Neurotoxicity of ß-HgS differs from environmental mercury pollutants (MeHgCl and HgCl2) in Neuro-2a cell.

ß-HgS, differing from environmental mercury pollutants (MeHgCl and HgCl2) in chemical form, is used as traditional medicine in Asian countries for thousands of years. In this study, Neuro-2a cells were exposed to ß-HgS, MeHgCl and HgCl2 (5 µM) for 6-24 h. The cell viability of ß-HgS was higher than MeHgCl with 25.9% and 72.4% in 12 h and 24 h respectively. As the incubation time increased, MeHgCl had obvious damage to cell morphology, decreased the ratio of Bcl-2 and Bak and increased the expressions of TNF-α, IL-6 and IL-1ß significantly. Furthermore, the expressions of IL-1ß and IL-6 in HgCl2 group were increased significantly in 6 h and 24 h. The apoptotic rates in MeHgCl and HgCl2 group were respectively higher than ß-HgS with 32.2% and 7.30% in 24 h. Our findings indicate that ß-HgS is much less neurotoxicity than MeHgCl and HgCl2 in Neuro-2a cells.

Ayurvedic processing of α-HgS gives novel physicochemistry and distinct toxicokinetics in zebrafish.

Rasasindura (RS) is an Ayurvedic medicine, which contains ∼99% α-HgS. It is used as a rejuvenating agent and commonly used to treat diseases such as syphilis, insomnia, high fever, and nervous disorders. Cinnabar ore (α-HgS) is a well-known mineral, which is readily available. Despite it, Ayurvedic practitioners adopted an involved and tedious procedure for the preparation of RS. In this study, three samples, one was Ayurvedic (RS), the second one was the commercial (HGS), and the third one was cinnabar ore (CN), were physiochemically examined. Zebrafish model was employed for toxicity study with an oral dose of 100 mg/kg/day for the three samples for 10 days. We found that RS conferred novel physicochemical properties, which were not seen in HGS and CN. Significantly, the average crystallite size of RS was lowest (26 nm) as compared to HGS (31 nm) and CN (34 nm), and the rate of increase of crystallite size with temperature was lowest in RS. RS did not show any significant behavioral toxicity in zebrafish, which was seen with the HGS-and CN-treated zebrafish. HGS-and CN-treated zebrafish showed a significantly high (∗∗∗p < 0.001) decrease (77 ± 7.6% and 51 ± 6.5%, respectively) of glutathione (GSH) levels in the brain, however, for RS-treated zebrafish, the change of GSH was insignificant (26 ± 2.5%, p > 0.05). Interestingly, HGS significantly altered the γ-aminobutyric acid (GABA) in brain tissue. Therefore, among all three samples, RS exhibited the lowest toxicity, which can be credited to the distinct toxicokinetics by these samples.

Superoxide Dismutase and Pseudocatalase Increase Tolerance to Hg(II) in Thermus thermophilus HB27 by Maintaining the Reduced Bacillithiol Pool.

Mercury (Hg) is a widely distributed, toxic heavy metal with no known cellular role. Mercury toxicity has been linked to the production of reactive oxygen species (ROS), but Hg does not directly perform redox chemistry with oxygen. How exposure to the ionic form, Hg(II), generates ROS is unknown. Exposure of Thermus thermophilus to Hg(II) triggered ROS accumulation and increased transcription and activity of superoxide dismutase (Sod) and pseudocatalase (Pcat); however, Hg(II) inactivated Sod and Pcat. Strains lacking Sod or Pcat had increased oxidized bacillithiol (BSH) levels and were more sensitive to Hg(II) than the wild type. The ΔbshA Δsod and ΔbshA Δpcat double mutant strains were as sensitive to Hg(II) as the ΔbshA strain that lacks bacillithiol, suggesting that the increased sensitivity to Hg(II) in the Δsod and Δpcat mutant strains is due to a decrease of reduced BSH. Treatment of T. thermophilus with Hg(II) decreased aconitase activity and increased the intracellular concentration of free Fe, and these phenotypes were exacerbated in Δsod and Δpcat mutant strains. Treatment with Hg(II) also increased DNA damage. We conclude that sequestration of the redox buffering thiol BSH by Hg(II), in conjunction with direct inactivation of ROS-scavenging enzymes, impairs the ability of T. thermophilus to effectively metabolize ROS generated as a normal consequence of growth in aerobic environments.IMPORTANCEThermus thermophilus is a deep-branching thermophilic aerobe. It is a member of the Deinococcus-Thermus phylum that, together with the Aquificae, constitute the earliest branching aerobic bacterial lineages; therefore, this organism serves as a model for early diverged bacteria (R. K. Hartmann, J. Wolters, B. Kröger, S. Schultze, et al., Syst Appl Microbiol 11:243-249, 1989, https://doi.org/10.1016/S0723-2020(89)80020-7) whose natural heated habitat may contain mercury of geological origins (G. G. Geesey, T. Barkay, and S. King, Sci Total Environ 569-570:321-331, 2016, https://doi.org/10.1016/j.scitotenv.2016.06.080). T. thermophilus likely arose shortly after the oxidation of the biosphere 2.4 billion years ago. Studying T. thermophilus physiology provides clues about the origin and evolution of mechanisms for mercury and oxidative stress responses, the latter being critical for the survival and function of all extant aerobes.

