Evaluation of mRNA markers in differentiating human SH-SY5Y cells for estimation of developmental neurotoxicity.

Current guidelines for developmental neurotoxicity (DNT) evaluation are based on animal models. These have limitations so more relevant, efficient and robust approaches for DNT assessment are needed. We have used the human SH-SY5Y neuroblastoma cell model to evaluate a panel of 93 mRNA markers that are frequent in Neuronal diseases and functional annotations and also differentially expressed during retinoic acid-induced differentiation in the cell model. Rotenone, valproic acid (VPA), acrylamide (ACR) and methylmercury chloride (MeHg) were used as DNT positive compounds. Tolbutamide, D-mannitol and clofibrate were used as DNT negative compounds. To determine concentrations for exposure for gene expression analysis, we developed a pipeline for neurite outgrowth assessment by live-cell imaging. In addition, cell viability was measured by the resazurin assay. Gene expression was analyzed by RT-qPCR after 6 days of exposure during differentiation to concentrations of the DNT positive compounds that affected neurite outgrowth, but with no or minimal effect on cell viability. Methylmercury affected cell viability at lower concentrations than neurite outgrowth, hence the cells were exposed with the highest non-cytotoxic concentration. Rotenone (7.3 nM) induced 32 differentially expressed genes (DEGs), ACR (70 µM) 8 DEGs, and VPA (75 µM) 16 DEGs. No individual genes were significantly dysregulated by all 3 DNT positive compounds (p < 0.05), but 9 genes were differentially expressed by 2 of them. Methylmercury (0.8 nM) was used to validate the 9 DEGs. The expression of SEMA5A (encoding semaphorin 5A) and CHRNA7 (encoding nicotinic acetylcholine receptor subunit α7) was downregulated by all 4 DNT positive compounds. None of the DNT negative compounds dysregulated any of the 9 DEGs in common for the DNT positive compounds. We suggest that SEMA5A or CHRNA7 should be further evaluated as biomarkers for DNT studies in vitro since they also are involved in neurodevelopmental adverse outcomes in humans.

Methylmercury effects on avian brains.

Methylmercury (MeHg) is a concerning contaminant due to its ubiquity and harmful effects on organisms. Although birds are important models in the neurobiology of vocal learning and adult neuroplasticity, the neurotoxic effects of MeHg are less understood in birds than mammals. We surveyed the literature on MeHg effects on biochemical changes in the avian brain. Publication rates of papers related to neurology and/or birds and/or MeHg increased with time and can be linked with historical events, regulations, and increased understanding of MeHg cycling in the environment. However, publications on MeHg effects on the avian brain remain relatively low across time. The neural effects measured to evaluate MeHg neurotoxicity in birds changed with time and researcher interest. The measures most consistently affected by MeHg exposure in birds were markers of oxidative stress. NMDA, acetylcholinesterase, and Purkinje cells also seem sensitive to some extent. MeHg exposure has the potential to affect most neurotransmitter systems but more studies are needed for validation in birds. We also review the main mechanisms of MeHg-induced neurotoxicity in mammals and compare it to what is known in birds. The literature on MeHg effects on the avian brain is limited, preventing full construction of an adverse outcome pathway. We identify research gaps for taxonomic groups such as songbirds, and age- and life-stage groups such as immature fledgling stage and adult non-reproductive life stage. In addition, results are often inconsistent between experimental and field studies. We conclude that future neurotoxicological studies of MeHg impacts on birds need to better connect the numerous aspects of exposure from molecular physiological effects to behavioural outcomes that would be ecologically or biologically relevant for birds, especially under challenging conditions.

Methylmercury Induces Mitochondria- and Endoplasmic Reticulum Stress-Dependent Pancreatic ß-Cell Apoptosis via an Oxidative Stress-Mediated JNK Signaling Pathway.

