MeHg Tolerance Research Articles

Occurrence of methylmercury in aerobic environments: Evidence of mercury bacterial methylation based on simulation experiments

Methylmercury (MeHg) is mainly produced by anaerobic δ-proteobacteria such as sulfate-reducing bacteria (SRB). However, mercury bio-methylation has also been found to occur in the aerobic soil of the Three Gorges Reservoir (TGR). Using γ-proteobacterial TGR bacteria (TGRB) and δ-proteobacterial Desulfomicrobium escambiense strains, the efficiency of mercury methylation and demethylation was evaluated using an isotope tracer technique. Kinetics simulation showed that the bacterial Hg methylation rate (km) of TGRB3 was 4.36 × 10−9 pg·cell−1·h−1, which was significantly lower than that of D. escambiense (170.74 ×10−9 pg·cell−1·h−1) under anaerobic conditions. Under facultative and/or aerobic conditions, D. escambiense could not survive, while the km of TGRB3 were 0.35 × 10−9 and 0.29 × 10−9 pg·cell−1·h−1, respectively. Furthermore, the bacterial MeHg tolerance threshold of TGRB3 was 3.47 × 10−9 pg·cell−1, which was 98.6-fold lower than that of D. escambiense under anaerobic conditions. However, the MeHg tolerance threshold of TGRB3 remained at 0.50–0.52 × 10−9 pg·cell−1 under facultative and/or aerobic conditions. Notably, bacterial Hg methylation rates (km) were higher than the corresponding bacterial MeHg demethylation rates (kd1). These results establish the contribution of some aerobic and/or facultative anaerobic bacteria to net environmental MeHg production in terrestrial ecosystems and provide a novel understanding of the biogeochemical cycle of MeHg. SynopsisHg methylation of facultative and/or aerobic bacteria may contribute to the net production of environmental methylmercury in terrestrial ecosystems.

The efficiencies of inorganic mercury bio-methylation by aerobic bacteria under different oxygen concentrations

Limited information is available about the bio-methylation of inorganic mercury (iHg) under aerobic conditions. In this study, two γ-proteobacteria strains (P. fluorescens TGR-B2 and P. putida TGR-B4) were obtained from the soil of The Three Gorges Reservoir (TGR), demonstrating effective aerobic transformation capacities of iHg into methylmercury (MeHg). Based on periodical changes in soil oxygen content of the TGR, a culture system was established, in which 300 ng Hg (II) L–1 and O2 were set at 7%, 14%, and 21%, respectively. Results indicated that the two strains differed significantly in bacterial growth rate and MeHg production. The kinetic model of MeHg showed typical characteristics of a “two-staged” process: The first stage was dominated by bio-methylation, which was shown by increasing of net MeHg content. Moreover, the second stage was dominated by bio-demethylation, which decreased net MeHg content. Thus, we hypothesized that the mechanism of aerobic bacterial iHg bio-methylation: (1) should inefficiency compared to anaerobic bacteria i.e.SRB, which were regulated by hgcA/B gene clusters, (2) might be regarded as a passive stress response and depended on the bacterial iHg intoxication threshold and MeHg tolerance threshold.

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.

Editor's Highlight: Glutathione S-Transferase Activity Moderates Methylmercury Toxicity During Development in Drosophila.

Glutathione (GSH) pathways play a central role in methylmercury (MeHg) metabolism and elimination, largely due to formation of a more readily transported MeHg-GSH conjugate. Glutathione S-transferases (GSTs) have therefore been proposed to facilitate MeHg elimination by catalyzing MeHg-GSH conjugation. A role for human GSTP1 in MeHg disposition is suggested by the association of two common polymorphisms in the coding region (Ile105Val and Ala114Val) with Hg levels in either blood or hair. In this study, we investigated a functional role for GSTs in modulating MeHg toxicity during development. Using the Drosophila model to execute targeted manipulations of both endogenous GSTs and introduced human GSTP1 variants we correlate gene and protein expression levels with GST activity and also with MeHg body burden and developmental outcomes. RNAi knockdown of endogenous GSTD1, GSTE1, or GSTS1, individually, increased susceptibility to MeHg during pupal development resulting in a reduced rate of adult eclosion. Exogenous expression of human GSTP1 in developing flies resulted in increased MeHg tolerance relative to control flies as seen with elevated eclosion rates when reared on MeHg containing food. Furthermore, the GSTP1105 and GSTP1114 variants showed a reduced enzyme activity relative to wild-type GSTP1 (GSTP1wt). Finally, we observed a trend whereby Hg body burden was inversely related to the levels of GST activity. However, in some instances GSTP1 expression resulted in increased eclosion rates without reducing Hg body burden suggesting that GSTs interact with MeHg via both toxicokinetic and toxicodynamic mechanisms. These findings indicate that GSTs moderate MeHg toxicity during development in our experimental model.

