Review: myogenic and muscle toxicity targets of environmental methylmercury exposure.

A number of environmental toxicants are noted for their activity that leads to declined motor function. However, the role of muscle as a proximal toxicity target organ for environmental agents has received considerably less attention than the toxicity targets in the nervous system. Nonetheless, the effects of conventional neurotoxicants on processes of myogenesis and muscle maintenance are beginning to resolve a concerted role of muscle as a susceptible toxicity target. A large body of evidence from epidemiological, animal, and in vitro studies has established that methylmercury (MeHg) is a potent developmental toxicant, with the nervous system being a preferred target. Despite its well-recognized status as a neurotoxicant, there is accumulating evidence that MeHg also targets muscle and neuromuscular development as well as contributes to the etiology of motor defects with prenatal MeHg exposure. Here, we summarize evidence for targets of MeHg in the morphogenesis and maintenance of skeletal muscle that reveal effects on MeHg distribution, myogenesis, myotube formation, myotendinous junction formation, neuromuscular junction formation, and satellite cell-mediated muscle repair. We briefly recapitulate the molecular and cellular mechanisms of skeletal muscle development and highlight the pragmatic role of alternative model organisms, Drosophila and zebrafish, in delineating the molecular underpinnings of muscle development and MeHg-mediated myotoxicity. Finally, we discuss how toxicity targets in muscle development may inform the developmental origins of health and disease theory to explain the etiology of environmentally induced adult motor deficits and accelerated decline in muscle fitness with aging.

Assessing the role of the gut microbiome in methylmercury demethylation and elimination in humans and gnotobiotic mice.

The risk of methylmercury (MeHg) toxicity following ingestion of contaminated foodstuffs (e.g., fish) is directly related to the kinetics of MeHg elimination among individuals. Yet, the factors driving the wide range of inter-individual variability in MeHg elimination within a population are poorly understood. Here, we investigated the relationship between MeHg elimination, gut microbiome demethylation activity, and gut microbiome composition using a coordinated human clinical trial and gnotobiotic mouse modeling approach together with metagenomic sequence analysis. We first observed MeHg elimination half-lives (t1/2) ranging from 28 to 90 days across 27 volunteers. Subsequently, we found that ingestion of a prebiotic induced changes in the gut microbiome and mixed effects (increased, decrease, and no effect) on elimination in these same individuals. Nonetheless, elimination rates were found to correlate with MeHg demethylation activity in cultured stool samples. In mice, attempts to remove the microbiome via generation of germ-free (GF) animals or through antibiotic (Abx) treatment both diminished MeHg demethylation to a similar extent. While both conditions substantially slowed elimination, Abx treatment resulted in significantly slower elimination than the GF condition, indicating an additional role for host-derived factors in supporting elimination. Human fecal microbiomes transplanted to GF mice restored elimination rates to that seen in control mice. Metagenomic sequence analysis of human fecal DNA did not identify genes encoding proteins typically involved in demethylation (e.g., merB, organomercury lyase). However, the abundance of several anaerobic taxa, notably Alistipes onderdonkii, were positively correlated with MeHg elimination. Surprisingly, mono-colonization of GF free mice with A. onderdonkii did not restore MeHg elimination to control levels. Collectively, our findings indicate the human gut microbiome uses a non-conventional pathway of demethylation to increase MeHg elimination that relies on yet to be resolved functions encoded by the gut microbes and the hostClinical Trial NCT04060212, prospectively registered 10/1/2019.

Organomercurial Lyase (MerB)-Mediated Demethylation Decreases Bacterial Methylmercury Resistance in the Absence of Mercuric Reductase (MerA).

