In vitro study on antagonism mechanism of glutathione, sodium selenite and mercuric chloride.

It has been broadly recognized that the antagonism between selenium (Se) and mercury (Hg) can reduce the toxicity of mercury in organism. Glutathione (GSH) can participate in the metabolism of Se and Hg in vivo and promote the formation of low-toxic Hg-Se complexes, which is a vital way of detoxification for Hg. In this paper, the reaction mechanism of GSH-Se(IV) binary system, GSH-Hg(II) binary system and GSH-Se(IV)-Hg(II) ternary system were systematically studied from the aspects of stoichiometry, thermodynamics and kinetics, via hyphenated techniques including high performance liquid chromatography (HPLC)-ultraviolet (UV) detection, HPLC-inductively coupled plasma mass spectrometry (ICP-MS) and HPLC-electrospray ionization mass spectrometry (ESI-MS). For GSH-Se(IV) binary system, selenodiglutathione (GSSeSG) was the crucial intermediate; the reaction was exothermic and irreversible at constant pressure; it followed second-order kinetics with a fast kinetics (rate constant (k)=4534.2mol-1Ls-1). For GSH-Se(IV)-Hg(II) ternary system, GSSeSeSG would form by the extremely weak dissociation of two molecules of GSSeSG; Hg(II) would rapidly coordinate with GSSeSeSG to generate (HgxSey)n(GS)m precipitates. The mechanism of GSH-Se(IV)-Hg(II) antagonism system involves two processes, the competitive combination of Hg and Se with GSH and the formation of (HgxSey)n(GS)m complexes.

Pretreatment with diallylsulphide modulates mercury-induced neurotoxicity in male rats.

Many studies have reported on the toxicity and related oxidative stress of mercury. Antioxidants play an important role in counteracting metal-induced neurotoxicity under in vivo conditions. In this study, the effect of diallylsulphide (DAS) was evaluated on mercuric chloride induced activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione content in brains of rats. Pretreatment of rats with DAS in the Hg-treated group also inhibited an increase in lipid peroxidation and elevated acetyl cholinesterase and glutathione content. Activities of antioxidant enzymes were also restored concomitantly when compared to the control rats after DAS administration. DAS also caused a decrease in tumor necrosis factor-α level which was higher in HgCl2-treated group. The results indicate that DAS augments antioxidant defense with anti-inflammatory response against HgCl2-induced neurotoxicity. The increased level of antioxidant enzymes enhances the antioxidant potential of the organ to reduce oxidative stress.

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.

Is the lobster cockroach Nauphoeta cinerea a valuable model for evaluating mercury induced oxidative stress?

Organic and inorganic forms of mercury are highly neurotoxic environmental contaminants. The exact mechanisms involved in mercury neurotoxicity are still unclear. Oxidative stress appears to play central role in this process. In this study, we aimed to validate an insect-based model for the investigation of oxidative stress during mercury poisoning of lobster cockroach Nauphoeta cinerea. The advantages of using insects in basic toxicological studies include the easier handling, rapid proliferation/growing and absence of ethical issues, comparing to rodent-based models. Insects received solutions of HgCl2 (10, 20 and 40mgL(-1) in drinking water) for 7d. 24h after mercury exposure, animals were euthanized and head tissue samples were prepared for oxidative stress related biochemical determinations. Mercury exposure caused a concentration dependent decrease in survival rate. Cholinesterase activity was unchanged. Catalase activity was substantially impaired after mercury treatment 40mgL(-1). Likewise, GST had a significant decrease, comparing to control. Peroxidase and thioredoxin reductase activity was inhibited at concentrations of 20mgL(-1) and 40mgL(-1) comparing to control. These results were accompanied by decreased GSH levels and increased hydroperoxide and TBARS formation. In conclusion, our results show that mercuric compounds are able to induce oxidative stress signs in insect by modulating survival rate as well as inducing impairments on important antioxidant systems. In addition, our data demonstrates for the first time that Nauphoeta cinerea represents an interesting animal model to investigate mercury toxicity and indicates that the GSH and thioredoxin antioxidant systems plays central role in Hg induced toxicity in insects.