Genome-wide association analysis of tolerance to methylmercury toxicity in Drosophila implicates myogenic and neuromuscular developmental pathways.

Methylmercury (MeHg) is a persistent environmental toxin present in seafood that can compromise the developing nervous system in humans. The effects of MeHg toxicity varies among individuals, despite similar levels of exposure, indicating that genetic differences contribute to MeHg susceptibility. To examine how genetic variation impacts MeHg tolerance, we assessed developmental tolerance to MeHg using the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP). We found significant genetic variation in the effects of MeHg on development, measured by eclosion rate, giving a broad sense heritability of 0.86. To investigate the influence of dietary factors, we measured MeHg toxicity with caffeine supplementation in the DGRP lines. We found that caffeine counteracts the deleterious effects of MeHg in the majority of lines, and there is significant genetic variance in the magnitude of this effect, with a broad sense heritability of 0.80. We performed genome-wide association (GWA) analysis for both traits, and identified candidate genes that fall into several gene ontology categories, with enrichment for genes involved in muscle and neuromuscular development. Overexpression of glutamate-cysteine ligase, a MeHg protective enzyme, in a muscle-specific manner leads to a robust rescue of eclosion of flies reared on MeHg food. Conversely, mutations in kirre, a pivotal myogenic gene identified in our GWA analyses, modulate tolerance to MeHg during development in accordance with kirre expression levels. Finally, we observe disruptions of indirect flight muscle morphogenesis in MeHg-exposed pupae. Since the pathways for muscle development are evolutionarily conserved, it is likely that the effects of MeHg observed in Drosophila can be generalized across phyla, implicating muscle as an additional hitherto unrecognized target for MeHg toxicity. Furthermore, our observations that caffeine can ameliorate the toxic effects of MeHg show that nutritional factors and dietary manipulations may offer protection against the deleterious effects of MeHg exposure.

Drosophila CYP6g1 and its human homolog CYP3A4 confer tolerance to methylmercury during development

Methylmercury (MeHg) is a persistent environmental toxicant that is commonly encountered through dietary fish and seafood. While the fetal nervous system is a well-known primary target for MeHg toxicity, the risks of MeHg exposures that are commonly experienced today through diet and environmental exposure remain uncertain. Despite knowledge of numerous cellular processes that are affected by MeHg, the mechanisms that ultimately influence tolerance or susceptibility to MeHg in the developing fetus are not well understood. Using transcriptomic analyses of developing brains of MeHg tolerant and susceptible strains of Drosophila, we previously identified members of the cytochrome p450 (CYP) family of monooxygenases/oxidoreductases as candidate MeHg tolerance genes. While CYP genes encode Phase I enzymes best known for xenobiotic metabolism in the liver, several classes of CYPs are required for synthesis or degradation of essential endobiotics, such as hormones and fatty acids, that are critical to normal development. We now demonstrate that variation in expression CYP genes can strongly influence MeHg tolerance in the developing fly. Importantly, modulating expression of a single CYP, CYP6g1, specifically in neurons or the fat body (liver equivalent) is sufficient to rescue development in the presence of MeHg. We also demonstrate a conserved function for CYP3A4, a human homolog of CYP6g1, in conferring MeHg tolerance to flies. Finally, we show that pharmacological induction of CYPs with caffeine parallels an increase in tolerance to MeHg in developing flies. These findings establish a previously unidentified role for CYPs in MeHg toxicity and point to a potentially conserved role of CYP genes to influence susceptibility to MeHg toxicity across species.

Methylmercury tolerance is associated with the humoral stress factor gene Turandot A

Methylmercury (MeHg) is an environmental neurotoxicant that targets the developing nervous system. In an effort to understand mechanisms of MeHg toxicity we have identified candidate genes that confer tolerance to MeHg using a Drosophila model. Whole genome transcript profiling of developing larval brains of MeHg-tolerant and non-tolerant flies has identified Turandot A (TotA) as a potential MeHg tolerance gene. TotA is a secreted humoral stress response factor in Drosophila that is a direct target of conserved innate immunity signaling pathways. Here we characterize TotA expression in newly generated isogenic lines (isolines) of flies derived from our previously established MeHg-tolerant and non-tolerant populations. TotA mRNA transcript and protein expression is seen to be higher in the tolerant isolines than the non-tolerant lines. Elevated TotA expression in the tolerant lines was seen to span all the larval developmental stages pointing toward a difference in the TotA gene regulation between the MeHg tolerant and non-tolerant strains. We show that TotA is most highly expressed in the fat body (liver equivalent) and is selectively upregulated in the fat body of tolerant flies relative to brain and gut tissues. Fat body-specific transgenic expression of TotA invokes MeHg tolerance as seen by enhanced development of flies reared on MeHg food. In addition, cell based assays show that high TotA expressing C6 cells are more tolerant to MeHg than the low TotA expressing S2 cells. Knockdown of TotA in the C6 cells trends toward a reduction in MeHg tolerance. Identification of TotA as a MeHg tolerance gene suggests a role for conserved cytokine/immune signaling pathways in modulating MeHg toxicity.

