GENETIC VARIATION IN ABC-TRANSPORTERS MODIFY METHYLMERCURY DISPOSITION
Methylmercury (MeHg) metabolism differs between individuals and may explain differences in MeHg susceptibility. One explanation for this variation might be that genetic pre-disposition affecting MeHg kinetics can result in individual differences in MeHg body burden despite similar MeHg intake. The glutathione (GSH) pathway has a central role in MeHg metabolism since GSH-MeHg conjugation is a prerequisite for elimination of MeHg from target organs and the body. GSH-MeHg transport occurs via ATP-binding cassette (ABC) transporters, also known to act as multidrug resistance-like proteins. ABC transporters have been associated with MeHg metabolism and toxicity in experimental and animal models, and there is also recent evidence for a role for ABC transporters in MeHg transport in humans. In studies of mother-child cohorts from Italy, Greece and Spain, we found that the childs genotype having single nucleotide polymorphisms (SNPs) in the ABC transporters ABCB1, ABCC1 and ABCC2 can modify the association between maternal fish intake and child MeHg levels (measured in cord blood). Consistent results were found in a Seychellois mother-child cohort in which we observed associations of SNPs in ABCB1, ABCC1 and ABCC2 with maternal MeHg (measured in hair), with one SNP in the ABCC1 gene also showing association with child neurodevelopment. These human studies support the experimental animal models that ABC transporters play a major role for the kinetics of MeHg in the body. Moreover, variation in ABC genes may influence the MeHg dose in the developing child and thus could be relevant to neurodevelopmental outcomes.
ALTERNATIVE TESTING METHODS: COMPARING TEST SENSITIVITIES BETWEEN FISH EMBRYO AND FISH CELL MODELS
Mercury is an intensely studied element and compound family. Regulations pertaining to environmental concentrations, occupational exposures, and dietary consumptions should be scrutinized and refined as the state of the art progresses. The Tox 21 initiative challenges the scientific community to progress to methods that are at once, higher throughput, less consumptive of animal life, and more sensitive than classical toxicity testing methods. In current standard methods apical endpoints used for zebrafish embryos (coagulation, somite formation, and tail detachment) are often surrogates for mortality. Basic biochemical assays, when used in concert, may form the basis of a more sensitive and informative series of endpoints than measures that are stand-ins for mortality. In addition, there is a need to compare in vitro and in vivo results. Here, a systems approach was taken to assess methylmercury toxicity using enzyme activities as indicators of redox stress in both a zebrafish (Danio rerio) embryo model, and a zebrafish derived liver cell line (ATCC CRL-2643). Zebrafish cells were exposed to a range (0.01 µM to 1 µM) of methylmercury-chloride. A similar design was used to expose zebrafish embryos to aqueous methylmercury-chloride (0.01 µM to 10 µM). Current standard endpoints (OECD TG 236) were assessed along with a battery of biochemical assays. Enzymatic activity assays performed in vitro and in vivo included monoamine oxidase, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, peroxiredoxin, thioredoxin reductase, and oxoglutarate dehydrogenase. Uptake by embryos and cells was quantified by using GC-CVAFS, and direct mercury AA. In vitro model results were compared to in vivo exposure of zebrafish embryos to similar concentrations of methylmercury. A comparison of the sensitivity of endpoints between standard test methods and the biochemical endpoints will be discussed. Further discussion will include the responses of in vitro exposures using these biochemical endpoints in contrast with in vivo responses. These studies are expected to narrow the gap between mercury in vitro models and in vivo observations in teleost fish. Future work will include studies investigating the effects of exposure among three forms of mercury: inorganic mercury, methylmercury-chloride and methylmercury-cysteine.
METHYLMERCURY-INDUCED OXIDATIVE DAMAGE IN LIVER, KIDNEY AND BRAIN OF RATS: PROTECTION BY N-ACETYL CYSTEINE AND SELENIUM
Methylmercury is a ubiquitous and hazardous material in the environment due to the circulation of inorganic mercury in the environment which is dissolved into rivers and sea, condensed through the food chain, ingested by humans, and consequently affects human health. Therefore, methylmercury deserves the attention of environmental health experts. It is an emerging problem in the world as its concentration is rising continuously due to increased industrial, medicinal and domestic uses. It is bio transformed to highly toxic free metabolites thus eliciting tissue and blood biochemical alterations and oxidative stress. The aim of present study describes the protective effect of N-acetyl cysteine (NAC) either alone or in combination with selenium against chronic mercuric chloride poisoning. The experiment was carried out in male albino Sprague Dawley rats (N=30) which was divided into ﬁve groups. Group 1 served as control were received vehicle for twelve weeks (once daily for 7 days). Groups 25 were administered methylmercury (MeHg: 1.0 mg/kg, p.o.) for twelve weeks (once daily for 5 days), group 2 served as experimental control. Animals of groups 3, 4 and 5 were received N-acetyl cysteine (NAC: 0.6 mg/kg, i.p.) and selenium (Se: 0.5 mg/kg, p.o.) and NAC with Se in combination for twelve weeks (after toxicant administration for remaining 2 days only). Chronic MeHg exposure altered various biochemical parameters, including transaminases, alkaline phosphatase, lactate dehydrogenase, bilirubin, gamma-glutamyl transferase, triglycerides and cholesterol, urea, creatinine, uric acid and blood urea nitrogen contents with concomitant decline in protein and albumin concentration in serum. In addition, a signiﬁcant rise in lipid peroxidation level and mercury ion concentration with concomitant decrease in reduced glutathione content and the antioxidant enzymes activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase after chronic MeHg exposure. Results of the present investigation clearly showed that combination therapy with NAC+Se provide maximum protection against chronic mercury toxicity than monotherapy (alone treated groups) by preventing oxidative degradation of biological membrane from metal mediated free radical attacks. The activities of comet assay and MTT assay significantly recovered the damage towards normal after combined treatment (NAC+ Se). Biochemical data were well supported by histopathological findings. In conclusion, combination therapy may be an ideal choice for long term mercury toxicity and oxidative stress.
