Papers of the Month
By Douglas Ganini da Silva, Mahita Kadmiel, Gabriel Knudsen, Emily Mesev, and Qing Xu
NTP finds high levels of MCHM cause skin irritation
Pure and crude 4-methylcyclohexanemethanol (MCHM) can cause skin irritation, according to scientists at the National Toxicology Program (NTP). MCHM is used in coal beneficiation, a method that improves the quality of raw coal. The evaluation followed a 2014 chemical spill in which crude MCHM contaminated 15 percent of West Virginia’s tap water.
Approximately 1,900 West Virginia residents reported dermal irritation after crude MCHM was released from a storage tank into the Elk River, a water source for a large portion of the Charleston area. The authors assessed the ability of pure and crude MCHM to produce skin irritation by measuring ear swelling in mice after localized exposure to MCHM. Skin sensitization was assessed by measuring lymphocyte proliferation in the draining lymph node, an indicator of an immune response.
Although both pure and crude MCHM produced skin irritation in mice, pure MCHM appeared to be a stronger irritant. In contrast, only crude MCHM, not pure, was shown to act as a skin sensitizer. The study speculated that these differences might arise from variations in isomer concentration or in the presence of other chemicals mixed into crude MCHM. However, neither pure nor crude MCHM at levels similar to those in the tap water after the Elk River spill produced irritation or sensitization in mice. (EM)
Johnson VJ, Auerbach SS, Luster MI, Waidyanatha S, Masten SA, Wolfe MS, Burleson FG, Burleson GR, Germolec DR. 2017. Evaluation of 4-methylcyclohexanemethanol (MCHM) in a combined irritancy and local lymph node assay (LLNA) in mice. Food Chem Toxicol 105:99−105.
Membrane protein orchestrates dialogue between fetus and mother
Epithelial membrane protein 2 (EMP2) has a novel role in regulating proteins involved in placental development, according to NIEHS scientists and their collaborators. The work may help researchers understand interactions that could result in pregnancy loss or complications in pregnancy.
The authors established that EMP2 plays a critical role in placental development by generating a mouse model that lacked EMP2. Female mice without EMP2 (EMP2 null), but carrying fetuses with at least 50 percent of EMP2, had no pregnancy complications. However, EMP2 null mothers carrying fetuses without EMP2 had reduced litter sizes. The finding suggested that the fetal dose of EMP2 was crucial and that the maternal EMP2 was not.
Through elegant breeding experiments, the authors demonstrated that EMP2 that originated from the fetal side of the placenta was critical for pregnancy. Global gene expression profiling revealed that genes implicated in vascular changes, coagulation, and oxidative stress were dysregulated in EMP2 null placentas. These data were further supported by histological changes in the placentas, including abnormal blood vessel development. The authors also found that EMP2 expression was reduced in human placentas obtained from pregnancies with growth-restricted babies. This observation implied that the significance of EMP2 in pregnancy development is pertinent to human health. (MK)
Williams CJ, Chu A, Jefferson WN, Casero D, Sudhakar D, Khurana N, Hogue CP, Aryasomayajula C, Patel P, Sullivan P, Padilla-Banks E, Mohandessi S, Janzen C, Wadehra M. 2017. Epithelial membrane protein 2 (EMP2) deficiency alters placental angiogenesis, mimicking features of human placental insufficiency. J Pathol; doi: 10.1002/path.4893 [Online 14 March 2017].
Fluorescent proteins can cause oxidative stress in cells
NIEHS researchers recently determined the mechanism that fluorescent proteins use to induce oxidative stress in cells. Fluorescent proteins are a powerful tool used in biomedical research for protein labeling and cell tracing. Enhanced green fluorescent protein (eGFP) is the most frequently used protein. Although fluorescent proteins are believed to be harmless, the authors showed that eGFP continuously generates free radicals in mammalian and bacterial cells.
