Early life exposure to low levels of AHR agonist PCB126 (3,3’,4,4’,5- pentachlorobiphenyl) reprograms gene expression in adult brain

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Toxicological Sciences 160 (2017): 386-397, doi:10.1093/toxsci/kfx192.Early life exposure to environmental chemicals can have long-term consequences that are not always apparent until later in life. We recently demonstrated that developmental exposure of zebrafish to low, non-embryotoxic levels of 3,3’,4,4’,5-pentachlorobiphenyl (PCB126) did not affect larval behavior, but caused changes in adult behavior. The objective of this study was to investigate the underlying molecular basis for adult behavioral phenotypes resulting from early life exposure to PCB126. We exposed zebrafish embryos to PCB126 during early development and measured transcriptional profiles in whole embryos, larvae and adult male brains using RNA-sequencing. Early life exposure to 0.3 nM PCB126 induced cyp1a transcript levels in 2-dpf embryos, but not in 5-dpf larvae, suggesting transient activation of aryl hydrocarbon receptor with this treatment. No significant induction of cyp1a was observed in the brains of adults exposed as embryos to PCB126. However, a total of 2209 and 1628 genes were differentially expressed in 0.3 nM and 1.2 nM PCB126-exposed groups, respectively. KEGG pathway analyses of upregulated genes in the brain suggest enrichment of calcium signaling, MAPK and notch signaling, and lysine degradation pathways. Calcium is an important signaling molecule in the brain and altered calcium homeostasis could affect neurobehavior. The downregulated genes in the brain were enriched with oxidative phosphorylation and various metabolic pathways, suggesting that the metabolic capacity of the brain is impaired. Overall, our results suggest that PCB exposure during sensitive periods of early development alters normal development of the brain by reprogramming gene expression patterns, which may result in alterations in adult behavior

Role of DNA methylation of AHR1 and AHR2 promoters in differential sensitivity to PCBs in Atlantic Killifish, Fundulus heteroclitus

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Aquatic Toxicology 101 (2011): 288-294, doi:10.1016/j.aquatox.2010.10.010.Atlantic killifish (Fundulus heteroclitus) inhabiting the PCB-contaminated Superfund site in New Bedford Harbor (MA, USA) have evolved genetic resistance to the toxic effects of these compounds. They also lack induction of cytochrome P4501A (CYP1A) and other aryl hydrocarbon receptor (AHR)-dependent responses after exposure to AHR agonists, suggesting an overall down-regulation of the AHR signaling pathway. In this study, we hypothesized that the genetic resistance is due to altered AHR expression resulting from hypermethylation of DNA in the promoter region of AHR genes in fish inhabiting New Bedford Harbor. To test this hypothesis, we cloned and sequenced AHR1 and AHR2 promoter regions and employed bisulfite conversion-polymerase chain reaction (BS-PCR) followed by clonal analysis to compare the methylation status of CpG islands of AHR1 and AHR2 in livers of adult killifish collected from New Bedford Harbor and a reference site (Scorton Creek, MA). No significant differences in methylation profiles were observed in either AHR1 or AHR2 promoter regions between NBH and SC fish. However, hypermethylation of the AHR1 promoter correlated with low expression of transcripts in the liver in both populations. In comparison to AHR1, hepatic mRNA expression of AHR2 is high and its promoter is hypomethylated. Taken together, our results suggest that genetic resistance to contaminants in NBH fish is not due to altered methylation of AHR promoter regions, but that promoter methylation may control tissue-specific expression of AHR genes in killifish.This work is funded in part by the Superfund Basic Research Program at Boston University to MEH (NIH Grant P42ES007381) and the postdoctoral scholar program at WHOI, with funding provided by the Dr. George D. Grice Postdoctoral Scholarship Fund to NA

Role of DNA methylation in altered gene expression patterns in adult zebrafish (Danio rerio) exposed to 3, 3’, 4, 4’, 5-pentachlorobiphenyl (PCB 126)