MERCURY-INDUCED EPIGENETIC TRANSGENERATIONAL INHERITANCE OF ABNORMAL NEUROBEHAVIOR IS CORRELATED WITH SPERM EPIMUTATIONS AND DYSREGULATED BRAIN AND RETINA GENE EXPRESSION IN ZEBRAFISH
Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant. Developmental exposure of zebrafish to MeHg is known to alter their neurobehavior in a manner similar to other vertebrates. The current study investigated the direct and transgenerational effects of MeHg, at tissue doses similar to those detected in exposed human populations, on neurobehavior (i.e., visual startle and spontaneous locomotion), sperm epimutations (i.e., differential DNA methylation regions [DMRs]), and transcriptomics in zebrafish, an established human health model. F0 generation embryos were exposed to MeHg (0, 1, 3, 10, 30, and 100 nM) for 24 hours ex vivo. F0 generation control and MeHg-exposed lineages were reared to adults and bred to yield the F1 generation, which was subsequently bred to the F2 generation. Direct exposure (F0 generation) and transgenerational actions (F2 generation) were then evaluated. Hyperactivity and visual deficit were observed in the unexposed descendants (F2 generation) of the MeHg-exposed lineage compared to control. An increase in F2 generation sperm epimutations was observed relative to the F0 generation. Investigation of the DMRs in the F2 generation MeHg-exposed lineage sperm revealed associated genes in the neuroactive ligand-receptor interaction and actin-cytoskeleton pathways being effected, which correlate to the observed neurobehavioral phenotypes. Analysis of dysregulated genes revealed via sequencing of total RNA isolated from F2 generation MeHg-exposed zebrafish retina and brain (compared to control) showed significant enrichment in pathways associated with vision, circadian rhythm, and neurological disorders. Collectively, this evidence supports that developmental MeHg exposure can induce epigenetic transgenerational inheritance of abnormal neurobehavior correlated with dysregulated brain and retina gene expression and sperm epimutations in F2 generation adult zebrafish. Therefore, mercury has the ability to promote the epigenetic transgenerational inheritance of disease in zebrafish, which significantly impacts its environmental health considerations in all species, including humans. Further study of the transgenerational health effects of MeHg is required to better understand the risks for human populations, which, if transgenerational effects are ignored, are dramatically underestimated.
PHYSIOLOGICALLY-BASED PHARMACOKINETIC MODELING OF METHYLMERCURY IN YOUNG RINGED SEALS
Methylmercury (MeHg) is a potent, bioaccumulative neurotoxicant that is particularly harmful to mammals at early developmental stages. Previously published studies have measured concentrations of MeHg in young ringed seals but have made little effort to quantitatively explain variability in the data. We constructed a physiologically-based pharmacokinetic model to examine the relative importance of body growth, changing diet, and internal demethylation to explain changes in MeHg burdens in various biological compartments. The model is evaluated with measured MeHg and total mercury (Hg) concentrations in young-of-the-year ringed seal tissues from Labrador, Canada that were harvested between 2012 and 2015. Most seals were less than one year of age. Measured mean Hg concentrations (wet weight) in muscle and tissues from 52 individuals were: 0.095 mg/g (n = 52, s = 0.081 mg/g), and 0.67 mg/g (s = 1.06 mg/g), respectively. Mean MeHg concentration (wet weight) in muscle was 0.11 (n = 35, s = 0.089 mg/g) and is 0.12 mg/g (n = 52, s = 0.15 mg/g) in liver. Model outputs show that the dynamics of mercury disposition and accumulation in the first year can be largely attributed to dietary shifts. This model can be further modified and applied to other marine mammals to study early-life exposure patterns for species of interest, including those consumed as country foods by Inuit communities in Arctic and Subarctic regions.
THE EFFECTS OF METHYLMERCURY ON BREAST CANCER CELLS
Metalloestrogens are small ionic metals that activate the estrogen receptor (ER). Studies have shown that when metalloestrogens bind to the ER, there is an increase in transcription and expression of estrogen-regulated genes, which induces proliferation of estrogen-dependent breast cancer. Methylmercury (MeHg), a metalloestrogen, is present in the environment and is toxic at moderate to high concentrations. However, at lower concentrations MeHg may promote the proliferation of ER-positive breast cancers and protect cells against pro-apoptotic signals. To investigate the effects of MeHg treatment on breast cancer cells in vitro, MCF7 breast cancer cells were treated with concentrations of MeHg ranging from 1 nM to 100 mM. MeHg analysis was used to quantify intracellular mercury concentrations and cell proliferation and apoptosis were determined by cell counting and Annexin-V staining, respectively. We defined a protocol that maximizes cellular exposure to mercury and we found that treatment of human ER-positive breast cancer cells with 1 nM MeHg promoted proliferation, while treatment with a concentration of 100 nM induced apoptosis. Investigations into the effects of dietary MeHg treatment of zebrafish on breast cancer growth and progression are currently underway. Clarifying the effects of MeHg on breast cancer will improve our understanding of how environmental toxins affect tumor progression and may lead to the development of future therapeutic strategies.
