A systems toxicology approach identifies Lyn as a key signaling phosphoprotein modulated by mercury in a B lymphocyte cell model.

Network and protein-protein interaction analyses of proteins undergoing Hg²âº-induced phosphorylation and dephosphorylation in Hg²âº-intoxicated mouse WEHI-231 B cells identified Lyn as the most interconnected node. Lyn is a Src family protein tyrosine kinase known to be intimately involved in the B cell receptor (BCR) signaling pathway. Under normal signaling conditions the tyrosine kinase activity of Lyn is controlled by phosphorylation, primarily of two well known canonical regulatory tyrosine sites, Y-397 and Y-508. However, Lyn has several tyrosine residues that have not yet been determined to play a major role under normal signaling conditions, but are potentially important sites for phosphorylation following mercury exposure. In order to determine how Hg²âº exposure modulates the phosphorylation of additional residues in Lyn, a targeted MS assay was developed. Initial mass spectrometric surveys of purified Lyn identified 7 phosphorylated tyrosine residues. A quantitative assay was developed from these results using the multiple reaction monitoring (MRM) strategy. WEHI-231 cells were treated with Hg²âº, pervanadate (a phosphatase inhibitor), or anti-Ig antibody (to stimulate the BCR). Results from these studies showed that the phosphoproteomic profile of Lyn after exposure of the WEHI-231 cells to a low concentration of Hg²âº closely resembled that of anti-Ig antibody stimulation, whereas exposure to higher concentrations of Hg²âº led to increases in the phosphorylation of Y-193/Y-194, Y-501 and Y-508 residues. These data indicate that mercury can disrupt a key regulatory signal transduction pathway in B cells and point to phospho-Lyn as a potential biomarker for mercury exposure.

Therapeutic potential of N-acetyl cysteine with antioxidants (Zn and Se) supplementation against dimethylmercury toxicity in male albino rats.

Mercury (Hg) is currently one of the most prevalent pollutants in the environment. Many studies have examined its effects on the health of both humans and animals. Experimental studies have shown that sulfur-containing nutrients play an important role as detoxification and protecting cell against the detrimental properties of mercury. The present study was undertaken to elucidate the toxicity induced by dimethylmercury in male rats through the activities of transaminases, alkaline phosphatase, lactate dehydrogenase in serum and oxidative damage as acetyl cholinesterase activity in different regions of brain and lipid peroxidation, reduced glutathione content, mean DNA damage in liver, kidney and brain of rats given dimethylmercury (10 mg/kg, p.o., once only) along with combination therapy of N-acetyl cysteine (2 mM/kg, i.p.), zinc (2 mM/kg, p.o.) and selenium (0.5 mg/kg, p.o.) for 3 days. In the dimethylmercury group, activities of transaminases, alkaline phosphatase, lactate dehydrogenase in serum, level of lipid peroxidation, mean DNA damage and mercury ion concentration were significantly higher whereas reduced glutathione content and the activity of acetyl cholinesterase were significantly lower compared to controls (P≤0.05). Combined treatment of zinc and selenium with N-acetyl cysteine to dimethylmercury-exposed rats showed a substantial reduction in the levels of DMM-induced oxidative damage and comet tail length. In conclusion, the results of this study support that the supplementation of zinc and selenium with N-acetyl cysteine can improve the DMM induced blood and tissue biochemical oxidative stress and molecular alterations by recoupment in mean DNA damage.

Protective role of melatonin against the mercury induced oxidative stress in the rat thyroid.

Present study investigated the protective role of melatonin (MLT, 5mg/kg body wt., ip) against the long term effects of mercuric chloride (MC; 2 and 4 mg/kg body wt., po) in the thyroid gland of the rats through certain antioxidative indices like superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione (GSH), catalase (CAT) and lipid peroxidation (LPO), other biochemical parameters such as succinate dehydrogenase (SDH), adenosine triphosphatase (ATPase), acid phosphatase (ACPase) and alkaline phosphatase (ALPase) were also measured. Antioxidative enzymes and other parameters showed a significant reduction while LPO and mercury levels increased significantly in a dose dependent manner in MC treated animals as compared to control groups. Co-treatment with MLT revealed no significant effect on antioxidative and metabolic indices in the thyroid gland of rats. The results of present study thus strongly suggest that mercury affected antioxidant defense system and other metabolic enzymes of thyroid. Co-administration of melatonin exerted a protective effect against mercury induced endocrine toxicity.

l-Arginine normalizes NOS activity and zinc-MT homeostasis in the kidney of mice chronically exposed to inorganic mercury.