[Research on mercury methylation by Geobacter sulfurreducens and its influencing factors].

Mercury methylation by Geobacter sulfurreducens and the effects of environmental factors were studied under laboratory conditions. The results showed that G. sulfurreducens could grow well in the presence of low concentrations of mercuric chloride, but its growth was inhibited to a certain extent, mainly expressed in the prolonged lag phase. G. sulfurreducens could transform inorganic mercury into methylmercury, and this process was affected by many environmental factors. The efficiency of mercury methylation reached 38% under anaerobic conditions with 1 mg x L(-1) HgCl2 and 0.9% salinity at 35 degrees C, pH 6.0. Increasing the initial HgCl2 concentration or salinity in an appropriate manner improved mercury methylation, but the concentration of methylmercury reduced when the concentrations of HgCl2 and salinity were too high. The efficiency of mercury methylation increased with the increasing temperature in range of 4-35 degrees C. Weakly acidic environment was more beneficial to mercury methylation than acidic, neutral or alkaline conditions. In addition, the efficiency of mercury methylation was also affected by humic acid and cysteine. Humic acid inhibited mercury methyaltion, whereas cysteine could improve the efficiency of mercury methylation. This study provided a direct evidence for mercury methylation mediated by iron-reducing bacteria in the natural aquatic ecosystem.

Mercuric compounds induce pancreatic islets dysfunction and apoptosis in vivo.

Mercury is a toxic heavy metal that is an environmental and industrial pollutant throughout the world. Mercury exposure leads to many physiopathological injuries in mammals. However, the precise toxicological effects of mercury on pancreatic islets in vivo are still unclear. Here, we investigated whether mercuric compounds can induce dysfunction and damage in the pancreatic islets of mice, as well as the possible mechanisms involved in this process. Mice were treated with methyl mercuric chloride (MeHgCl, 2 mg/kg) and mercuric chloride (HgCl(2), 5 mg/kg) for more than 2 consecutive weeks. Our results showed that the blood glucose levels increased and plasma insulin secretions decreased in the mice as a consequence of their exposure. A significant number of TUNEL-positive cells were revealed in the islets of mice that were treated with mercury for 2 consecutive weeks, which was accompanied by changes in the expression of the mRNA of anti-apoptotic (Bcl-2, Mcl-1, and Mdm-2) and apoptotic (p53, caspase-3, and caspase-7) genes. Moreover, plasma malondialdehyde (MDA) levels increased significantly in the mice after treatment with mercuric compounds for 2 consecutive weeks, and the generation of reactive oxygen species (ROS) in the pancreatic islets also markedly increased. In addition, the mRNA expression of genes related to antioxidation, including Nrf2, GPx, and NQO1, were also significantly reduced in these islets. These results indicate that oxidative stress injuries that are induced by mercuric compounds can cause pancreatic islets dysfunction and apoptosis in vivo.

Hg L3 XANES study of mercury methylation in shredded Eichhornia crassipes.

