Expression and function of ryanodine receptor related pathways in PCB tolerant Atlantic killifish (Fundulus heteroclitus) from New Bedford Harbor, MA, USA

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Aquatic Toxicology 159 (2015): 156-166, doi:10.1016/j.aquatox.2014.12.017.Atlantic killifish (Fundulus heteroclitus) thrive in New Bedford Harbor (NBH), MA, highly contaminated with polychlorinated biphenyls (PCBs). Resident killifish have evolved tolerance to dioxin-like (DL) PCBs, whose toxic effects through the aryl hydrocarbon receptor (AhR) are well studied. In NBH, non-dioxin like PCBs (NDL PCBs), which lack activity toward the AhR, vastly exceed levels of DL congeners yet how killifish counter NDL toxic effects has not been explored. In mammals and fish, NDL PCBs are potent activators of ryanodine receptors (RyR), Ca2+ release channels necessary for a vast array of physiological processes. In the current study we compared the expression and function of RyR related pathways in NBH killifish with killifish from the reference site at Scorton Creek (SC, MA). Relative to the SC fish, adults from NBH displayed increased levels of skeletal muscle RyR1 protein, and increased levels of FK506-binding protein 12 kDa (FKBP12), an accessory protein essential for NDL PCB-triggered changes in RyR channel function. In accordance with increased RyR1 levels, NBH killifish displayed increased maximal ligand binding, increased maximal response to Ca2+ activation and increased maximal response to activation by the NDL PCB congener PCB 95. Compared to SC, NBH embryos and larvae had increased levels of mtor and ryr2 transcripts at multiple stages of development, and generations, while levels of serca2 were decreased at 9 days post-fertilization in the F1 and F2 generations. These findings suggest that there are compensatory and heritable changes in RyR mediated Ca2+ signaling proteins or potential signaling partners in NBH killifish.Funding was provided through the NIEHS Superfund Research Program UC Davis (INP and EBF; P42-ES004699) and Boston University (JJS and JVG; P42-ES007381). Support was supplied via the UC Davis NHLBI Training Grant (T32-HL086350, EBF). Additional support came from NIEHS 1R01-ES014901, 1R01-ES017425, the UC Davis Center for Children’s Environmental Health (1P01-ES011269, U.S. Environmental Protection Agency Grant 8354320), and an unrestricted JB Johnson Foundation gift grant.2015-12-1

Triclosan Impairs Swimming Behavior and Alters Expression of Excitation-Contraction Coupling Proteins in Fathead Minnow (Pimephales promelas)

Triclosan (TCS), a high volume chemical widely used in consumer products, is a known aquatic contaminant found in fish inhabiting polluted watersheds. Mammalian studies have recently demonstrated that TCS disrupts signaling between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR), two proteins essential for excitation-contraction (EC) coupling in striated muscle. We investigated the swimming behavior and expression of EC coupling proteins in larval fathead minnows (Pimephales promelas) exposed to TCS for up to 7 days. Concentrations as low as 75 mu g L-1 significantly altered fish swimming activity after 1 day; which was consistent after 7 days of exposure. The mRNA transcription and protein levels of RyR and DHPR (subunit CaV1.1) isoforms changed in a dose and time dependent manner. Crude muscle homogenates from exposed larvae did not display any apparent changes in receptor affinity toward known radioligands. In nonexposed crude muscle homogenates, TCS decreased the binding of [H-3]PN20-110 to the DHPR and decreased the binding of [H-3]-ryanodine to the RyR, demonstrating a direct impact at the receptor level. These results support TCS's impact on muscle function in vertebrates further exemplifying the need to re-evaluate the risks this pollutant poses to aquatic environments

Transcriptomic profiling permits the identification of pollutant sources and effects in ambient water samples

Contaminant exposure is one possible contributor to population declines of endangered fish species in the Sacramento-San Joaquin Estuary, California, including the endangered delta smelt (Hypomesus transpacificus). Herein we investigated transcriptional responses in larval delta smelt resulting from exposure to water samples collected at the Department of Water Resources Field Station at Hood, a site of concern, situated upstream of known delta smelt habitat and spawning sites and downstream of the Sacramento Regional Wastewater Treatment Plant (SRWTP). Microarray assessments indicate impacts on energy metabolism, DNA repair mechanisms and RNA processing, the immune system, development and muscle function. Transcription responses of fish exposed to water samples from Hood were compared with exposures to 9% effluent samples from SRWTP, water from the Sacramento River at Garcia Bend (SRGB), upstream of the effluent discharge, and SRGB water spiked with 2 mg/L total ammonium (9% effluent equivalent). Results indicate that transcriptomic profiles from Hood are similar to 9% SRWTP effluent and ammonium spiked SRGB water, but significantly different from SRGB. SRGB samples however were also significantly different from laboratory controls, suggesting that SRWTP effluent is not solely responsible for the responses determined at Hood, that ammonium exposure likely enhances the effect of multiple-contaminant exposures, and that the observed mortality at Hood is due to the combination of both effluent discharge and contaminants arising from upstream of the tested sites. (C) 2013 Elsevier B.V. All rights reserved