Methylmercury (MeHg), a long-lasting organic pollutant, is known to induce cytotoxic effects in mammalian cells. Epidemiological studies have suggested that environmental exposure to MeHg is linked to the development of diabetes mellitus (DM). The exact molecular mechanism of MeHg-induced pancreatic ß-cell cytotoxicity is still unclear. Here, we found that MeHg (1-4 µM) significantly decreased insulin secretion and cell viability in pancreatic ß-cell-derived RIN-m5F cells. A concomitant elevation of mitochondrial-dependent apoptotic events was observed, including decreased mitochondrial membrane potential and increased proapoptotic (Bax, Bak, p53)/antiapoptotic (Bcl-2) mRNA ratio, cytochrome c release, annexin V-Cy3 binding, caspase-3 activity, and caspase-3/-7/-9 activation. Exposure of RIN-m5F cells to MeHg (2 µM) also induced protein expression of endoplasmic reticulum (ER) stress-related signaling molecules, including C/EBP homologous protein (CHOP), X-box binding protein (XBP-1), and caspase-12. Pretreatment with 4-phenylbutyric acid (4-PBA; an ER stress inhibitor) and specific siRNAs for CHOP and XBP-1 significantly inhibited their expression and caspase-3/-12 activation in MeHg-exposed RIN-mF cells. MeHg could also evoke c-Jun N-terminal kinase (JNK) activation and reactive oxygen species (ROS) generation. Antioxidant N-acetylcysteine (NAC; 1mM) or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox; 100 µM) markedly prevented MeH-induced ROS generation and decreased cell viability in RIN-m5F cells. Furthermore, pretreatment of cells with SP600125 (JNK inhibitor; 10 µM) or NAC (1 mM) or transfection with JNK-specific siRNA obviously attenuated the MeHg-induced JNK phosphorylation, CHOP and XBP-1 protein expression, apoptotic events, and insulin secretion dysfunction. NAC significantly inhibited MeHg-activated JNK signaling, but SP600125 could not effectively reduce MeHg-induced ROS generation. Collectively, these findings demonstrate that the induction of ROS-activated JNK signaling is a crucial mechanism underlying MeHg-induced mitochondria- and ER stress-dependent apoptosis, ultimately leading to ß-cell death.

MicroRNA Expression Influences Methylmercury-Induced Lipid Accumulation and Mitochondrial Toxicity in Caenorhabditis elegans.

Metabolic effects of methylmercury (MeHg) are gaining wider attention. We have previously shown that MeHg causes lipid dysregulation in Caenorhabditis elegans (C. elegans), leading to altered gene expression, increased triglyceride levels and lipid storage, and altered feeding behaviors. Transcriptional regulators, such as transcription factors and microRNAs (miRNAs), have been shown to regulate lipid storage, serum triglycerides, and adipogenic gene expression in human and rodent models of metabolic diseases. As we recently investigated adipogenic transcription factors induced by MeHg, we were, therefore, interested in whether MeHg may also regulate miRNA sequences to cause metabolic dysfunction. Lipid dysregulation, as measured by triglyceride levels, lipid storage sites, and feeding behaviors, was assessed in wild-type (N2) worms and in transgenic worms that either were sensitive to miRNA expression or were unable to process miRNAs. Worms that were sensitive to the miRNA expression were protected from MeHg-induced lipid dysregulation. In contrast, the mutant worms that were unable to process miRNAs had exacerbated MeHg-induced lipid dysregulation. Concurrent with differential lipid homeostasis, miRNA-expression mutants had altered MeHg-induced mitochondrial toxicity as compared to N2, with the miRNA-sensitive mutants showing mitochondrial protection and the miRNA-processing mutants showing increased mitotoxicity. Taken together, our data demonstrate that the expression of miRNAs is an important determinant in MeHg toxicity and MeHg-induced metabolic dysfunction in C. elegans.

Selective dopaminergic effects on attention and memory in male mice exposed to Methylmercury during adolescence.

Gestational exposure to methylmercury disrupts dopamine-mediated behavior and produces heightened sensitivity to monoamine agonists later in life. This has been reported and replicated following both pre- and post-natal exposure. Impacts of methylmercury when exposure occurs during the sensitive period of adolescence, a key period of dopaminergic development, remain underexplored. There have been variable results thus far in studies investigating links between adolescent exposure to methylmercury and alterations in executive function and altered sensitivity to monoamine agonists. The current study was designed to investigate adolescent exposure by exposing male mice to 0, 0.3, or 3 ppm methylmercury during adolescence and training them in a hybrid task to assess two executive functions, attention and memory, in adulthood. Behavior in these animals was probed with a range of doses of the dopamine agonist, d-amphetamine, and the norepinephrine agonist, desipramine. Attention and memory in these mice were sensitive to disruption by d-amphetamine and interacted with methylmercury exposure. Choice latencies were also longer in the MeHg-exposed mice. Desipramine did not affect behavior in these animals nor did it interact with methylmercury. It is concluded that methylmercury-related inhibition of behavior observed in this study were differentially sensitive to acute disruption in dopamine, but not norepinephrine, neurotransmission.

