

Zuotai (mainly ß-HgS) and Zhusha (also called as cinnabar, mainly α-HgS) are used in traditional medicines in combination with herbs or even drugs in the treatment of various disorders, while mercury chloride (HgCl2) and methylmercury (MeHg) do not have known medical values but are highly toxic. This study aimed to compare the effects of mercury sulfides with HgCl2 and MeHg on hepatic drug processing gene expression. Mice were orally administrated with Zuotai (ß-HgS, 30mg/kg), α-HgS (HgS, 30mg/kg), HgCl2 (33.6mg/kg), or MeHg (3.1mg/kg) for 7days, and the expression of genes related to phase-1 drug metabolism (P450), phase-2 conjugation, and phase-3 (transporters) genes were examined. The mercurials at the dose and duration used in the study did not have significant effects on the expression of cytochrome P450 1-4 family genes and the corresponding nuclear receptors, except for a slight increase in PPARα and Cyp4a10 by HgCl2. The expressions of UDP-glucuronosyltransferase and sulfotransferase were increased by HgCl2 and MeHg, but not by Zuotai and HgS. HgCl2 decreased the expression of organic anion transporter (Oatp1a1), but increased Oatp1a4. Both HgCl2 and MeHg increased the expression of multidrug resistance-associated protein genes (Mrp1, Mrp2, Mrp3, and Mrp4). Zuotai and HgS had little effects on these transporter genes. In conclusion, Zuotai and HgS are different from HgCl2 and MeHg in hepatic drug processing gene expression; suggesting that chemical forms of mercury not only affect their disposition and toxicity, but also affect their effects on the expression of hepatic drug processing genes.

Renal oxidative stress and renal CD8(+) T-cell infiltration in mercuric chloride-induced nephropathy in rats: role of angiotensin II.

Mercuric chloride (HgCl2) induces kidney damage, in part, through oxidative stress. A role for angiotensin II (Ang II) in pro-inflammatory events in a model of acute HgCl2-induced nephropathy was reported. Ang II is a potent oxidative stress inducer; however, its role in oxidative/anti-oxidative events in HgCl2-induced nephropathy remains unknown. The aim of this study was to determine the role of Ang II in the oxidative stress and renal infiltration of CD8(+) T-cells after an acute HgCl2 intoxication. Three groups of Sprague Dawley rats were treated with a single subcutaneous dose of 2.5 mg/kg HgCl2: for 3 days prior to and for 4 days after that injection, rats in one group received Losartan (30 mg/kg), in another group Enalapril (30 mg/kg) or normal saline in the last group. Two other groups of drug-treated rats received saline in place of HgCl2. A final group of rats received saline in place of HgCl2 and the test drugs. All treatments were via gastric gavage. At 96 h after the vehicle/HgCl2 injection, blood and kidney samples were harvested. Renal sections were homogenized for measures of malondialdehyde (MDA), reduced glutathione (GSH) and catalase activity. Frozen sections were studied for the presence of superoxide anion ([Formula: see text]) and CD8(+) T-cells. HgCl2-treated rats had increased interstitial and tubular expression of [Formula: see text], high levels of MDA, normal catalase activity and GSH content, increased levels of interstitial CD8(+) T-cells and an increased percentage of necrotic tubules. Anti-Ang II treatments diminished the HgCl2-induced increases in interstitial [Formula: see text], CD8(+) T-cells and tubular damage and increased catalase and GSH expression above that due to HgCl2 alone; the HgCl2-induced high MDA levels were unaffected by the drugs. These data provide new information regarding the potential role of Ang II in the oxidative stress and renal CD8(+) T-cell infiltration that occur during HgCl2 nephropathy.

Safety evaluation of mercury based Ayurvedic formulation (Sidh Makardhwaj) on brain cerebrum, liver & kidney in rats.

