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Environmental Factor, December 2015

Intramural papers of the month

By Robert Brown, John House, Mahita Kadmiel, Emily Mesev, and Qing Xu

NTP finds toxicity associated with organophosphorus flame retardants

A team led by National Toxicology Program scientists found that many organophosphorus flame retardants (OPFRs) might pose health hazards similar to those from brominated flame retardants (BFRs). Manufacturers are turning away from the latter, because of their toxicity and environmental persistence. This study suggests that many OPFRs might have comparable toxicity and must be further evaluated if they are to be considered safe replacements.

OPFRs share structural similarities with organophosphate insecticides, which raise concerns about their effects in the human body. Researchers in this study used cell-based in vitro assays and in vivo model systems using lower organisms, such as the zebrafish and the roundworm C. elegans. They evaluated the potential for developmental toxicity and neurotoxicity among eight OPFRs, covering a wide range of structures, and compared their activities with two well-characterized BFRs, tetrabromobisphenol A and tetrabromodiphenyl ether.

Assays revealed that exposure to many OPFRs caused adverse effects at equal or greater potency than BFRs. For example, many of the compounds led to developmental deaths and malformation of zebrafish embryos; decreased growth and larval development in C. elegans; decreased proliferation of human neuroprogenitor cells and decreased neurite outgrowth in human neurons; and decreased spontaneous electrical firing in primary rat neuronal cells.

However, not all OPFRs were active in the assays used to test these chemicals. Notably, tris(2-chloroethyl)phosphate was negative for activity up to the maximum concentration tested. These findings suggest that OPFRs differ in their health effects and lays the groundwork for further characterization of these compounds. (EM)

Citation Behl M, Hsieh JH, Shafer TJ, Mundy WR, Rice JR, Boyd WA, Freedman JH, Hunter ES 3rd, Jarema K, Padilla S, Tice RR 2015. Use of alternative assays to identify and prioritize organophosphorus flame retardants for potential developmental and neurotoxicity. Neurotoxicol Teratol; doi:10.1016/ [Online 16 September 2015].

High resolution structure of the APE1-DNA complex

Using X-ray crystallography, NIEHS researchers have generated a molecular snapshot that shows how noncoding DNA apurinic-apyrimidinic (AP) sites are recognized and cleaved by AP endonuclease 1 (APE1), an enzyme involved in DNA base excision repair. Since the expression of APE1 is increased in some aggressive tumors, the findings may provide insights into targeting APE1 for cancer treatment.

AP sites are DNA locations without a purine or a pyrimidine. They frequently occur in cells either spontaneously or from oxidative DNA damage. APE1 prepares the DNA lesions to be patched by DNA polymerases, by incising, or nicking, the AP sites. Previous studies provided limited information about the nicking process, but in this study, the researchers identified the molecular features that contribute to APE1 cleavage.

By characterizing the crystallized structure of the APE1-DNA complex, the researchers determined the metal-binding site that stabilized the complex, the water molecule that acted as the donor of electrons required for DNA strand incision, and the arginine sites that facilitated DNA binding. More importantly, they found that a thymine-guanine (T-G) mispair, which often arose from methylated CpG islands within mammalian promoters, distorted the neighboring AP site and reduced the activity of APE1. These results imply a crucial role of APE1 in DNA repair and genome stability. (QX)

Citation Freudenthal BD, Beard WA, Cuneo MJ, Dyrkheeva NS, Wilson SH . 2015. Capturing snapshots of APE1 processing DNA damage. Nat Struct Mol Biol 22(11):924-931.

Role of a T-type Ca2+ channel in fertility

NIEHS scientists and their collaborators were the first to demonstrate that the T-type Ca2+ channel CaV3.2 has an essential role in oocyte maturation and embryonic development in mice. Ca2+ acts as an important second messenger during oocyte maturation, fertilization, and embryonic development for mammals. Prior to this report, the ion channels responsible for the fertilization-induced influx of Ca2+ were entirely unknown.

To demonstrate the importance of CaV3.2 for Ca2+ homeostasis in the oocyte and fertilized egg, researchers used genetically modified mice, as well as pharmacological inhibitors that target the CaV3.2 Ca2+ channel. These studies reveled that a loss of CaV3.2 function resulted in oocytes with reduced cellular stores of Ca2+ and fertilized eggs with less Ca2+ response. Not only do these findings document an important and previously unrecognized function of a T-type channel, but they also have further implications for infertile couples, because the therapeutic use of Ca2+ channel blockers in women could negatively impact fertility. (RB)

Citation Bernhardt ML, Zhang Y, Erxleben CF, Padilla-Banks E, McDonough CE, Miao YL, Armstrong DL, Williams CJ. 2015. CaV3.2 T-type channels mediate Ca2+ entry during oocyte maturation and following fertilization. J Cell Sci; doi:10.1242/jcs.180026 [Online 19 October 2015].