DHA ameliorates MeHg­induced PC12 cell apoptosis by inhibiting the ROS/JNK signaling pathway.

Recent studies have reported that methylmercury (MeHg) induces neuronal apoptosis, which is accompanied by abnormal neurological development. Despite the important role of docosahexaenoic acid (DHA) in maintaining the structure and function of the brain, as well as improving neuronal apoptosis induced by MeHg, the exact mechanism remains unknown. The present study hypothesized that the reactive oxygen species (ROS)­mediated JNK signaling pathway may be associated with the protective effect of DHA against MeHg­induced PC12 cell apoptosis. Cell Counting Kit­8, TUNEL staining, flow cytometry, ROS detection, PCR and western blot analysis were performed. The results demonstrated that MeHg inhibited the activity of PC12 cells, causing oxidative damage and promoting apoptosis; however, DHA significantly attenuated this effect. Mechanistic studies revealed that MeHg increased intracellular ROS levels and JNK protein phosphorylation, and decreased the expression levels of the anti­apoptotic protein Bcl­2, whereas DHA reduced ROS levels and JNK phosphorylation, and increased Bcl­2 expression. In addition, the ROS inhibitor N­acetyl­l­cysteine (NAC) was used to verify the experimental results. After pretreatment with NAC, expression levels of Bcl­2, Bax, phosphorylated­JNK and JNK were assessed. Bcl­2 protein expression was increased and the Bcl­2/Bax ratio was increased. Moreover, the high expression levels of phosphorylated­JNK induced by MeHg were significantly decreased. Based on the aforementioned results, the present study indicated that the effects of DHA against MeHg­induced PC12 cell apoptosis may be mediated via the ROS/JNK signaling pathway.

RNA sequencing and proteomic profiling reveal different alterations by dietary methylmercury in the hippocampal transcriptome and proteome in BALB/c mice.

Methylmercury (MeHg) is a highly neurotoxic form of mercury (Hg) present in seafood. Here, we recorded and compared proteomic and transcriptomic changes in hippocampus of male BALB/c mice exposed to two doses of MeHg. Mice were fed diets spiked with 0.28 mg MeHg kg-1, 5 mg MeHg kg-1, or an unspiked control diet for 77 days. Total mercury content was significantly (P < 0.05) increased in brain tissue of both MeHg-exposed groups (18 ± 2 mg Hg kg-1 and 0.56 ± 0.06 mg Hg kg-1). Hippocampal protein and ribonucleic acid (RNA) expression levels were significantly altered both in tissues from mice receiving a low dose MeHg (20 proteins/294 RNA transcripts) and a high dose MeHg (61 proteins/876 RNA transcripts). The majority but not all the differentially expressed features in hippocampus were dose dependent. The combined use of transcriptomic and proteomic profiling data provided insight on the influence of MeHg on neurotoxicity, energy metabolism, and oxidative stress through several regulated features and pathways, including RXR function and superoxide radical degradation.

Amino Acid Transporter LAT1 (SLC7A5) Mediates MeHg-Induced Oxidative Stress Defense in the Human Placental Cell Line HTR-8/SVneo.

The placental barrier can protect the fetus from contact with harmful substances. The potent neurotoxin methylmercury (MeHg), however, is very efficiently transported across the placenta. Our previous data suggested that L-type amino acid transporter (LAT)1 is involved in placental MeHg uptake, accepting MeHg-L-cysteine conjugates as substrate due to structural similarity to methionine. The aim of the present study was to investigate the antioxidant defense of placental cells to MeHg exposure and the role of LAT1 in this response. When trophoblast-derived HTR-8/SVneo cells were LAT1 depleted by siRNA-mediated knockdown, they accumulated less MeHg. However, they were more susceptible to MeHg-induced toxicity. This was evidenced in decreased cell viability at a usually noncytotoxic concentration of 0.03 µM MeHg (~6 µg/L). Treatment with ≥0.3 µM MeHg increased cytotoxicity, apoptosis rate, and oxidative stress of HTR-8/SVneo cells. These effects were enhanced under LAT1 knockdown. Reduced cell number was seen when MeHg-exposed cells were cultured in medium low in cysteine, a constituent of the tripeptide glutathione (GSH). Because LAT1-deficient HTR-8/SVneo cells have lower GSH levels than control cells (independent of MeHg treatment), we conclude that LAT1 is essential for de novo synthesis of GSH, required to counteract oxidative stress. Genetic predisposition to decreased LAT1 function combined with MeHg exposure could increase the risk of placental damage.