Identification of Potential Long Noncoding RNA Biomarker of Mercury Compounds in Zebrafish Embryos.

Heavy metal pollution elicits severe environmental concern and health problem worldwide. Mercury is considered as a ubiquitous pollutant due to its versatile application in medicine, industry, and cosmetics. Long noncoding RNAs (lncRNAs) are transcripts greater than 200 nt without protein-encoding function. However, little is known about the mechanism of heavy metals-induced noncoding RNA changes in aquatic organisms. To reveal the epigenetic mechanism of mercury toxicity in zebrafish embryos and explore novel specific mercury-toxicological biomarkers, several well-studied lncRNAs were screened by real-time PCR, and the spatial-temporal expression of lncRNAs biomarker was evaluated by in situ hybridization. The nerve systems of zebrafish embryos were evaluated by detecting locomotor behavior and the expression of neuro-genes. We identified a mercury responsive lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (malat1), among five candidate lncRNAs. HgCl2, MeHg, PbCl2, CdCl2, and K2CrO4 exposure assay showed that malat-1 was a mercury specific induced lncRNAs. Malat1 was highly expressed in the brain region, eyes, and notochord of developing zebrafish embryos after exposure to mercury compounds. HgCl2 showed neurobehavior disturbance and changed neuro-genes expression pattern in zebrafish larvae. This study provides a biological method to detect inorganic or organic mercury using malat1 as a novel biomarker of mercury contamination and also clues for the exploration of neurotoxicity mechanism of mercury compounds.

Effect of the Tibetan Medicine Zuotai on Degranulation and Inflammatory Mediator Release in RBL-2H3 Cells.

Zuotai is a drug containing mercury considered to be the king of Tibetan medicine. The biosafety of Zuotai led people's attention and so far little is known about the toxicity of Zuotai to mast cells. RBL-2H3 cells which used as an alternative model of mast cells were treated with Zuotai, ß-HgS and positive drug Compound 48/80 respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the toxicity of drugs to RBL-2H3 cells. The degranulation of RBL-2H3 cells was studied from ß-hexosaminidase, histamine, interleukin (IL)-4 and tumor necrosis factor-α (TNF-α). The result showed that Zuotai can affect the cytotoxicity and degranulation of RBL-2H3 cells and the results can provide reference for the toxicity evaluations of Tibetan medicine Zuotai.

Hepatorenal protective effects of medicinal herbs in An-Gong-Niu-Huang Wan (AGNH) against cinnabar- and realgar-induced oxidative stress and inflammatory damage in mice.

An-Gong-Niu-Huang Wan (AGNH) is a famous traditional Chinese medicine prescription that contains cinnabar (HgS) and realgar (As2S2); the clinical practice of AGNH is hindered because both mercury and arsenic are hepatorenal toxic metalloids. It is noted that the cinnabar and realgar in AGNH are not used alone, but rather combined with different kinds of medicinal herbs as a formula to use. In this study, we evaluated the hepatorenal protective effects of the medicinal herbs in AGNH after co-exposure to cinnabar and realgar for 4 weeks in mice. The combination of the herbs in AGNH alleviated cinnabar and realgar-induced histopathological alterations and oxidative stress in the liver and kidneys. Furthermore, in cinnabar and realgar-treated mice, the increased expression levels of inducible enzymes (COX-2 and iNOS) and proinflammatory mediators (IL-1ß, TNF-α, PGE2 and NO) in the liver and kidneys were consistently down-regulated when medicinal herbs were combined as a formula. We also found that the herbs could reduce the inflammatory response by the inactivation of the MAPK and PI3K/Akt signaling pathway and the resulting blockade of NF-κB activation. Overall, our data indicates that the herbal medicines in AGNH attenuate cinnabar and realgar-induced hepatorenal toxicity by improving antioxidant competence and suppressing inflammatory injury.

Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury.

Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems - glutathione (GSH) and thioredoxin (Trx) systems - are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5µM of inorganic mercury (Hg2+), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells.

Oral administration of Moringa oleifera oil but not coconut oil prevents mercury-induced testicular toxicity in rats.

This study was conducted to compare the effects of administration of coconut oil (CO) and Moringa oleifera oil (MO) on testicular oxidative stress, sperm quality and steroidogenesis parameters in rats treated with mercury chloride (HgCl2 ). After 15 days of oral administration of CO (2 ml kg-1 body weight) and MO (2 ml kg-1 body weight) along with intraperitoneal (i.p.) administration of HgCl2 (5 mg kg-1 body weight) alone or in combination, we found that CO treatment did not protect against HgCl2 -induced poor sperm quality (motility, count) as well as decreased testosterone level and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) activity. Treatment with CO alone decreased glutathione (GSH), and glutathione peroxidase (GSH-Px) activities and increased malondialdehyde (MDA) level in rat's testis, whereas MO did not change these parameters. Cotreatment with MO prevented HgCl2 -induced testicular catalase (CAT) and superoxide dismutase (SOD) activities, poor sperm quality and low testosterone level and also blocks the adverse effect of CO+HgCl2 (2 ml kg-1 body weight + 5 mg kg-1 body weight) on the investigated endpoints. In conclusion, MO and not CO decreased the deleterious effects of HgCl2 on sperm quality and steroidogenesis in rats and also strengthen the antioxidant defence of the testes. Therefore, MO is beneficial as an antioxidant in HgCl2 -induced oxidative damage.

Mercury sulfides are much less nephrotoxic than mercury chloride and methylmercury in mice.

Mercury sulfides (α-HgS, ß-HgS) are frequently included in traditional medicines. Mercury is known for nephrotoxicity, their safety is of concern. To address this question, mice were orally administrated with Zuotai (54% ß-HgS, 30mg/kg), α-HgS (HgS, 30mg/kg), HgCl2 (33.6mg/kg), or MeHgCl (3.1mg/kg) for 7days, and nephrotoxicity was examined. Animal body weights were decreased by HgCl2 and to a lesser extent by MeHg, but unaltered after Zuotai and HgS. HgCl2 and MeHg produced renal tubular vacuolation, interstitial inflammation and cell degeneration with protein cysts in the tubular lumen, while these pathological lesions were mild in Zuotai and HgS-treated mice. Electron microscopy showed that HgCl2 and MeHg produced spotted swelling endothelium reticulum, while these lesions were mild or absent in Zuotai and HgS-treated mice. Renal Hg contents reached 250-300ng/mg kidney in HgCl2 and MeHg groups as compared to 2-3ng/mg in Zuotai and HgS groups. The expression of kidney injury biomarkers, kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (Ngal), were increased after HgCl2 and MeHg, but unaltered after Zuotai and HgS. The expression of renal influx transporters Oat3 and Oatp4c1 was decreased, while the expression of renal efflux transporter such as Mrp2, Mrp4, and Mate2 was increased following HgCl2 and MeHg. These gene expressions were unchanged after Zuotai and HgS. In summary, both α-HgS and ß-HgS are less nephrotoxic than HgCl2 and MeHg, indicating that chemical forms of mercury are a major determinant of mercury disposition and toxicity.

The Tibetan medicine Zuotai differs from HgCl2 and MeHg in producing liver injury in mice.