Eichhornia crassipes (water hyacinth) is a non-native plant found in abundance in the Sacramento-San Joaquin River Delta (hereafter called Delta). This species has become a problem, clogging waterways and wetlands. Water hyacinth are also known to accumulate mercury. Recent attempts to curb its proliferation have included shredding with specialized boats. The purpose of this research is to better understand the ability of water hyacinth to phytoremediate mercury and to determine the effect of shredding and anoxic conditions on mercury speciation in plant tissue. In the field assessment, total mercury levels in sediment from the Dow Wetlands in the Delta were found to be 0.273 +/- 0.070 ppm Hg, and levels in hyacinth roots and shoots from this site were 1.17 +/- 0.08 ppm and 1.03 +/- 0.52 ppm, respectively, indicating bioaccumulation of mercury. Plant samples collected at this site were also grown in nutrient solution with 1 ppm HgCl2 under (1) aerobic conditions, (2) anaerobic conditions, and (3)with shredded plant material only. The greatest accumulation was found in the roots of whole plants. Plants grown in these conditions were also analyzed at Stanford Synchrotron Radiation Laboratory using Hg L3 X-ray Absorption Near Edge Spectroscopy (XANES), a method to examine speciation that is element-specific and noninvasive. Least-squares fitting of the XANES data to methylated and inorganic mercury(II) model compounds revealed that in plants grown live and aerobically, 5 +/- 3% of the mercury was in the form of methylmercury, in a form similar to methylmercury cysteine. This percentage increased to 16 +/- 4% in live plants grown anaerobically and to 22 +/- 6% in shredded anaerobic plants. We conclude that shredding of the hyacinth plants and, in fact, subjection of plants to anaerobic conditions (e.g., as in normal decay, or in crowded growth conditions) increases mercury methylation. Mechanical removal of the entire plant is significantly more expensive than shredding, but it may be necessary to avoid increased biomagnification of mercury in infested areas.

Cysteine 155 plays an important role in the assembly of Mycobacterium tuberculosis FtsZ.

The assembly of FtsZ plays an important role in bacterial cell division. Mycobacterium tuberculosis FtsZ (MtbFtsZ) has a single cysteine residue at position 155. We have investigated the role of the lone cysteine residue in the assembly of MtbFtsZ using different complimentary approaches, namely chemical modification by a thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or a cysteine-chelating agent HgCl(2), and site-directed mutagenesis of the cysteine residue. HgCl(2) strongly reduced the polymerized mass of MtbFtsZ while it had no detectable effect on the polymerization of Escherichia coli FtsZ, which lacks a cysteine residue. HgCl(2) inhibited the protofilamentous assembly of MtbFtsZ and induced the aggregation of the protein. Further, HgCl(2) perturbed the secondary structure of MtbFtsZ and increased the binding of a hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) with MtbFtsZ, indicating that the binding of HgCl(2) altered the conformation of MtbFtsZ. Chemical modification of MtbFtsZ by DTNB also decreased the polymerized mass of MtbFtsZ. Further, the mutagenesis of Cys-155 to alanine caused a strong reduction in the assembly of MtbFtsZ. Under assembly conditions, the mutated protein formed aggregates instead of protofilaments. Far-UV CD spectroscopy and ANS binding suggested that the mutated MtbFtsZ has different conformation than that of the native MtbFtsZ. The effect of the mutation or chemical modification of Cys-155 on the MtbFtsZ assembly has been explained considering its location in the MtbFtsZ crystal structure. The results together suggest that the cysteine residue (Cys-155) of MtbFtsZ plays an important role in the assembly of MtbFtsZ into protofilaments.

Scalp hair as a biomarker in environmental and occupational mercury exposed populations: suitable or not?

Hair is a well-established and widely used matrix for measuring mercury exposure of an individual. Although a variety of washing procedures to remove external mercury contamination have been proposed, no standardized procedures are available yet. In this study, different washing reagents like l-cysteine (Cys), 2-mercaptoethanol (ME), and disodium diaminoethanetetra acetate (EDTA) were used to find out if it is possible to remove mercury contamination from human scalp hair spiked with HgCl2 solutions at different concentrations. It was found that the external mercury contamination could not be fully washed off even using reagents with high affinity to mercury like l-cysteine and ME. However, for the well-pulverized CRM hair samples some of the endogenous mercury was washed off. It suggests that hair is not a suitable biomarker for evaluation of total mercury exposure especially in people like mercury miners or gold miners/burners associated with serious external Hg exposure. However, hair still can be used as an indicator for methyl mercury exposure because, generally, there is almost no exogenous contamination of methyl mercury in hair.

Sonolytic desorption of mercury from aluminum oxide: effects of pH, chloride, and organic matter.