IMPACT OF METHYLMERCURY IN HYPOSTOMUS GYMNORHYNCHUS AND HOPLIAS AIMARA HARVESTED IN PRISTINE AND CONTAMINATED SITES FROM THE OYAPOCK RIVER IN FRENCH GUIANA
A multidisciplinary approach has been conducted to compare toxicity mechanisms of methylmercury (MeHg) previously obtained in zebrafish (Danio rerio) during experimental condition to those obtained in fish harvested in the field in French Guiana. For this purpose, genes known to be involved in antioxidant defences, metal chelation, active efflux of organic compounds, mitochondrial metabolism, DNA repair and apoptosis have been cloned and characterized for two typical fish: Hypostomus gymnorhynchus (a grazer fish) and Hoplias aimara (a top predator). Fish were obtained from three different sites; a pristine site (3 sauts) and two mercury-contaminated sites (Oyapock and Camopi). Expression levels were determined on brain, muscle and liver as well as ultrastructural analyses by electronic microscopy. Moreover, quantification of mercury bioaccumulation and of metallothionein (MT) proteins have been done in the same tissue. Results obtained for H. gymnorhynchus evidenced that genes involved in detoxification, oxidative stress response and mitochondrial metabolism were overexpressed in contaminated sites compared to pristine one in all the tissue. Histological analyses showed that damages in mitochondria were also observed in fish from the contaminated sites. Indeed, in contaminated muscles a decrease of the inter-bundle surface, mitochondria with variable shapes, sizes and cristae disorganization, also decreasing the surface area and inter-bundle surfaces were observed. In liver from the contaminated fish, an increased quantification of MT levels were evidenced. All these results confirmed those obtained in laboratory-controlled experiment with D. rerio.
TOXICOKINETICS OF MERCURY IN THE HUMAN PLACENTA: PRELIMINARY DATA ON RELATIONSHIPS BETWEEN GENOTYPE AND PHENOTYPE IN HEALTHY AND DISEASED PLACENTA
Although it has been known for decades that the organic compound methyl mercury (MeHg) is neurotoxic and passes the placenta unhindered, our knowledge of the transport mechanisms is extremely low.
The placenta is a transient organ that persists just nine months but has a significant influence on health and development of the offspring. Dysfunction of the placenta often leads to pregnancy complications such as gestational diabetes (GDM), pre-eclampsia (PE), and intrauterine growth retardation (IUGR).
The development of placental dysfunction is multifactorial. The placenta is well suited for the investigation of genotype-phenotype associations because the organ is easily available, protein expression and function are readily measurable, and primary cells can be isolated therefrom.
In preliminary studies, we have investigated which proteins are involved in placental toxicokinetics (transport and metabolism) of mercury. A number of these proteins are also involved in placenta dysfunctions. They include system L amino acid transporters, enzymes of the anti-oxidative glutathione system and efflux transporters from the family of ABC transporters.
The main questions are: Which proteins are involved in placental dysfunction? To what extent does the genetic background (sequence variants) explain abundance or activity of these proteins? Is the infant genotype linked to the placental phenotype?
In total 170 mother-child pairs (100 healthy, 70 with GDM, PE, IUGR) will be genotyped. Expression and activity of the candidate proteins are examined in healthy and dysfunctional placentas using MeHg as a model substrate. By researching placental mercury toxicokinetics, we gain insights that are not only relevant for reproductive toxicology, but also contribute to a better understanding of the individually different placenta (patho) physiology. The aim is to identify genetic variants predisposing to pregnancy complications. The identification of such genetic markers is a very big step towards individual treatment and counselling during pregnancy. Preliminary data on selected candidate proteins will be discussed.
CHARACTERIZATION OF MERCURY-BINDING PROTEINS IN RAT BLOOD
Blood proteins play important roles in mercury transport, toxicity and metabolism. Characterization of mercury-binding proteins, especially proteins with low abundance is critical for understanding their trafficking and underlined mechanisms of toxicity. Till now, very limited proteins, i.e., albumin, 2-macroglobin, and transferrin had been identified as mercury-binding proteins in plasma. For erythrocyte, hemoglobin was the only protein known to associate with mercury. In this study, mercury-binding proteins in rat plasma and erythrocyte were characterized in vitro by continuous flow gel electrophoresis coupled with inductively coupled plasma mass spectrometry (GE-ICP-MS). It was found that most of Hg were associated with proteins, in which around 60% of mercury was bound to albumin and 25% to hemoglobin. Apart from the high abundance proteins, ten mercury-binding proteins with low abundance in blood were identified. There were four proteins in plasma, which were glutathione peroxidase 3, apolipoprotein E, alpha-1-antiproteinase, and transferrin, respectively. For erythrocyte samples, six mercury containing proteins were detected. Mercury preferentially binds to hemoglobin (8 %) followed by peroxiredoxin-2 (5 %), while the other three proteins bound 3 % mercury respectively. Our finding provided useful information to further investigate health effects and toxicities related to Hg exposure.
