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Environmental Factor, February 2015

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Intramural papers of the month

By Deacqunita Diggs, Gabriel Knudsen, Deepa Singh, Shannon Whirledge, and Annah Wyss

NTP uses new tool for analysis of rodent mammary gland development

Scientists from the National Toxicology Program (NTP) Laboratory have successfully adapted the Sholl analysis as a method for assessing alterations in rodent mammary gland development following chemical exposure. The Sholl analysis is traditionally used to measure the complexity of branching neuronal dendrites, but this study demonstrates that it can also be used as an accurate, simple, quick, and cost-effective tool for examining changes in epithelial branching in the rodent breast.

Mammary gland whole mounts were obtained from a previous study, in which timed pregnant rats were exposed to vehicle alone or ethinyl estradiol (EE), which is an endogenous estrogen used in birth control pills. Although histopathological analysis by the National Center for Toxicologic Research pathologists did not report any adverse effects in the rat mammary gland, the Sholl analysis revealed growth and developmental changes. The Sholl analysis indicated greater epithelial growth and enhanced branching density throughout the mammary epithelium of EE-treated rats compared with those of vehicle-exposed rats.

Because laboratories use a variety of techniques for measuring mammary gland development, the authors propose the use of the Sholl analysis as a reliable, standardized method for assessing changes in mammary gland development following chemical exposure. (DD)

CitationStanko JP, Easterling MR, Fenton SE. 2014. Application of Sholl analysis to quantify changes in growth and development in rat mammary gland whole mounts. Reprod Toxicol; doi:10.1016/j.reprotox.2014.11.004 [Online 15 November 2014].

Discontinuation of tamoxifen may blunt benefit of breast cancer prevention

For the majority of women using tamoxifen in the Sister Study, the benefit of breast cancer prevention appeared to outweigh the risks of not using the medication. However, a sizeable proportion of users lacked evidence that benefit exceeded risks, according to NIEHS epidemiologists. Tamoxifen has been approved by the Food and Drug Administration to both prevent and treat breast cancer.       

The researchers identified 788 tamoxifen users among the 50,884 women in the Sister Study, which is a prospective cohort study of initially breast cancer-free women who have a sister with previously diagnosed breast cancer. Based on a risk-benefit index developed at the National Cancer Institute, these women were classified as having no, moderate, or strong evidence that the chemopreventive benefits of using tamoxifen outweighed the risk of side effects.

The majority, or 74 percent, of tamoxifen users had moderate or strong evidence that benefits exceeded risks, whereas 20 percent lacked such evidence. Women with an intact uterus, women older than 50 years of age, and African American women appeared to have less favorable risk-benefit indexes. Nearly half, or 46 percent of users, stopped taking tamoxifen before the recommended 5 years. Women with a sister diagnosed with breast cancer before tamoxifen initiation or who reported genetic testing appeared more likely to discontinue use.

Examination of tamoxifen use for breast cancer prevention in nonclinical trial settings, such as the Sister Study, provides important insight into risk-benefit profiles in the general population. Although many women who are at higher risk for breast cancer may benefit from taking tamoxifen, high levels of discontinuation can mitigate potential gains. (AW)

CitationNichols HB, DeRoo LA, Scharf DR, Sandler DP. 2014. Risk-benefit profiles of women using tamoxifen for chemoprevention. J Natl Cancer Inst 107(1):354.

Estrogen directs uterine biology in cell-type manner

In an article published in Biology of Reproduction, NIEHS researchers used genetically modified mouse models to discover how the cell-type specific responses in the uterus govern the biological response to estrogen. This study demonstrated how estrogen signaling in neighboring cells of the uterus mediates distinct physiological responses in adjacent tissues. Uncovering the transcriptional profiles of specific uterine cell populations highlighted the complex signaling network that regulates female reproductive biology and implicated dysregulation of these networks in infertility.

Estrogen released from the ovary targets estrogen receptors in the uterus to regulate growth and proliferation during female reproductive events. The uterus is a heterogeneous tissue composed of cell types with specific functions following estrogen exposure. Building from previous discoveries( in which conditionally ablating the estrogen receptor from the epithelial layer of the uterus resulted in infertility and impaired estrogen action, the researchers used whole genome transcriptional analysis to identify those genes that require an epithelial estrogen receptor compared with genes that signal through neighboring stromal cells.