The mer operon encodes enzymes that transform and detoxify methylmercury (MeHg) and/or inorganic mercury [Hg(II)]. Organomercurial lyase (MerB) and mercuric reductase (MerA) can act sequentially to demethylate MeHg to Hg(II) and reduce Hg(II) to volatile elemental mercury (Hg0) that can escape from the cell, conferring resistance to MeHg and Hg(II). Most identified mer operons encode either MerA and MerB in tandem or MerA alone; however, microbial genomes were recently identified that encode only MerB. However, the effects of potentially producing intracellular Hg(II) via demethylation of MeHg by MerB, independent of a mechanism to further detoxify or sequester the metal, are not well understood. Here, we investigated MeHg biotransformation in Escherichia coli strains engineered to express MerA and MerB, together or separately, and characterized cell viability and Hg detoxification kinetics when these strains were grown in the presence of MeHg. Strains expressing only MerB are capable of demethylating MeHg to Hg(II). Compared to strains that express both MerA and MerB, strains expressing only MerB exhibit a lower MIC with MeHg exposure, which parallels a redistribution of Hg from the cell-associated fraction to the culture medium, consistent with cell lysis occurring. The data support a model whereby intracellular production of Hg(II), in the absence of reduction or other forms of demobilization, results in a greater cytotoxicity than the parent MeHg compound. Collectively, these results suggest that in the context of MeHg detoxification, MerB must be accompanied by an additional mechanism(s) to reduce, sequester, or redistribute generated Hg(II). IMPORTANCE Mercury is a globally distributed pollutant that poses a risk to wildlife and human health. The toxicity of mercury is influenced largely by microbially mediated biotransformation between its organic (methylmercury) and inorganic [Hg(II) and Hg0] forms. Here, we show in a relevant cellular context that the organomercurial lyase (MerB) enzyme is capable of MeHg demethylation without subsequent mercuric reductase (MerA)-mediated reduction of Hg(II). Demethylation of MeHg without subsequent Hg(II) reduction results in a greater cytotoxicity and increased cell lysis. Microbes carrying MerB alone have recently been identified but have yet to be characterized. Our results demonstrate that mer operons encoding MerB but not MerA put the cell at a disadvantage in the context of MeHg exposure, unless subsequent mechanisms of reduction or Hg(II) sequestration exist. These findings may help uncover the existence of alternative mechanisms of Hg(II) detoxification in addition to revealing the drivers of mer operon evolution.

Delivery Mode and Child Development at 20 Months of Age and 7 Years of Age in the Republic of Seychelles.

OBJECTIVE: To determine if cesarean delivery is adversely associated with child neurodevelopment as measured at 20 months and 7 years. METHODS: In a prospective cohort study (n = 1328) in the Republic of Seychelles, we examined the association between mode of delivery and 22 measures of child neurodevelopment spanning multiple domains: cognition, executive and psychomotor function, language development, behavior, scholastic achievement, and social communication. Using multivariable linear regression, we evaluated the relationship between delivery mode (Cesarean/vaginal delivery) and each developmental outcome, while controlling for relevant covariates including child sex and age, maternal age, maternal IQ, whether both parents lived with the child, and Hollingshead socioeconomic status. RESULTS: At 20 months, children born via cesarean delivery had slightly higher scores (ß = 0.11, 95% confidence interval: 0.00, 0.21) on the Infant Behavior Questionnaire-Revised Positive Affectivity/Surgency subtest, a measure of infant temperament, as compared to vaginal delivery. Delivery mode was not associated with any of the 7-year developmental outcomes. CONCLUSIONS FOR PRACTICE: Our study does not support the notion that cesarean delivery is associated with child neurodevelopmental outcomes.

Developmental Toxicology of Metal Mixtures in Drosophila: Unique Properties of Potency and Interactions of Mercury Isoforms.

Mercury ranks third on the U.S. Agency of Toxic Substances and Disease Registry priority list of hazardous substances, behind only arsenic and lead. We have undertaken uncovering the mechanisms underlying the developmental toxicity of methylmercury (MeHg), inorganic mercury (HgCl2), lead acetate (Pb), and sodium arsenite (As). To probe these differences, we used the Drosophila model, taking advantage of three developmental transitions-pupariation, metamorphosis, and eclosion-to differentiate potentially unique windows of toxicity. We elaborated dose response profiles for each individual metal administered in food and accounted for internal body burden, also extending analyses to evaluate combinatorial metal mixture effects. We observed all four metals producing larval lethality and delayed pupariation, with MeHg being most potent. Compared to other metals, MeHg's potency is caused by a higher body burden with respect to dose. MeHg uniquely caused dose-dependent failure in eclosion that was unexpectedly rescued by titrating in HgCl2. Our results highlight a unique developmental window and toxicokinetic properties where MeHg acts with specificity relative to HgCl2, Pb, and As. These findings will serve to refine future studies aimed at revealing tissue morphogenesis events and cell signaling pathways, potentially conserved in higher organisms, that selectively mediate MeHg toxicity and its antagonism by HgCl2.

Tissue-specific Nrf2 signaling protects against methylmercury toxicity in Drosophila neuromuscular development.