BACKGROUND & OBJECTIVES: Sidh Makardhwaj (SM) is a mercury based Ayurvedic formulation used in rheumatoid arthritis and neurological disorders. However, toxicity concerns due to mercury content are often raised. Therefore, the present study was carried out to evaluate the effect of SM on brain cerebrum, liver and kidney in rats. METHODS: Graded doses of SM (10, 50, 100 mg/kg), mercuric chloride (1 mg/kg) and normal saline were administered orally to male Wistar rats for 28 days. Behavioural parameters were assessed on days 1, 7, 14 and 28 using Morris water maze, passive avoidance, elevated plus maze and rota rod. Liver and kidney function tests were done on day 28. Animals were sacrificed and brain cerebrum acetylcholinesterase activity, levels of malondialdehyde (MDA), reduced glutathione (GSH) in brain cerebrum, liver, kidney were estimated. The levels of mercury in brain cerebrum, liver and kidney were estimated and histopathology of these tissues was also performed. RESULTS: SM in the doses used did not cause significant change in neurobehavioural parameters, brain cerebrum AChE activity, liver (ALT, AST, ALP bilirubin) and kidney (serum urea and creatinine) function tests as compared to control. The levels of mercury in brain cerebrum, liver, and kidney were found to be raised in dose dependent manner. However, the levels of MDA and GSH in these tissues did not show significant changes at doses of 10 and 50 mg/kg. Also, there was no histopathological change in cytoarchitecture of brain cerebrum, liver, and kidney tissues at doses of 10 and 50 mg/kg. INTERPRETATION & CONCLUSIONS: The findings of the present study suggest that Sidh Makardhwaj upto five times the equivalent human dose administered for 28 days did not show any toxicological effects on rat brain cerebrum, liver and kidney.

Chronic exposure to low doses of HgCl2 avoids calcium handling impairment in the right ventricle after myocardial infarction in rats.

Right ventricle systolic dysfunction is a major risk factor for death and heart failure after myocardial infarction (MI). Heavy metal exposure has been associated with the development of several cardiovascular diseases, such as MI. The aim of this study was to investigate whether chronic exposure to low doses of mercury chloride (HgCl2) enhances the functional deterioration of right ventricle strips after MI. Male Wistar rats were divided into four groups: Control (vehicle); HgCl2 (exposure during 4 weeks- 1st dose 4.6 µg/kg, subsequent dose 0.07 µg/kg/day, i.m. to cover daily loss); MI surgery induced and HgCl2-MI groups. One week after MI, the morphological and hemodynamic measurements and isometric tension of right ventricle strips were investigated. The chronic HgCl2 exposure did not worsen the injury compared with MI alone in the morphological or hemodynamic parameters evaluated. At basal conditions, despite similar maximum isometric force at L-max, relaxation time was increased in the MI group but unaffected in the HgCl2-MI compared to the Control group. Impairment of the sarcoplasmic reticulum (SR) function and reduction in the sarcolemmal calcium influx were observed in MI group associated with SERCA2a reduction and increased PLB protein expression. Induction of MI in chronic HgCl2 exposed rats did not cause any alteration in the developed force at L-max, lusitropic function or -dF/dt except for a tendency of a reduction SR function. These findings could be partially explained by the normalization in the sarcolemmal calcium influx and the increase in NCX protein expression observed only in this group. These results suggest that chronic exposure to low doses of HgCl2 prevents the impaired SR function and the reduced sarcolemmal calcium influx observed in MI likely by acting on NCX, PLB and SERCA2a protein expression.

Pomegranate (Punica granatum) peel extract provides protection against mercuric chloride-induced oxidative stress in Wistar strain rats.