DIFFERENT EFFECTS OF METHYLMERCURY EXPOSURE ON LOCOMOTOR BEHAVIORS IN ADULT MALE AND FEMALE ZEBRAFISH (DANIO RERIO)
Methylmercury (MeHg) is a widely distributed environmental neurotoxin with established effects on the central nervous system, yet the subtle behavioral effects of MeHg toxicity are less well studied. Previous work from this group has demonstrated that MeHg exposure disrupts locomotor behavior in adult male mice, specifically behaviors related to coordination, balance and strength. Other groups have shown that the deleterious effects of MeHg can be prevented in male mice with estrogen treatment, suggesting a sex difference in susceptibility to MeHg toxicity. The goal of the present study is to further examine sex differences in MeHg-induced behavioral changes using an aquatic animal model. This study investigated the impact of MeHg exposure on several swimming behaviors in adult male and female zebrafish. MeHg was administered daily in food over a three-week period. The daily MeHg dose (0.1 ug/g/day) was comparable to known environmental exposure levels. Swimming behaviors (swim distance, swim speed and line crossings) were assessed before treatment and then weekly during the treatment period. Preliminary findings suggest that swimming behaviors were unaffected in female zebrafish exposed to MeHg at this dose. However, MeHg exposure increased swimming activity in males by week three of treatment. Behavioral differences will be linked to changes in antioxidant enzyme activities in tissues. Future studies will focus on the potential neuroprotective effects of estrogen on MeHg toxicity in zebrafish.
ARE MATERNAL STOOL SAMPLES A VALID BIOMARKER FOR PRENATAL METHYLMERCURY EXPOSURE?
Abstract not available.
DEVELOPMENT OF AN ECOPHYSIOLOGICAL DAPHNIA MODEL TO EXAMINE THE INTERACTIVE EFFECTS OF NUTRITION AND METHYLMERCURY TOXICITY
In spite of efforts to address issues of mercury in the environment, levels continue to rise. Rapid and reliable tools are imperative in water quality management to mitigate potentially catastrophic ecosystem shifts. Through the integration of ecosystem-level models with chemical analysis tools, our research proposes the development of a novel bioindicator technique that will connect the physiological stress of individual organisms to ecosystem dynamics. Daphnia, a crustacean zooplankton, represents an essential compartment in aquatic ecosystems at the autotroph-heterotroph interface, connecting phytoplankton to many fish species, and has been suggested as a keystone species. We have developed a model that aims to elucidate the interactive effects of mercury toxicity and nutrition (in terms of food quality and quantity) on the relationship between phytoplankton and Daphnia through an understanding of both mass and energetic consequences. Generally, zooplankton modelling studies on food quality have examined a limited range of nutrients, typically macronutrients such as carbon, nitrogen and phosphorus; we have expanded our scope of nutrition to include fatty acids, amino acids, saturated fats, and other nutrients. Our model stipulates a physiological hierarchy, prioritizing different physiological processes in the order of neurological functions, bio-energetics, osmoregulatory maintenance, waste management, and growth investments. Daphnia resiliency is controlled by both food quantity and quality and each physiological process is modulated by the somatic levels of different metabolites. With the enrichment of food with congeners associated with neurological and energetic functions (e.g., tryptophan, phosphorus), effects of toxicity can be countered. Our work highlights that healthy dietary patterns can be critical in ameliorating toxicity, and therefore biotic populations cannot be maintained solely through contaminant management. Our modelling framework links physiological and biochemical level processes to ecosystem patterns to provide a deeper understanding of the interactions between toxicity and food quality and whether food quantity and quality can mitigate the impacts of mercury toxicity in Daphnia and improve resistance and resilience against mercury. We offer the prospective of quantitative response indicators in long-term water monitoring efforts and a framework that can accommodate parameterization from values derived through metabolomics experiments. Understanding the responses and linkages between these ecosystem drivers may improve the management and conservation of the ecosystems contaminated with mercury.
ELUCIDATING THE BIOCHEMICAL EFFECTS OF MERCURY EXPOSURE IN AN ENVIRONMENTAL SENTINEL WITH METABOLOMICS AND EPIGENETICS TECHNIQUES
Mercury (Hg) is a toxic environmental pollutant with a ubiquitous, worldwide distribution. The toxic effects of methylmercury (MeHg) exposure are well documented; and include neurodegenerative diseases (e.g. Minamata Disease), muscular disorders, and lowered reproductive success. However, determination of the sub-lethal effects of MeHg exposure is complicated due to the lack of symptoms prior to the onset of degenerative diseases. In an effort to classify some of the underlying biochemical effects of chronic MeHg exposure, we investigated physiological and epigenetic changes associated with two doses of chronic MeHg exposure over a two-year time period on an ecologically important reptilian sentinel, the diamondback terrapin (Malaclemys terrapin). Erythrocyte samples were analyzed for total mercury content and subsequently for global DNA methylation by LC-MS/MS and plasma was analyzed by 1H NMR for metabolomic effects. DNA methylation is a well-studied epigenetic modification that controls gene expression. When DNA methylation patterns are changed, as a result of external stimuli, altered gene expression and dysregulation of certain cellular processes can occur and be characterized. Analyzing the plasma metabolic profile of organisms exposed to MeHg in a controlled setting allows the identification of metabolites and their corresponding pathways that change in response to the exposure. Plasma metabolomic profiles were analyzed by multivariate and univariate techniques across doses, gender, and annual time scales. This study aimed to identify small molecules that change in response to chronic MeHg exposure, as well as investigate changes in epigenetic modifications that are likely exacerbated as a result of exposure. Both of these tools may lend insight to the underlying cellular mechanism of MeHg exposure and allows for a greater understanding of organism-environment interactions.