Zuotai is composed mainly of ß-HgS, while cinnabar mainly contains α-HgS. Both forms of HgS are used in traditional medicines and their safety is of concern. This study aimed to compare the hepatotoxicity potential of Zuotai and α-HgS with mercury chloride (HgCl2) and methylmercury (MeHg) in mice. Mice were orally administrated with Zuotai (30 mg/kg), α-HgS (HgS, 30 mg/kg), HgCl2 (33.6 mg/kg), or CH3HgCl (3.1 mg/kg) for 7 days, and liver injury and gene expressions related to toxicity, inflammation and Nrf2 were examined. Animal body weights were decreased by HgCl2 and to a less extent by MeHg. HgCl2 and MeHg produced spotted hepatocyte swelling and inflammation, while such lesions are mild in Zuotai and HgS-treated mice. Liver Hg contents reached 45-70 ng/mg in HgCl2 and MeHg groups; but only 1-2 ng/mg in Zuotai and HgS groups. HgCl2 and MeHg increased the expression of liver injury biomarker genes metallothionein-1 (MT-1) and heme oxygenase-1 (HO-1); the inflammation biomarkers early growth response gene (Egr1), glutathione S-transferase (Gst-mu), chemokine (mKC) and microphage inflammatory protein (MIP-2), while these changes were insignificant in Zuotai and HgS groups. However, all mercury compounds were able to increase the Nrf2 pathway genes NAD(P)H: quinone oxidoreductase 1 (Nqo1) and Glutamate-cysteine ligase, catalytic subunit (Gclc). In conclusion, the Tibetan medicine Zuotai and HgS are less hepatotoxic than HgCl2 and MeHg, and differ from HgCl2 and MeHg in hepatic Hg accumulation and toxicological responses.

Phenylthiourea alters toxicity of mercury compounds in zebrafish larvae.

In recent years larval stage zebrafish have been emerging as a standard vertebrate model in a number of fields, ranging from developmental biology to pharmacology and toxicology. The tyrosinase inhibitor 1-phenyl-2-thiourea (PTU) is used very widely with larval zebrafish to generate essentially transparent organisms through inhibition of melanogenesis, which has enabled many elegant studies in areas ranging from neurological development to cancer research. Here we show that PTU can have dramatic synergistic and antagonistic effects on the chemical toxicology of different mercury compounds. Our results indicate that extreme caution should be used when employing PTU in toxicological studies, particularly when studying toxic metal ions.

Mitochondrial thioredoxin reductase inhibition, selenium status, and Nrf-2 activation are determinant factors modulating the toxicity of mercury compounds.

The thioredoxin system has essential functions in the maintenance of cellular redox homeostasis in the cytosol, nucleus, and mitochondria. Thioredoxin (Trx) and thioredoxin reductase (TrxR) are targets for mercury compounds in vitro and in vivo. This study aimed at understanding mechanistically how the mitochondrial and cytosolic thioredoxin systems were affected by mercurials, including the regulation of TrxR transcription. The effects of coexposure to selenite and mercurials on the thioredoxin system were also addressed. Results in HepG2 cells showed that TrxR1 expression was enhanced by Hg(2+), whereas exposure to MeHg decreased expression. Selenite exposure also increased the expression of TrxR1 and resulted in higher specific activity. Coexposure to 2 µM selenite and up to 5 µM Hg(2+) increased even further TrxR1 expression. This synergistic effect was not verified for MeHg, because TrxR1 expression and activity were reduced. Analysis of Nrf-2 translocation to the nucleus and TrxR mRNA suggests that induction of TrxR1 transcription was slower upon exposure to MeHg in comparison to Hg(2+). Subcellular fractions showed that MeHg affected the activity of the thioredoxin system equally in the mitochondria and cytosol, whereas Hg(2+) inhibited primarily the activity of TrxR2. The expression of TrxR2 was not upregulated by any treatment. These results show important differences between the mechanisms of toxicity of Hg(2+) and MeHg and stress the narrow range of selenite concentrations capable of antagonizing mercury toxicity. The results also highlight the relevance of the mitochondrial thioredoxin system (TrxR2 and Trx2) in the development of mercury toxicity.

Exposure to low dose of cinnabar (a naturally occurring mercuric sulfide (HgS)) caused neurotoxicological effects in offspring mice.