Triclosan Impairs Swimming Behavior and Alters Expression of Excitation-Contraction Coupling Proteins in Fathead Minnow (<i>Pimephales promelas</i>)

Triclosan (TCS), a high volume chemical widely used in consumer products, is a known aquatic contaminant found in fish inhabiting polluted watersheds. Mammalian studies have recently demonstrated that TCS disrupts signaling between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR), two proteins essential for excitation-contraction (EC) coupling in striated muscle. We investigated the swimming behavior and expression of EC coupling proteins in larval fathead minnows (<i>Pimephales promelas</i>) exposed to TCS for up to 7 days. Concentrations as low as 75 μg L^–1 significantly altered fish swimming activity after 1 day; which was consistent after 7 days of exposure. The mRNA transcription and protein levels of RyR and DHPR (subunit CaV1.1) isoforms changed in a dose and time dependent manner. Crude muscle homogenates from exposed larvae did not display any apparent changes in receptor affinity toward known radioligands. In nonexposed crude muscle homogenates, TCS decreased the binding of [³H]­PN20-110 to the DHPR and decreased the binding of [³H]-ryanodine to the RyR, demonstrating a direct impact at the receptor level. These results support TCS’s impact on muscle function in vertebrates further exemplifying the need to re-evaluate the risks this pollutant poses to aquatic environments

Ryanodine receptor and FK506 binding protein 1 in the Atlantic killifish (Fundulus heteroclitus): A phylogenetic and population-based comparison

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 Aquatic Toxicology 192 (2017): 105-115, doi:10.1016/j.aquatox.2017.09.002.Non-dioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine receptors (RyR), microsomal Ca2+ channels of broad significance. Teleost fish may be important models for NDL PCB neurotoxicity, and we used sequencing databases to characterize teleost RyR and FK506 binding protein 12 or 12.6 kDa (genes FKBP1A; FKBP1B), which promote NDL PCB-triggered Ca2+ dysregulation. Particular focus was placed on describing genes in the Atlantic killifish (Fundulus heteroclitus) genome and searching available RNA-sequencing datasets for single nucleotide variants (SNV) between PCB tolerant killifish from New Bedford Harbor (NBH) versus sensitive killifish from Scorton Creek (SC), MA. Consistent with the teleost whole genome duplication (tWGD), killifish have six RyR genes, corresponding to a and b paralogs of mammalian RyR1, 2 and 3. The presence of six RyR genes was consistent in all teleosts investigated including zebrafish. Killifish have four FKBP1; one FKBP1b and three FKBP1a named FKBP1aa, FKBP1ab, likely from the tWGD and a single gene duplicate FKBP1a3 suggested to have arisen in Atherinomorphae. The RyR and FKBP1 genes displayed tissue and developmental stage-specific mRNA expression, and the previously uncharacterized RyR3, herein named RyR3b, and all FKBP1 genes were prominent in brain. We identified a SNV in RyR3b encoding missense mutation E1458D. In NBH killifish, 57% were heterozygous and 28% were homozygous for this SNV, whereas almost all SC killifish (94%) lacked the variant (n≥39 per population). The outlined sequence differences between mammalian and teleost RyR and FKBP1 together with outlined population differences in SNV frequency may contribute to our understanding of NDL PCB neurotoxicity.This research was supported by the KC Donnelly Research Externship made possible by the National Institute of Environmental Health Sciences’ Superfund Research Program (EBH) and the Superfund Research Programs at UC Davis (INP and EBH; P42ES004699) and Boston University (JJS, JVG, MEH, SIK; P42ES007381). Additional support was provided by the National Institute of Health (INP; R01 ES014901; and P01 AR052354) and by National Science Foundation collaborative research grants (MEH and SIK; DEB-1265282 and DEB-1120263). This research was also supported in part by an appointment (to BC) with the Postdoctoral Research Program at the U.S. Environmental Protection (US EPA) Office of Research and Development administered by the Oak Ridge Institute for Science and Education (ORISE) through Interagency Agreement No. DW92429801 between the U.S. Department of Energy and the US EPA