Inorganic mercury (HgCl2) exposure provokes damage in many organs, especially kidney. Inducible nitric oxide synthase (iNOS) expression, total NOS activity and the profiles of zinc (Zn), copper (Cu) and Hg as well as their distribution when bound to specific intracellular proteins, including metallothioneins (MT), were studied during HgCl2 exposure and after l-arginine treatment in C57BL/6 mouse kidney. HgCl2 exposure modulates differently iNOS expression and NOS activity, increasing iNOS expression but, conversely, decreasing total NOS activity in the mouse kidney. Moreover, during Hg exposure an increased MT production occurs. The kidney damage leads to a loss of urinary proteins, increased plasma creatinine and high Zn mobilization with consequent increased urinary Zn excretion. l-arginine treatment recovers NOS activity and induces a normalization of MT induction, plasma creatinine values and urinary proteins excretion, suggesting that l-arginine may limit kidney damages by Hg exposure.

Evaluation of protective efficacy of Spirulina fusiformis against mercury induced nephrotoxicity in Swiss albino mice.

The toxicity of mercury to animals and man is well established and this depends greatly on the form of the mercury compounds. In most animals' species, including man, the kidney is the main site of deposition of inorganic mercury and target organ for its toxicity. In the present study Spirulina fusiformis (a cyanobacterium, belongs to family--Oscillatoriaceae) has been investigated as a possible modifier of mercury induced renal damages in Swiss albino mice. Animals were divided into four groups. (i) Control group--only vehicle (0.9% NaCl) was administered as i.p. (ii) HgCl(2) treated group--5.0 mg/kg b.wt. HgCl(2) was administered as i.p. (iii) Spirulina treated group--800 mg/kg b.wt. Spirulina extract was administered orally. (iv) Combination group--S. fusiformis was administered 10 days before mercuric chloride administration and continued upto 30 days after mercuric chloride administration (5.0 mg/kg b.wt.). The animals were autopsied on 1, 3, 7, 15 and 30 days after treatment and the activity of alkaline phosphatase (ALP), acid phosphatase (ACP), lactate dehydrogenase (LDH) and MDA (malondialdehyde) level were measured in kidney homogenates. The results indicated that there was a time-dependent significant enhancement in MDA content and ACP activity and decrease in LDH and ALP activity observed after HgCl(2) treatment. Mercury intoxication also induces pathological alterations in the kidney such as degeneration of glomerulus, proximal and distal tubules. A dose-dependent mortality was also observed following administration of different doses of HgCl(2). In combined treatment of Spirulina with HgCl(2), a significant decrease in MDA content and ACP activity and elevation in LDH and ALP activity was observed as compared to HgCl(2) treated group. Spirulina pre- and post-treatment with mercury also significantly reduces pathological alterations in kidney. Thus, the results from the present study suggest that S. fusiformis can significantly modify the renal damages against mercuric chloride induced toxicity.

Short-interfering RNA-mediated silencing of thioredoxin reductase 1 alters the sensitivity of HeLa cells toward cadmium.

The mammalian thioredoxin reductase (TrxR) is a selenocysteine-containing flavoprotein that regulates the thioredoxin system, one of the major systems that maintain the intracellular redox balance. We previously reported that cytosolic TrxR (TrxR1), one of three mammalian TrxR isozymes, was induced by treatment with cadmium. In the present study, to study the role of cadmium-induced TrxR1 in cellular defense, we silenced the expression of TrxR1 in HeLa cells by using small interfering RNA and examined the effect of TrxR1 silencing on the sensitivity of the cells toward cadmium. We found that the gene silencing of TrxR1 had a dual effect on cadmium-induced cell death, depending on the concentration of cadmium. The TrxR1 silencing increased the sensitivity toward a low dose (less than 10 microM) of cadmium but decreased the sensitivity toward a high dose of cadmium. These results suggested that TrxR1 might play an important role in the cellular defense system against cadmium in two ways. TrxR1 might rescue the cells from a low dose of cadmium-induced moderate injury, while it might promote the death of cells severely injured by a high dose of cadmium.

Toxicity induced by Hg2+ on choline acetyltransferase activity from E. electricus (L.) electrocytes: the protective effect of 2,3 dimercapto-propanol (BAL).

