Papers of the Month
By Janelle Weaver
DNTP study shows that zebrafish screening protocols affect toxicity outcomes
A quantitative assessment of sources of variability in the design of zebrafish experiments could set the groundwork for global efforts to harmonize protocols, according to researchers from the NIEHS Division of the National Toxicology Program (DNTP).
Zebrafish embryos have been widely used as toxicity screening tools due to advantages such as transparency and rapid development. But data obtained from independent laboratories can vary vastly, complicating the use of zebrafish screening for regulatory decisions.
To investigate this issue, DNTP researchers analyzed zebrafish toxicity data from three independent laboratories using the same set of 87 compounds. They estimated the concentrations that elicited death, developmental toxicity, and impairment of movement responses.
For laboratories that used similar assay protocol parameters, the concordance reached as high as 86% for developmental toxicity results and as high as 68% for neurological toxicity results. By contrast, the concordance dropped for laboratories that used different protocol parameters. Strikingly, the concentration required to produce developmental and neurological toxicity ranged, on average, up to four-fold and six-fold, respectively.
Factors that potentially contributed to this variability included the fish strain, whether the outermost membrane surrounding the embryo had been removed, the exposure scenario and volume, and behavioral testing time. According to the authors, the findings may guide ongoing efforts to incorporate zebrafish as a complementary model for drug development and toxicity testing.
Citation: Hsieh JH, Behl M, Parham F, Ryan K. 2022. Exploring the influence of experimental design on toxicity outcomes in zebrafish embryo tests. Toxicol Sci; doi:10.1093/toxsci/kfac053 [Online 27 May 2022].
Early biomarkers may predict lung disease in preterm infants
Molecular biomarkers present at birth are linked to the later development of a chronic respiratory illness called bronchopulmonary dysplasia (BPD), according to NIEHS researchers and their collaborators.
BPD primarily affects infants born before 30 weeks gestational age with birthweights less than 1,000 grams, and who receive more than four weeks of therapeutic oxygen supplementation in the neonatal intensive care unit. The condition is characterized by impaired lung development that persists into later life. Cases have been increasing and therapeutic options for prevention and treatment are limited. In addition, the mechanisms that lead to the disease are not fully understood.
To address this knowledge gap, the researchers collected umbilical cord blood samples from 107 preterm newborns, 14 of whom developed BPD. They analyzed epigenetic patterns – chemical modifications that could affect gene activity in the absence of changes in the underlying DNA sequence. The results revealed a higher number of epigenetic mutations that altered DNA methylation, as well as differences in gene activity, in newborns who later developed BPD compared to those who did not.
Specifically, BPD risk was associated with altered cord blood DNA methylation profiles in genes involved in lung and alveolar development, and hematopoiesis. Taken together, the results shed light on the biological pathways involved in BPD and suggest several potential epigenetic biomarkers that might indicate higher susceptibility to BPD at birth. Co-first authors Xuting Wang, Ph.D., and Hye-Youn Cho, Ph.D., from the Environmental Epigenomics and Disease Group, indicate that further studies are needed to determine whether CpG sites (regions where DNA methylation occurs) they identified will prove to be clinically relevant.
Citation: Wang X, Cho HY, Campbell MR, Panduri V, Coviello S, Caballero MT, Sambandan D, Kleeberger SR, Polack FP, Ofman G, Bell DA. 2022. Epigenome-wide association study of bronchopulmonary dysplasia in preterm infants: results from the discovery-BPD program. Clin Epigenetics 14(1):57.
Scientists assess the epigenetics of lung function across ancestries
A large-scale, multi-ancestry study by NIEHS researchers and their collaborators provides a comprehensive evaluation of epigenome-wide DNA methylation in relation to lung function.
Lung function is affected by both genetics and environmental exposures. Large genome-wide association studies have identified more than 300 loci related to lung function, but much of the variability remains unexplained. Epigenetic marks, such as methylation (chemical modifications that can alter gene expression without affecting the underlying DNA sequence), are influenced by genetics and the environment and may contribute. Epigenome-wide studies have revealed some associations between lung function and cytosine-phosphate-guanine (CpG) sites, which are regions where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases. But replication has been limited, most research focused on European populations, and no large-scale multi-ancestry study had been published.
To overcome these limitations, the researchers conducted epigenome-wide analyses of blood DNA methylation and lung function in 17,503 adults from European, African, and Hispanic/Latino ancestries. Lung function was related to DNA methylation at 1,267 CpG sites spanning 1,042 genes. The vast majority of these genes had never been implicated in lung function. Although most signals were consistent across ancestries, some were potentially unique to individuals of African ancestry. Some implicated genes are targets of approved or investigational drugs.
