Papers of the Month
By Anika Dzierlenga, Mahita Kadmiel, Cody Nichols, Kelly Shipkowski, and Salahuddin Syed
NTP develops screening method for androgen receptor pathway activation
Researchers from the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods, working with the U.S. Environmental Protection Agency, designed an efficient and cost-effective computational model to determine whether a chemical harbors androgen receptor (AR) pathway activity. The team validated the model by building a database of reference chemicals that included a wide range of AR activity, which ultimately allowed the testing strategy to be incorporated into a regulatory setting.
The researchers integrated both experimental and computational data from a suite of 11 ToxCast and Tox21 in vitro high-throughput screening assays that map key biological events along the AR pathway. Use of these assays was supplemented with an antagonist confirmation assay to distinguish true activity from cytotoxicity. At the same time, the scientists conducted a systematic literature review to externally validate the method, compiling a library of 158 reference chemicals with pre-existing experimental data. They determined that the new pathway model was more than 95 percent accurate for detecting both agonist and antagonist activity. An agonist is substance that initiates a biochemical action, and an antagonist is a substance that inhibits a biochemical action.
After screening 1,855 chemicals, the researchers predicted that 220 chemicals had clear AR pathway activity and 174 chemicals had weak AR pathway activity. Future efforts will connect AR pathway disruption by these chemicals to relevant human exposures and implications for human health. (AD)
Citation: Kleinstreuer NC, Ceger P, Watt ED, Martin M, Houck K, Browne P, Thomas RS, Casey WM, Dix DJ, Allen D, Sakamuru S, Xia M, Huang R, Judson R. 2016. Development and validation of a computational model for androgen receptor activity. Chem Res Toxicol; doi: 10.1021/acs.chemrestox.6b00347 [Online 18 November 2016].
New technique measures genomic mutations
In collaboration with scientists from across the United States, NIEHS researchers developed a new approach to measure mutations that accumulate during the lifetime of healthy individuals. The results demonstrated that the amount of DNA damage in skin cells caused by ultraviolet (UV) light was similar to the amount due to naturally occurring processes in the body. The findings suggested that the mechanisms involved in cancer development also occur in healthy cells. The research might help scientists design better preventative and therapeutic strategies to address cancer and other genetic diseases.
The scientists took skin biopsies from the arms and hips of two healthy adult humans and evaluated the number and type of mutations in the samples. They determined that every cell contained at least a single chromosomal rearrangement, which is one type of mutation, and 600 to 13,000 DNA base substitutions. Also, because the sun-exposed skin had a higher number of mutations than the hip samples, which had been protected from sunlight by clothes, the researchers were able to determine specific changes induced by UV exposure.
Although the authors stressed that a larger study using more cell types and volunteers is needed to refine estimates of mutation rates, they maintained this work helped them understand how the environment and naturally occurring processes contribute to cancer. (KS)
Citation: Saini N, Roberts SA, Klimczak LJ, Chan K, Grimm SA, Dai S, Fargo DC, Boyer JC, Kaufman WK, Taylor JA, Lee E, Cortes-Ciriano I, Park PJ, Schurman SH, Malc EP, Mieczkowski PA, Gordenin DA. 2016. The impact of environmental and endogenous damage on somatic mutation load in human skin fibroblasts. PLoS Genet 12(10):e1006385.
Crystal structure of a protein essential for ribosomal biogenesis
Scientists at NIEHS solved the crystal structure of a protein called Nop15. Nop15 is an RNA-binding protein known to be critical for the formation of ribosomes, which are the free or membrane-bound organelles that serve as protein factories of the cell. Nop15, like other RNA binding proteins, can bind RNA via a region called an RNA recognition motif domain. However, variations in the sequence neighboring the RNA recognition motif dictate the binding specificity between Nop15 and RNA.
X-ray crystallography studies revealed that Nop15 from yeast contains a core RNA recognition motif that is flanked by a C-terminal sequence. The authors found that in the absence of RNA, the RNA recognition site is masked by the C-terminal sequence, thereby controlling the availability of Nop15 to interact with its target RNA. By using additional structural approaches and binding assays, the authors determined that the C-terminal sequence, and not the N-terminal sequence, of Nop15 is important for successful RNA recognition and interaction. Upon pre-ribosomal RNA binding, the C-terminal region refolds and forms new interactions to stabilize the RNA structure. These findings shed light on the highly complex process of ribosome biogenesis. (MK)
Citation: Zhang J, Gonzalez LE, Hall TM. 2016. Structural analysis reveals the flexible C-terminus of Nop15 undergoes rearrangement to recognize a pre-ribosomal RNA folding intermediate. Nucleic Acids Res; doi: 10.1093/nar/gkw961 [Online 26 October 2016].
Link between cytochrome c and alpha-synuclein in Parkinson's disease
Using a pesticide-induced model of Parkinson’s disease (PD) in mice, researchers at NIEHS showed for the first time that a small protein known as cytochrome c is heavily involved in promoting the neuronal death seen in PD. According to their findings, cytochrome c, a mitochondrial protein involved in energy generation, oxidizes a neuronal protein called alpha-synuclein to free radicals, which ultimately contributes to neuronal death. The scientists used the fungicide Maneb and herbicide paraquat in their studies, because these chemicals are commonly used together in agriculture. The chemicals have also been documented to trigger PD symptoms in mice and to increase the risk of PD in humans.
The authors demonstrated that mitochondrial dysfunction leads to the release of cytochrome c into the cytosol. Once there, the protein acts as a peroxidase, which means it catalyzes the formation of free radicals in the presence of hydrogen peroxide. In this paper, alpha-synuclein free radicals generated by cytochrome c were detected in the brains of pesticide-exposed mice using an immuno-spin trapping method. Microarray results of alpha-synuclein knockout mice exposed to pesticides suggested that these mice were protected from PD. These data indicate the importance of cytochrome c and alpha-synuclein in PD development. (SS)
Citation: Kumar A, Ganini D, Mason RP. 2016. Role of cytochrome c in alpha-synuclein radical formation: implications of alpha-synuclein in neuronal death in Maneb- and paraquat-induced model of Parkinson’s disease. Mol Neurodegener 11(1):70.
Early-life farm exposures may protect against allergy sensitization in adults
Based on blood analysis of study participants, NIEHS scientists and their collaborators found that early-life farm exposures were strongly associated with a reduced risk of atopy, or allergic sensitization to common environmental allergens. Previous studies, primarily from small European farms, showed that early life farm exposures protect against atopy in childhood. This study in adults showed that this protection persists into adulthood. Early farm exposures had little or no link to the development of asthma. The work helps researchers better understand how the environment shapes the human immune system.
The authors analyzed data from the NIEHS Agricultural Lung Health Study, a large case-control study of asthma in farmers and farm spouses, nested within the Agricultural Health Study. Participants provided information about their mother’s exposure while pregnant and their own early-life exposures to the farming environment. The strongest signal appeared to come from maternal exposure to the farming environment during pregnancy.
The authors found that early-life farm exposures, particularly maternal farming activities while pregnant, were strongly associated with reduced risk of atopy in adults. These results extend previous work done primarily in children and suggest that protective associations of early-life exposure to the farming environment endure across the lifecourse. (CN)
Citation: House JS, Wyss AB, Hoppin JA, Richards M, Long S, Umbach DM, Henneberger PK, Beane Freeman LE, Sandler DP, Long O’Connell E, Barker-Cummings C, London SJ. 2016. Early-life farm exposures and adult asthma and atopy in the Agricultural Lung Health Study. J Allergy Clin Immunol; doi: 10.1016/j.jaci.2016.09.036 [Online 12 November 2016].