CONTEXT: Pomegranate [Punica granatum L. (Lythraceae)] is rich in antioxidants. OBJECTIVE: The present work was undertaken to evaluate the protective effects of methanol extract of pomegranate peel extract (PPE) against a rat model of oxidative stress generated by treatment with mercuric chloride (HgCl(2)). MATERIALS AND METHODS: Rats were injected with HgCl(2) (5 mg/kg body weight in 0.9% NaCl) for developing oxidative stress. The methanol extract of pomegranate peel (PPE) was suspended in 0.5% sodium carboxymethylcellulose and fed to rats at 50 mg/kg body weight/day up to 30 days after HgCl(2) treatment. Biomarkers of oxidative stress including erythrocyte plasma membrane redox system (PMRS), lipid peroxidation, advanced oxidation protein products (AOPP), intracellular glutathione, and plasma antioxidant capacity as FRAP values were measured after PPE supplementation. RESULTS: HgCl(2)-treated rats showed increased oxidative stress as evidenced by increased activity of erythrocyte PMRS 153%, lipid peroxidation 31%, and AOPP 290%. There was a significant (p < 0.01) decrease (25%) in plasma antioxidant capacity and intracellular glutathione (75%). Supplementation with PPE protected rats from HgCl(2) induced oxidative damage. PPE supplementation to control rats increased antioxidant defense. DISCUSSION AND CONCLUSION: The results clearly demonstrate that PPE treatment augments the antioxidant defense mechanism in mercuric chloride-induced oxidant toxicity. To the best of our knowledge this is the first report of the increase in erythrocyte PMRS activity in rats after supplementation with any kind of antioxidant supplementation. This study emphasizes the need for characterizing PPE for potential unknown antioxidant compounds.

Short-term mercury exposure on Na(+)/K(+)-ATPase activity and ionoregulation in gill and brain of an Indian major carp, Cirrhinus mrigala.

Recently mercury pollution has been increased considerably in aquatic resources throughout the world and it is a growing global concern. In this study, the 96 h LC50 value of waterborne mercuric chloride for Cirrhinus mrigala was found to be 0.34 mg/L (with 95% confidence limits). Fingerlings of C. mrigala were exposed to 0.068 and 0.034 mg/L of mercuric chloride for 96 h to assess the Na(+)/K(+)-ATPase activity and ionoregulation (Na(+), K(+) and Cl(-)) in gill and brain. Results showed that Na(+)/K(+)-ATPase activity and ionic levels (Na(+), K(+) and Cl(-)) in gill and brain of fish exposed to different concentrations of mercuric chloride were found to be significantly (p<0.05) decreased throughout the study period. Mercury inactivates many enzymes by attaching to sulfur atoms in which the enzyme Na(+)/K(+)-ATPase is highly sensitive to mercury. The inhibition of gill and brain Na(+)/K(+)-ATPase activity might have resulted from the physicochemical alteration of the membrane due to mercury toxicity. Moreover, inhibition of Na(+)/K(+)-ATPase may affect the ion transport and osmoregulatory function by blocking the transport of substances across the membrane by active transport. The present study indicates that the alterations in these parameters can be used in environmental biomonitoring of mercury contamination in aquatic ecosystem.

MRP2 and the handling of mercuric ions in rats exposed acutely to inorganic and organic species of mercury.

Mercuric ions accumulate preferentially in renal tubular epithelial cells and bond with intracellular thiols. Certain metal-complexing agents have been shown to promote extraction of mercuric ions via the multidrug resistance-associated protein 2 (MRP2). Following exposure to a non-toxic dose of inorganic mercury (Hg²+), in the absence of complexing agents, tubular cells are capable of exporting a small fraction of intracellular Hg²+ through one or more undetermined mechanisms. We hypothesize that MRP2 plays a role in this export. To test this hypothesis, Wistar (control) and TR(-) rats were injected intravenously with a non-nephrotoxic dose of HgCl2 (0.5 µmol/kg) or CH3HgCl (5 mg/kg), containing [²°³Hg], in the presence or absence of cysteine (Cys; 1.25 µmol/kg or 12.5mg/kg, respectively). Animals were sacrificed 24 h after exposure to mercury and the content of [²°³Hg] in blood, kidneys, liver, urine and feces was determined. In addition, uptake of Cys-S-conjugates of Hg²+ and methylmercury (CH3Hg+) was measured in inside-out membrane vesicles prepared from either control Sf9 cells or Sf9 cells transfected with human MRP2. The amount of mercury in the total renal mass and liver was significantly greater in TR⁻ rats than in controls. In contrast, the amount of mercury in urine and feces was significantly lower in TR⁻ rats than in controls. Data from membrane vesicles indicate that Cys-S-conjugates of Hg²+ and CH3Hg+ are transportable substrates of MRP2. Collectively, these data indicate that MRP2 plays a role in the physiological handling and elimination of mercuric ions from the kidney.

