Identification and Characterization of Dioctyl Sodium Sulfosuccinate (DOSS) as an Obesogen and Metabolic Disruptor

The National obesity epidemic has reached a point where 36.5% of the adults are obese. While diet, exercise and genetics are major factors driving obesity, recent studies also implicate environmental chemical agents known as ‘Obesogens’. Research efforts have begun to identify obesogens, which promote obesity and metabolic syndrome and are especially potent during fetal and childhood development. The research presented herein provides a logical framework for the identification, characterization and validation of obesogens in environmental samples. This framework was developed based on potential obesogens present in crude oil (MC252 oil) and dispersant (Corexit) from the Deepwater Horizon oil spill given the magnitude of the spill and cleanup efforts and that components of oil have been previously implicated as obesogens. Receptor transactivation assays for nuclear receptors that regulate metabolic pathways previously implicated were used to identify obesogenic activities. Corexit components were identified to have obesogenic activities, including dioctyl sodium sulfosuccinate (DOSS), Span 80 and Tween 80. These candidate obesogens were then validated in vitro using adipogenic differentiation assays. DOSS increased adipogenesis in both mouse and human pre-adipocytes and stem cells. DOSS is a ubiquitous chemical used as a food additive and stool softener often prescribed to pregnant women. As such, DOSS was evaluated in vivo using mice in a scenario imitating human pregnancy-associated exposure. Significantly, DOSS exposure to pregnant dams produced increased weight gain and adiposity, glucose intolerance, decreased bone area, altered adipokine production, a proinflammatory state and dyslipidemia in the male F1 population. Together, these studies provide a framework for investigation of obesogens. Furthermore, the data suggest that DOSS can act as an obesogen in vivo at physiologically relevant doses and prompt further research into the safe use of DOSS during pregnancy

Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation

Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM

Application of the Key Characteristics of Carcinogens to Per and Polyfluoroalkyl Substances

Per- and polyfluoroalkyl substances (PFAS) constitute a large class of environmentally persistent chemicals used in industrial and consumer products. Human exposure to PFAS is extensive, and PFAS contamination has been reported in drinking water and food supplies as well as in the serum of nearly all people. The most well-studied member of the PFAS class, perfluorooctanoic acid (PFOA), induces tumors in animal bioassays and has been associated with elevated risk of cancer in human populations. GenX, one of the PFOA replacement chemicals, induces tumors in animal bioassays as well. Using the Key Characteristics of Carcinogens framework for cancer hazard identification, we considered the existing epidemiological, toxicological and mechanistic data for 26 different PFAS. We found strong evidence that multiple PFAS induce oxidative stress, are immunosuppressive, and modulate receptor-mediated effects. We also found suggestive evidence indicating that some PFAS can induce epigenetic alterations and influence cell proliferation. Experimental data indicate that PFAS are not genotoxic and generally do not undergo metabolic activation. Data are currently insufficient to assess whether any PFAS promote chronic inflammation, cellular immortalization or alter DNA repair. While more research is needed to address data gaps, evidence exists that several PFAS exhibit one or more of the key characteristics of carcinogens

Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system

Bis-seq showing strong ASM in the <i>ELK3</i>, <i>STEAP3-C2orf76</i>, <i>C3orf27-RPN1</i>, and <i>VTRNA2-1</i> index regions.

<p><b>A,</b> Gene map and bis-seq of the index amplicon containing SNP rs2302902 in the first intron of the <i>ELK3</i> gene. The strong asymmetry in CpG methylation, with the G allele consistently hypermethylated in the PBL samples, indicates haplotype-dependent non-imprinted ASM (additional data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-t001" target="_blank">Table 1</a>; haplotype map in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen.1003622.s005" target="_blank">Figure S5</a>). There is some tissue-specificity, with biallelic hypermethylation in the HMEC sample. The grey (lower) bars indicate the index amplicons for initial bis-seq, each tagged by the index SNP that showed recurrent ASM in the MSNP data. The green bars indicate CGIs and the black rectangles are exons. The X's indicate polymorphic CpG sites. <b>B,</b> Gene map and bis-seq of the index amplicon containing SNP rs1530562, between the <i>STEAP3</i> and <i>C2orf76</i> genes in chromosome band 2q14, showing strong ASM with the G allele consistently hypermethylated in multiple tissues consistent with haplotype-dependent non-imprinted ASM, but with biallelic hypermethylation in sperm DNA (additional data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-t001" target="_blank">Table 1</a>). <b>C,</b> Gene map and bis-seq of the index amplicon containing SNP rs2811488 located downstream of the last exon of the <i>RPN1</i> gene in chromosome band 3q21, showing strong ASM in placenta, with the G allele or A allele hypermethylated, depending on parent-of-origin, as proven in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-g002" target="_blank">Figure 2</a>. For this imprinted DMR the ASM is highly tissue-specific, being seen in placenta but not in PBL, liver, lung, brain, HMEC or sperm (additional data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-t001" target="_blank">Table 1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen.1003622.s002" target="_blank">Figure S2</a>). <b>D,</b> Gene map and bilsufite sequencing of the index amplicon containing SNP rs2346019 downstream of the <i>VTRNA2-1</i> vault-family RNA, located in chromosome band 5q31. Strong ASM is observed in multiple tissues with the A allele or G allele methylated, consistent with imprinting, which is proven by the data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-g002" target="_blank">Figure 2</a>.</p

