Efficient Arsenic Metabolism — The <em>AS3MT</em> Haplotype Is Associated with DNA Methylation and Expression of Multiple Genes Around <em>AS3MT</em>

<div><p>Arsenic is a very potent toxicant. One major susceptibility factor for arsenic-related toxicity is the efficiency of arsenic metabolism. The efficiency, in turn, is associated with non-coding single nucleotide polymorphisms (SNPs) in the arsenic methyltransferase <em>AS3MT</em> on chromosome 10q24. However, the mechanism of action for these SNPs is not yet clarified. Here, we assessed the influence of genetic variation in <em>AS3MT</em> on DNA methylation and gene expression within 10q24, in people exposed to arsenic in drinking water. DNA was extracted from peripheral blood from women in the Argentinean Andes (N = 103) and from cord blood from new-borns in Bangladesh (N = 127). <em>AS3MT</em> SNPs were analyzed with Sequenom or Taqman assays. Whole genome epigenetic analysis with Infinium HumanMethylation450 BeadChip was performed on bisulphite-treated DNA. Whole genome gene expression analysis was performed with Illumina DirectHyb HumanHT-12 v4.0 on RNA from peripheral blood. Arsenic exposure was assessed by HPLC-ICPMS. In the Argentinean women, the major <em>AS3MT</em> haplotype, associated with more efficient arsenic metabolism, showed increased methylation of <em>AS3MT</em> (p = 10⁻⁶) and also differential methylation of several other genes within about 800 kilobasepairs: <em>CNNM2</em> (p<10⁻¹⁶), <em>NT5C2</em> (p<10⁻¹⁶), <em>C10orf26</em> (p = 10⁻⁸), <em>USMG5</em> (p = 10⁻⁵), <em>TRIM8</em> (p = 10⁻⁴), and <em>CALHM2</em> (p = 0.038) (adjusted for multiple comparisons). Similar, but weaker, associations between <em>AS3MT</em> haplotype and DNA methylation in 10q24 were observed in cord blood (Bangladesh). The haplotype-associated altered CpG methylation was correlated with reduced expression of <em>AS3MT</em> and <em>CNNM2</em> (r_s = −0.22 to −0.54), and with increased expression of <em>NT5C2</em> and <em>USMG5</em> (r_s = 0.25 to 0.58). Taking other possibly influential variables into account in multivariable linear models did only to a minor extent alter the strength of the associations. In conclusion, the <em>AS3MT</em> haplotype status strongly predicted DNA methylation and gene expression of <em>AS3MT</em> as well as several genes in 10q24. This raises the possibility that several genes in this region are important for arsenic metabolism.</p> </div

The relationship between <i>CNNM2</i> DNA methylation and <i>AS3MT</i> gene expression, stratified for <i>AS3MT</i> haplotype.

<p>Scatterplot depicting the relationship between DNA methylation (expressed as fraction of methylated CpG) for <i>CNNM2</i> (X-axis) and gene expression for <i>AS3MT</i> (3′UTR; expression relative to the median for individuals with 2 copies of the <i>AS3MT</i> haplotype) (Y-axis), where individuals are stratified for <i>AS3MT</i> haplotype (total N  = 48). Outliers are denoted by *; the outlier to the left has a relative gene expression of 22.8 and the outlier to the right has a relative gene expression of 24.9.</p

Univariable and multivariable regression analyses comprising DNA methylation sites that were significantly correlated with <i>AS3MT</i> haplotype in either Argentina or in Bangladesh.

*<p>The β–coefficient refers to the slope of the regression line (change in methylation level) with increasing numbers of copies of the haplotype.</p>†<p>β and p-value from univariable regression. The models look as follows: Methylation level = α+β₁×copies of <i>AS3MT</i> haplotype.</p>‡<p>β and p-value from multivariable regression (Argentina). The model look as follows: Methylation level = α+β₁×copies of <i>AS3MT</i> haplotype+β₂×total urinary arsenic (natural ln transformed)+β₃×age+β₄×use of coca leaves (yes/no).</p>§<p>β and p-value from multivariable regression (Bangladesh). The model look as follows: Methylation level = α+β₁×copies of <i>AS3MT</i> haplotype+β₂× total arsenic in maternal urine around gestational weeks 6–8 (natural ln transformed)+β₃×maternal age+β₄×sex of child.</p

Statistically significant correlations between <i>AS3MT</i> haplotype and DNA methylation in either Argentina or in Bangladesh, as well as descriptions of the DNA methylation sites.