Na-nitroprusside and HgCl2 modify the water permeability and volume of human erythrocytes.

The passage of water through the aquaporin-1 (AQP1) transmembrane channel protein of the human erythrocyte is known to be inhibited by organic mercurials such as p-chloromercuribenzoate (pCMB), which react with the free SH-group of the critical cysteine (Cys189) located near the constriction of the AQP1 water-specific channel. Sodium nitroprusside (SNP), which is known as a nitric oxide (NO) donor in interactions with SH-containing molecules, is shown here to suppress the diffusional water permeability (P(d)) of the erythrocyte membrane, presumably as a result of reaction with the Cys189 of the human erythrocyte AQP1 water channels. Further, treatment of erythrocytes with HgCl(2) is found to result in a cell volume decrease that can be related to activation of membrane K(+)-selective Gárdos channels and subsequent loss of intracellular K(+) and cell shrinkage. The variations in P(d) and volume of the erythrocyte were deduced from induced variations in the measured proton ((1)H) nuclear magnetic resonance (NMR) transverse (T(2)) relaxation functions of water exchanging between diamagnetic intracellular and paramagnetic extracellular compartments of the 20-25% hematocrit samples. The extracellular solvent contained 10 mM membrane-impermeable paramagnetic Mn(2+) ions. The (1)H-T(2) NMR technique allows determination of the time constant tau(exch) (for exchange of the erythrocyte intracellular water) that is inversely proportional to the permeability coefficient P(d) when the intracellular water volume is left unmodified, as in the case of SNP-treated erythrocytes. However, for HgCl(2)-treated erythrocytes, this technique showed simultaneous variation of both tau(exch) and the volume ratio V(in)/V(out) of intracellular and extracellular water in proportions suggesting that P(d) was left unmodified. The HgCl(2) effect has been found to be partly reversible by the reducing activity of added mercaptoethanol.

Endogenous antioxidant defence system in rat liver following mercury chloride oral intoxication.

Mercury is a highly toxic metal which induces oxidative stress. Superoxide dismutases, catalase, and glutathion peroxidase are proteins involved in the endogenous antioxidant defence system. In the present study rats were administered orally, by gavage, a single daily dose of HgCl2 for three consecutive days. In order to find a relation between the proteins involved in the antioxidant defence and mercury intoxication, parameters of liver injury, redox state of the cells, as well as intracellular protein levels and enzyme activities of Mn-dependent superoxide dismutase (MnSOD), Cu-Zn-dependent superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase (GPx) were assayed both in blood and in liver homogenates. HgCl2 at the doses of 0.1 mg/kg produced liver damage which that was detected by a slight increase in serum alanine aminotransferase and gamma glutamyl transferase. Hepatic GSH/GSSG ratio was assayed as a parameter of oxidative stress and a significant decrease was detected, as well as significant increases in enzyme activities and protein levels of hepatic antioxidant defence systems. Changes in both MnSOD and CuZnSOD were parallel to those of liver injury and oxidative stress, while the changes detected in catalase and GPx activities were progressively increased along with the mercury intoxication. Other enzyme activities related to the glutathione redox cycle, such as glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), also increased progressively. We conclude that against low doses of mercury that produce a slight oxidative stress and liver injury, the response of the liver was to induce the synthesis and activity of the enzymes involved in the endogenous antioxidant system. The activities of all the enzymes assayed showed a rapidly induced coordinated response.