These studies indicate that the cell-specific response to estrogen in the uterus occurs in a temporal manner, where stromal estrogen receptor signaling is required for the initial response to estrogen, but the epithelial estrogen receptor is required for the subsequent later-phase transcriptional response in the epithelium. An understanding of the precise mechanisms by which estrogens regulate uterine biology offers insight into the etiology of infertility and oncogenesis in women. (SW)

CitationWinuthayanon W. Hewitt SC, Korach KS. 2014. Uterine epithelial cell estrogen receptor alpha-dependent and –independent genomic profiles that underlie estrogen responses in mice. Biol Reprod 91(5):110.

New class of PUF protein family as revealed via its structure

NIEHS scientists and their collaborators have obtained the first crystal structure of the human Puf-A protein. The Puf-A protein exhibits an alternative fold for the PUF-family proteins, with 11 Pumilio (PUM) alpha-helical repeats that are arranged in an L-like shape. The structure is in contrast to the classical PUF proteins that only have eight PUM repeats, which are arranged in a crescent shape. The PUM repeats are important for binding of PUF proteins to RNA with high sequence specificity. 

Based on a second crystal structure of the Puf-A and DNA complex, along with biochemical studies, the scientists demonstrated that Puf-A and its yeast homologue Puf6 can bind to both RNA and DNA without any sequence specificity. The researchers also demonstrated that a conserved basic surface of the yeast homologue, Puf6, is important for RNA binding, pre-rRNA processing, and mRNA localization.

Overall, the authors believe that, similar to the many alpha-helical repeat protein families, PUM repeats can also be assembled into folds that are different from that of the classical PUF proteins, thereby possibly facilitating binding to different structured nucleic acids. (DS)

CitationQiu C, McCann KL, Wine RN, Baserga SJ, Hall TM. 2014. A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization. Proc. Natl Acad Sci U S A 111(52):18554-18559.

NIEHS researchers explore new mechanisms of arsenic toxicity

NIEHS researchers recently explored a novel mechanism by which low-level arsenic exposures can lead to cellular toxicity. Inorganic arsenic salts are common environmental pollutants that may be encountered in water, food, drugs, dust, or smoke. Naturally high levels of inorganic arsenic in groundwater are a source of exposure to millions of people worldwide and present a significant public health problem. Long-term arsenic exposures are associated with a wide variety of deleterious health effects, including cancers, diabetes, cardiovascular disease, nervous disorders, and reproductive toxicities.

Human fibroblasts were used to assess genome-wide mRNA expression patterns following a short-term exposure of 24 hours to sodium arsenite, a water-soluble arsenic salt. Noncytotoxic concentrations of arsenite were used in an effort to avoid secondary effects on gene expression. Using a combination of microarray analyses and real-time polymerase chain reaction, the researchers found that exposure to environmentally relevant arsenic doses actually stabilized the mRNA coding for delta-aminolevulinate synthase 1 (ALAS1), which is the rate-limiting enzyme in heme biosynthesis. This stabilization is expected to lead to increased ALAS1 enzyme expression, which would then maintain intracellular heme levels despite the parallel induction of heme oxygenase 1 (HMOX1). It had been previously assumed that changes in gene transcription were the mechanism behind arsenic-induced changes in mRNA levels, but this new research suggests that a change in mRNA stability can be a factor in some cases. (GK)

CitationQiu LQ, Abey S, Harris S, Shah R, Gerrish KE, Blackshear PJ. 2014. Global analysis of posttranscriptional gene expression in response to sodium arsenite. Environ Health Perspect; doi:10.1289/ehp.1408626 [Online 21 November 2014].

(Deacqunita Diggs, Ph.D., is a National Health and Environmental Effects Laboratory fellow in the Environmental Protection Agency Developmental Toxicity Branch. Gabriel Knudsen, Ph.D., is an Intramural Research Training Award fellow in the National Cancer Institute Center for Cancer Research Laboratory of Toxicology and Toxicokinetics. Deepa Singh, Ph.D., is a visiting fellow in the NIEHS Mechanisms of Mutation Group. Shannon Whirledge, Ph.D., is a research fellow in the NIEHS Molecular Endocrinology Group. Annah Wyss, Ph.D., is a research fellow with the NIEHS Genetics, Environment, and Respiratory Disease Group.)

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