Cinnabar, a naturally occurring mercuric sulfide (HgS), has long been used in Chinese mineral medicine for more than 2000 years. Although mercury is well-known for its toxicity, whether cinnabar induces neurotoxicity, especially in infants and children, is unknown. The purpose of this study was to explore the neurotoxic effects of low-dose of cinnabar (10 mg/kg/day) on developing mice. The results revealed neurobehavioral defects in F1-C-Cin group, which were associated with Hg accumulation, increased NO(x) levels in whole blood, and Na(+)/K(+)-ATPase activities in brain tissues. F1- and F2-Cin-V groups were found to increase brain Hg contents and prominent neurobehavioral defects compared with F1-C-V group, suggesting that the fetal brain was more susceptible to irreversible effects for cinnabar-induced damage. Moreover, F1- and F2-Cin-Cin groups had severely neurobehavioral dysfunctions, closely correlated with the further alteration of NO(x) levels and Na(+)/K(+)-ATPase activities than F1- and F2-C-Cin groups. Effects in F2-Cin-Cin group were more significant than those in F1-Cin-Cin group. In conclusion, this study demonstrates that exposure to low-dose of cinnabar during the perinatal and developmental stages results in irreversible and severe injuries of the neurotoxicity in offspring, and NO(x) and Na(+)/K(+)-ATPase activities may exist potential and useful biomarkers for neurotoxicity-induced by low-doses of mercuric compounds.

Low-dose inorganic mercury increases severity and frequency of chronic coxsackievirus-induced autoimmune myocarditis in mice.

Mercury is a widespread environmental contaminant with neurotoxic impacts that have been observed over a range of exposures. In addition, there is increasing evidence that inorganic mercury (iHg) and organic mercury (including methyl mercury) have a range of immunotoxic effects, including immune suppression and induction of autoimmunity. In this study, we investigated the effect of iHg on a model of autoimmune heart disease in mice induced by infection with coxsackievirus B3 (CVB3). We examined the role of timing of iHg exposure on disease; in some experiments, mice were pretreated with iHg (200 µg/kg, every other day for 15 days) before disease induction with virus inoculation, and in others, they were treated with iHg after the acute (viral) phase of disease but before the development of dilated cardiomyopathy (DCM). iHg alone had no effect on heart pathology. Pretreatment with iHg before CVB3 infection significantly increased the severity of chronic myocarditis and DCM compared with control animals receiving vehicle alone. In contrast, treatment with iHg after acute myocarditis did not affect the severity of chronic disease. The increased chronic myocarditis, fibrosis, and DCM induced by iHg pretreatment were not due to increased viral replication in the heart, which was unaltered by iHg treatment. iHg pretreatment induced a macrophage infiltrate and mixed cytokine response in the heart during acute myocarditis, including significantly increased interleukin (IL)-12, IL-17, interferon-γ, and tumor necrosis factor-α levels. IL-17 levels were also significantly increased in the spleen during chronic disease. Thus, we show for the first time that low-dose Hg exposure increases chronic myocarditis and DCM in a murine model.

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

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

Cell-density-dependent methylmercury susceptibility of cultured human brain microvascular pericytes.

The knowledge of vascular toxicity is important for understanding the neurotoxicity of methylmercury. In the present study, we investigated the cell-density-dependent susceptibility of human brain microvascular pericytes to methylmercury-induced toxicity by using a cell-culture system. The susceptibility of sparse pericyte cultures to methylmercury was greater than that of the dense cultures. In addition, the sparse cultures were more susceptible to methylmercury than to inorganic mercury and cadmium. The intracellular accumulation of methylmercury in the sparse cells was significantly higher than that in the dense cells. Methylmercury is transported through the L-type large neutral amino acid transporter (LAT 1) in the form of a complex with cysteine. The mRNA- and protein-level expressions of LAT 1 in the sparse cells were markedly higher than those in the dense cells; in addition, the LAT 1 expression was increased by methylmercury. However, there was no reduction in the levels of glutathione and metallothionein, which are involved in the defense mechanisms against methylmercury, in the sparse cells. The present data revealed that pericytes are markedly susceptible to methylmercury-induced cytotoxicity at low cell densities. The susceptibility of the sparse pericytes is postulated to be due to the not only constitutively higher but also methylmercury-induced expression of LAT 1, which increased the intracellular accumulation of methylmercury.

Environmental toxicants inhibit neuronal Jak tyrosine kinase by mitochondrial disruption.