Methylmercury (MeHg) can elicit cognitive and motor deficits due to its developmental neuro- and myotoxic properties. While previous work has demonstrated that Nrf2 antioxidant signaling protects from MeHg toxicity, in vivo tissue-specific studies are lacking. In Drosophila, MeHg exposure shows greatest developmental toxicity in the pupal stage resulting in failed eclosion (emergence of adults) and an accompanying 'myosphere' phenotype in indirect flight muscles (IFMs). To delineate tissue-specific contributions to MeHg-induced motor deficits, we investigated the potential of Nrf2 signaling in either muscles or neurons to moderate MeHg toxicity. Larva were exposed to various concentrations of MeHg (0-20 µM in food) in combination with genetic modulation of the Nrf2 homolog cap-n-collar C (CncC), or its negative regulator Keap1. Eclosion behavior was evaluated in parallel with the morphology of two muscle groups, the thoracic IFMs and the abdominal dorsal internal oblique muscles (DIOMs). CncC signaling activity was reported with an antioxidant response element construct (ARE-GFP). We observed that DIOMs are distinguished by elevated endogenous ARE-GFP expression, which is only transiently seen in the IFMs. Dose-dependent MeHg reductions in eclosion behavior parallel formation of myospheres in the DIOMs and IFMs, while also increasing ARE-GFP expression in the DIOMs. Modulating CncC signaling via muscle-specific Keap1 knockdown and upregulation gives a rescue and exacerbation, respectively, of MeHg effects on eclosion and myospheres. Interestingly, muscle-specific CncC upregulation and knockdown both induce lethality. In contrast, neuron-specific upregulation of CncC, as well as Keap1 knockdown, rescued MeHg effects on eclosion and myospheres. Our findings indicate that enhanced CncC signaling localized to either muscles or neurons is sufficient to rescue muscle development and neuromuscular function from a MeHg insult. Additionally, there may be distinct roles for CncC signaling in myo-morphogenesis.

Perspectives on the Drosophila melanogaster Model for Advances in Toxicological Science.

The use of Drosophila melanogaster for studies of toxicology has grown considerably in the last decade. The Drosophila model has long been appreciated as a versatile and powerful model for developmental biology and genetics because of its ease of handling, short life cycle, low cost of maintenance, molecular genetic accessibility, and availability of a wide range of publicly available strains and data resources. These features, together with recent unique developments in genomics and metabolomics, make the fly model especially relevant and timely for the development of new approach methodologies and movements toward precision toxicology. Here, we offer a perspective on how flies can be leveraged to identify risk factors relevant to environmental exposures and human health. First, we review and discuss fundamental toxicologic principles for experimental design with Drosophila. Next, we describe quantitative and systems genetics approaches to resolve the genetic architecture and candidate pathways controlling susceptibility to toxicants. Finally, we summarize the current state and future promise of the emerging field of Drosophila metabolomics for elaborating toxic mechanisms. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Postnatal methylmercury exposure and neurodevelopmental outcomes at 7 years of age in the Seychelles Child Development Study Nutrition Cohort 2.

BACKGROUND: Consumption of fish yields many nutritional benefits, but also results in exposure to methylmercury (MeHg). The developing brain is known to be particularly susceptible to MeHg toxicity in high doses. However, the potential impact of low-level environmental exposure from fish consumption on children's neurodevelopment remains unclear. METHODS: We investigated postnatal MeHg exposure at 7 years and its association with a battery of 17 neurodevelopmental outcomes in a subset of children (n = 376) from 1535 enrolled mother-child pairs in Nutrition Cohort 2 of the Seychelles Child Development Study (SCDS NC2). Each outcome was modeled in relation to postnatal MeHg exposure using linear regression, adjusting for prenatal MeHg exposure, levels of maternal polyunsaturated fatty acids (PUFA), and several other covariates known to be associated with neurodevelopmental outcomes. RESULTS: Median postnatal MeHg exposure at 7 years was 2.5 ppm, while the median prenatal MeHg exposure was 3.5 ppm. We found no statistically significant associations between postnatal MeHg exposure and any of the 17 neurodevelopmental outcomes after adjusting for prenatal MeHg exposure and other covariates. CONCLUSIONS: These findings are consistent with previous cross-sectional analyses of the SCDS Main Cohort. Continued follow-up of the entire NC2 cohort at later ages with repeated exposure measures is needed to further confirm these findings.

KEAP1 polymorphisms and neurodevelopmental outcomes in children with exposure to prenatal MeHg from the Seychelles Child Development Study Nutrition Cohort 2.