The Role of Human LRRK2 in Methylmercury-Induced Inhibition of Microvesicle Formation of Cephalic Neurons in Caenorhabditis elegans.

In a previous study, we have shown that methylmercury (MeHg) exposure causes focal aggregation of intracellular transgenic mCherry protein in dendrites of cephalic (CEP) neurons in Caenorhabditis elegans (C. elegans). However, the underlying mechanism is unknown. We hypothesized that reduced cellular release of mCherry via extracellular vesicles by MeHg contributes to its accumulation and intracellular aggregation. Thus, we characterized vesicular structures in CEP dendrites, which were 1-3 µm in diameter and could readily bud off from the plasma membrane of the dendrites. Chronic treatment of C. elegans with MeHg (5 µM, 4-10 days) reduced the number of vesicles attached to CEP dendrites (attached vesicles) and vesicles unattached to CEP dendrites (unattached vesicles), as well as the presence of extracellular mCherry, supporting the hypothesis that release of mCherry by microvesicle formation is inhibited by MeHg. Leucine-rich repeat kinase 2 (LRRK2) has an important function in membrane biology. Further investigation showed that the effects of MeHg were modified by human LRRK2. In worms with the wild-type LRRK2, the vesicle numbers were significantly reduced by MeHg (0.5 and 5 µM). The effects of MeHg on the presence of extracellular mCherry and attached vesicles were modified by the human wild-type LRRK2. Independent of MeHg treatment, the G2019S mutant LRRK2 showed reduced number of unattached vesicles; however, the levels of extracellular mCherry were increased. Knockdown of C. elegans irk-1, the homolog of human LRRK2, reduced the number of attached vesicles, corroborating that LRRK2 plays an important role in the formation of microvesicles.

Low Doses of Methylmercury Induce the Proliferation of Thyroid Cells In Vitro Through Modulation of ERK Pathway.

Exposure to environmental endocrine disruptors has been associated with an increased frequency of thyroid pathology. In this study, we evaluated the effects of various concentrations of methylmercury (MeHg) on immortalized, non-tumorigenic thyroid cells (Nthy-ori-3-1). Exposure to MeHg at 2.5 and 5 µM for 24 h caused a reduction in cell viability with a decrease of the cell population in sub-G0 phase, as detected by MTT and flow cytometry. Conversely, MeHg at the lower concentration of 0.1 µM increased the cell viability with a rise of G2/M phase. An immunoblot analysis showed higher expression levels of phospho-ERK and not of phospho-Akt. Further enhancement of the cell growth rate was observed after a prolonged exposure of the cells up to 18 days to MeHg 0.1 µM. The present findings demonstrate the toxicity of high concentrations of MeHg on thyroid cells, while showing that treatment with lower doses of Hg, as may occur after prolonged exposure to this environmental contaminant, exerts a promoting effect on thyroid cell proliferation, by acting on the ERK-mediated pro-oncogenic signal transduction pathway.

Direct Observation of Methylmercury and Auranofin Binding to Selenocysteine in Thioredoxin Reductase.

Selenoenzymes, containing a selenocysteine (Sec) residue, fulfill important roles in biology. The mammalian thioredoxin reductase selenoenzymes are key regulators of antioxidant defense and redox signaling and are inhibited by methylmercury species and by the gold-containing drug auranofin. It has been proposed that such inhibition is mediated by metal binding to Sec in the enzyme. However, direct structural observations of these classes of inhibitors binding to selenoenzymes have been few to date. Here we therefore have used extended X-ray absorption fine structure as a direct structural probe to investigate binding to the selenium site in recombinant rat thioredoxin reductase 1 (TrxR1). The results demonstrate for the first time the direct and complete binding of the metal atom of the inhibitors to the selenium atom in TrxR1 for both methylmercury and auranofin, indicating that TrxR1 inhibition indeed can be attributed to such direct metal-selenium binding.

Methyl and Ethylmercury elicit oxidative stress and unbalance the antioxidant system in Saccharomyces cerevisiae.