Cadmium, mercury and rotenone are environmental pollutants whose neurotoxic mechanisms are not fully understood. We have shown previously that exposure of nerve cells to these agents produces oxidative stress which reversibly blocks growth factor and cytokine-mediated Janus kinase (Jak)/signal transducer and activator of transcription (STAT) signaling. Here we determined a critical role for mitochondrial dysfunction in inhibiting Jak/STAT activity in human BE(2)-C neuroblastoma cells. Exposure of BE(2)-C cells to the heavy metals CdCl(2) and HgCl(2) and to the mitochondrial complex I inhibitor rotenone inhibited interleukin-6, interferon-gamma and ciliary neurotrophic factor-mediated Jak/STAT signaling, reduced Jak1 and Jak2 auto-phosphorylation and induced Jak tyrosine nitration. However, identical exposure of HepG2 hepatoma cells produced no inhibition of these cytokine responses. In contrast, mitochondria in both BE(2)-C and HepG2 cells showed reduced mitochondrial membrane potential and increased superoxide production after exposure to CdCl(2), HgCl(2) and rotenone. Further, in an in vitro Jak auto-phosphorylation assay Jak2 isolated from either BE(2)-C or HepG2 cells was equally inhibited by mitochondria made dysfunctional by treatment with CdCl(2), HgCl(2) and rotenone. Each of these pro-oxidant effects was reversed by the mitochondrial antioxidant alpha-lipoic acid. The actions of cadmium were also blocked by the mitochondrial complex III bypass agent, 2,6-dichloroindophenol. Therefore, in BE(2)-C cells CdCl(2), HgCl(2) and rotenone disrupt mitochondria to increase intracellular ROS, which directly inhibits neuronal Jak tyrosine kinase activity. Non-neuronal cells such as HepG2 cells that are resistant to oxidative stress-mediated inhibition of cytokine signaling possess some as yet unknown mechanism that protects Jak kinases from oxidative insults. Pro-oxidant-induced mitochondrial dysfunction resulting in selective neuronal Jak inhibition provides a potential mechanism for environmental agents to promote neurodegeneration.

Relationship between proline and Hg2+-induced oxidative stress in a tolerant rice mutant.

There has been little agreement regarding the mechanism by which proline reduces heavy metal stress. The present work examines the relationship between Hg(2+)-induced oxidative stress and proline accumulation in rice and explores the possible mechanisms through which proline protects against Hg(2+) stress. The effect of proline on alleviation of Hg(2+) toxicity was studied by spectrophotography and enzymatic methods. Hg(2+) induced oxidative stress in rice by increasing lipid peroxidation. Pretreatment of the rice with 2 mM proline for 12 h profoundly alleviated Hg(2+)-induced lipid peroxidation and minimized H(2)O(2) accumulation. Proline pretreatment significantly reduced (p < 0.01) the Hg(2+) content in rice leaves. A comparison of the effects of proline pretreatment on H(2)O(2) accumulation by Hg(2+) and aminotrazole suggested that proline protected cells from Hg(2+)-induced oxidative stress by scavenging reactive oxygen species. The present work demonstrates a protective effect of proline on Hg(2+) toxicity through detoxifying reactive oxygen species, rather than chelating metal ions or maintaining the water balance under Hg(2+) stress.

Inhibition of the human thioredoxin system. A molecular mechanism of mercury toxicity.

Mercury toxicity mediated by different forms of mercury is a major health problem; however, the molecular mechanisms underlying toxicity remain elusive. We analyzed the effects of mercuric chloride (HgCl(2)) and monomethylmercury (MeHg) on the proteins of the mammalian thioredoxin system, thioredoxin reductase (TrxR) and thioredoxin (Trx), and of the glutaredoxin system, glutathione reductase (GR) and glutaredoxin (Grx). HgCl(2) and MeHg inhibited recombinant rat TrxR with IC(50) values of 7.2 and 19.7 nm, respectively. Fully reduced human Trx1 bound mercury and lost all five free thiols and activity after incubation with HgCl(2) or MeHg, but only HgCl(2) generated dimers. Mass spectra analysis demonstrated binding of 2.5 mol of Hg(2+) and 5 mol of MeHg(+)/mol of Trx1 with the very strong Hg(2+) complexes involving active site and structural disulfides. Inhibition of both TrxR and Trx activity was observed in HeLa and HEK 293 cells treated with HgCl(2) or MeHg. GR was inhibited by HgCl(2) and MeHg in vitro, but no decrease in GR activity was detected in cell extracts treated with mercurials. Human Grx1 showed similar reactivity as Trx1 with both mercurial compounds, with the loss of all free thiols and Grx dimerization in the presence of HgCl(2), but no inhibition of Grx activity was observed in lysates of HeLa cells exposed to mercury. Overall, mercury inhibition was selective toward the thioredoxin system. In particular, the remarkable potency of the mercury compounds to bind to the selenol-thiol in the active site of TrxR should be a major molecular mechanism of mercury toxicity.