Mechanism of nuclear uptake of a DNA repair scaffold protein revealed

NIEHS scientists used X-ray crystallography to identify and characterize the nuclear localization signal (NLS) of XRCC1, a scaffold protein involved in DNA repair. Cooperative binding of the NLS major and minor motifs helps explain how it meets the inherently conflicting requirements of high affinity and high flux that are needed for efficient nuclear import.

Due to the complexity of DNA damage, repair often requires involvement of several repair enzymes, which bind to a scaffold protein such as XRCC1. The XRCC1 protein enters the nucleus due to its NLS and also co-transports other repair enzymes, such as DNA ligase 3-alpha. Classical nuclear import involves binding of the XRCC1 NLS to Importin alpha, enabling it to translocate into the nucleus.

In addition to X-ray crystallography, the authors used fluorescence imaging and peptide binding assays to show that certain regions in the NLS sequence major and minor motifs were critical for efficient nuclear import of XRCC1. The researchers also suggested a model in which partial dissociation of XRCC1 NLS from Importin alpha facilitates unloading of the XRCC1 cargo from Importin alpha, allowing other proteins to bind. This action allows a more efficient nuclear transport system. (MK)

Citation Kirby TW, Gassman NR, Smith CE, Pedersen LC, Gabel SA, Sobhany M, Wilson SH, London RE. . 2015. Nuclear localization of the DNA repair scaffold XRCC1: uncovering the functional role of a bipartite NLS. Sci Rep 5:13405.

Refined mining of GWAS data reveals new insights into airway disease

Genome-wide association studies (GWAS) for pulmonary function and airway obstruction have identified numerous associated single nucleotide polymorphisms (SNPs). Yet, combined, these SNPs explain a small fraction of the variability seen. To utilize the oft-ignored information in GWAS from SNPs with moderate association, the researchers combined GWAS data with gene-ontology (GO) methodology. They analyzed discovery and replication meta-GWAS data sets for pulmonary function, to identify 131 overlapping enriched gene sets. These gene-enrichment sets mapped to distinct biological modules, such as development, cell adhesion, cell migration, tissue remodeling, and immunity, and surprisingly, were driven by SNPs below genome-wide significance thresholds.

They next analyzed airflow obstruction with a combined GWAS data set to reveal, as expected, significant overlap with lung function GO categories, as well as new GO categories, including extracellular matrix (ECM) remodeling, collagen processing, and integrins. Further analysis of the ECM module revealed MMP10 as a candidate for follow-up, and subsequent work in a mouse emphysematous smoking model demonstrated the module’s involvement in the pathogenesis of cigarette smoke-induced airway disease.

This work provides a novel way to use information below significance thresholds, usually discarded from GWAS, to gain additional understanding of critical biological processes involved in the pathogenesis of complex traits. (JH)

Citation Gharib SA, Loth DW, Soler Artigas M, Birkland TP, Wilk JB, Wain LV, Brody JA, Obeidat M, Hancock DB, Tang W, Rawal R, Boezen HM, Imboden M, Huffman JE, Lahousse L, Alves AC, Manichaikul A, Hui J, Morrison AC, Ramasamy A, Smith AV, Gudnason V, Surakka I, Vitart V, Evans DM, Strachan DP, Deary IJ, Hofman A, Glaser S, Wilson JF, North KE, Zhao JH, Heckbert SR, Jarvis DL, Probst-Hensch N, Schulz H, Barr RG, Jarvelin MR, O’Connor GT, Kahonen M, Cassano PA, Hysi PG, Dupuis J, Hayward C, Psaty BM, Hall IP, Parks WC, Tobin MD, London SJ, CHARGE Consortium, SpiroMeta Consortium. 2015. Integrative pathway genomics of lung function and airflow obstruction. Hum Mol Genet 24(23):6836-6848.

(Robert Brown, Ph.D., is an Intramural Research and Training Award (IRTA) fellow in the NIEHS Cell Biology Group. John House, Ph.D., is an IRTA fellow in the NIEHS Genetic Epidemiology Group. Mahita Kadmiel, Ph.D., is a visiting fellow in the NIEHS Molecular Endocrinology Group. Emily Mesev is an IRTA postbaccalaureate fellow in the NIEHS Intracellular Regulation Group. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)

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