BACKGROUND: The effect of mercury (Hg(2+)) on the activity of choline acetyltransferase (ChAT) from electrocytes of Electrophorus electricus (L.) was studied due to the importance of this enzyme and acetylcholine in many neurochemical functions such as arousal, learning, and memory. MATERIAL/METHODS: Mercury, which has affinity to thiol groups, acted as a potent inhibitor of ChAT, which was obtained by differential centrifugation and ammonium sulfate precipitation, at 80%, from the main electric organ homogenate. RESULTS: Mercury inhibition presents different kinetic behaviors for both enzyme substrates: noncompetitive to choline and of mixed type to AcCoA, with inhibition constants on the order of 0.5 to 1.0 microM. The enzyme activity was recovered using 2,3 dimercapto-propanol (BAL), a well-known chelate for sulphydryl groups and metals, which acted as a protecting agent and was able to revert the Hg(2+) inhibition at a concentration of 10 (-6) M. After treatment with this metal and in the presence of 2,3 dimercapto-propanol, 70% of the enzyme activity was recovered for AcCoA and 80% for choline. CONCLUSIONS: The observed inhibition is likely due to direct protein interaction, because the addition of BAL reversed the effects of HgCl(2) on ChAT activity. The results cast new light on the mechanisms of mercurial neurotoxicity.

Change in the iso-enzyme profiles of urinary N-acetyl-beta-D-glucosoaminidase in workers exposed to mercury.

The iso-enzyme profiles of urinary N-acetyl-beta-D-glucosoaminidase (NAG) were studied in the workers from a chloro-alkaline electrolysis plant that had been continuously exposed to elemental mercury. The same workers then had a work break of four months and were later re-exposed to mercury for another five months. The activities of urinary NAG in workers exposed to mercury before and after the work break and in the control group were 2.09 +/- 1.03 IU/L; 0.90 +/- 0.52 IU/L and 1.13 +/- 0.35 IU/L, respectively. The fraction of the dominant A form activity in the total activity, in the workers exposed to mercury after a work break (85.31 +/- 5.32%), was almost equal to the fraction of A in the control group (84.64 +/- 2.75%). The percent fraction of A forms (A and A2) during continuous exposure was almost equal to the sum of the fractions A and A2 in the control group and for exposure after the work break. The fraction of the B form activity in the total activity during exposure to elemental mercury for five months after the work break (7.41 +/- 3.45%) was lower (P < 0.1) than the fraction of the B form in the control group (8.62 +/- 2.19%). The decrease in the fraction of the B form compared with the control group was more significant (P < 0.001) in the case of continuously exposed workers (5.25 +/- 2.55%). Beside the major A and B iso-enzymes of NAG, the A2 form was also isolated, and its fraction in the control group (6.73 +/- 2.15%) was not negligible. For workers exposed to mercury for five months, it was 7.28 +/- 3.52%. It is concluded that mercury affected the increased exocytosis of iso-enzyme A and the inhibition of B iso-enzyme of NAG. The increase in activity of urinary NAG in subjects exposed to mercury was a consequence of the increased excretion of A form. It was established also that the four months work break was sufficient for the repair of renal damages that occurred during continuous exposure, and which were monitored by the activity of NAG. Thus, the effects of mercury seem to have been reversible.

Relationship between allozyme genotype and sensitivity to stressors in the western mosquitofish Gambusia affinis detected for elevated temperature but not mercury.

Previous studies with Gambusia holbrooki have found associations of allozyme genotype with tolerance to metals, pesticides, heat, and salinity. To examine the generality of these relationships, we looked for similar associations of mercury and heat tolerance with allozyme genotype at the GPI-2, MDH-1 and MDH-2 loci in its sister species Gambusia affinis. This was done to assess if the loci themselves or closely linked loci were associated with mercury tolerance, because weaker linkage associations would be unlikely to persist across species boundaries. Moreover, the use of two very different types of stress allowed us to determine if the higher tolerance of particular allozyme genotypes is specific to a certain stress or reflects a higher tolerance to stress in general. Associations between genotype and tolerance to mercury and heat were determined in laboratory exposures of about 875 fish in each of two exposures where tolerance was measured as time-to-death, followed by electrophoresis on cellulose acetate gels. For none of the three loci did we find an association of genotype with tolerance to mercury. This contrasts with reports of such an association for GPI-2 and MDH-1 in G. holbrooki, so our results do not support the hypothesis that observed associations between allozyme genotypes and mercury tolerance are due to the allozymes or closely linked loci. However, our comparison was weakened by a scarcity of the GPI-2 genotypes reported to be mercury sensitive in G. holbrooki. Furthermore, rapid mortality in our mercury exposure may have affected the ability to detect genotypic differences in survival. The MDH-1 heterozygote showed higher tolerance to heat stress compared to homozygotes, although this difference was only significant for the most common homozygous genotype. No such relationship between MDH-1 and heat stress has been reported in G. holbrooki. We found no evidence that associations between allozyme genotype and tolerance are similar for different types of stresses, which could be an advantage for using allozymes as an indicator of exposure history to a stressor. Our study also showed that G. affinis sex and weight influence tolerance to mercury and heat.