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Environmental Epigenetics 4 (2018): dvy005, doi:10.1093/eep/dvy005.There is growing evidence that environmental toxicants can affect various physiological processes by altering DNA methylation patterns. However, very little is known about the impact of toxicant-induced DNA methylation changes on gene expression patterns. The objective of this study was to determine the genome-wide changes in DNA methylation concomitant with altered gene expression patterns in response to 3, 3’, 4, 4’, 5-pentachlorobiphenyl (PCB126) exposure. We used PCB126 as a model environmental chemical because the mechanism of action is well-characterized, involving activation of aryl hydrocarbon receptor, a ligand-activated transcription factor. Adult zebrafish were exposed to 10 nM PCB126 for 24 h (water-borne exposure) and brain and liver tissues were sampled at 7 days post-exposure in order to capture both primary and secondary changes in DNA methylation and gene expression. We used enhanced Reduced Representation Bisulfite Sequencing and RNAseq to quantify DNA methylation and gene expression, respectively. Enhanced reduced representation bisulfite sequencing analysis revealed 573 and 481 differentially methylated regions in the liver and brain, respectively. Most of the differentially methylated regions are located more than 10 kilobases upstream of transcriptional start sites of the nearest neighboring genes. Gene Ontology analysis of these genes showed that they belong to diverse physiological pathways including development, metabolic processes and regeneration. RNAseq results revealed differential expression of genes related to xenobiotic metabolism, oxidative stress and energy metabolism in response to polychlorinated biphenyl exposure. There was very little correlation between differentially methylated regions and differentially expressed genes suggesting that the relationship between methylation and gene expression is dynamic and complex, involving multiple layers of regulation.This work was supported by the National Institute of Health Outstanding New Environmental Scientist Award to NA (NIH R01ES024915) and Woods Hole Center for Oceans and Human Health [National Institutes of Health (NIH) grant P01ES021923 and National Science Foundation Grant OCE-1314642 to M. Hahn, J. Stegeman, NA and SK]

Nrf2 and Nrf2-related proteins in development and developmental toxicity : insights from studies in zebrafish (Danio rerio)

© The Author(s), 2015. This is the author's version of the work and is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Free Radical Biology and Medicine 88B (2015): 275-289, doi:10.1016/j.freeradbiomed.2015.06.022.Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap’n’collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects.This work was supported in part by National Institutes of Health grants R01ES016366 (MEH), R01ES015912 (JJS), and F32ES017585 (ART-L).2016-06-2

Numerical prediction of diffusion and electric field-induced iron nanoparticle transport

Zero valent iron nanoparticles (nZVI) are considered very promising for the remediation of contaminated soils and groundwaters. However, an important issue related to their limited mobility remains unsolved. Direct current can be used to enhance the nanoparticles transport, based on the same principles of electrokinetic remediation. In this work, a generalized physicochemical model was developed and solved numerically to describe the nZVI transport through porous media under electric field, and with different electrolytes (with different ionic strengths). The model consists of the Nernst–Planck coupled system of equations, which accounts for the mass balance of ionic species in a fluid medium, when both the diffusion and electromigration of the ions are considered. The diffusion and electrophoretic transport of the negatively charged nZVI particles were also considered in the system. The contribution of electroosmotic flow to the overall mass transport was included in the model for all cases. The nZVI effective mobility values in the porous medium are very low (10−7–10−4 cm2 V−1 s−1), due to the counterbalance between the positive electroosmotic flow and the electrophoretic transport of the negatively charged nanoparticles. The higher the nZVI concentration is in the matrix, the higher the aggregation; therefore, low concentration of nZVI suspensions must be used for successful field application.This work has been funded by the research grant SFRH/BD/76070/2011, by project PTDC/AGR-AAM/101643/2008 NanoDC under Portuguese National funds through “Fundação para a Ciência e a Tecnologia” and by FP7-PEOPLE-IRSES-2010-269289-ELECTROACROSS. The Department of Civil and Environmental Engineering at Lehigh University is acknowledged for the funding of equipment development, testing and analysis of the nZVI transport experiments