BACKGROUND: Humans differ in the metabolism of the neurotoxicant methyl mercury (MeHg). This variation may be partially due to variation in genes encoding the transcription factor Nuclear factor E2-related factor 2 (NRF2) and its negative regulator Kelch-like ECH-Associated Protein 1 (KEAP1), which regulate glutathione and related transporter and antioxidant proteins that play a role in the metabolism and neurotoxicity of MeHg. AIM: To elucidate a potential risk from genetic variation in NFE2L2 (encoding NRF2) and KEAP1 toward prenatal mercury exposure and child neurodevelopmental outcomes at 20 months and 7 years of age in a population with variable prenatal exposure to MeHg from maternal fish consumption. MATERIAL AND METHODS: Nutrition Cohort 2 is a mother-child cohort in the Republic of Seychelles. Children were genotyped for NFE2L2 (rs2364723, rs13001694) and KEAP1 (rs8113472, rs9676881) polymorphisms (N = 1285 after removing siblings). Total mercury (Hg) was measured in cord blood as a biomarker for prenatal MeHg exposure. Child neurodevelopmental outcomes included the Bayley Scales of Infant Development II administered at 20 months of age, and outcomes across multiple neurodevelopmental domains from 14 tests administered in children and 3 instruments completed by parents when children were 7 years of age. RESULTS: The mean cord blood MeHg concentration was 34 (95% CI 11, 75) µg/L. None of the four polymorphisms had a significant association (p < 0.05) with either cord MeHg or neurodevelopmental test results at 20 months. There were no significant associations between either NFE2L2 polymorphism and any developmental test scores. At 7 years, children carrying KEAP1 rs8113472 CA showed significantly worse performance on psychomotor function than children with the CC variant (finger tapping, dominant hand: ß - 1.19, SE 0.34; finger tapping, non-dominant hand: ß - 0.92, SE 0.31) and worse social communication (SCQ Total: ß 0.65, SE 0.27). Children carrying rs8113472 AA, versus children with CC, showed significantly better performance on social communication (SRS Total: ß - 8.88, SE 3.60). Children carrying KEAP1 rs9676881 AG, versus children with GG, showed significantly worse performance on psychomotor function (trailmaking A time: ß 8.66, SE 3.37) and cognition (KBIT Matrices: ß - 0.96, SE 0.36). CONCLUSION: No associations between NFE2L2 and KEAP1 polymorphisms and MeHg concentration were identified. However, at 7 years, KEAP1 polymorphisms were associated with differences in neurodevelopmental outcomes in children from a population with high fish intake.

Targeted Intracellular Demethylation of Methylmercury Enhances Elimination Kinetics and Reduces Developmental Toxicity in Transgenic Drosophila.

Methylmercury (MeHg) persists today as a priority public health concern. Mechanisms influencing MeHg metabolism, kinetics, and toxicity outcomes are therefore essential knowledge for informing exposure risks. Evidence points to different toxic potencies of MeHg and inorganic mercury (Hg2+), highlighting the role for biotransformation (demethylation) in regulating MeHg toxicokinetics/dynamics. Whereas microbial MeHg demethylation in the gut is seen to influence elimination kinetics, the potential for systemic demethylation in tissues and target organs to influence MeHg toxicity remains uncertain. To investigate the consequences of systemic MeHg demethylation across development, we engineered transgenic Drosophila to express the bacterial organomercurial lyase enzyme (merB) in a targeted and tissue-specific manner. With all combinations of merB-induced demethylation, ubiquitously (via an actin promoter) or in a tissue-specific manner (ie, gut, muscle, neurons), we observe a rescue of MeHg-induced eclosion failure at the pupal to adult transition. In MeHg-fed larvae with ubiquitous or targeted (gut and muscle) merB expression, we see a significant decrease in MeHg body burden at the pupal stage relative to control flies. We also observe a significant increase in the MeHg elimination rate with merB demethylation induced in adults (control, t1/2 = 7.2 days; merB flies, t1/2 = 3.1 days). With neuronal-specific merB expression, we observe a rescue of MeHg-induced eclosion failure without a decrease in Hg body burden, but a redistribution of Hg away from the brain. These results demonstrate the previously unidentified potential for intracellular MeHg demethylation to promote transport and elimination of Hg, and reduce developmental MeHg toxicity. Impact Statement: These findings demonstrate the potential for MeHg demethylation in situ to contribute significantly to the MeHg elimination and distribution kinetics of whole animals and thereby affords a means of protection against the toxic insult of MeHg. Therefore, this study reveals important insight into processes that can determine an individual's resistance or susceptibility to MeHg and provides rationale for therapies targeting a novel metabolism-based pathways to alleviate toxicity risk stemming from MeHg exposure.