Methylmercury (MeHg) and Ethylmercury (EtHg) are toxic to the central nervous system. Human exposure to MeHg and EtHg results mainly from the consumption of contaminated fish and thimerosal-containing vaccines, respectively. The mechanisms underlying the toxicity of MeHg and EtHg are still elusive. Here, we compared the toxic effects of MeHg and EtHg in Saccharomyces cerevisiae (S. cerevisiae) emphasizing the involvement of oxidative stress and the identification of molecular targets from antioxidant pathways. Wild type and mutant strains with deleted genes for antioxidant defenses, namely: γ-glutamylcysteine synthetase, glutathione peroxidase, catalase, superoxide dismutase, mitochondrial peroxiredoxin, cytoplasmic thioredoxin, and redox transcription factor Yap1 were used to identify potential pathways and proteins from cell redox system targeted by MeHg and EtHg. MeHg and EtHg inhibited cell growth, decreased membrane integrity, and increased the granularity and production of reactive species (RS) in wild type yeast. The mutants were predominantly less tolerant of mercurial than wild type yeast. But, as the wild strain, mutants exhibited higher tolerance to MeHg than EtHg. Our results indicate the involvement of oxidative stress in the cytotoxicity of MeHg and EtHg and reinforce S. cerevisiae as a suitable model to explore the mechanisms of action of electrophilic toxicants.

Neuroprotective activation of astrocytes by methylmercury exposure in the inferior colliculus.

Methylmercury (MeHg) is well known to induce auditory disorders such as dysarthria. When we performed a global analysis on the brains of mice exposed to MeHg by magnetic resonance imaging, an increase in the T1 signal in the inferior colliculus (IC), which is localized in the auditory pathway, was observed. Therefore, the purpose of this study is to examine the pathophysiology and auditory dysfunction induced by MeHg, focusing on the IC. Measurement of the auditory brainstem response revealed increases in latency and decreases in threshold in the IC of mice exposed to MeHg for 4 weeks compared with vehicle mice. Incoordination in MeHg-exposed mice was noted after 6 weeks of exposure, indicating that IC dysfunction occurs earlier than incoordination. There was no change in the number of neurons or microglial activity, while the expression of glial fibrillary acidic protein, a marker for astrocytic activity, was elevated in the IC of MeHg-exposed mice after 4 weeks of exposure, indicating that astrogliosis occurs in the IC. Suppression of astrogliosis by treatment with fluorocitrate exacerbated the latency and threshold in the IC evaluated by the auditory brainstem response. Therefore, astrocytes in the IC are considered to play a protective role in the auditory pathway. Astrocytes exposed to MeHg increased the expression of brain-derived neurotrophic factor in the IC, suggesting that astrocytic brain-derived neurotrophic factor is a potent protectant in the IC. This study showed that astrogliosis in the IC could be an adaptive response to MeHg toxicity. The overall toxicity of MeHg might be determined on the basis of the balance between MeHg-mediated injury to neurons and protective responses from astrocytes.

Evidences of possible influences of methylmercury concentrations on condition factor and maturation of Lophius vomerinus (Cape monkfish).

Muscle and liver tissues of Lophius vomerinus off the coast of Namibia were analysed to investigated the influence of MeHg on the biological parameters of L.vomerinus by (i) determining if the variability in total MeHg concentrations is influenced by length, maturity status and sex, and (ii) assessing if there is a relationship between biological indices (Condition factor (K), Gonadosomatic Index (GSI) Hepatosomatic Index (HSI)) and MeHg concentrations. Correlations between total MeHg concentrations and fish length, K and HSI were observed. A weak positive correlation was observed between total MeHg and GSI for combined sex. Total MeHg concentration in tissues of L. vomerinus is significantly dependent on the maturity stages (p < 0.05). K was significantly inversely correlated with total MeHg in tissues of L. vomerinus. The evidence presented in this study suggests that MeHg in L. vomerinus tissues could be detrimental to both its physiology and population dynamics.

Neurodevelopmental and Metabolomic Responses from Prenatal Coexposure to Perfluorooctanesulfonate (PFOS) and Methylmercury (MeHg) in Sprague-Dawley Rats.