*<p>According to NCBI Ref. Sequence: NC_000010.10, H sapiens chromosome 10, GRCh37.p5 Primary Assembly <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053732#pone.0053732-NCBI2" target="_blank">[42]</a>.</p>†<p>Situated in a CpG island, shore, or shelf according to Emboss CpGPlot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053732#pone.0053732-Rice1" target="_blank">[41]</a>.</p>‡<p>Enhancer elements were defined according to Illumina’s annotations (<a href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL13534" target="_blank">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL13534</a>).</p>§<p>According to <a href="http://www.rforge.net/IMA/snpsites.txt" target="_blank">http://www.rforge.net/IMA/snpsites.txt</a>. The numbers in parentheses indicate the distance of the SNP from the CpG site.</p>¶<p><i>r</i> = Pearson product-moment correlation coefficient.</p>∥<p>Beta (β)-values represent the fraction of methylation and hence range from 0 (unmethylated) to 1 (methylated).</p><p>Abbreviations: FDR; False Discovery Rate, SNP = Single nucleotide polymorphism.</p

Correlations^* and effect estimates (β) ^† between total urinary arsenic (As), DNA methylation and gene expression in Argentina, as well as the influence of haplotype on gene expression.

<p>A. denotes CpG sites that were positively associated with the <i>AS3MT</i> haplotype and B. denotes CpG sites that were inversely associated with the haplotype.</p>*<p><i>r</i>_s and p-value from Spearman correlation.</p>†<p>β and p-value from multivariable regression.</p>‡<p>Model (the β stated in the table is β₁): Gene expression = α+β₁× total urinary arsenic (natural ln transformed)+β₂× RIN.</p>§<p>Model (the β stated in the table is β₁): Gene expression = α+β₁×copies of <i>AS3MT</i> haplotype+β₂× total urinary arsenic (natural ln transformed)+β₃×RIN.</p>¶<p>Model (the β stated in the table is β₁): Gene expression = α+β₁×methylation level+β₂× total urinary arsenic (natural ln transformed)+β₃×RIN.</p

Characteristics of the study subjects in Argentina and Bangladesh.

<p>For comparison, also the full study population in Argentina <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053732#pone.0053732-Engstrom1" target="_blank">[12]</a> is included.</p>*<p>Median stratified for haplotype. N = 7 (0 copies), 41 (1 copy), and 45 (2 copies) in Argentina (one individual missing haplotype data), N = 90 (0 copies), 32 (1 copy), 5 (2 copies) in Bangladesh, and N = 17 (0 copies), 71 (1 copy), and 84 (2 copies) in the full study population in Argentina.</p>†<p>P-values from non-parametric Kruskal-Wallis test, numbers of haplotype copies is the grouping variable.</p>‡<p>Adjusted for specific weight. Measured at gestational week 6–8 in Bangladesh.</p>§<p>NA =  not applicable, since no RNA was available for analysis in the Bangladeshi study population.</p

Map of genes in chromosome band 10q24 associated with <i>AS3MT</i> haplotype-related DNA methylation.

<p>Positions of DNA methylation and gene expression assays are also shown. Black circles denote DNA methylation sites statistically significantly associated with <i>AS3MT</i> haplotype in Argentina and the white circle denotes the DNA methylation site statistically significantly associated with <i>AS3MT</i> haplotype in Bangladesh. A grey box around the DNA methylation ID denotes DNA methylation sites statistically significantly associated with <i>AS3MT</i> haplotype in both Argentina and Bangladesh.</p

Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages

<div><p>Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated <i>versus</i> dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs.</p></div

Phylogenetic analysis of AS3MT proteins from animal, fungal, green and red algal, and other eukaryotic lineages, as well as archaeal and bacterial lineages demonstrates that AS3MT is present in a range of kingdoms.

<p>The tree shown is the consensus tree derived by Bayesian inference using MrBayes, as described in Experimental procedures. MrBayes posterior probabilities are shown for branches with >0.5 MrBayes posterior probability support; branches with a value of 1 are shown by a filled circle. Bootstrap values of the same tree, derived by maximum likelihood using the program RAxML, are shown at key branches. AS3MT is phylogenetically split into one major group (I) divided into one subgroup of bacteria, SAR (stramenopiles, alveolata, rhizaria), and animals; and another major group (II) of bacteria, archaea, ascomycote and basidiomycote fungi, and <i>Hydra magnipapillata</i>. The species names and the database accession numbers are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175422#pone.0175422.s008" target="_blank">S1 Table</a>. Species that have two isoforms are shown with numbers 1 and 2 for the respective isoform.</p

Suggested horizontal gene transfer events between bacteria and eukaryotes (dashed lines labelled I-V) for <i>AS3MT</i> throughout evolution.

<p>Schematic tree of life originally based on Woese [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175422#pone.0175422.ref090" target="_blank">90</a>] and updated by Forterre [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175422#pone.0175422.ref091" target="_blank">91</a>].</p