The effects of pH, Cl-, and humic acid (HA) on sonolytic desorption of Hg(II) from aluminum oxide were examined. Results showed that Hg(II) desorption was achieved by lowering the pH from 7.0 to 4.0. Ultrasound enhanced Hg(II) release at short times compared to both hydrodynamic mixing and that expected on the basis of the pH-dependent Hg(II) adsorption curve. However, prolonged sonication led to decreases in Hg(II) desorption due to occlusion by aluminum hydroxide precipitation induced by ultrasound. The presence of Cl- greatly improved Hg(II) desorption at pH 4.0 due to the formation of stable nonadsorbing HgCl2(0) complexes at low pH, reducing free Hg(II) ion in solution. However, Cl- did not affect Hg(ll) desorption at pH 8.0, where Hg(OH)2(0) is the dominant Hg species rather than HgCl2(0). Hg(ll) desorption from HA-laden Al2O3 was dominated by HA. The greater the desorption of HA, the greater the desorption of Hg(II). Ultrasound enhanced the initial Hg(II) release by facilitating HA desorption. However, decreases in Hg(II) desorption were observed over longer sonication times due to the sonochemically induced reassociation of desorbed HA back onto Al2O3. Information obtained in this study provides insight into understanding sonolytic release of Hg from Hg-contaminated particles and sediments.

Mechanisms of cholinesterase inhibition by inorganic mercury.

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.

Structural basis of aquaporin inhibition by mercury.

The aquaporin family of channels was defined based on the inhibition of water transport by mercurial compounds. Despite the important role of mercurials, little is known about the structural changes involved upon mercury binding leading to channel inhibition. To elucidate the mechanism we designed a mutant, T183C, of aquaporin Z (AqpZ) patterned after the known mercury-sensitive site of aquaporin 1 (AQP1) and determined the X-ray crystal structures of the unbound and mercury blocked states. Superposition of the two structures shows no conformational rearrangement upon mercury binding. In the blocked structure, there are two mercury sites, one bound to Cys183 and occluding the pore, and a second, also bound to the same cysteine but found buried in an interstitial cavity. To test the mechanism of blockade we designed a different mutant, L170C, to produce a more effective mercury block at the pore site. In a dose-response inhibition study, this mutant was 20 times more sensitive to mercury than wild-type AqpZ and four times more sensitive than T183C. The X-ray structure of L170C shows four mercury atoms at, or near, the pore site defined in the T183C structure and no structural change upon mercury binding. Thus, we elucidate a steric inhibition mechanism for this important class of channels by mercury.

The adsorptive capacity of vapor-phase mercury chloride onto powdered activated carbon derived from waste tires.

Injection of powdered activated carbon (PAC) upstream of particulate removal devices (such as electrostatic precipitator and baghouses) has been used effectively to remove hazardous air pollutants, particularly mercury-containing pollutants, emitted from combustors and incinerators. Compared with commercial PACs (CPACs), an alternative PAC derived from waste tires (WPAC) was prepared for this study. The equilibrium adsorptive capacity of mercury chloride (HgCl2) vapor onto the WPAC was further evaluated with a self-designed bench-scale adsorption column system. The adsorption temperatures investigated in the adsorption column were controlled at 25 and 150 degrees C. The superficial velocity and residence time of the flow were 0.01 m/sec and 4 sec, respectively. The adsorption column tests were run under nitrogen gas flow. Experimental results showed that WPAC with higher Brunauer-Emmett-Teller (BET) surface area could adsorb more HgCl2 at room temperature. The equilibrium adsorptive capacity of HgCl2 for WPAC measured in this study was 1.49 x 10(-1) mg HgCl2/g PAC at 25 degrees C with an initial HgCI2 concentration of 25 microg/m3. With the increase of adsorption temperature < or = 150 degrees C, the equilibrium adsorptive capacity of HgCl2 for WPAC was decreased to 1.34 x 10(-1) mg HgCl2/g PAC. Furthermore, WPAC with higher sulfur contents could adsorb even more HgCl2 because of the reactions between sulfur and Hg2+ at 150 degrees C. It was demonstrated that the mechanisms for adsorbing HgCl2 onto WPAC were physical adsorption and chemisorption at 25 and 150 degrees C, respectively. Experimental results also indicated that the apparent overall driving force model appeared to have the good correlation with correlation coefficients (r) > 0.998 for HgCl2 adsorption at 25 and 150 degrees C. Moreover, the equilibrium adsorptive capacity of HgCl2 for virgin WPAC was similar to that for CPAC at 25 degrees C, whereas it was slightly higher for sulfurized WPAC than for CPAC at 150 degrees C.