2,3-Dimercapto-1-propanol does not alter the porphobilinogen synthase inhibition but decreases the mercury content in liver and kidney of suckling rats exposed to HgCl2.

Heavy metals have received great attention as environmental pollutants mainly because once introduced in the biological cycle they are incorporated in the food chain. Especially the mercury toxicity due to a diversity of effects caused by different chemical species should be emphasized. Heavy metal intoxication has been treated with chelating agents such as 2,3-dimercapto-1-propanol (BAL). However, the efficacy of this treatment is questionable due to the lack of specific effect on the toxic metal. The present study examined the effects of HgCl2 exposure (five doses of 5.0 mg/kg between ages 8 to 12 days) on physiological parameters, on porphobilinogen synthase activity, and on mercury content in liver, kidneys and brain from suckling rats. The effect of BAL (one dose of 12.5-75 mg/kg) applied 24 hr after mercury intoxication on these parameters was also investigated. The results demonstrate that HgCl2 intoxication induced a decrease of corporal weight gain as well as brain weight and an increase in renal weight. The inhibition of porphobilinogen synthase from liver and kidney, is still significant and was not modified by subsequent BAL treatment. However, BAL altered two effects induced by mercury: increase in death percentage and decrease in mercury contents in liver and kidney. The increase of mortality induced by mercury was not promoted by metal redistribution to brain nor by the increase of porphobilinogen synthase inhibition induced by metal. More investigations are necessary to determine if the different effects of BAL on intoxication by metals are possibly related to other tissues and/or if the probable metal-chelating complex formed is more toxic than the metal itself.

Costimulation requirements of induced murine systemic autoimmune disease.

Costimulation between T cells and APC is required for productive immune responses. A number of receptor/ligand pairs have been shown to mediate costimulation, including CD28/B7 molecules (CD80 and CD86), CD40/CD40 ligand (CD40L, CD154), and LFA-1 (CD18)/ICAM-1 (CD54). T-B cell costimulation also plays a significant role in autoimmune diseases such as systemic lupus erythematosus. Murine HgCl2-induced autoimmunity (mHgIA) is a T cell-dependent systemic autoimmune disease that shares a number of common pathogenic mechanisms with idiopathic lupus. In this report, the significance of costimulation in mHgIA is examined by attempting to induce disease in mice deficient in either CD40L, CD28, or ICAM-1. Unlike absence of ICAM-1, homozygous deficiencies in either CD40L or CD28 significantly reduced the development of mHgIA. CD40L displayed a gene dosage effect as heterozygous mice also showed reduction of autoantibody responses and immunopathology. Markers of T cell activation such as CD44 and CTLA-4 were associated with disease expression in wild-type and ICAM-1-deficient mice but not in CD40L- or CD28-deficient mice. Absence of CTLA-4 expression in CD40L-/- mice suggests that signaling via both CD28 and CD40L is important for T cell activation and subsequent autoimmunity in mHgIA. Attempts to circumvent the absence of CD40L by increasing CD28 signaling via agonistic Ab failed to elicit CTLA-4 expression. These findings indicate that breaking of self-tolerance in mHgIA requires signaling via both the CD28/B7 and CD40/CD40L pathways.

Importance of functional EGF receptors in recovery from acute nephrotoxic injury.

Previous studies have demonstrated increased renal expression of EGF receptor (EGFR) and EGFR ligands in response to acute toxic or ischemic renal tubular injury and have indicated that exogenous administration of EGF accelerates recovery from such injury. However, no studies to date have proved definitively an essential role for EGFR-mediated responses in regeneration after tubule injury. To this end, waved-2 (wa-2) mice, which contain a point mutation in EGFR that reduces receptor tyrosine kinase activity by >90%, were studied. These mice have a mild phenotype (wavy coat, curly whiskers, and runted stature) and normally developed kidneys. Acute nephrotoxic injury was induced in wa-2 and wild-type mice with HgCl(2). One day after HgCl(2) injection, functional renal compromise was comparable in wild-type and wa-2 mice. However, the rates of recovery of serum blood urea nitrogen and creatinine levels were markedly slower in wa-2 mice. Histologic evidence of tubular injury also was more severe and persisted longer in wa-2 mice. Furthermore, their kidneys demonstrated reduced levels of DNA synthesis and increased TdT-mediated dUTP nick-end labeling staining. These studies indicate that functional EGFR activity is an essential component of the kidney's ability to recover from acute injury and that EGFR may regulate genes involved in growth, repair, and cell survival in the kidney.