Variation in Methylmercury Metabolism and Elimination in Humans: Physiological Pharmacokinetic Modeling Highlights the Role of Gut Biotransformation, Skeletal Muscle, and Hair.

The biological half-life (t1/2) of methylmercury (MeHg) shows considerable individual variability (t1/2 < 30 to > 120 days), highlighting the importance of mechanisms controlling MeHg metabolism and elimination. Building on a prior physiologically based pharmacokinetic (PBPK) model, we elucidate parameters that have the greatest influence on variability of MeHg t1/2 in the human body. Employing a dataset of parameters for mean organ volumes and blood flow rates appropriate for man and woman (25-35 years) and child (4 - 6 years), we demonstrate model fitness by simulating data from our prior controlled study of MeHg elimination in people. Model predictions give MeHg t1/2 of 46.9, 38.9, and 31.5 days and steady-state blood MeHg of 2.6, 2.6, and 2.3 µg/l in man, woman, and child, respectively, subsequent to a weekly dose of 0.7 µg/kg body weight. The major routes of elimination are biotransformation to inorganic Hg in the gut lumen (73% in adults, 61% in child) and loss of MeHg via excretion within growing hair (13% in adults, 24% in child). Local and global sensitivity analyses of model parameters reveal that variation in biotransformation rate in the gut lumen, and rates of transport between gut lumen and gut tissue, have the greatest influence on MeHg t1/2. Volume and partition coefficients for skeletal muscle (SM) and gut tissue also show significant sensitivity affecting model output of MeHg t1/2. Our results emphasize the role of gut microbiota in MeHg biotransformation, transport kinetics at the level of the gut, and SM mass as moderators of MeHg kinetics in the human body.

Latent effects of early-life methylmercury exposure on motor function in Drosophila.

The developmental toxicant, methylmercury (MeHg), can elicit motor deficits that last well into adulthood. Recent studies using Drosophila showed that the developing musculature is sensitive to high doses of MeHg, where a larval feeding paradigm resulted in compromised myotendinous junction (MTJ) formation during development, by a mechanism involving the NG2 homologue, kon-tiki (kon). Low-dose exposures to MeHg that do not produce muscle pathology during development, nevertheless result in impaired flight behavior later in adult life. The present study evaluated the potential for relatively low-dose exposure to produce latent adult muscle pathology and motor impairments, as assayed by climbing and flight, as well as to evaluate molecular mechanisms that may contribute to motor deficits. Wildtype larvae were fed 0, 2, 2.5, or 5 µM MeHg laden food until eclosion. The effect of 5 µM MeHg on MTJ-related gene expression during pupal development was assessed via quantitative RT-qPCR analysis. Upon eclosion, adults were transferred to standard food bottles for 4, 11, or 30 days prior to motor testing. Survivorship (%) was determined from a subset of 200 flies per treatment. Average climbing speed (cm/s) was quantified 4-days post-eclosion (PE). Flight ability was assayed 11- or 30-days PE by measuring landing height (cm) of flies dropped into an adhesive-lined vertical column. In parallel, total body mercury was measured to estimate the influence of residual MeHg at the time of motor testing. Muscle morphology was assessed using immuno-fluorescence microscopy. Exposure to 5uM MeHg significantly reduced climbing speed, and flight ability 4 and 11 - days PE, respectively. While age-related flight deficits were seen in each sex, flight deficits due to MeHg persisted to 30-day PE timepoints exclusively in males. Expression of kon was upregulated across the window of pupal development essential to establishing adult MTJ. However, experimentally restricting the induction of comparable levels of kon to muscle during the same periods did not recapitulate the flight deficits, indicating that muscle-specific induction of kon alone is not sufficient to contribute to latent flight impairments. Adult flight muscle morphology of 11-day PE flies treated with 5 µM MeHg was indistinct from controls, implying muscle structure is not grossly perturbed to impair flight. Collectively, the current data suggest that developmental exposure to 5 µM MeHg reduces flight ability in each sex at 11 day-PE and that latent deficits at 30-day PE are male-specific. It remains to be determined whether the developing MTJ of Drosophila is a sensitive target of MeHg, and whether or not kon acts in conjunction with additional MTJ factors to constitute a MeHg target.