Determination of the adsorptive capacity and adsorption isotherm of vapor-phase mercury chloride on powdered activated carbon using thermogravimetric analysis.

This study investigated the use of thermogravimetric analysis (TGA) to determine the adsorptive capacity and adsorption isotherm of vapor-phase mercury chloride on powdered activated carbon (PAC). The technique is commonly applied to remove mercury-containing air pollutants from gas streams emitted from municipal solid waste incinerators. An alternative form of powdered activated carbon derived from a pyrolyzed tire char was prepared for use herein. The capacity of waste tire-derived PAC to adsorb vapor-phase HgCl2 was successfully measured using a self-designed TGA adsorption system. Experimental results showed that the maximum adsorptive capacities of HgCl2 were 1.75, 0.688, and 0.230 mg of HgCl2 per gram of powdered activated carbon derived from carbon black at 30, 70, and 150 degrees C for 500 microg/m3 of HgCl2, respectively. Four adsorption isotherms obtained using the Langmuir, Freundlich, Redlich-Peterson, and Brunauer-Emmett-Teller (BET) models were used to simulate the adsorption of HgCl2. The comparison of experimental data associated with the four adsorption isotherms indicated that BET fit the experimental results better than did the other isotherms at 30 degrees C, whereas the Freundlich isotherm fit the experimental results better at 70 and 150 degrees C. Furthermore, the calculations of the parameters associated with Langmuir and Freundlich isotherms revealed that the adsorption of HgCl2 by PAC-derived carbon black favored adsorption at various HgCl2, concentrations and temperatures.

Mercury analysis of acid- and alkaline-reduced biological samples: identification of meta-cinnabar as the major biotransformed compound in algae.

The biotransformation of Hg(II) in pH-controlled and aerated algal cultures was investigated. Previous researchers have observed losses in Hg detection in vitro with the addition of cysteine under acid reduction conditions in the presence of SnCl2. They proposed that this was the effect of Hg-thiol complexing. The present study found that cysteine-Hg, protein and nonprotein thiol chelates, and nucleoside chelates of Hg were all fully detectable under acid reduction conditions without previous digestion. Furthermore, organic (R-Hg) mercury compounds could not be detected under either the acid or alkaline reduction conditions, and only beta-HgS was detected under alkaline and not under acid SnCl2 reduction conditions. The blue-green alga Limnothrix planctonica biotransformed the bulk of Hg(II) applied as HgCl2 into a form with the analytical properties of beta-HgS. Similar results were obtained for the eukaryotic alga Selenastrum minutum. No evidence for the synthesis of organomercurials such as CH3Hg+ was obtained from analysis of either airstream or biomass samples under the aerobic conditions of the study. An analytical procedure that involved both acid and alkaline reduction was developed. It provides the first selective method for the determination of beta-HgS in biological samples. Under aerobic conditions, Hg(II) is biotransformed mainly into beta-HgS (meta-cinnabar), and this occurs in both prokaryotic and eukaryotic algae. This has important implications with respect to identification of mercury species and cycling in aquatic habitats.

Zinc(II), cadmium(II) and mercury(II) complexes of the vitamin B1 antagonist oxythiamine.