UNVEILING COPING STRATEGIES WITH MERCURY IN AQUATIC AND SEMI-AQUATIC INHABITANTS
Despite the credible information on environmental hazards identification/characterization, risk assessment and coping strategies in aquatic and semi-aquatic inhabitants, lacunae on mechanistic aspect entangling the previously mentioned issues are perceptible in literature. Thus, the current work aimed to assess mercurys potential toxicity on biota and discusses organisms survival strategies under natural environmental conditions. Primary consumers (bivalve- Scrobicularia plana) were selected since they comprise the baseline of the food web and serve as a conduit for bottom-up energy transfer, whereas the salt marshes plants (Halimione portulacoides and Juncus maritimus) were considered keeping in view their status as essential ecosystem base components. Area chosen for this study was Laranjo Basin, Ria de Aveiro, Portugal, where a well defined mercury gradient has been reported due to chloralkali plant.
In bivalves, inter-age and organ-specific approaches were applied by using different annual age classes (2+, 3+, 4+ and 5+ year old) and assessing specific organs (gills, digestive gland), respectively. Besides total and organic mercury accumulation, endpoints combining lipid peroxidation, as damage sign, and antioxidant protection were determined. It was revealed that the use of whole-body analyses could be particularly compromising when enzymatic antioxidants are addressed. Besides the contribution to understand mercury toxicodynamics, specific organs approach was strongly recommended in order to avoid misinterpretations. Analyses of non-enzymatic antioxidants revealed that the evolution of the adaptive skills of S.plana over time depends on the contamination extent. Hence, under a moderate contamination scenario, the intervention of the different antioxidants (measured in whole-body) took place harmoniously, evidencing an adjustment capacity increasing with the age. In opposition, under a higher contamination degree S.plana failed to efficiently cope with mercury threat.
Salt marsh plants H. portulacoides and J. maritimus exhibited the highest and the least mercury-remediation/accumulation potential, respectively. Salt marshes dominated by J. maritimus exhibited a stronger capacity to retain and phytostabilize mercury in belowground than those dominated by H. portulacoides. Conversely, extensively H. portulacoides-colonized salt marshes are expected to translocate more mercury to aboveground parts. It can be possible to phytostabilize metals in rhizosediments (using J. maritimus) or phytoextract metal by accumulating in aboveground plant tissue for subsequent plant removal (using H. portulacoides). Moreover, mercury-induced oxidative stress was differentially counteracted by ascorbate-glutathione cycle-components in the studied plants. These changes were strongly substantiated with the changes at proteome level. Conclusively, coping strategies in both animal and plant revealed their significance as biomarkers for environmental hazards safety screening.
THE BIOCHEMISTRY OF THE FIVE “SOS” MECHANISMS OF MERCURY TOXICITY
Methylmercury (MeHg) toxicity is characterized by: 1.) long latency of onset of symptoms following toxic exposure, 2.) neurological tissue specificity of pathological effects, 3.) inhibition of selenoenzymes in brain tissues, 4.) oxidative damage in the affected tissues, 5.) accentuated fetal vulnerability, and, 6.) preventative/rescue effects accompanying supplemental dietary selenium (Se) commonly referred to as the Se-protective effect. These aspects had previously been difficult to explain, but understanding of MeHg toxicology has improved tremendously in the past decade. Since we now recognize that Hgs affinity for selenium (Se) is ~1 million times greater than its affinity for sulfur, its second best binding partner, it has become obvious that Se, not sulfur, is the molecular target of MeHg toxicity. Because of its high binding affinity for Se, MeHg inhibits synthesis and activities of Se-dependent enzymes that are required for the health, function, and development of brain tissues. The 25 genetically distinct and functionally elite enzymes expressed in humans perform essential functions in brain physiology including; preventing and reversing oxidative damage to lipids and proteins, enabling DNA synthesis, and regulating thyroid hormone metabolism, calcium homeostasis, and cell signaling pathways. The toxic effects of high MeHg exposures arise from its unique abilities to selectively and irreversibly inhibit selenoenzyme activities. This understanding MeHgs effects provides a unifying perspective that consistently explains all aspects of MeHg toxicity. The involvement of Se-physiology in mechanisms of MeHg toxicity were not initially recognized because selenoenzyme metabolism and its unique roles in brain and endocrine tissues were generally unknown. However, in recent years, the Se-dependent aspects of their physiology are becoming better understood. As a result, the characteristic features of MeHg toxicity have become much easier to understand. High MeHg exposures occur through a sequence of biochemical reactions referred to here as SOS mechanisms. These include; Synergies of Sequestration (SOS-1), Silencing of Selenoenzymes (SOS-2), Sequestration of Selenium (SOS-3), Suicide of Selenium-Deprived Cells (SOS-4) and Sustained Oblivion of Sec Synthesis (SOS-5). These disruptions have consequences that increase in severity as tissue MeHg concentrations approach, and especially as they exceed equimolar stoichiometry with tissue Se. The effects of SOS mechanisms on Se physiology and biochemistry coincide with features of MeHg toxicity which were previously difficult to explain. Predictions based on these mechanisms coincide with observations in cell culture and animal models as well as in epidemiological studies of the effects of MeHg exposures on neurodevelopmental and cardiovascular outcomes.