Using electron spin resonance spectroscopy and other biochemical assays, the scientists found that immature forms of eGFP and a related red fluorescent protein, TagRFP, generated the free radical superoxide anion and hydrogen peroxide in the presence of a compound called NADH, which is found in all living organisms. Analyses with real-time polymerase chain reaction and a DNA microarray showed that gene expression of cells was altered by eGFP, such as an increase in hypoxia inducible factor-1alpha in mammalian cells and activation of SoxR/S in bacteria.
The authors concluded that cells that expressed eGFP generated continuous hydrogen peroxide at sufficient levels to act as a global messenger and induce oxidative stress response. Because superoxide anion and hydrogen peroxide are known to be involved in a variety of biological functions, the authors suggested studies that use fluorescent proteins should validate results with alternative methods. (GK)
Ganini D, Leinisch F, Kumar A, Jiang J, Tokar EJ, Malone CC, Petrovich RM, Mason RP. 2017. Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells. Redox Biol 12:462−468.
ER alpha-mediated transcription protects against fatty liver
NIEHS researchers revealed that DNA binding and transcription mediated by estrogen receptor alpha (ERalpha) is required for estrogen-dependent protection against nonalcoholic fatty liver disease (NAFLD), a disorder with excess fat in the liver. The findings provide new insights into the development and treatment of NAFLD and the associated metabolic syndrome.
NAFLD affects up to 25 percent of people in the U.S. and may lead to liver damage if left untreated. Estrogen has been shown to guard against development of NAFLD in menopausal women, but may cause side effects, including increased risk for cancer. Better options include therapies that target specific pathways.
As a main mediator of estrogen actions, ERalpha controls gene transcription through DNA binding. To identify crucial mechanisms responsible for the protective effects of estrogen, researchers in this study analyzed several ERalpha-related mouse models.
They found that a high fat diet (HFD) in female mice devoid of ERalpha or in those with a mutated ERalpha DNA-binding domain led to elevated liver fat accumulation and increased liver impairment compared with control mice. Interestingly, HFD feeding did not affect liver-specific ERalpha knockout mice, which suggested the importance of nonhepatic ERalpha signaling. The results demonstrated an essential role of ERalpha-mediated direct transcription in protection against fatty liver. (QX)
Hart-Unger S, Arao Y, Hamilton KJ, Lierz SL, Malarkey DE, Hewitt SC, Freemark M, Korach KS . 2017. Hormone signaling and fatty liver in females: analysis of estrogen receptor alpha mutant mice. In J Obes (Lond); doi: 10.1038/ijo.2017.50 [Online 21 March 2017].
Examining rhythmic signals and oscillatory patterns in biological systems
Led by an NIEHS scientist, a research team created a computer program called Order Restricted Inference for Oscillatory Systems (ORIOS). ORIOS, which is available from the NIEHS website, is a methodology for accurately detecting rhythmic signals in oscillatory systems. Examples of oscillatory systems in living organisms include circadian clock, cell cycles, menstrual cycles, and sleep patterns.
The authors demonstrated the usefulness of ORIOS by analyzing global gene expression data of the liver and pituitary gland in mice, and of the human cell lines U2OS and NIH3T3. Using computer simulations, they also demonstrated that, compared with two popular methods, ORIOS had a higher true positive rate, which refers to the frequency of correctly declaring a rhythmic signal to be rhythmic. It also had a higher true negative rate, or frequency of a nonrhythmic signal to be nonrhythmic. ORIOS performed better because it did not use mathematical models, but relied on the underlying mathematical inequalities that describe a rhythmic pattern.
The ability of ORIOS to detect rhythmic signals with high accuracy will allow researchers to discover novel genes that are potentially governed by the circadian clock, and hence, may have important applications. (DGS)
Larriba Y, Rueda C, Fernandez MA, Peddada SD. 2016. Order restricted inference for oscillatory systems for detecting rhythmic signals. Nucleic Acids Res 44(22):e163.