Methylmercury (MeHg) and perfluorooctanesulfonate (PFOS) are major contaminants of human blood that are both common in dietary fish, thereby raising questions about their combined impact on human development. Here, pregnant Sprague-Dawley rats ingested a daily dose, from gestational day 1 through to weaning, of either 1 mg/kg bw PFOS (PFOS-only), 1 mg/kg MeHg (MeHg-only), a mixture of 0.1 mg/kg PFOS and 1 mg/kg MeHg (Low-Mix), or of 1 mg/kg of PFOS and 1 mg/kg MeHg (High-Mix). Newborns were monitored for physical milestones and reflexive developmental responses, and in juveniles the spontaneous activity, anxiety, memory, and cognition were assessed. Targeted metabolomics of 199 analytes was applied to sectioned brain regions of juvenile offspring. Newborns in the High-Mix group had decreased weight gain as well as delayed reflexes and innate behavioral responses compared to controls and individual chemical groups indicating a toxicological interaction on early development. In juveniles, cumulative mixture effects increased in a dose-dependent manner in tests of anxiety-like behavior. However, other developmental test results suggested antagonism, as PFOS-only and MeHg-only juveniles had increased hyperactivity and thigmotaxic behavior, respectively, but fewer effects in Low-Mix and High-Mix groups. Consistent with these behavioral observations, a pattern of antagonism was also observed in neurochemicals measured in rat cortex, as PFOS-only and MeHg-only juveniles had altered concentrations of metabolites (e.g., lipids, amino acids, and biogenic amines), while no changes were evident in the combined exposures. The cortical metabolites altered in PFOS-only and MeHg-only exposed groups are involved in inhibitory and excitatory neurotransmission. These proof-of-principle findings at relatively high doses indicate the potential for toxicological interaction between PFOS and MeHg, with developmental-stage specific effects. Future mixture studies at lower doses are warranted, and prospective human birth cohorts should consider possible confounding effects from PFOS and mercury exposure on neurodevelopment.

Methyl-mercury induces apoptosis through ROS-mediated endoplasmic reticulum stress and mitochondrial apoptosis pathways activation in rat cortical neurons.

Methyl-mercury (MeHg) is a dangerous environmental contamination biotransformed from mercury or inorganic mercury compounds in waterways, which induces severe toxic effects in central nervous system. Oxidative stress is involved in various ways of intracellular physiological or pathological processes including neuronal apoptosis. For understanding the ways that oxidative stress participating in MeHg-induced apoptosis, the current study attempted to explore the effects of oxidative stress on endoplasmic reticulum (ER) and mitochondria function, especially focussing on ER stress followed by unfold protein response (UPR), as well as mitochondrial apoptosis pathways activation in primary cultured cortical neurons. Cells were exposed to 0, 0.25, 0.5, or 1 µM MeHg for 1-6 h, respectively, followed by cell viability quantification. For further experiments, 100 µM of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) pre-treatment for 3 h followed by 1 µM MeHg for 6 h were performed for evaluation of oxidative stress, neuronal apoptosis, ER stress, UPR activation, and mitochondrial dysfunction. Results showed that MeHg induced neuronal oxidative stress, apoptosis, up-regulating glucose-regulated protein (GRP78, GRP94), spliced Xbp1, activating transcription factor 4 (ATF4) mRNA, with activation of UPR including PKR-like ER kinase-eIF2α, inositol-requiring enzyme 1, and ATF6 pathways, as well as C/EBP homologous transcription factor protein and cleaved caspase-12 up-regulation. In addition, mitochondrial function was disrupted by MeHg, which was supported by caspase-3 and caspase-9 activation, and high levels of cytoplasm cytochrome C and apoptosis induce factor. Trolox pre-treatment significantly blocked neuronal apoptosis, ER stress, UPR activation, as well as mitochondrial dysfunction, in addition to the direct anti-oxidation. In conclusion, MeHg induces neuronal apoptosis through ER and mitochondria pathway, oxidative stress plays important roles in mediating apoptosis pathways activation.

Hormesis of methylmercury-human serum albumin conjugate on N9 microglia via ERK/MAPKs and STAT3 signaling pathways.