PROTEOME OF PRIMARY ASTROCYTES INDICATE A CENTRAL ROLE OF OXIDATIVE INJURY EXPOSED BY MEHG
Methylmercury (MeHg) is a neurotoxicant that posed a health risk in both wildlife and human. Cerebellum is the main target of mercury toxicity. The toxic effects of MeHg on specific brain cell type is still unclear. In this study, cerebellar primary astrocytes were analyzed for cellular reactive oxygen species (ROS), total Hg accumulation, and whole cell proteome after exposed with MeHg (1 µM and 5 µM) for 24 hours. ROS and intracellular Hg concentrations showed a dose-dependent increase (p<0.05). In proteomic analysis, a total of 3230 proteins were identified, among them 436 proteins were significantly changed (One Way ANOVA, p<0.05). Biological processes enrichment analysis showed that small molecule metabolic process, energy derivation by oxidation of organic compounds, cellular respiration, oxidation-reduction process and positive regulation of DNA biosynthetic process are the main functions affected by MeHg. Oxidation-reduction process is the most significantly changed category with 75 proteins were involved. After correction of the p value (Permutation-based FDR method), only 17 and 35 proteins were differentially expressed in 1 µM and 5 µM exposed MeHg, respectively. Oxidoreduction coenzyme metabolic process was significantly changed in both treatment groups. Oxidoreductase complex, mitochondrial respiratory chain, and membrane raft are the main celluar components affected in 5 µM MeHg but not in 1 µM MeHg (p<0.05). KEGG Pathway analysis showed Huntington’s disease (HD) was enriched (p<0.05) in 1 µM MeHg exposed astrocytes, which is further confirmed our previous publication on a MeHg dosed marmoset model. These results indicated that oxidation and reduction process is a major event occurred in MeHg exposed astrocytes. MeHg exposure was associated with neurodegenerative disease such as HD. Increasing dose of MeHg can disrupt energy generation which is evidenced by the significant changes of mitochondrial respiratory chain system.
EFFECTS ON PROLACTIN (PRL) ON CYTOTOXITY INDUCED BY METHYLMERCURY IN HUMAN LYMPHOCYTES AND HUMAN PROMYELOCYTIC LEUKEMIA CELL HL60, BOTH IN VITRO.
Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well known and humans are exposed by consumption of contaminated fish, shellfish and algae. Methylmercury has been an environmental concern to public health and regulatory agencies for over 50 years because of its neurotoxicity. Its association with nervous system toxicity in adults and infants near Minamata Bay, Japan, in the 1950’s initiated environmental health research inquiries that continue to this day. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes; and after that evaluate Prolactina's action (PRL) on the organomercurial (CH3HgCl) activity in human lymphocytes and human promyelocytic leukemia cell HL60, both in vitro. The cultures were incubated to 37ºC for 48 h for both biological systems and the treatments were realize 9h after the beginning stimulating cells. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl (1, 10, 100, and 1000 µg/l) when compared to control, whether alone or in an evident sinergistic combination with HgCl2. In the second experimental block, CH3HgCl when alone treaty (50, 100, 500 and 1000 μM) it was cytotoxic also, however its reduced when treaty together with PRL (1,10 and 100 nM). The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2, however reduced been in PRL presence. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 µg/l alone, and at 1, 10, 100, and 1000 µg/l when combined with HgCl2, showing a synergistic cytotoxic effect, the same results presented in the second experimental block too, however the PRL presents a protector effect over that. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic and the PRL is a potent antimutagenic agent in this biological systems.
TOXICOKINETICS OF MERCURY DEMETHYLATION: MERCURY ACCUMULATION AND SPECIATION IN THE LIVERS OF MARINE AND FRESHWATER FISH SPECIES
Mercury is a global contaminant that is atmospherically deposited from a variety of anthropogenic and natural sources. In aquatic systems, bacteria convert inorganic mercury (Hg (II)) into the more bioaccumulative and toxic form methylmercury (MeHg). Fish store mercury mainly in muscle, liver and kidney tissues. In the liver, MeHg is metabolized (demethylated) to form Hg (II). In many fish species, it is unknown if there is a threshold concentration at which demethylation occurs or whether demethylation is rate limited. It has been postulated that phylogenetic differences among fishes in immune organization may determine demethylation potential and thus the ability of a species to distribute mercury amongst tissues. The objective of this study was to examine mercury accumulation and speciation in several marine (n= 12) and freshwater (n=14) fish species. Liver and muscle tissue samples were taken and total mercury concentrations determined using a Milestone DMA-80 direct Hg analyzer. Toluene extraction or GC-CVAFS was used to remove MeHg from liver tissues and determine concentrations of MeHg and Hg (II). A percent MeHg was calculated for each individual as well as a ratio of total muscle mercury to total liver mercury as a proxy for examining sequestration tissues. Mean total muscle Hg concentrations across all species ranged from 0.28 (Ladyfish) 5.00 (Bowfin) mg\kg dw, and mean total liver Hg ranged from 0.24 (Bonnethead) 13.51 (Spotted Gar) mg\kg dw. The highest concentrations for both muscle and liver were seen in freshwater species. Liver total Hg was positively correlated with liver MeHg for salmonids, whereas for all other species, there seems to be a threshold effect. Our results show multiple trends to describe the relationship between liver total Hg and % MeHg. These results will add important information to the knowledge of how phylogeny potentially influences mercury demethylation, and the toxicokinetics of mercury demethylation amongst fishes to help develop a next-generation risk assessment that incorporates species sensitivity.