Neuroligin-1 Is a Mediator of Methylmercury Neuromuscular Toxicity.

Methylmercury (MeHg) is a developmental toxicant capable of eliciting neurocognitive and neuromuscular deficits in children with in utero exposure. Previous research in Drosophila melanogaster uncovered that developmental MeHg exposure simultaneously targets the developing musculature and innervating motor neuron in the embryo, along with identifying Drosophila neuroligin 1 (nlg1) as a gene associated with developmental MeHg sensitivity. Nlg1 and its transsynaptic partner neurexin 1 (Nrx1) are critical for axonal arborization and NMJ maturation. We investigated the effects of MeHg exposure on indirect flight muscle (IFM) morphogenesis, innervation, and function via flight assays and monitored the expression of NMJ-associated genes to characterize the role of Nlg1 mediating the neuromuscular toxicity of MeHg. Developmental MeHg exposure reduced the innervation of the IFMs, which corresponded with reduced flight ability. In addition, nlg1 expression was selectively reduced during early metamorphosis, whereas a subsequent increase was observed in other NMJ-associated genes, including nrx1, in late metamorphosis. Developmental MeHg exposure also resulted in persistent reduced expression of most nlg and nrx genes during the first 11 days of adulthood. Transgenic modulation of nlg1 and nrx1 revealed that developing muscle is particularly sensitive to nlg1 levels, especially during the 20-36-h window of metamorphosis with reduced nlg1 expression resulting in adult flight deficits. Muscle-specific overexpression of nlg1 partially rescued MeHg-induced deficits in eclosion and flight. We identified Nlg1 as a muscle-specific, NMJ structural component that can mediate MeHg neuromuscular toxicity resulting from early life exposure.

Associations of prenatal methylmercury exposure and maternal polyunsaturated fatty acid status with neurodevelopmental outcomes at 7 years of age: results from the Seychelles Child Development Study Nutrition Cohort 2.

BACKGROUND: Fish is a primary source of protein and n-3 PUFA but also contains methylmercury (MeHg), a naturally occurring neurotoxicant to which, at sufficient exposure levels, the developing fetal brain is particularly sensitive. OBJECTIVES: To examine the association between prenatal MeHg and maternal status of n-3 and n-6 PUFA with neurodevelopment, and to determine whether PUFA might modify prenatal MeHg associations with neurodevelopment. METHODS: We examined the Seychelles Child Development Study Nutrition Cohort 2 (NC2) at age 7 y. We used a sophisticated and extensive neurodevelopmental test battery that addressed 17 specific outcomes in multiple neurodevelopmental domains: cognition, executive and psychomotor function, language development, behavior, scholastic achievement, and social communication. Analyses were undertaken on 1237 mother-child pairs with complete covariate data (after exclusions) and a measure of at least 1 outcome. We examined the main and interactive associations of prenatal MeHg exposure (measured as maternal hair mercury) and prenatal PUFA status (measured in maternal serum at 28 weeks' gestation) on child neurodevelopmental outcomes using linear regression models. We applied the Bonferroni correction to account for multiple comparisons and considered P values <0.0029 to be statistically significant. RESULTS: Prenatal MeHg exposure and maternal DHA and arachidonic acid (20:4n-6) (AA) status were not significantly associated with any neurodevelopmental outcomes. Findings for 4 outcomes encompassing executive function, cognition, and linguistic skills suggested better performance with an increasing maternal n-6:n-3 PUFA ratio (P values ranging from 0.004 to 0.05), but none of these associations were significant after adjusting for multiple comparisons. No significant interaction between MeHg exposure and PUFA status was present. CONCLUSIONS: Our findings do not support an association between prenatal MeHg exposure or maternal DHA and AA status with neurodevelopmental outcomes at age 7 y. The roles of n-6 and n-3 PUFA in child neurodevelopment need further research.

Methylmercury modifies temporally expressed myogenic regulatory factors to inhibit myoblast differentiation.