Sequence and functional characterization of hypoxia inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus

Author Posting. © The Author(s), 2016. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in American Journal of Physiology-Regulatory Integrative and Comparative Physiology, 312 (2017): R412-R425, doi:10.1152/ajpregu.00402.2016.The hypoxia inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus occurs in habitats prone to hypoxia, it responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, 2α, and 3α independently formed complexes with the β subunit (ARNT) to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α, HIF2αb, a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations.This research was supported in part by the National Science Foundation (IBN-0236494 and DEB-1120263) and by National Institute of Environmental Health Sciences (NIEHS) grant P42ES007381 (Superfund Basic Research Program at Boston University). Data interpretation was aided by reference to a preliminary draft of the F. heteroclitus genome sequence, which was supported by funding from the National Science Foundation (collaborative research grants DEB-1120512, DEB-1265282, DEB-1120013, DEB-1120263, DEB-1120333, DEB-1120398)

Eltrombopag for the treatment of immune thrombocytopenia: The aegean region of Turkey experience

Objective: Immune thrombocytopenia (ITP) is an immune-mediated disease characterized by transient or persistent decrease of the platelet count to less than 100x109/L. Although it is included in a benign disease group, bleeding complications may be mortal. With a better understanding of the pathophysiology of the disease, thrombopoietin receptor agonists, which came into use in recent years, seem to be an effective option in the treatment of resistant cases. This study aimed to retrospectively assess the efficacy, long-term safety, and tolerability of eltrombopag in Turkish patients with chronic ITP in the Aegean region of Turkey. Materials and Methods: Retrospective data of 40 patients with refractory ITP who were treated with eltrombopag in the Aegean region were examined and evaluated. Results: The total rate of response was 87%, and the median duration of response defined as the number of the platelets being over 50x109/L was 19.5 (interquartile range: 5-60) days. In one patient, venous sinus thrombosis was observed with no other additional risk factors due to or related to thrombosis. Another patient with complete response and irregular follow-up for 12 months was lost due to sudden death as the result of probable acute myocardial infarction. Conclusion: Although the responses to eltrombopag were satisfactory, patients need to be monitored closely for overshooting platelet counts as well as thromboembolic events. © 2015 Turkish Society of Hematology. All rights reserved

Transcriptomic assessment of resistance to effects of an aryl hydrocarbon receptor (AHR) agonist in embryos of Atlantic killifish (Fundulus heteroclitus) from a marine Superfund site

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Genomics 12 (2011): 263, doi:10.1186/1471-2164-12-263.Populations of Atlantic killifish (Fundulus heteroclitus) have evolved resistance to the embryotoxic effects of polychlorinated biphenyls (PCBs) and other halogenated and nonhalogenated aromatic hydrocarbons that act through an aryl hydrocarbon receptor (AHR)-dependent signaling pathway. The resistance is accompanied by reduced sensitivity to induction of cytochrome P450 1A (CYP1A), a widely used biomarker of aromatic hydrocarbon exposure and effect, but whether the reduced sensitivity is specific to CYP1A or reflects a genome-wide reduction in responsiveness to all AHR-mediated changes in gene expression is unknown. We compared gene expression profiles and the response to 3,3',4,4',5-pentachlorobiphenyl (PCB-126) exposure in embryos (5 and 10 dpf) and larvae (15 dpf) from F. heteroclitus populations inhabiting the New Bedford Harbor, Massachusetts (NBH) Superfund site (PCB-resistant) and a reference site, Scorton Creek, Massachusetts (SC; PCB-sensitive). Analysis using a 7,000-gene cDNA array revealed striking differences in responsiveness to PCB-126 between the populations; the differences occur at all three stages examined. There was a sizeable set of PCB-responsive genes in the sensitive SC population, a much smaller set of PCB-responsive genes in NBH fish, and few similarities in PCB-responsive genes between the two populations. Most of the array results were confirmed, and additional PCB-regulated genes identified, by RNA-Seq (deep pyrosequencing). The results suggest that NBH fish possess a gene regulatory defect that is not specific to one target gene such as CYP1A but rather lies in a regulatory pathway that controls the transcriptional response of multiple genes to PCB exposure. The results are consistent with genome-wide disruption of AHR-dependent signaling in NBH fish.This work was supported in part by National Institutes of Health grants P42ES007381 (Superfund Basic Research Program at Boston University) and by a grant from the WHOI Ocean Life Institute