Long-range methylation mapping of the non-imprinted <i>STEAP3-C2orf76</i> region in 8 types of human tissues shows that small discrete DMRs can be present in loci with non-imprinted ASM.

<p>A map from the UCSC genome browser and an ASM heat map derived from long read bis-seq in the <i>STEAP3-C2orf76</i> region. In the genome browser map, the index amplicon that provided the starting point for the mapping is in red, and CTCF binding sites, GC percentage, CGIs and H3K4me1 activating mark intensities are shown. The presence and strength of ASM is color-coded as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003622#pgen-1003622-g003" target="_blank">Figure 3</a>. This region shows a single discrete epicenter of ASM (DMR) which overlaps with the initial index fragment discovered using MSNP data. ASM in this domain is present in 6 tissues. The red stippled cells indicate tissues with a greater than 0.30 average difference in fractional methylation of the two alleles <i>but</i> with only one heterozygous sample or with less than half of the samples showing significant ASM (p≤0.05, using the t-test). The grey stippled cells indicate amplicons with insufficient read depth (<10).</p

Non-imprinted ASM in the <i>STEAP3-C2orf76</i> region and imprinted ASM in the <i>RPN1-C3orf27</i> regions are both associated with ASE.

<p><b>A,</b> ASE of the <i>C2orf76</i> gene, associated with the <i>C2orf76-STEAP3</i> DMR, is shown in duplicate for four PBL samples (rs6542522). In each case the C allele is preferentially expressed in the cDNA when compared to the genomic DNA, consistent with the sequence dependent nature of ASM in this locus. Overall, 12 informative PBL samples were analyzed in duplicate for ASE and 7 PBL samples showed preferential expression of the C allele. Likewise, preferential expression of the C allele was identified in 7 out of the 22 informative liver samples (data not shown). <b>B,</b> ASE of the lncRNA corresponding to the AK097792 EST, overlapping the index SNP rs2811488 in the <i>RPN1-C3orf27</i> intergenic DMR are shown on the left and those overlapping two additional informative SNPs, i.e. rs35604103 and rs61112519, are shown on the right for four placenta samples. Overall, the assays were performed in duplicates for 13 informative placenta samples. Ten of them showed a definite or complete ASE. In each of 6 informative samples assayed for both ASE and ASM, the hypermethylated maternal allele was the relatively repressed one.</p

ASM downstream of the <i>RPN1</i> gene and in the <i>VTRNA2-1</i> gene is due to genomic imprinting with hypermethylation of the maternal allele.

<p>Trios consisting of matched parents (PBL) and offspring (placental chorionic villi from the fetal surface) were analyzed for ASM to determine the parental origin of the hypermethylated and hypomethylated alleles in the <i>C3orf27-RPN1</i> and <i>VTRNA2-1</i> index regions. <b>A, </b><i>Hpa</i>II-predigestion/PCR/RFLP assay for ASM at non-polymorphic <i>Hpa</i>II sites in an amplicon including SNP rs12487604 (located immediately downstream of <i>RPN1</i>) which creates a <i>Bts</i>I restriction site. Undigested genomic DNA and genomic DNA pre-digested with the methylation-sensitive restriction enzyme <i>Hpa</i>II were amplified by PCR and then digested with <i>Bts</i>I. In the samples pre-digested with <i>Hpa</i>II (+) one allele drops out, indicating hypomethylation. A homozygous case for each allele is shown, as well as four heterozygotes; two cases show T-allele hypermethylation and two show A-allele hypermethylation. <b>B,</b> Trios analyzed by this assay showing parental imprinting of the index region downstream of <i>RPN1</i>. A total of 13 informative trios (two shown) were analyzed and each showed hypermethylation of the maternally-derived allele in the placenta (p<0.00311). <b>C,</b> Examples of bis-seq of trios for the index amplicon including SNP rs2346019 in the <i>VTRNA2-1</i> locus. The hypermethylated allele was found to be maternally derived in each of 10 informative trios (p<0.001565).</p