Papers of the Month
By Yu-Wei Chen, Katie Glenn, Kiri Hoff, Gabriel Knudsen, and Alicia Richards
NTP develops novel method for visualizing breast cancer progression
Scientists at the National Toxicology Program (NTP) developed a method for converting mammary gland whole mounts to hematoxylin and eosin−stained sections. The new inexpensive procedure can help researchers identify frequently undetected lesions, and it complements standard histological methods. The research team suggested that the approach could provide valuable information on how chemical and environmental exposures alter normal mammary growth. A video of the scientists performing the technique appeared in the Journal of Visualized Experiments.
Mammary glands from rodents are usually collected and prepared for normal histological evaluation, but evaluation of tissue from aged animals can result in inconclusive findings in the absence of a second evaluation technique. The researchers showed that whole-mounted mammary glands collected from mice at various stages of development, from adolescence through reproductive senescence, could be performed with minimal additional effort using common laboratory reagents and supplies.
The authors concluded that when this technique is used in combination with standard histological evaluation of mammary glands, it reduces the chance of false negative findings in investigations of possible mammary carcinogens. Current and future studies are evaluating known endocrine active chemicals to demonstrate its applicability. (GK)
Tucker DK, Foley JF, Bouknight SA, Fenton SE. 2017. Sectioning mammary gland whole mounts for lesion identification. J Vis Exp; doi: 10.3791/55796 [Online 24 July 2017].
High vitamin D levels associated with decreased breast cancer risk
Researchers at NIEHS found evidence that increased blood levels of vitamin D reduced the risk of breast cancer. Vitamin D is derived naturally from sunlight exposure, nutritional supplements, and dietary sources, and is known to have certain effects that could reduce the risk for cancer. The authors analyzed enrollment data and serum samples from the NIEHS Sister Study, which is a study that examines environmental and genetic contributions to health outcomes in more than 50,000 women who each have a sister with breast cancer.
The scientists measured the vitamin D prehormone 25(OH)D in serum samples from 1,611 participants who later developed breast cancer and 1,843 randomly selected participants. They found a statistically significant association between high levels of 25(OH)D and lower breast cancer risk, with the strongest association among postmenopausal and obese women. Evidence in the larger cohort also found a reduced risk among postmenopausal women who regularly took vitamin D supplements. The authors reported some limitations to this study, such as the limited number of minority participants and unavoidable inaccuracies in participant-reported vitamin D exposure. Nevertheless, although confirmation in other studies will be needed, this work supports the use of vitamin D supplementation for breast cancer risk reduction. (KG)
O’Brien KM, Sandler DP, Taylor JA, Weinberg CR. 2017. Serum vitamin D and risk of breast cancer within five years. Environ Health Perspect 125(7):077004.
Mouse embryos are not female by default
NIEHS researchers and their colleagues at Baylor College of Medicine discovered that COUP-TFII, a nuclear receptor present in developing organs of embryos, is responsible for sexual differentiation. Their findings challenge the prevailing theory that the lack of androgen determines which reproductive tract is maintained in the embryo.
Using a mouse model, the researchers found that female embryos deficient in COUP-TFII retained both the male and female reproductive tracts. When the mouse embryos were exposed to an androgen antagonist, the knockout embryos still exhibited both reproductive tracts, indicating that a lack of androgen is not responsible for the regression of the male duct. Using reverse transcription polymerase chain reaction, the researchers found higher expression of fibroblast growth factor (FGF) in the mesonephroi of the knockout embryos compared with the controls. The mesonephros is the embryo’s excretory organ. It is also the compartment where the male duct develops and COUP-TFII is housed. COUP-TFII inhibits the pathway that FGF activates, causing regression of the male duct in the embryo.