PROTEOMIC AND TRANSCRIPTOMIC INSIGHTS INTO CELLULAR AND MOLECULAR DAMAGE AND RECOVERY FROM INORGANIC AND ORGANIC HG EXPOSURE IN A MODEL MICROORGANISM
Bacteria are the most vigorous and versatile transformers of Hg in all global ecosystems, some making the very lipid soluble forms, Hg(0) and MeHg(I), and others converting these to highly reactive ionic forms Hg(II) and MeHg(I). Such processes, long studied in the external environment, are increasingly recognized also to operate within commensal microbial ecosystems (microbiota) of humans, and other wildlife exposed to Hg environmentally or iatrogenically. All macroscale Hg transformations have at their base the enzymes within individual cells which catalyze those transformations. Most often those cells are bacterial, simply because they outnumber all other cell forms on Earth and occupy niches in every temperature, humidity, salinity, and oxygen range on the planet, even those with highly toxic metal values.
Knowledge of the many ways in which diverse bacteria are affected by and then transform Hg is essential for understanding the macroscale processes on which environmental remediation, stewardship and risk analyses are based. This is true not only on the scale of terrestrial geology, oceanography, glaciology, atmospheric science, etc but also directly in the plant and animal victims of Hgs toxicity. Surprisingly, there is limited understanding of the distinctly different ways in which inorganic (Hg) and organic (RHg) mercury compounds affect subcellular biochemistry. Most monitoring of exposure to these compounds is by proxies such as behavioral or neurological measures and/or a few signature proteins or metabolites.
Moving our vision to the interior of living cells, we investigate key subcellular targets of mercurials in a common gut commensal, E. coli. Well describe the following studies on growing E.coli cells: (1) biophysical analyses quantifying disruption of electrolyte, metal, and biothiol homeostases by RHg and especially Hg compounds; (2) a novel global LC/MS-MS proteomics method identifying >300 proteins variously vulnerable to stable RHg or Hg modification, many with highly conserved human homologs, especially in mitochondria; and (3) a longitudinal RNA-seq systems level comparison of gene expression showing strikingly different responses immediately after exposure to and during recovery from RHg or Hg compounds. Our findings provide foundations for (a) developing specific biomarkers for RHg/Hg exposure, (b) understanding long-term Hg accumulation in higher vertebrates, (c) investigating the mechanisms of hormesis i.e. use of subtoxic stressors as adaptogens, and (d) assessing nutritional, pharmaceutical or environmental interventions to recruit bacteria in support of recovery from chronic or acute RHg/Hg exposure in individual animals or humans or in external environmental settings.
EVALUATION OF NEUROBEHAVIORAL DISORDERS IN METHYLMERCURY-EXPOSED KK-AY MICE BY DYNAMIC WEIGHT BEARING TEST
Methylmercury (MeHg) is known to cause neurobehavioral disorders in humans and experimental animals. We are studying MeHg toxicity in type 2 diabetic KK-Ay mice to clarify the effect of glucose metabolism disturbance on MeHg toxicity. In a previous study, we observed that exposing 4-week-old KK-Ay mice to 5 mg Hg/kg MeHg resulted in severe neurobehavioral disorders such as hindlimb clasping. Neurobehavioral disorders due to MeHg exposure in rodents have been mostly evaluated by observation of hindlimb crossing, rota-rod performance test, and footprint test. However, it is difficult to quantitatively evaluate the neurobehavioral disturbance of MeHg-treated KK-Ay mice because of their obesity. In this study, we administrated MeHg (5 mg Hg/kg/day p.o.) to male KK-Ay mice three times per week for 5 weeks beginning at 12 weeks of age, and examined neurobehavioral disturbances using the dynamic weight bearing (DWB) test, which has been used to assess pain. MeHg-treated mice began to lose body weight approximately 4 weeks after treatment was initiated. Seven of 9 MeHg-treated mice showed neurological symptoms such as ataxia and gait disturbance in the final stage of the experiment. The weight measured using the DWB test was lower for the forelimb than for the hindlimb before MeHg treatment and until 1 week after treatment was initiated. At weeks 24 of MeHg exposure, the DWB load on the forelimb became similar to that on the hindlimb. After 5 weeks of exposure, the DWB load on the forelimb exceeded that on the hindlimb. Injury of sciatic nerves in MeHg-exposed KK-Ay mice with neurobehavioral disorders was detected using the Masson-Goldner staining. This finding indicates that the DWB test can be useful for semi-quantitative evaluation of neurobehavioral disorders in MeHg-exposed rodents.
MATERNAL TRANSFER OF MERCURY IN THE BLOOD OF ORGANISMS STANDING ON THE TOP OF TROPHIC CHAIN
Marine mammals are exposed to mercury mainly through the diet. Being top predators they accumulate high levels of pollutants as the consequence of biomagnification process. After absorbtion, toxic substances, as methylmercury, are distributed throughout the organism via circulatory system. Blood is an excellent transport channel for methylmercury. Percentage of this form in blood may exceed 90%. Females exposure to methylmercury during pregnancy may be extremely dangerous to the fetus. because physiological barrier such as placenta is transparent for this compound and cannot provide protection against it.
The object of examination was the grey seal (Halichoerus grypus) females and their pups blood. Seals stay in Hel Marine Station (Institute of Oceanography, University of Gdansk, Poland). 4 females located there every year give a birth to pups which after 3 weeks of nursing are being prepared to live independently in their natural environment. After this, all the pups are released to the Baltic Sea due to grey seal reintroduction programme. During 3 years of sampling 9 pups were born in sealarium.
In all blood samples total (HgTOT) and organic (HgORG) myercury concentrations were measured using atomic absorption spectrometer AMA 254. Wherein analysis of the HgORG included its previous extraction and transferring it to a hydrophobic carrier.
The aim of the study was not only checking the relationship between MeHg concentration in seal females and their pups blood, but also the analysis of changes in Hg concentration in females and their pups blood during the first weeks after birth. To verify whether the transfer processes between mother and offspring occur in a similar way in humans, studies were supplemented with the analysis of human blood (women and their newborn children).
