NTP high-throughput assay better classifies cytotoxicity

National Toxicology Program (NTP) researchers and their colleagues developed a real-time screening assay that allows researchers to understand how compounds become toxic and kill living cells. They hope the assay will be used to study the safety of chemicals in cellular models and determine methods that may translate into animal models.

The scientists exposed two cellular models to more than 10,000 chemicals for different lengths of time to assess the time course of toxic outcomes. These cells were evaluated using two real-time assays that measured the metabolic activity of the cells and the integrity of the cell membrane. The data were used to group the chemicals based on the time course of the observed cytotoxic outcomes. Researchers then identified important underlying biological processes and grouped the chemicals by their mechanism of action, which can be used to predict toxicological outcomes.

Previously, translating the screening of chemicals for potentially toxic endpoints between cell lines, animal models, and different cell lines was problematic. With the development of this assay and informatics methods, future studies related to chemical-induced outcomes could be much quicker and more reliable. (CN)

Citation: Hsieh JH, Huang R, Lin JA, Sedykh A, Zhao J, Tice RR, Paules RS, Xia M, Auerbach SS. 2017. Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage. PLoS One 12(5):e0177902.

Changes in biomarkers linked to PAH exposure in pregnant women

An NIEHS epidemiologist was part of a team that discovered associations between exposure to polycyclic aromatic hydrocarbons (PAHs) and biomarkers of inflammation, angiogenesis, and oxidative stress in pregnant women. PAHs, which are formed during the combustion of organic matter, may come from coal-fired plants, tobacco smoke, and grilled meat. Previous studies have found that PAH exposure is associated with several adverse health outcomes, such as impaired neurodevelopment, childhood asthma, and eye, skin, and respiratory tract inflammation.

Using isotope dilution liquid chromatography with tandem mass spectrometry (LC-MS/MS), the scientists measured eight PAH metabolites in urine samples from 200 pregnant women in their third trimester. The participants were part of the prospective LIFECODES birth cohort study conducted at Brigham and Women’s Hospital. Immunoassays and LC-MS/MS were used to detect 13 biomarkers of inflammation, oxidative stress, and angiogenesis from blood or urine.

The largest positive associations were for 8-isoprostane, a biomarker of oxidative stress, and C-reactive protein, a biomarker of inflammation. No single metabolite stood out as uniformly associated with the array of biomarkers. The study also found that women with private health insurance and higher levels of education tended to have lower levels of PAH metabolites in their urine. (SSR)

Citation: Ferguson KK, McElrath TF, Pace GG, Weller D, Zeng L, Pennathur S, Cantonwine DE, Meeker JD. 2017. Urinary polycyclic aromatic hydrocarbon metabolite associations with biomarkers of inflammation, angiogenesis, and oxidative stress in pregnant women. Environ Sci Technol 51(8):4652−4660.

Oxidative stress associated with lower breast cancer risk

Oxidative stress, which is an overabundance of reactive oxygen species, may reduce the risk of breast cancer among premenopausal women according to a study from NIEHS scientists and their collaborators. Findings from this work, which represents the largest sample of premenopausal women to date, are consistent with a protective effect of apoptosis, or programmed cell death, and senescence, or aging, induced by oxidative stress among younger women.

The scientists conducted a nested case-control study that involved nearly 1,000 premenopausal women who were part of the NIEHS Sister Study. Using urine samples that were collected when women joined the study — before any of the women developed breast cancer — the research team measured two biomarkers, F2-isoprostane and its metabolite, to quantify oxidative stress. The researchers adjusted for variables, including body mass index, waist circumference, smoking, physical activity, income, nutrition, and use of antioxidants.

The researchers determined that higher levels of oxidative stress, as measured by the F2-isoprostane metabolite, were associated with lower breast cancer risk among women who remained premenopausal or were aged younger than 46 years at diagnosis. They did not find a relationship between oxidative stress and breast cancer risk in women who reached menopause before diagnosis. (HV)

Citation: Nichols HB, Anderson C, White AJ, Milne GL, Sandler DP. 2017. Oxidative stress and breast cancer risk in premenopausal women. Epidemiology; doi: 10.1097/EDE.0000000000000685 [Online 17 May 2017].

Crystal structure solved for ribosomal assembly factor Nsa1/WDR74

By determining the crystal structure of the Saccharomyces cerevisiae ribosome assembly factor Nsa1, NIEHS researchers have discovered that a particular region of Nsa1 serves as a platform to recruit another ribosomal assembly factor, Rix7, to pre-60S particles. pre-60S particles are the precursors to the large ribosome subunit, part of the ribosomal machinery that translates mRNA into proteins. Previous studies have linked Nsa1 and Rix7 with nucleolar pre-60S particles, but scientists have not been able to detect a direct interaction between these two proteins.

Previous studies have linked improper ribosomal assembly with cancer and a class of diseases known as ribosomopathies. In this study, researchers found that Rix7, known as NVL2 in mammals, recognizes an N-terminal domain in Nsa1, or WDR74 in mammals, called a WD40 domain. Crystal structure analysis of Nsa1/WDR74 determined that the WD40 domain contains a seven-bladed beta-propeller, and within it, a conserved loop that can associate with the alpha-beta region of the D1 domain of Rix7/NVL2.

Structural and functional analyses also showed that the flexible C-terminus of Nsa1/WDR74 contains many lysine residues and extends away from the WD40 domain. This feature appears to be necessary for nucleolar localization. The findings suggest that association and nucleolar targeting of Nsa1/WRD74 and Rix7/NVL2 are important for assembly of the large ribosomal subunit. (EM)

Citation: Lo YH, Romes EM, Pillon MC, Sobhany M, Stanley RE. 2017. Structural analysis reveals features of ribosome assembly factor Nsa1/WDR74 important for localization and interaction with Rix7/NVL2. Structure 25(5):762−772.e4.

Cnot3 promotes cardiac cell growth by degrading cell cycle inhibitors

According to a team of scientists from NIEHS and China, Cnot3, a protein that is part of a multi-subunit complex called Ccr4-Not, regulates the process in which human embryonic stem cells (hESCs) differentiate, or change, into heart muscle cells. The researchers are the first to reveal that Cnot3 encourages the proliferation, or rapid increase, in the number of these cells by binding to cell cycle inhibitors. This binding promotes the degradation of gene products that would otherwise stop the formation and growth of cardiac cells. The work suggests that Cnot3 may provide a way for scientists to produce large numbers of cardiac cells that could be used to treat patients who have experienced heart damage due to disease.

The scientists used an hESCs-based cardiac differentiation model, as well as cultured human heart muscle cells and cells taken from rodents that had heart attacks. They found that human and mouse heart muscle cells that exhibited high levels of proliferation contained increased levels of Cnot3. Removing Cnot3 from these cells reduced the number of heart cells being produced, whereas overexpressing the Cnot3 gene led to a greatly enhanced proliferation of heart muscle cells. (RA)

Citation: Zhou B, Liu J, Ren Z, Yao F, Ma J, Song J, Bennett B, Zhen Y, Wang L, Hu G, Hu S. 2017. Cnot3 enhances human embryonic cardiomyocyte proliferation by promoting cell cycle inhibitor mRNA degradation. Sci Rep 7(1):1500.