These findings uncover the mechanism behind sexual dimorphism, challenging the long-held notion that embryos are female by default. Future research will reevaluate the role that androgen plays in sexual development. (AR)
Zhao F, Franco HL, Rodriguez KF, Brown PR, Tsai MJ, Tsai SY, Yao HH. 2017. Elimination of the male reproductive tract in the female embryo is promoted by COUP-TFII in mice. Science 357(6352):717−720. (Story)
TNF needed for one type of allergic airway inflammation
New findings from NIEHS scientists suggest that toll-like receptor (TLR) ligands and proteases in the environment act through different molecular pathways to promote airway inflammation. The work may help explain why only some asthma patients benefit from drugs that inhibit tumor necrosis factor (TNF), a protein secreted during inflammatory responses.
Asthma, a chronic inflammatory disease of the airways, is associated with exposure to a variety of adjuvants, or substances, that promote allergic inflammatory responses. Before this study, it was unclear whether different types of adjuvants induced asthma through similar or distinct mechanisms. Using two types of adjuvants, TLR ligands and proteases, the researchers characterized the signaling pathways these adjuvants triggered during allergic responses.
Not only did they find that TLR ligands and proteases promoted distinct forms of airway inflammation, they also observed that only TLR ligands increased the level of TNF. The scientists further identified mechanisms through which TNF induced airway inflammation, and confirmed that TNF is an indispensable component in the TLR ligand-mediated, but not protease-mediated, form of asthma. The results suggested that analysis of patients’ environmental exposures might help predict their responses toward anti-TNF therapy and also reveal novel molecules in the TNF pathway that could be targeted for therapeutic intervention. (YWC)
Whitehead GS, Thomas SY, Shalaby KH, Nakano K, Moran TP, Ward JM, Flake GP, Nakano H, Cook DN. 2017. TNF is required for TLR ligand-mediated but not protease-mediated allergic airway inflammation. J Clin Invest 127(9):3313–3326.
Adjustable DNA pol beta catalyzes an efficient reverse reaction
Pyrophosphorolysis, a process catalyzed by DNA polymerase beta that breaks down DNA, can be modulated by using a pyrophosphate analogue, imidodiphosphate, according to NIEHS researchers. The finding provides a tool for understanding how DNA polymerase beta maintains its fidelity during its role in DNA repair and regulates its sensitivity to chain-terminating nucleoside drugs that are used to treat cancer.
Biochemical studies revealed that a pyrophosphate (PPi) mimic can be used to alter the ratio that DNA polymerase beta uses for its forward and reverse reactions. However, the forward reaction is typically favored by changing the bridging oxygen to nitrogen within PPi, which also increases the reverse reaction. Using a technique known as time-lapse x-ray crystallography, the researchers were able to identify how changing the oxygen to nitrogen affected the reverse reaction.
The crystallography studies demonstrated that two magnesium ions were bound within the catalytic and metal binding sites of the enzyme. This binding led to the repositioning of the active oxygen closer to its target phosphate, offering an explanation for why this pyrophosphate mimic alters the reverse reaction. Future studies will apply this discovery to understanding the effects on other important polymerases, such as polymerase gamma and HIV-1 reverse transcriptase. (KH)
Shock DD, Freudenthal BD, Beard WA, Wilson SH. 2017. Modulating the DNA polymerase beta reaction equilibrium to dissect the reverse reaction. Nat Chem Biol 13(10):1074−1080.
(Yu-Wei Chen, Ph.D., is a research fellow in the NIEHS Developmental Neurobiology Group. Katie Glenn, Ph.D., is an Intramural Research and Training Award (IRTA) fellow in the Mechanism of Mutation Group. Kiri Hoff, Ph.D., is an IRTA fellow in the Mitochondrial DNA Replication Group. Gabriel Knudsen, Ph.D., is a Cancer Research Training Award fellow in the National Cancer Institute, Center for Cancer Research, Laboratory of Toxicology and Toxicokinetics. Alicia Richards, is a pregraduate fellow in the National Cancer Institute Laboratory of Toxicology and Toxicokinetics.)