The reaction of oxythiamine chloride hydrochloride (HOxTCl x HCl) with ZnCl2, CdCl2 and HgCl2 in ethanol yielded the complexes [ZnCl3(HOxT)], [CdCl3(HOxT)] and [HgCl3(HOxT)]. In water, the reaction with CdCl2 afforded [CdCl2(OxT)], but reaction with ZnCl2 or HgCl2 yielded unidentified products. The four new complexes were characterized by mass spectrometry and IR spectroscopy in the solid state and by 1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy in hexadeuterated dimethylsulfoxide (DMSO-d6), and three were also studied by X-ray diffractometry. In [ZnCl3(HOxT)] and [HgCl3(HOxT)] the oxythiamine ligand is bound to the metal via N(1') and adopts the V conformation exhibited by thiamine in biological contexts. The infrared (IR) spectrum of [CdCl3(HOxT)] suggests a similar coordination mode. In [CdCl2(OxT)] each OxT zwitterion coordinates to one Cd(II) ion via its N(1') atom and to another via its N(3') and O atoms, giving rise to a polymeric chain along the x-axis. The coordination number of the metal is made up to six by Cdc...Cl interactions, two of which link the polymeric chains in pairs. This seems to be the first metal complex containing the oxythiamine ligand as a zwitterion, with the N(3')-H/O(4'alpha)-H group deprotonated. Neither HOxTCl nor its zinc(II) complex showed any significant activity in vitro against HeLa cells.

Mercurophilic interaction in novel polynuclear Hg(II)-2-aminoethanethiolates.

The reaction of 2-aminoethanethiol hydrochloride with Hg(2)Cl(2) in water yielded elemental mercury and one-dimensional polynuclear compounds [{Hg(3)Cl(5)(SCH(2)CH(2)NH(3))(3)}Cl](n) and [HgCl(SCH(2)CH(2)NH(2))(H(2)O)(2)](n) (2). The coordination environment around Hg in 1 and 2 is quite variable despite similar reaction conditions. The formation of a five-membered S/N chelate in 2 can be attributed to the use of base to produce a neutral ligand. Three independent types of Hg atoms, both three- and four-coordinate are observed in 1, whereas in 2, the Hg atom is tetracoordinate with S, N and Cl atoms in the primary coordination sphere. Despite distinct structural chemistry, the coordination environments in 1 and 2 are fairly similar with repeating units connected with bridged thiolate S atoms in addition to a terminal Cl attached to Hg. Intermolecular hydrogen-bonding involving amine protons, Cl and water molecules are responsible for a three-dimensional network in both 1 and 2. A short Hg...Hg distance of 3.564 A, indicates the presence of a mercurophilic interaction in 1. The compounds have been characterized by (1)H and (13)C NMR, UV-Vis, FT-IR, Raman, mass spectrometry, TGA and single X-ray crystallography.

Mercury impairment of mouse thymocyte survival in vitro: involvement of cellular thiols.