Evaluation of ppk-specified polyphosphate as a mercury remedial tool.

To evaluate the utility of polyphosphate kinase gene (ppk)-specified polyphosphate in mercury remediation, a fusion plasmid, pMK27, with ppk from Klebsiella aerogenes and mercury transport genes, merT and merP, from Pseudomonas K-62, was constructed. The transcription and translation of ppk, merT and merP were found to be mercury-inducible. The ppk-specified polyphosphate was identified in cells preinduced by Hg2+, but not in cells without mercury induction, suggesting that the synthesis of polyphosphate is regulated by merR. The hypersensitive phenotype to Hg2+, shown by bacteria with pMRD141, which contains merT and merP, was almost completely restored to its original levels when the ppk was introduced into the plasmid, suggesting that the Hg2+-toxicity was reduced by the polyphosphate, probably via chelation formation. Bacteria with pMK27 accumulated approximately 6-fold more mercury than the bacteria with cloning vector, pUC119. These results clearly demonstrate that the polyphosphate is capable of retaining mercury in the cells without taxing the cells. Based on the results obtained in the present study, the fusion plasmid pMK27 may serve as a strategy for mercury remediation.

Increased levels of glutathione S transferases and appearance of novel alpha class isoenzymes in kidneys of mice exposed to mercuric chloride. I. Biochemical and immunohistochemical studies.

Glutathione S transferases (GST) are a family of enzymes that detoxify electrophilic xenobiotics. This enzyme family was examined in kidneys of mice exposed to mercuric chloride, a known nephrotoxin, because GST have been shown to protect cells against toxicant-induced damage and may serve as biomarkers for toxicant exposure. The purpose of this study was to determine the effect of mercuric chloride on GST activity, isoenzyme levels, and cellular localization in the kidney of Swiss Webster mice. The cellular localizations of alpha, mu, and pi class GST in the kidneys of control and mercuric chloride treated mice were studied immunohistochemically. The GST isoenzyme levels were measured by high-performance liquid chromatography. The mice treated with mercuric chloride had (1) increased amounts of GSTA1/A2 protein in kidney homogenates as compared with controls when analyzed by chromatography and electrophoresis; (2) two new isoforms of the alpha isoenzyme in kidney as demonstrated by Western blot, polyacrylamide gel electrophoresis, and high-performance liquid chromatography, and (3) increased reactivity between antibodies, against GSTA1/A2 or GSTM1 isoenzymes, and cells in the proximal and distal renal tubules as shown by immunohistochemical techniques. The authors conclude that the GSTA1/A2 may protect those cells in the proximal and distal tubules of the renal cortex from toxicant effects of mercuric chloride. This would be one general mechanism for cell protection against a wide variety of toxicants including heavy metals and halogenated aromatics.

Catalase and superoxide dismutase activities as biomarkers of oxidative stress in workers exposed to mercury vapors.

For this article we investigated the role of three blood antioxidant enzyme activities and total antioxidant status (TAS) as biological markers of oxidative stress in workers exposed to mercury (Hg(o)) vapors. Twenty-two female workers took part in the study. The examination included a questionnaire on age, educational level, occupational history, actual health status, previous accidents and diseases, smoking and dietary habits, and alcohol consumption. Blood and urine sampling for biological analyses completed this examination. The workers were classified into three subgroups according to their creatinine-corrected Hg concentration in urine. Blood antioxidant enzyme activities and TAS were compared between groups with nonparametric distribution-free methods. A significant difference existed in catalase activity and a slight, but not significant, difference existed in Cu2+/Zn2+ superoxide dismutase (Cu2+/Zn2+ SOD) activity between the three groups. No differences were observed in either the glutathione peroxidase activity or the TAS between these groups. Catalase and Cu2+/Zn2+ SOD activities were increased in the groups of workers with higher creatinine-corrected urinary Hg concentrations when compared with the group of lower creatinine-corrected urinary Hg concentrations. Catalase activity was positively correlated with the creatinine-corrected concentration of Hg in urine, and Cu2+/Zn2+ SOD activity was slightly correlated with the creatinine-corrected concentration of Hg in urine. The role of erythrocyte catalase and Cu2+/Zn2+ SOD activities we have measured is in agreement with the hypothesis of the involvement of reactive oxygen species production as an important event in chronic exposure to Hg(o) vapors in humans. In spite of the small size of the sample, these results indicate that erythrocyte catalase and Cu2+/Zn2+ SOD activities could be considered as markers of biological effect in workers exposed to Hg(o) vapors.