Methylmercury (MeHg) is a pervasive environmental toxicant, with known detrimental effects on neurodevelopment. Despite a longstanding paradigm of neurotoxicity, where motor deficits are prevalent among those developmentally exposed, consideration of muscle as a MeHg target has received minimal investigation. Recent evidence has identified muscle-specific gene networks that modulate developmental sensitivity to MeHg toxicity. One such network is muscle cell differentiation. Muscle cell differentiation is a coordinated process regulated by the myogenic regulatory factors (MRFs): Myf5, MyoD, MyoG, and MRF4. A previous study demonstrated that MeHg inhibits muscle cell differentiation in vitro, concurrent with reduced MyoG expression. The potential for MeHg to modify the temporal expression of the MRFs to alter differentiation, however, has yet to be fully explored. Using the C2C12 mouse myoblast model, we examined MRF expression profiles at various stages subsequent to MeHg exposure to proliferating myoblasts. MeHg was seen to persistently alter myoblast differentiation capacity, as myod, myog, and mrf4 gene expression were all affected. Myog exhibited the most robust changes in expression across the various culture conditions, while myf5 was unaffected. Following MeHg exposure to myoblasts, where elevated p21 expression indicated departure from proliferation, cells failed to subsequently differentiate, even in the absence of MeHg, as reflected by a concurrent reduction in MRF4 and myosin heavy chain (MHC), markers of terminal differentiation. Our results indicate that within a brief window of exposure MeHg can disrupt the intrinsic myogenic differentiation program of proliferative myoblasts.

Methylmercury myotoxicity targets formation of the myotendinous junction.

Methylmercury (MeHg) is a ubiquitous environmental contaminant and developmental toxicant known to cause a variety of persistent motor and cognitive deficits. While previous research has focused predominantly on neurotoxic MeHg effects, emerging evidence points to a myotoxic role whereby MeHg induces defects in muscle development and maintenance. A genome wide association study for developmental sensitivity to MeHg in Drosophila has revealed several conserved muscle morphogenesis candidate genes that function in an array of processes from myoblast migration and fusion to myotendinous junction (MTJ) formation and myofibrillogenesis. Here, we investigated candidates for a role in mediating MeHg disruption of muscle development by evaluating morphological and functional phenotypes of the indirect flight muscles (IFMs) in pupal and adult flies following 0, 5, 10, and 15 µM MeHg exposure via feeding at the larval stage. Developmental MeHg exposure induced a dose-dependent increase in muscle detachments (myospheres) within dorsal bundles of the IFMs, which paralleled reductions eclosion and adult flight behaviors. These effects were selectively phenocopied by altered expression of kon-tiki (kon), a chondroitin sulfate proteoglycan 4/NG2 homologue and a central component of MTJ formation. MeHg elevated kon transcript expression at a crucial window of IFM development and transgene overexpression of kon could also phenocopy myosphere phenotypes and eclosion and flight deficits. Finally, the myosphere phenotype resulting from 10 µM MeHg was partially rescued in a background of reduced kon expression using a targeted RNAi approach. Our findings implicate a component of the MTJ as a MeHg toxicity target which broaden the understanding of how motor deficits can emerge from early life MeHg exposure.

Developmental exposure to methylmercury and resultant muscle mercury accumulation and adult motor deficits in mice.

Developmental methylmercury (MeHg) exposure can have lasting consequences on neural development and motor function across the lifespan. Recent evidence for MeHg targeting of myogenic pathways has drawn attention to the possibility that developing skeletal muscle plays a role in the motor deficits stemming from early life MeHg exposure. In this study we examined a potential role for muscle in influencing MeHg developmental toxicity in offspring of female mice exposed to MeHg via drinking water. Dams had access to 0, 0.5 or 5.0 ppm MeHg chloride in drinking water from two weeks prior to mating through weaning. Blood, brain and muscle tissue was harvested from dams at weaning and pups at postnatal days (PND) 6, 21 and 60 for analysis of total Hg. Muscle tissue sections were examined with histological stains. Behavioral testing of offspring was conducted at PND 60 and included locomotor activity, inverted screen, grip strength and rotarod tests to assess motor function. Total Hg (tHg) levels in dam muscles at weaning were 1.7-3-fold higher than Hg levels in blood or brain. In PND6 male and female pups, muscle and brain tHg levels were 2 to 4-fold higher than blood tHg. Brain tHg levels decreased more rapidly than muscle tHg levels between PND 6 and 21. Premised on modeling of growth dilution, brain tissue demonstrated an elimination of tHg while muscle tissue exhibited a net uptake of tHg between PND 6 and 21. Despite overall elevated Hg levels in developing muscle, no gross morphological or cytological phenotypes were observed in muscle at PND 60. At the higher MeHg dose, grip strength was reduced in both females and males at PND 60, whereas only male specific deficits were observed in locomotor activity and inverted screen tests with marginally significant deficits on rotarod. These findings highlight a potential role for developing skeletal muscle in mediating the neuromuscular insult of early life MeHg exposure.