Neonatal tolerance to a Th2-mediated autoimmune disease generates CD8+ Tc1 regulatory cells.

Immunological tolerance can be achieved in animals by exposure of newborn to a foreign antigen. Depending on the dose and timing of the antigenic challenge, tolerance has been reported to result in clonal deletion, anergy or active suppression. In this latter case, regulatory T cells prevent autoimmunity by suppressing the reactivity of pathogenic self-reactive T cells. We have previously reported the generation of a neonatal, mercury-specific, and dominant tolerance to autoimmunity induced by mercury salts in rats. Chronic exposure to mercury salts can lead to SLE-like autoimmune responses, mediated by autoreactive CD4+ Th2 cells, that regulate and are followed by a resistant state mediated by protective CD8+ T cells. The aim of the study was to compare the resistance to the neonatal tolerance to mercury disease, and to further characterize the CD8+ T cells endowed with regulatory capacity in the neonatal tolerance model. We report here that resistance to mercury disease is long lasting and not mercury-specific, suggesting that different CD8+ T cells are involved in resistance and neonatal tolerance, and that regulatory CD8+ Tc1 cells generated in tolerance are required to control the CD8- cell population from developing Th2-mediated autoimmunity. Upon mercury recall, CD8+ CD45RC(high) T cells, that represent the Tc1 subset in the rat, expanded and were polarized towards IFNgamma production. Interestingly, identical results were obtained with the CD8+ CD25+T cell population. Substantial amounts of FasL gene expression were detected in CD8+ T lymphocytes upon recall with the tolerogen. AICD may be one of the regulatory mechanisms used by these regulatory CD8+ Tc1 cells that control neonatal tolerance to a Th2-mediated autoimmune disorder.

Inhibitory signal override increases susceptibility to mercury-induced autoimmunity.

After exposure to subtoxic doses of heavy metals such as mercury, H-2(s) mice develop an autoimmune syndrome consisting of the rapid production of IgG autoantibodies that are highly specific for nucleolar autoantigens and a polyclonal increase in serum IgG1 and IgE. In this study, we explore the role of two inhibitory immunoreceptors, CTLA-4 and FcgammaRIIB, in the regulation of mercury-induced autoimmunity. In susceptible mice treated with mercuric chloride (HgCl(2)), administration of a blocking anti-CTLA-4 Ab resulted in a further increase in anti-nucleolar autoantibodies and in total serum IgG1 levels. Furthermore, in some DBA/2 mice, which are normally resistant to heavy metal-induced autoimmunity, anti-CTLA-4 treatment leads to the production of anti-nucleolar Abs, thereby overcoming the genetic restriction of the disease. In mice deficient for the FcgammaRIIB, HgCl(2) administration did not trigger autoantibody production, but resulted in an increase in IgE serum levels. Taken together, these results indicate that different inhibitory mechanisms regulate various manifestations of this autoimmune syndrome.

Subcellular recruitment of fibrillarin to nucleoplasmic proteasomes: implications for processing of a nucleolar autoantigen.