Influence of mercury and cadmium intoxication on hepatic microsomal CYP2E and CYP3A subfamilies.

The effects of subchronic mercuric acetate and of acute mercuric acetate and cadmium chloride administration to rat hepatic microsomal protein and total cytochrome P450, as well as on p-nitrophenol hydroxylase (CYP2E) and erythromycin N-demethylase (CYP3A) activities were examined. It was found that Hg2+ and Cd2+ intoxication resulted in a significant decrease of total cytochrome P450 content. Acute Hg2+ and Cd2+ exposure decreased microsomal protein level. These metals also reduced CYP2E1 activity, while they did not seem to alter CYP3A1-mediated drug metabolism. This effect on CYP2E1 may be connected to free radical generation during Hg2+ and Cd2+ intoxication. Investigation is in progress using more P450 markers for elucidation of the effect of Hg2+ and Cd2+ on P450 activities.

[The criterial significance of the histochemical manifestations of the nephrotoxic effect of mercury in its complex uptake into the body]. / Kriterial'naia znachimost' gistokhimicheskikh proiavlenii nefrotoksicheskogo éffekta rtuti pri kompleksnom postuplenii ee v organizm.

Certain aspects of kidney metabolism under a complex organism exposure to mercury have been studied. Different kinds of the heavy metal effect on various chains of metabolism have been established, dependent on the concentration and duration of mercury intake, which is evidence of the many mechanisms and ways of detoxication process. Oxido-reduction enzymes activity is changed due to direct mercury exposure, as well as due to disturbances in boundness of metabolic processes in which these enzymes are involved. The peculiarity of thiolysis mercury effect is the influence of mercury on protein metabolism enzymes--glutamate dehydrogenase (GDG) and NADF-dependent GDG, which is closely related to restructuring of oxidation-reduction kidney functions.

Increased urinary enzyme excretion in workers exposed to nephrotoxic chemicals.

Nephrotoxic chemicals are commonly present in the environment, particularly in the workplace. The level of occupational exposure to these chemicals has been so reduced that exposure to these agents now rarely causes clinically evident acute renal disease. A sensitive indicator of renal injury, urinary excretion of N-acetyl-beta-glucosaminidase, was utilized to evaluate persons exposed in the workplace to lead, mercury, or organic solvents, for evidence of renal effects from this exposure. None of the persons had clinically evident renal disease by history, none had hypertension, and all had normal findings on urinalysis. When compared with appropriate control populations, workers exposed to lead, workers exposed to mercury, and two of three groups of workers exposed to organic solvents had significant increases in urinary acetyl glucosaminidase activity. The third group of laboratory workers with low exposure to organic solvents had no increase in urinary acetyl glucosaminidase activity. It is concluded that exposure to environmental nephrotoxins at levels currently considered safe can produce renal effects as manifested by elevations of urinary acetyl glucosaminidase excretion. It is speculated that these renal effects are not always innocuous.

[Experimental chronic poisoning by mercury and alcohol: histopathologic, ultrastructural findings and toxicologic and enzymatic evaluations]. / Intossicazione cronica sperimentale da mercurio ed alcool: reperti istopatologici, ultrastrutturali e dosaggi tossicologici ed enzimatici.

Rats were treated with HgCl2 and/or alcohol per os in a standard diet as to get a chronic intoxication. Histological and ultrastructural examination of the liver, GPT, GOT, LDH enzymatic activities, and toxicological mercury evaluation were performed. Liver cell degenerative changes with focal necrosis, and structural alteration and fibrosis were demonstrated in the group of rats treated with HgCl2. The combined administration of HgCl2 and alcohol did not result in more advanced lesions, even if steatosis could be demonstrated in the liver. An increase of the GPT, GOT and LDH enzymatic activities was demonstrated in the rats of both groups but it was higher in the rats treated with HgCl2 and alcohol combined. On the contrary, the liver and kidney mercury storage was higher in the rats treated with HgCl2 alone.