Maternal polymorphisms in glutathione-related genes are associated with maternal mercury concentrations and early child neurodevelopment in a population with a fish-rich diet

Introduction: Glutathione (GSH) pathways play a key role the metabolism and elimination of the neurotoxicant methylmercury (MeHg). We hypothesized that maternal genetic variation linked to GSH pathways could influence MeHg concentrations in pregnant mothers and children and thereby also affect early life development. Methods: The GCLM (rs41303970, C/T), GCLC (rs761142, T/G) and GSTP1 (rs1695, A/G) polymorphisms were genotyped in 1449 mothers in a prospective study of the Seychellois population with a diet rich in fish. Genotypes were analyzed in association with maternal hair and blood Hg, fetal blood Hg (cord blood Hg), as well as children's mental (MDI) and motor development (PDI; MDI and PDI assessed by Bayley Scales of Infant Development at 20 months). We also examined whether genotypes modified the association between Hg exposure and developmental outcomes. Results: GCLC rs761142 TT homozygotes showed statistically higher mean maternal hair Hg (4.12 ppm) than G carriers (AG 3.73 and GG 3.52 ppm) (p = 0.037). For the combination of GCLC rs761142 and GCLM rs41303970, double homozygotes TT + CC showed higher hair Hg (4.40 ppm) than G + T carriers (3.44 ppm; p = 0.018). No associations were observed between GSTP1 rs1695 and maternal hair Hg or between any genotypes and maternal blood Hg or cord blood Hg. The maternal GSTP1 rs1695 rare allele (G) was associated with a lower MDI among children (β = −1.48, p = 0.048). We also observed some interactions: increasing Hg in maternal and cord blood was associated with lower PDI among GCLC rs761142 TT carriers; and increasing Hg in hair was associated with lower MDI among GSTP1 rs1695 GG carriers. Conclusions: Maternal genetic variation in genes involved in GSH synthesis is statistically associated with Hg concentrations in maternal hair, but not in maternal or fetal blood. We observed interactions that suggest maternal GSH genetics may modify associations between MeHg exposure and neurodevelopmental outcomes

Glutathione S-transferase activity moderates methylmercury toxicity during development in Drosophila

Glutathione (GSH) pathways play a central role in methylmercury (MeHg) metabolismand 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

ANOVA table for caffeine effect.

<p>Phenotype  =  (hatching rate for each replicate at 10 µM MeHg + Caffeine normalized to the line means of hatching rate at 0 µM MeHg) – (line means of hatching rate at 10 µM MeHg normalized to the line means of hatching rate at 0 µM MeHg). Residuals are weighted by the square of number of flies assayed.</p><p>ANOVA table for caffeine effect.</p

GeneMANIA network analyses reveals both overlapping and unique biological functions among GWA genes from MeHg alone and MeHg+caffeine.

<p>GeneMANIA derived biological network integration results from MeHg and MeHg+caffeine GWA genes. Overlapping networks with muscle related function (bold text)and receptor signaling/tyrosine kinase function (italicized bold text) are indicated. Also shown are False discovery rate (FDR) and coverage indicated by the number of the genes in the ‘Network’ with a given function relative to all the genes in the ‘Genome’ identified with that function.</p><p>GeneMANIA network analyses reveals both overlapping and unique biological functions among GWA genes from MeHg alone and MeHg+caffeine.</p

Overlap of genes identified by common polymorphic markers for variance in MeHg and MeHg+caffeine treatments.

<p>Candidate genes were identified from one or more associated polymorphisms in GWA analyses. A total of 145 and 106 genes were identified for MeHg and MeHg+caffeine, respectively. In common between the two treatments are 5 genes: <i>pumilio</i>, <i>Synaptotagmin</i> β, <i>Glut4EF</i>, <i>pHCl</i>, and <i>CG9005</i>.</p

Altered MeHg tolerance in <i>kirre</i> mutant flies.

<p>(A) Schematic representation of mutant lines targeting <i>kirre</i>. <i>kirre^M107148</i> is located at <i>X</i>: 2921499 targeting <i>kirre</i> transcripts E, F, B and G. <i>kirre</i>^MI00678 is located at <i>X</i>: 2952881 targeting E, F, B and G transcripts. <i>kirre</i>^G1566 is located at <i>X</i>: 3025507 in Exon 10 targeting all 7 splice variants of <i>kirre</i>. Gray arrows depict the location of the forward and reverse primers used for qRT-PCR analysis. <i>(</i>B) Eclosion assays of <i>kirre</i> mutant lines (MiMIC and EP) compared to <i>y¹w^67c23</i> control line. Statistical analyses done by z<i>-</i> test. <i>N</i> = 300 flies/bar. * <i>p</i><0.01, **<i>p</i><0.001 and *** <i>p</i><0.0001 relative to <i>y¹w^67c23.</i> (C) Expression of <i>kirre</i> mRNA in P12 pupae thoracic RNA extracts (pooled sample of n = 20 pupae) measured via qRT-PCR.</p

MeHg disruption of DLM muscle development.

<p>(A, B) <i>Mef2>RFP</i> pupae reared on indicated concentration of MeHg to stage P6 (A) or P12 (B) and imaged by bright light and red fluorescence to reveal DLM morphology. (C) Close-up image of selected panel from A and B. The solid arrow indicates reduced DLM bundle size and defects in DLM bundle splitting with MeHg. Open arrows indicate displacement of attachment sites of DVM bundles. Asterisks (*) indicates failure of extension and anchoring of the DLM resulting in myofibers coalesced in a ball. The development of eyes and bristles appear unaffected by MeHg treatment.</p