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Environmental Factor, September 2015

Intramural papers of the month

By Robert Brown, Greg Buchold, Tara Ann Cartwright, Simone Otto, and Qing Xu

NTP team identifies aromatase inhibitors in Tox21 library

Using a high-throughput screening system called the AroER tri-screen assay, a research team led by scientists from the National Toxicology Program (NTP) identified environmental chemicals in the Tox21 10K library that inhibit aromatase activity. Aromatase is found in a cellular structure known as the endoplasmic reticulum. It triggers the conversion of testosterone to estradiol, and androstenedione to estrone, in the final step of the steroid biosynthesis pathway. More importantly, aromatase plays a key role in maintaining balance between androgen and estrogen, which helps maintain human endocrine health.

The AroER tri-screen assay identified 113 possible compounds that selectively interact with aromatase. Mechanistic studies demonstrated that 10 of these, including both drugs and fungicides, had novel structures. A reversibility assay confirmed that four of the compounds — trovafloxacin, imazalil, erlotinib, and amlodipine besylate — irreversibly inhibit aromatase in a concentration-dependent manner. Enzyme kinetic analysis further revealed that erlotinib, imazalil sulfate, and trovafloxacin mesylate are noncompetitive inhibitors, whereas amlodipine besylate is a competitive inhibitor.

Taken together, this study suggests that exposure to a number of natural or synthetic environmental chemicals, through diet, medication, or fungicides used in agriculture, may significantly affect human endocrine health. (TAC)

CitationChen S, Hsieh JH, Huang R, Sakamuru S, Hsin LY, Xia M, Shockley KR, Auerbach S, Kanaya N, Lu H, Svoboda D, Witt KL, Merrick BA, Teng CT, Tice RR. 2015. Cell-based high-throughput screening for aromatase inhibitors in the Tox21 10K library. Toxicol Sci; doi:10.1093/toxsci/kfv141 [Online 3 July 2015].

RGS2 protein required for proper fertilization in mice

NIEHS scientists and collaborators found that a compound called regulator of G-protein signaling 2 (RGS2) blocks premature calcium signaling in a fertilizable egg, or oocyte. The release of calcium signals the egg to start developing, which can prevent a proper sperm-egg union if it is too early. Other research has suggested that RGS2 may be used as a therapeutic agent for cardiovascular disorders, but this study is the first to determine its role in reproduction.

Team members discovered that expression of RGS2 is particularly high in mature eggs. To determine its function, they depleted eggs of RGS2. They then used in vitro fertilization, calcium imaging, and immunoblots to investigate changes in the depleted oocytes. The fertility of the RGS2 knockout mice was also analyzed during the study.

When RGS2 expression was depleted, exposure to acidic pH or acetylcholine caused premature activation of the egg. In addition, the zona pellucida, a protein matrix that surrounds the egg, was cleaved prematurely in both RGS2-depleted eggs and eggs from RGS2 knockout mice. These effects are caused by calcium signaling and do not occur if calcium signaling is blocked by RGS2. The zona pellucida changes may explain why RGS2 knockout mice are subfertile. (SO)

CitationBernhardt ML, Lowther KM, Padilla-Banks E, McDonough CE, Lee KN, Evsikov AV, Uliasz TF, Chidiac P, Williams CJ, Mehlmann LM. 2015. Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs. Development 142(15):2633-2640. (Story)

Cholesterol trafficking protein could provide therapeutic target for inflammatory diseases

NIEHS researchers determined that the cholesterol transporter ATP binding cassette A1 (ABCA1) controls the repertoire of proteins, known as the proteome, within macrophage rafts, which are membrane structures that serve as platforms for signal transduction. They also found that stomatin-like protein (SLP)-2 is an ABCA1-regulated raft protein that controls raft composition and raft signaling. How cells control which proteins localize to rafts has remained poorly understood. This study may help scientists understand the cell signaling mechanisms that underlie inflammatory diseases, including atherosclerosis and cardiovascular disease.