Methylmercury (MeHg+) is an extremely toxic organomercury cation that can induce severe neurological damage. Once it enters the body, methylmercury binds to amino acids or proteins containing free sulfhydryl groups. In particular, methylmercury is known to bind with human serum albumin (HSA) in human plasma; however, the effects of methylmercury-HSA conjugate (MeHg-HSA) on the central nervous system (CNS) are not fully understood. In the present study, we used the microglial cell line N9 as the target cells to evaluate the effect of MeHg-HSA on physiological function of the CNS preliminarily. The various factors in the cell culture were monitored by MTT assay, total lactate dehydrogenase assay, ELISA, qPCR, Western blot and flow cytometry techniques. The results showed that low-dose treatment with MeHg-HSA activated N9 cells, promoting cell proliferation and total cell number, enhancing NO and intracellular Ca2+ levels, and suppressing the release of TNFα and IL1ß without cytotoxic effects; while high-dose MeHg-HSA exhibited cytotoxic effects on N9 cells, including promoting cell death and increasing the secretion of TNFα and IL1ß. These results indicate that MeHg-HSA causes hormesis in microglia N9 cells. Furthermore, ERK/MAPKs and STAT3 signaling pathways related to the hormesis of MeHg-HSA on N9 cells. In addition, low dose of MeHg-HSA might be viewed as something very close to a lowest observed adverse effect level (LOAEL) for N9 cells. These findings will be useful for investigating the hormesis mechanism of MeHg+ and exploring the specific functions of MeHg-sulfhydryl conjugates on the central nervous system.

Low doses of methylmercury exposure during adulthood in rats display oxidative stress, neurodegeneration in the motor cortex and lead to impairment of motor skills.

Despite the vast distribution among tissues, the central nervous system (CNS) represents the main target of methylmercury (MeHg) toxicity. However, few studies have evaluated the effects of MeHg exposure on the CNS at equivalent doses to human environmental exposure. In our study, we evaluated the motor cortex, an important area of motor control, in adult rats chronically exposed to MeHg in a concentration equivalent to those found in fish-eating populations exposed to mercury (Hg). The parameters evaluated were total Hg accumulation, oxidative stress, tissue damage, and behavioral assessment in functional actions that involved this cortical region. Our results show in exposed animals a significantly greater level of Hg in the motor cortex; increase of nitrite levels and lipid peroxidation, associated with decreased antioxidant capacity against peroxyl radicals; reduction of neuronal and astrocyte density; and poor coordination and motor learning impairment. Our data showed that chronic exposure at low doses to MeHg is capable of promoting damages to the motor cortex of adult animals, with changes in oxidative biochemistry misbalance, neurodegeneration, and motor function impairment.

Brain methylmercury uptake in fetal, neonate, weanling, and adult rats.

Fetuses and neonates are known to be highly susceptible to methylmercury (MeHg) toxicity, but little is known about the relative uptake of MeHg from blood to the developing brain. We measured time-course changes in mercury (Hg) concentrations in the brain of fetal, neonate, weanling, and adult rats after an injection of 0.08 µg (0.4 nmol) Hg/g MeHg. In the prenatal experiment, MeHg was subcutaneously injected to pregnant dams on embryonic days 17, 18, 18.5, 19, 19.5, or 20, and Hg concentrations in tissues were measured in both mothers and fetuses on embryonic day 21 (1 day before parturition). Brain Hg levels in fetuses peaked 2 days after injection and were approximately 1.5 times higher than in mothers. In the postnatal experiment, the same MeHg dose was injected subcutaneously to male rats on postnatal days 1 (neonates), 35 (weanlings), or 56 (adults). Mercury concentrations in tissues were measured 1, 2, 3, 4, 5, or 6 days after the injection. Brain Hg levels peaked most rapidly in neonates, and were approximately 1.5 times higher than levels in weanlings or adults. Throughout the examined period, peak Hg levels in the brain and the Hg brain/blood ratio 24 h after injection were highest in fetuses, followed by the levels in neonates, and decreased with life stage. These findings suggest that relatively higher brain MeHg uptake is an important factor in the vulnerability of fetuses and neonates to MeHg exposure.

Methylmercury-l-Cysteine targeting L-type amino acid transporter conjugate cytotoxicity on C6 glioma cells.

Glioma is the most common primary tumor in the brain, accounting for about 40~50% of intracranial primary tumors. Most chemotherapeutic drugs have difficulty in penetrating the blood-brain barrier, and their clinical applications are greatly limited. We evaluated the effects of methylmercury-L-cysteine (MeHg-L-cys) and methylmercury chloride (MMC) on apoptosis of C6 glioma cells. L-type amino acid transporter (LAT1) was used to investigate the targeted transport function and cytotoxicity of MeHg- L-cys in glioma. MeHg-L-cys enhanced the ability of targeting glioma cells and reduced the adverse reactions to normal brain tissues. Therefore, it is significantly important to develop new anti-glioma drugs targeting the blood-brain barrier.