Down-regulation of Delta by proteolytic processing.

Notch signaling regulates cell fate decisions during development through local cell interactions. Signaling is triggered by the interaction of the Notch receptor with its transmembrane ligands expressed on adjacent cells. Recent studies suggest that Delta is cleaved to release an extracellular fragment, DlEC, by a mechanism that involves the activity of the metalloprotease Kuzbanian; however, the functional significance of that cleavage remains controversial. Using independent functional assays in vitro and in vivo, we examined the biological activity of purified soluble Delta forms and conclude that Delta cleavage is an important down-regulating event in Notch signaling. The data support a model whereby Delta inactivation is essential for providing the critical ligand/receptor expression differential between neighboring cells in order to distinguish the signaling versus the receiving partner.

Variation in the biological half-life of methylmercury in humans: Methods, measurements and meaning.

BACKGROUND: Understanding methylmercury (MeHg) toxicity requires a complete understanding of its fundamental toxicokinetic and toxicodynamic characteristics in the human body. The biological half-life (t1/2) of MeHg is a kinetic property that directly influences the body burden of Hg that results from repeated exposures such as can occur with fish and seafood consumption. The t1/2 of MeHg in humans is approximately 50 days, equivalent to an elimination rate (kel) of 0.014 day-1. However, numerous studies report a wide range of half-life values (t1/2 < 30 to >120 days), demonstrating that significant variation in the biological process of MeHg elimination exists. This variation is a source of considerable uncertainty in deriving a meaningful reference dose for MeHg applicable to all individuals in a population. SCOPE OF REVIEW: First, we summarize fundamentals of MeHg toxicokinetics, emphasizing the central role that biological half-life plays in MeHg dosimetry. We next present important considerations for how kinetic analyses are performed. We provide an example of how MeHg half-life variation directly influences the body burden and, in certain contexts, can result in MeHg levels exceeding the US EPA Reference Dose. We then survey existing studies that report MeHg half-life determinations in people. MAJOR CONCLUSIONS: Recent advances in methods of determining MeHg kinetics in people have made individualized assessment of MeHg elimination rates more accurate and readily obtainable. GENERAL SIGNIFICANCE: Characterization of MeHg half-life, particularly in vulnerable individuals, such as pregnant women and children, will diminish the remaining toxicokinetic uncertainty surrounding MeHg exposures and will better inform the risk assessment process.

Drosophotoxicology: Elucidating Kinetic and Dynamic Pathways of Methylmercury Toxicity in a Drosophila Model.

The risks of methylmercury (MeHg) toxicity are greatest during early life where it has long been appreciated that the developing nervous system is an especially sensitive target. Yet, understanding the discrete mechanisms of MeHg toxicity have been obscured by the wide variation in the nature and severity of developmental outcomes that are typically seen across individuals in MeHg exposed populations. Some insight has come from studies aimed at identifying a role for genetic background as a modifier of MeHg toxicity, which have predominantly focused on factors influencing MeHg toxicokinetics, notably, polymorphisms in genes related to glutathione (GSH) metabolism. For example, variants in genes encoding the catalytic and modifier subunits of glutamyl-cysteine ligase (GCLc and GCLm), the rate limiting enzyme for GSH synthesis, have been reported to associate with Hg body burden (Hg levels in blood or hair) in humans. However, GSH can facilitate both toxicokinetics and toxicodynamics of MeHg by forming MeHg-GSH conjugates, which are readily transported and excreted, and by acting indirectly as an anti-oxidant. In this study, we refine a model to distinguish kinetic and dynamic traits of MeHg toxicity using a paradigm of Drosophotoxicolgy. First, we identify that the pupal stage is selectively sensitive to MeHg toxicity. Using a protocol of larval feeding, measurements of Hg body burden, and assays of development to adulthood (pupal eclosion), we identify strain-dependent variation in MeHg elimination as a potential kinetic determinant of differential tolerance to MeHg. We also find that global upregulation of GSH levels, with GCLc trans-gene expression, can induce MeHg tolerance and reduce Hg body burden. However, we demonstrate that MeHg tolerance can also be achieved independently of reducing Hg body burden, in both wild-derived strains and with targeted expression of GCLc in developing neuronal and muscle tissue, pointing to a robust toxicodynamic mechanism. Our findings have important implications for understanding variation in MeHg toxic potential on an individual basis and for informing the process of relating a measurement of Hg body burden to the potential for adverse developmental outcome.