Identification of Methylmercury Tolerance Gene Candidates in Drosophila

Methylmercury (MeHg) is a ubiquitous environmental contaminant that preferentially targets the developing nervous system. Variable outcomes of prenatal MeHg exposure within a population point to a genetic component that regulates MeHg toxicity. We therefore sought to identify fundamental MeHg tolerance genes using the Drosophila model for genetic and molecular dissection of a MeHg tolerance trait. We observe autosomal dominance in a MeHg tolerance trait (development on MeHg food) in both wild-derived and laboratory-selected MeHg-tolerant strains of flies. We performed whole-genome transcript profiling of larval brains of tolerant (laboratory selected) and nontolerant (control) strains in the presence and absence of MeHg stress. Pairwise transcriptome comparisons of four conditions (+/-selection and +/-MeHg) identified a "down-down-up" expression signature, whereby MeHg alone and selection alone resulted in a greater number of downregulated transcripts, and the combination of selection + MeHg resulted in a greater number of upregulated transcripts. Functional annotation cluster analyses showed enrichment for monooxygenases/oxidoreductases, which include cytochrome P450 (CYP) family members. Among the 10 CYPs upregulated with selection + MeHg in tolerant strains, CYP6g1, previously identified as the dichlorodiphenyl trichloroethane resistance allele in flies, was the most highly expressed and responsive to MeHg. Among all the genes, Turandot A (TotA), an immune pathway-regulated humoral response gene, showed the greatest upregulation with selection + MeHg. Neural-specific transgenic overexpression of TotA enhanced MeHg tolerance during pupal development. Identification of TotA and CYP genes as MeHg tolerance genes is an inroad to investigating the conserved function of immune signaling and phase I metabolism pathways in MeHg toxicity and tolerance in higher organisms.

Methylmercury disruption of embryonic neural development in Drosophila

Methylmercury (MeHg) is a potent environmental neurotoxin that preferentially targets the developing embryonic nervous system. While a number of cytotoxic mechanisms of MeHg have been characterized in differentiated cells its mode of action in the developing nervous system in vivo is less clear. Studies in primate and rodent models demonstrate aberrant cell migration and disorganized patterning of cortical layers in the brain following MeHg exposure. However, defining the molecular and cellular pathways targeted by MeHg will require more genetically accessible animal models. In this study, we instigate a method of in vitro MeHg exposure using Drosophila embryos. We demonstrate dose-dependent inhibition of embryonic development with MeHg revealed by a failure of embryos to hatch to the larval stage. In addition, we document definitive phenotypes in neural development showing abnormalities in neuronal and glial cell patterning consistent with disrupted migration. We observe pronounced defects in neurite outgrowth in both central and peripheral neurons. Ectopic expression of the Nrf2 transcription factor in embryos, a core factor in the antioxidant response element (ARE) pathway, enhances embryonic development and hatching in the presence of MeHg, illustrating the power of this model for investigation of candidate MeHg tolerance genes. Our data establish a utility for the Drosophila embryo model as a platform for elucidating MeHg sensitive pathways in neural development.

A rapid change in methylmercury tolerance in a population of killifish, Fundulus heteroclitus, from a golf course pond

Embryos of killifish ( Fundulus heteroclitus) from Bullhead Bay, Southampton, New York, showed considerable variation in tolerance to methylmercury (meHg) in 1980, 1981 and 1983, in that some females produced eggs that were tolerant to the teratological effects of 0·05 ppm, while other females produced susceptible eggs. However, in 1982, over 40% of the females produced clutches of non-viable eggs (which could not be tested for meHg tolerance) and those fish which produced viable eggs generally produced tolerant ones. After hatching, the larvae were also more tolerant. Accompanying the shift in meHg tolerance was a trend of increasing fin ray count of the females, a parameter which has previously been correlated with the production of more tolerant embryos. In 1982, a nearby population exhibited a distribution of embryonic and larval tolerance and fin ray count comparable with that of the first population in the other years. We hypothesize that the unusually high rainfall in June, 1982 caused an inflow of pesticides and heavy metals from a golf course adjacent to the first site, and that the contaminants were responsible for the striking changes in reproductive success and meHg tolerance. Residues of chlorinated hydrocarbon insecticides were found in fish of the first, but not of the second, population. The variability in the original population may have permitted a segment of it to withstand the inflow of pollutants, while the susceptible individuals produced non-viable eggs. This may have been the cause of the rapid shift of overall tolerance in the population.