Interaction of Fish Aryl Hydrocarbon Receptor Paralogs (AHR1 and AHR2) with the Retinoblastoma Protein

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) and related compounds. In some mammalian cell lines, TCDD induces G1 cell cycle arrest, which depends on an interaction between the AHR and the retinoblastoma tumor suppressor (RB). Mammals possess one AHR, whereas fishes possess two or more AHR paralogs that differ in the domains important for AHR-RB interactions in mammals. To test the hypothesis that fish AHR paralogs differ in their ability to interact with RB, we cloned RB cDNA from Atlantic killifish, Fundulus heteroclitus, and studied the interactions of killifish RB protein with killifish AHR1 and AHR2. In coimmunoprecipitation experiments, in vitro-expressed killifish RB coprecipitated with both AHR1 and AHR2. Consistent with these results, both killifish AHR1 and AHR2 interacted with RB in mammalian two-hybrid assays. These results suggest that both fish AHR1 and AHR2 paralogs may have the potential to influence cell proliferation through interactions with RB

Multiple Orbitoides d’Orbigny lineages in the Maastrichtian? Data from the Central Sakarya Basin (Turkey) and Arabian Platform successions (Southeastern Turkey and Oman)

The standard reconstruction of species of Orbitoides d'Orbigny into a single lineage during the late Santonian to the end of the Maastrichtian is based upon morphometric data from Western Europe. An irreversible increase in the size of the embryonic apparatus, and the formation of a greater number of epi-embryonic chamberlets (EPC) with time, is regarded as the main evolutionary trends used in species discrimination. However, data from Maastrichtian Orbitoides assemblages from Central Turkey and the Arabian Platform margin (Southeastern Turkey and Oman) are not consistent with this record. The Maastrichtian Besni Formation of the Arabian Platform margin in Southeastern Turkey yields invariably biconvex specimens, with small, tri- to quadrilocular embryons and a small number of EPC, comparable to late Campanian Orbitoides medius (d'Archiac). The upper Maastrichtian Tarakli Formation from the Sakarya Basin of Central Turkey contains two distinct, yet closely associated forms of Orbitoides, easily differentiated by both external and internal features. Flat to biconcave specimens possess a small, tri- to quadrilocular embryonic apparatus of Orbitoides medius-type and a small number of EPC, whereas biconvex specimens possess a large, predominantly bilocular embryonic apparatus, and were assigned to Orbitoides ex. interc. gruenbachensis Papp-apiculatus Schlumberger based on morphometry. The flat to biconcave specimens belong to a long overlooked species Orbitoides pamiri Meric, originally described from the late Maastrichtian of the Tauride Mountains in SW Turkey. This species is herein interpreted to be an offshoot from the main Orbitoides lineage during the Maastrichtian, as are forms that we term Orbitoides 'medius', since they recall this species, yet are younger than normal occurrence with the accepted morphometrically defined lineage. The consistent correlation between the external and internal test features in O. pamiri implies that the shape of the test is not an ecophenotypic variation, but appears to be biologically controlled. We, therefore, postulate that more than one lineage of Orbitoides exists during the Maastrichtian, with a lineage that includes O. 'medius' and O. pamiri displaying retrograde evolutionary features