Heavy metals are well known to be able to induce immunotoxicity, but comparative metal studies related to apoptosis have not been conducted. In the present study, the effects of arsenic, cadmium, gold, lead, manganese, and mercury on thymocytes from BALB/c mice were analyzed. Thymic cells were cultured for 3-24 h in vitro in the absence or presence of metal, and markers of apoptosis or cell death, including annexin V binding, DNA loss/oligonucleosomal fragmentation, 7-amino-actinomycin D uptake (loss of impermeance), changes of the mitochondrial membrane potential (JC-1 fluorescence), and Western analysis of cellular thiols, were assayed. Mercury (Hg) was the only metal shown to be consistently toxic with the dose and times utilized. Cadmium (Cd) was the only other metal tested that also produced some significant level of DNA loss; however, the induction of apoptosis by Cd was not as consistent as that observed with Hg. When Hg was added with 2-mercaptoethanol (2-ME), Hg produced greater toxicity. Endogenous DNA synthesis by thymocytes was immediately inhibited by Hg and Hg + 2-ME. The Hg + 2-ME-induced apoptosis appeared to be associated with altered levels of cellular thiols, in that glutathione (GSH) depletion was significant in comparison to the non-metal control and Hg alone. The increased Hg-induced toxicity in the presence of 2-ME likely was due to the ability of 2-ME to enhance (10- to 20-fold) the cellular uptake of Hg. Western analysis with biotin maleimide demonstrated that Hg + 2-ME and to a lesser extent the positive control dexamethasone eliminated many reactive thiols; the major thiol-reactive protein still reactive with the maleimide probe had an approximate Molecular Mass of 45 kD. Surprisingly, Hg alone enhanced the expression of this thiol-expressing protein, which by Mass Spectrometry (MS)/MS analysis was shown to be beta-actin. Hg also produced the appearance of yet to be identified new proteins. Based on the results with Hg + 2-ME, it is suggested that numerous protein thiols participate in maintenance of cell survival and their loss is associated with apoptosis. The increased expression of new thiol-reactive proteins or thiol-reactive proteins with altered electrophoretic profiles needs to be further investigated. However, the enhanced toxicity attributed to Hg + 2-ME suggests that increased intracellular oxidative stress, observed as increased depletion of GSH, is responsible for the accelerated cell death.

Effect of subchronic treatment with mercury chloride on NTPDase, 5'-nucleotidase and acetylcholinesterase from cerebral cortex of rats.

The aim of the present investigation was to evaluate the effect of a subchronic treatment (30 days/30 doses) with subcutaneous injections (0.1 mg/kg) of HgCl2 on NTPDase (E.C. 3.6.1.5), 5'-nucleotidase (E.C 3.1.3.5) and acetylcholinesterase (AChE, E.C. 3.1.1.7) activities in brain from adult rats. NTPDase and 5'-nucleotidase were measured in cortical synaptosomal fraction and AChE was measured in the homogenate of cerebral cortex and hippocampus. After the subchronic treatment (30 days), NTPDase activity was enhanced approximately 35% (p < 0.05) with ATP and ADP as substrates and no difference was observed in 5'-nucleotidase activity (AMP hydrolysis). In addition, AChE activity was enhanced in the cerebral cortex (22%, p < 0.05) and hippocampus (26%, p < 0.05) after the subchronic treatment. Mercury deposited in brain was measured by cold vapor (atomic absorption spectrometry) and no difference between the control and the subchronically treated group was observed. Here we showed for the first time that exposure to low levels of Hg2+, which resembles occupational exposure to low levels of mercury, caused a marked increase in NTPDase and AChE activities. The relationship of these alterations with the neurotoxicity of inorganic mercury deserves further studies.

Microbial mercury transformation in anoxic freshwater sediments under iron-reducing and other electron-accepting conditions.

Potential rates of microbial methylation of inorganic mercury (added as HgCl2) and degradation of methyl mercury (MeHg) (added as CH3HgCl) were investigated in anoxic sediments from the Mobile Alabama River Basin (MARB) dominated by different terminal electron-accepting processes (TEAPs). Potential rates of methylation were comparable under methanogenic and sulfate-reducing conditions but suppressed under iron-reducing conditions, in slurries of freshwater wetland sediment In contrast, MeHg degradation rates were similar under all three TEAPs. Microbial Hg methylation and MeHg degradation were also investigated in surface sediment from three riverine sites, two of which had iron reduction and one sulfate reduction, as the dominant TEAP (as determined by 14C-acetate metabolism and other biogeochemical measurements). Methylation was active in sulfate-reducing sediments of a tributary creek and suppressed in iron-reducing, sandy sediments from the open river, whereas MeHg degradation was active at all three sites. Although iron-reducing conditions often suppressed methylation, some methylation activity was observed in two out of three replicates from iron-reducing sediments collected near a dam. Given that MeHg degradation was consistently observed under all TEAPs, our results suggest that the net flux of MeHg from iron-reducing surface sediments may be suppressed (due to inhibition of gross MeHg production) compared to sediments supporting other TEAPs.