A prerequisite for proteins to interact in a cell is that they are present in the same intracellular compartment. Although it is generally accepted that proteasomes occur in both, the cytoplasm and the nucleus, research has been focusing on cytoplasmic protein breakdown and antigen processing, respectively. Thus, little is known on the functional organization of the proteasome in the nucleus. Here we report that within the nucleus 20S and 26S proteasomes occur throughout the nucleoplasm and partially colocalize with splicing factor-containing speckles. Because proteasomes are absent from the nucleolus, a recruitment system was used to analyze the molecular fate of nucleolar protein fibrillarin: Subtoxic concentrations of mercuric chloride (HgCl(2)) induce subcellular redistribution of fibrillarin and substantial colocalization (33%) with nucleoplasmic proteasomes in different cell lines and in primary cells isolated from mercury-treated mice. Accumulation of fibrillarin and fibrillarin-ubiquitin conjugates in lactacystin-treated cells suggests that proteasome-dependent processing of this autoantigen occurs upon mercury induction. The latter observation might constitute the cell biological basis of autoimmune responses that specifically target fibrillarin in mercury-mouse models and scleroderma.

Mercury intoxication from skin ointment containing mercuric ammonium chloride.

OBJECTIVE: A one-year follow-up was performed of a 21-year-old man with a 16-year history of diabetes mellitus type I, who had been using ointment containing 10% mercuric ammonium chloride (hydrargyrum amidochloratum; HgNH(2)Cl) for eczema for approximately 3 weeks. Tiredness, fasciculations on the extremities and poor control of diabetes appeared after the end of the ointment treatment. Nephrotic syndrome and hypertension were diagnosed 1 month later. Two months after the ointment application the patient was very weak with tremors of the hands, almost unable to walk, and had lost 20 kg of body weight. He had severe neurasthenic symptoms and his behaviour suggested acute psychosis. METHODS: Internal, neurological and neuropsychological examinations were performed. Mercury in urine was determined by flameless atomic absorption spectrometry. RESULTS: The urine mercury level on admission was 252.0 microg/l. He was treated with Dimaval, sodium (2,3)-dimercaptopropane(-1)-sulphonate capsules for 12 days (total dose 6.3 g). The highest urine mercury excretion during antidote treatment was 2336.0 microg/24 h. The patient had proteinuria of up to 11.10 g/24 h, and renal biopsy revealed diffuse membranous glomerulonephritis of the 1st stage without apparent diabetic nephropathy. Similarly, neuropathy did not have typical signs of diabetic neuropathy. His clinical condition started to improve during the first 2 weeks. Further follow-up has shown slow normalisation of renal functions. After 1 year, proteinuria decreased to 0.62 g/24 h and body weight normalised. Neuropsychological and electromyographic findings became almost normal. CONCLUSION: Severe intoxication developed after a short period of ointment application. Most signs of damage disappeared in the course of 1 year, except mild proteinuria and neuropathy. The evolution was favourable and confirmed the primary role of mercury intoxication in the severe deterioration of the clinical status of the patient.

[Introduction: scope and purpose of the multicenter project " Assessment of effects due to low doses in inorganic mercury following environmental and occupational exposure: human and in vitro studies on specific toxicity mechanisms"]. / Introduzione: scopi e finalità del progetto multicentrico "Valutazione degli effetti conseguenti a basse dosi di mercurio inorganico da esposizioni ambientali ed occupazionali: studio dei meccanismi di tossicità specifica in vitro e nell'uomo".

The principal aims of the project financed by the Italian Ministry of University and Scientific and Technological Research were: to verify if at the current limit values early biological effects can be demonstrated; to identify the levels of internal dose that can cause early effects; to evaluate the non-occupational factors that can contribute to the levels of internal dose. In particular, the mercury intake derived from dental amalgams and fish consumption was considered. The internal dose was measured with the traditional biological indicators (urinary and blood mercury) and with the speciation of a large percentage of biological samples by ICP-MS. The central nervous system, neuroendocrine function, kidney and the immune system were considered as target organs and were examined using previously standardized indicators of effects. Two groups of subjects were included in the study: workers with occupational exposure to inorganic mercury in different industrial settings and control subjects identified from the general population. The first group was characterized by an exposure level to inorganic mercury clearly below the current limit values; whereas the HgU levels of a relevant number of control subjects were similar to those measured in the exposed subjects. The in vitro studies covered several issues: the percutaneous absorption of mercury using skin derived from human post-mortem samples in a standardized model; the release of the metal from dental amalgams in different physiological conditions of the oral cavity; the effects of increasing doses of mercury chloride on tubular renal cells. The project was realized with the cooperation of seven Research Units from six Italian Universities. Researchers belonging to Departments of Occupational Medicine, Industrial Hygiene, General Pathology, Biochemistry, Odontology, and Biostatistics were involved to achieve a multidisciplinary approach. The results of this research project are described and discussed in the following papers.

