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Environmental Factor, September 2012

Intramural papers of the month

By Mamta Behl, Anshul Pandya, and Sheila Yong

Clock gene expression regulated by ROR gamma

A recent study conducted by NIEHS researchers demonstrated that retinoic acid-related orphan receptors (ROR) alpha or ROR gamma significantly reduced the expression level of several genes involved in regulating the body’s clock. Since disruptions to a person’s 24-hour wake-sleep cycle increases the risk for type 2 diabetes, some cancers, and becoming obese, RORs play a role in maintaining good health.

The scientists examined whether loss of ROR alpha or ROR gamma affected clock gene expression in ROR knockout mice. They found that both RORs regulated the transcription of Cry1, Bmal1, Rev-Erb alpha, and E4bp4 directly, and that ROR antagonists inhibited this transcriptional activation. Furthermore, chromatin immunoprecipitation sequencing (ChIP-Seq) analysis indicated that RORs were associated with ROR response elements in vivo, supporting the conclusion that these clock genes were directly regulated by RORs. ChIP-Seq analysis also showed that ROR gamma bound to the regulatory regions of several metabolic genes, while ROR alpha either didn’t bind or exhibited weak binding. As a result, the research team was the first to report that ROR gamma was more important in clock regulation than ROR alpha, countering the view of many in the nuclear receptor community that ROR alpha was more relevant. (AP)

CitationTakeda Y, Jothi R, Birault V, Jetten AM. 2012. RORgamma directly regulates the circadian expression of clock genes and downstream targets in vivo. Nucleic Acids Res; doi:10.1093/nar/gks630 [Online 29 June 2012]. Story

Fertility drugs and young-onset breast cancer

Among participants in the NIEHS Two Sister Study, funded in part by Susan G. Komen for the Cure, women who had used ovary-stimulating fertility drugs, clomiphene citrate or follicle-stimulating hormone, without getting pregnant, had reduced risk of young-onset breast cancer.

Participants were pairs of sisters, one of whom had been diagnosed with breast cancer before the age of 50. They were categorized based on whether they had used ovulation-stimulating fertility drugs and whether pregnancy had resulted. Women who had been treated without conceiving showed a significantly reduced risk compared with non-treated women. The use of those drugs likely serves as a marker for the fertility problems for which they are prescribed, problems that may confer reduced risk. By contrast, women who conceived through fertility treatment had significantly elevated risk, comparable to that of women in the population. The use of ovulation-stimulating fertility drugs changes the hormonal profiles of the first trimester pregnancy in ways that may increase the risk of breast cancer.

Unlike previous studies, the Two Sister Study distinguished between fertility treatments that produced pregnancy versus those that did not. Moreover, use of sisters who are well-matched for many factors allows for a fair comparison. (SY)

CitationFei C, DeRoo LA, Sandler DP, Weinberg CR. 2012. Fertility drugs and young-onset breast cancer: Results from the Two Sister Study. J Natl Cancer Inst 104(13):1021-1027.

Study estimates the frequency of an autoimmunity biomarker in U.S.

Scientists from NIEHS, the University of Florida, and SRA International have determined that more than 32 million people in the United States have antinuclear antibodies (ANA), a group of proteins called autoantibodies produced by the immune system that target its own tissues. ANA are frequently measured biomarkers for detecting autoimmune diseases, but the presence of ANA does not necessarily mean a person will get an autoimmune disease. This study presents the first nationally representative estimates of ANA prevalence based on sociodemographic groups, and provides an important baseline for future autoimmunity research.

The research team used indirect immunofluorescence to analyze sera from 4,754 participants in the 1999-2004 U.S. National Health and Nutrition Examination Survey (NHANES). The results demonstrated that the prevalence of ANA in the U.S. population varied in different groups, being higher among females, older individuals, African-Americans, and those with a normal body weight. Furthermore, the team found no significant associations of ANA with education, family income, alcohol use, smoking history, serum levels of cotinine, or C-reactive protein, a protein that increases in blood in response to inflammation. This work should serve as a useful baseline for future studies looking at changes in ANA prevalence over time and the factors associated with ANA development. (MB)

CitationSatoh M, Chan EK, Ho LA, Rose KM, Parks CG, Cohn RD, Jusko TA, Walker NJ, Germolec DR, Whitt IZ, Crockett PW, Pauley BA, Chan JY, Ross SJ, Birnbaum LS, Zeldin DC, Miller FW. 2012. Prevalence and sociodemographic correlates of antinuclear antibodies in the United States. Arthritis Rheum 64(7):2319-2327.

Revolutionizing the detection of free radical DNA in cells

NIEHS researchers made a major breakthrough in DNA damage research by revealing a new and improved method for detecting intracellular free radical DNA resulting from oxidative stress.

Antibody-based assays for detecting free radical DNA oxidation products are inadequate, due to the difficulty in generating antibodies against specific nucleotide oxidation products, which, in general, differ from their reduced counterparts by only one oxygen atom. Furthermore, identification of primary free radicals by electron spin resonance (ESR) in cells is impossible due to their instability. To address these issues, the team previously developed the immuno-spin trapping technique (IST), which traps free radicals with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and is then oxidized to its stable nitrone products that can be detected by antibodies or other analytical methods.

The present study illustrates the ability of IST to detect DNA free radicals, while minimizing artifacts characteristic of traditional approaches. The resulting DMPO-DNA adducts may also be visualized by confocal microscopy, allowing researchers to determine where the damage occurs in the cell. More importantly, this work also described a time lapse experiment during which both the initial DNA damage and subsequent repair were observed. (SY)

CitationBhattacharjee S, Chatterjee S, Jiang J, Sinha BK, Mason RP. 2012. Detection and imaging of the free radical DNA in cells — Site-specific radical formation induced by Fenton chemistry and its repair in cellular DNA as seen by electron spin resonance, immuno-spin trapping and confocal microscopy. Nucleic Acids Res 40(12):5477-5486.

(Mamta Behl, Ph.D., is a contract neurotoxicologist in the NTP Toxicology Branch. Anshul Pandya, Ph.D., is an Intramural Research Training Award fellow in the NIEHS Laboratory of Neurobiology. Sheila Yong, Ph.D., is a visiting fellow in the NIEHS Laboratory of Signal Transduction.)

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