In macrophages, lipid rafts concentrate or organize cell surface signal transduction, and their specific collection of proteins dictates their level of responsiveness to inflammatory stimuli. In this study, scientists found that macrophages deleted for the gene that encodes ABCA1 had wide-ranging changes in the raft proteome. They also found that SLP-2, one of the proteins that increased in abundance in ABCA1-null macrophage rafts, was itself an important determinant of lipid raft composition, organization, and inflammatory response. In light of these findings, the investigators suggest that SLP-2 is an important regulator of the innate immune response of macrophages and may represent a promising new therapeutic target for a number of illnesses. (RB)

CitationChowdhury SM, Zhu X, Aloor JJ, Azzam KM, Gabor KA, Ge W, Addo KA, Tomer KB, Parks JS, Fessler MB. 2015. Proteomic analysis of ABCA1-null macrophages reveals a role for stomatin-like protein-2 in raft composition and Toll-like receptor signaling. Mol Cell Proteomics 14(7):1859-1870.

BPA and phthalate exposure was not associated with subfertility

Certain phthalates and bisphenol A (BPA) have shown reproductive effects in past animal studies. However, in this study, NIEHS scientists and their collaborators found little evidence that BPA and phthalate metabolites reduce fertility. They measured these rapidly-metabolized and ubiquitous environmental contaminants in archived urine samples from 221 women enrolled in the Early Pregnancy Study (EPS).

The scientists then analyzed the association between the environmental exposures and follicular- and luteal-phase lengths, time to pregnancy, and early pregnancy loss. Higher concentrations of both urinary monocarboxyoctyl phthalate and BPA were associated with a shortening of the luteal phase by approximately half a day. However, more problematic reproductive problems, including lowered progesterone, longer time to pregnancy, and increased early fetal losses, were not observed. This research with naturally-conceiving women suggests that estimates of risk based only on subfertile women undergoing in vitro fertilization may overestimate the risks to the general population. (GB)

CitationJukic AM, Calafat AM, McConnaughey DR, Longnecker MP, Hoppin JA, Weinberg CR, Wilcox AJ, Baird DD. 2015. Urinary concentrations of phthalate metabolites and bisphenol A and associations with follicular-phase length, luteal-phase length, fecundability, and early pregnancy loss. Environ Health Perspect; doi:10.1289/ehp.1408164 [Online 10 July 2015].

MicroRNAs and chromatin modifiers steer the fate of human ES cells

NIEHS researchers have demonstrated that gene-silencing microRNAs regulate chromatin-remodeling proteins to direct the differentiation of human embryonic stem (ES) cells. The findings provide a novel epigenetic mechanism for ES cell programming that may also apply in human embryogenesis.

During human embryonic development, ES cells differentiate into three primary germ layers — endoderm, mesoderm, and ectoderm. These three layers eventually produce all of the specialized organs and tissues in the body. Both microRNAs and chromatin modifiers are essential parts of a network involved in controlling ES cell proliferation and differentiation, but the connection between the two players is unclear.

In this study, the researchers found that microRNA-302 (MiR-302), which is highly expressed in ES cells, repressed the expression of chromatin remodeling proteins BAF170 and BAF53a by binding with corresponding target sites. BAF170 suppression was required for changes in downstream genes and signaling pathways during differentiation. When ES cells were differentiated into endoderm, BAF170 decreased along with an induction of MiR-302. Conversely, BAF170 overexpression drove the cells toward ectodermal differentiation and blocked the induction of mesodermal and endodermal markers during directed endodermal differentiation. These results suggest that MiR-302-mediated repression of BAF170 is required for effective endodermal differentiation in human ES cells. (QX)

CitationWade SL, Langer LF, Ward JM, Archer TK. 2015. MiRNA-mediated regulation of the SWI/SNF chromatin remodeling complex controls pluripotency and endodermal differentiation in human ESCs. Stem Cells; doi:10.1002/stem.2084 [Online 29 June 2015].

(Greg Buchold, Ph.D., is a former NIEHS postdoctoral fellow in the NIEHS Reproductive and Developmental Biology Laboratory. Robert Brown, Ph.D., is an Intramural Research and Training Award (IRTA) fellow in the NIEHS Cell Biology Group. Tara Ann Cartwright, Ph.D., is a former postdoctoral fellow in the NIEHS Intracellular Regulation Group. Simone Otto, Ph.D., is an IRTA fellow in the NIEHS Ion Channel Physiology Group. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)

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