[In vitro study of the nephrotoxic mechanism of mercuric chloride]. / Studio in vitro dei meccanismi di nefrotossicità del cloruro di mercurio.

OBJECTIVES: Mercury (Hg), one of the most diffused and hazardous organ-specific environmental contaminants, exists in a wide variety of physical and chemical states, each of which with unique characteristics of target organ specificity. Exposure to Hg vapour and to organic mercurials specifically affects the CNS, while the kidney is the target organ for inorganic Hg compounds. Despite the increasing number of studies, the molecular bases of the nephrotoxic potential of Hg has not, up to now, been clarified, even if there is evidence suggesting that the ability of the metal to interact with proteins (thiol groups) or to generate oxygen radicals may play a major role. Within this context, the aim of the present study was to investigate, in vitro, the mechanism(s) of the early nephrotoxic potential of mercury chloride (HgCl2), one of the most diffused and biologically active mercury (Hg2+) compounds. For this purpose, two kidney-derived in vitro systems (the MDCK and the LLC-PK1 cell lines) were tested for their sensitivity to the salt, and MDCK was chosen as the most suitable in vitro model for our study. As possible biological markers of the organ-specific toxicity of the metal we analysed: i) critical biochemical parameters related to oxidative stress conditions (effect of Hg2+ on the anti-oxidant status of the cell), and ii) gap-junctional function (GJIC). METHODS: Classical toxicity tests (MTT and NR) were used for assessing the sensitivity (IC50) of LLP-CK1 and MDCK cell lines to the mercuric salt. Complete solubilisation of the salt in the culture media was verified by inductively coupled plasma mass spectrometry (ICP-MS). The influence of the metal on cell growth rate and viability were evaluated by conventional proliferation assays. For the following mechanistic studies, cells were exposed for different time periods (4 to 72 hours) to non-cytotoxic (0.1-50 microM) HgCl2 concentrations. The biochemical analysis of the pro-oxidant properties of the mercuric compound was performed by the measurement of anti-oxidant cellular defences against H2O2 [catalase (Cat), glutathione peroxidase (Gpx), and total glutathione (GSH)]. The influence of the metal on the GJIC capacity of MDCK cells was assessed by the "microinjection/dye-coupling" assay. RESULTS: Among the two kidney-derived in vitro systems, MDCK cell line was the most specifically sensitive to the toxic effect of HgCl2: it was, consequently, chosen as a "tubular cell model" for the following experimental steps. Tested for various time periods at increasing concentrations, the HgCl2 effect on MDCK cell proliferation and viability was found to be time- and dose-related. For concentrations < or = 50 microM, HgCl2 inhibits MDCK cell growth rate, being this effect significant (> 50% in respect to untreated controls) from the 24th from the beginning of the treatment, while, for concentrations > 50 microM, the metal causes cell death. Concerning the influence of HgCl2 on MDCK anti-oxidant defences, the most interesting results were obtained by analysing the influence of the mercury salt on the GSH cell content and Gpx activity. Both were, in fact, significantly affected by the presence of the mercury ion. HgCl2 also induced a rapid, dose- and time-related inhibitory effect on the GJIC capacity of the cells. CONCLUSIONS: Even if further investigations are needed to better clarify the possible causal relationship between our findings, they indicate that: a) MDCK cells represent a suitable in vitro model for the study of Hg nephrotoxicity; b) GJIC function is, among those considered in our study, one of the most sensitive biological endpoints for investigating the mechanism(s) of Hg2+ specific toxicity.