These findings can be leveraged for understanding mechanisms that regulate lung function, developing diagnostic and prognostic biomarkers of respiratory disease, and identifying potential targets for preventative and therapeutic strategies.
Citation: Lee M, Huan T, McCartney DL, Chittoor G, de Vries M, Lahousse L, Nguyen JN, Brody JA, Castillo-Fernandez J, Terzikhan N, Qi C, Joehanes R, Min JL, Smilnak GJ, Shaw JR, Yang CX, Colicino E, Hoang TT, Bermingham ML, Xu H, Justice AE, Xu CJ, Rich SS, Cox SR, Vonk JM, Prokić I, Sotoodehnia N, Tsai PC, Schwartz JD, Leung JM, Sikdar S, Walker RM, Harris SE, van der Plaat DA, Van Den Berg DJ, Bartz TM, Spector TD, Vokonas PS, Marioni RE, Taylor AM, Liu Y, Barr RG, Lange LA, Baccarelli AA, Obeidat M, Fornage M, Wang T, Ward JM, Motsinger-Reif AA, Hemani G, Koppelman GH, Bell JT, Gharib SA, Brusselle G, Boezen HM, North KE, Levy D, Evans KL, Dupuis J, Breeze CE, Manichaikul A, London SJ; BIOS consortium, GoDMC. 2022. Pulmonary function and blood DNA methylation: A multi-ancestry epigenome-wide association meta-analysis. Am J Respir Crit Care Med; doi:10.1164/rccm.202108-1907OC [Online 10 May 2022].
Prenatal phthalate exposure may predispose children to obesity
Endocrine disruptors called phthalates are linked to both lower adiposity and weight at birth, and higher adiposity during childhood, according to NIEHS researchers and their collaborators.
Phthalates are ubiquitous in the environment, personal care products, food and beverage packaging materials, and vinyl products. Prenatal phthalate exposure has been associated with lower birth weight but also higher weight in childhood. Yet few studies have examined prenatal phthalate exposure in relation to weight or BMI measured from the prenatal period to childhood. This pattern of association could be important since lower birth weight combined with rapid weight gain in early life is a risk factor for later cardiometabolic disease.
To address this knowledge gap, the researchers analyzed a panel of phthalate metabolites in urine collected from 780 pregnant women during two visits at four sites in the United States. Prenatal exposure to several phthalates was linked to lower weight at birth but not during childhood. In addition, prenatal exposure to some phthalates was associated with low weight-to-length ratio at delivery, as well as high body mass index at three to six years of age. According to the authors, continued investigation of the role of such environmental exposures is warranted, given the high rates of childhood obesity in the United States.
Citation: Ferguson KK, Bommarito PA, Arogbokun O, Rosen EM, Keil AP, Zhao S, Barrett ES, Nguyen RHN, Bush NR, Trasande L, McElrath TF, Swan SH, Sathyanarayana S. 2022. Prenatal phthalate exposure and child weight and adiposity from in utero to 6 years of age. Environ Health Perspect 130(4):47006.
Structural snapshots shed light on the choreography of catalysis
Crystal structures of mysterious molecular complexes have provided atomic-level insight into the diversity of mechanisms underlying enzymatic reactions, according to NIEHS researchers and their collaborators.
Protein tyrosine phosphatases (PTPs) are enzymes that are essential for many cellular functions. It has not been clear how enzymatic reactions proceed for a subgroup of PTPs that lack the classical version of a prominent structural element called the WPD loop. Progress toward solving this puzzle requires high-resolution structures of members of this particular group of enzymes interacting with their natural substrates.
To address this need, the researchers provided crystal complexes for one such PTP outlier: Arabidopsis thaliana plant and fungi atypical dual specificity phosphatase 1 (AtPFA-DSP1). High-resolution crystal structures revealed the catalytic core of AtPFA-DSP1 interacting with signaling molecules called inositol pyrophosphates (PP-InsPs). By linking together individual structural datasets, the researchers elucidated how the WPD loop contributes to catalysis, despite lacking its typical peptide sequence. In plants, this type of reaction may impact drought tolerance and regulate molecular defenses against herbivorous larvae and pathogens.
Taken together, the findings represent a significant extension to the known chemical versatility of the WPD loop, thereby greatly expanding mechanistic understanding of its important contributions to tailoring reactivity profiles for individual PTP family members.
Citation: Wang H, Perera L, Jork N, Zong G, Riley AM, Potter BVL, Jessen HJ, Shears SB. A structural exposé of noncanonical molecular reactivity within the protein tyrosine phosphatase WPD loop. 2022. Nat Commun 13(1):2231.
(Janelle Weaver, Ph.D., is a contract writer for the NIEHS Office of Communications and Public Liaison.)