This study resulted in statement that lactation period has impact on changes in mercury concentration in maternal as well as offspring blood. Just after delivery, levels of mercury concentrations in blood of pup mother pairs were similar (~25 ng Hgg-1 w.w). The differences started to occur as the experiment progresses. The lowest concentrations in females blood were measured just after labour. In next 21 days of nursing, concentration of mercury in females increased (up to 48 ng Hgg-1 w.w). It was observed that lactation period affected reversely on mercury concentration in pups blood which decreased at the same time (down to 7 ng Hgg-1 w.w).
THE INFLUENCE OF LIFE HISTORY AND DIET ON MERCURY BIOACCUMULATION AND BIOMAGNIFICATION IN BLOOD OF BOTTLENOSE DOLPHINS, TURSIOPS TRUNCATUS, UNDER HUMAN CARE
Physiological and ecological factors, including age, sex, diet, and geographical location can influence mercury concentrations in and among bottlenose dolphin (Tursiops truncatus) populations. Dolphins under human care can represent free-ranging dolphins and account for data variability usually introduced by location, age, unknown diets, and individual feeding preferences. This study examines total mercury concentrations and nitrogen (δ15N) and carbon (δ13C) bulk stable isotopes in whole blood of bottlenose dolphins under human care and their whole prey to determine how life history and diet influence mercury bioaccumulation and biomagnification. This sample set represents the longest ongoing study with the greatest number of animals and samples collected in a controlled setting known to date. Blood samples (n = 199) were collected from 2011 to 2015 from eighteen bottlenose dolphins at Dolphin Quest Hawaii (DQH) and Oahu (DQO) as well as paired prey samples (n = 20). Total mercury was measured by atomic absorption spectrometry (AAS) and bulk stable isotopes were measured by isotope ratio mass spectrometry (IRMS). The average mercury concentration (± SD) in bottlenose dolphin whole blood was 48.1 ± 18.6 ng/g wet mass, which is approximately ten times lower than blood mercury concentrations measured in the Sarasota, Florida wild dolphin population. Sex and age class had a statistically significant influence on dolphin blood mercury concentrations. Adult females had significantly greater mercury concentrations than adult males (p = 0.0039), and pregnant females had lower mercury concentrations than lactating females (p = 0.0274). Calf mercury concentrations were significantly lower than adults (p < 0.0001) and subadults (p = 0.0005). The average daily mercury uptake accounted for all the variability observed among sex and age class. Although mercury concentrations in blood increased on average approximately 3.3 ± 3.8 ng/g wet mass per year, mercury concentrations in whole blood did not increase significantly over time for all animals. This study was also the first to model mercury biomagnification between bottlenose dolphins and their diet in a controlled setting using δ15N measured in dolphin blood and prey. There was a significant positive correlation between log10 mercury concentration and δ15N values in whole blood and prey of adult dolphins (p < 0.0001). The trophic magnification slope (TMS) was calculated between 0.18 and 0.23, which was similar to other observations in marine food webs.
FORMATION OF HGSE MICRO-DEPOSITS IN THE EYE AND INTERNAL EAR OF LONG-FINNED PILOT WHALES
Since the industrial revolution, mercury concentrations in air, water, soil, and living organisms have been on the rise. Methylmercury is the most bioaccumulative form of mercury in the environment. Depending on the level of exposure, effects of methylmercury on wildlife can include mortality, reduced fertility, slower growth and abnormal behavior that affects survival.
The sensory impairments reported in individuals consuming fish high in methylmercury were previously attributed to methylmercury injuring a specific region of the brain. However, recent results (Korbas et al. ACS Chem. Biol. 2013, 8: 2256-2263), which showed preferential accumulation of methylmercury in sensory organs such as the eye, indicate that a more direct damage of specific sensory cells may also take place.
Pilot whales are source of high mercury food for local population in the Faroe Islands and are notorious for stranding themselves on beaches. Several hypotheses have been proposed to account for this behavior, however methylmercury-induced sensory impairment has never been explored in great detail. To verify the sensory toxicity hypothesis, we have investigated mercury and selenium distribution and speciation in the eye (lens, cornea, retina, optic nerve) and internal ear (cochlea, vestibule) of long-finned pilot whales (Globicephala melas) using synchrotron based X-ray fluorescence mapping (XFM) and micro-X-ray absorption spectroscopy (micro-XAS), respectively.
Using XFM and micro-XAS, we have probed several different specimens for mercury, selenium and other metals and metalloids. The mercury distribution and levels significantly varied between tissues. Low levels of mercury were detected in the eye lens, cornea and cochlea. Interestingly, high mercury was always co-localized with high selenium in the form of small (5-10 microns) deposits. Densely distributed HgSe micro-deposits were observed in the vestibular nerve. Similar deposits but with lower spatial density were also mapped in the optic nerve and in the inner nuclear layer of the retina. The HgSe particles had various sizes and Hg:Se ratios. Since in XFM, the spatial resolution of the elemental maps is determined by the size of the X-ray beam (5 microns in this study), those particles could be significantly smaller than a few microns. Currently, using Transmission Electron Microscopy (TEM), we are determining the dimensions and subcellular localization of these micro-deposits. The results of this study will shed more light on methylmercury detoxification pathways and the role of selenium therein. Moreover, they should also contribute to better understanding of the extent of risk to the wildlife health associated with chronic exposure to mercury.