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Resistance to polycyclic aromatic hydrocarbon toxicity and associated bioenergetic consequences in a population of Fundulus heteroclitus.

ABSTRACT: Several locations in the Elizabeth River, VA, USA are highly contaminated with polycyclic aromatic hydrocarbons (PAHs) due to the release of creosote mixtures from wood treatment facilities. Interestingly, some populations of Atlantic killifish (Fundulus heteroclitus) inhabiting the Elizabeth River (ER) are resistant to PAH-induced teratogenesis. However, evolutionary resistance to PAHs due to chronic PAH exposure is associated with reduced fitness and increased susceptibility to other environmental stressors in at least one PAH-resistant ER killifish population. More specifically, wild-caught and first generation PAH-resistant juvenile killifish have altered metabolic demands when compared to non-resistant fish. Herein, we investigated this association further by examining a previously under-studied population captured from the creosote-contaminated site Republic Creosoting (Rep). We assessed PAH toxicity and effects on energy metabolism in Rep killifish in comparison with killifish from the reference site Kings Creek (KC). Following exposures to simple and complex PAH mixtures, Rep killifish exhibited several phenotypes associated with PAH resistance including decreased incidences of developmental cardiovascular deformities and recalcitrant cytochrome P450 1A (CYP1A) activity. We evaluated bioenergetics in killifish embryos throughout development and found elevated basal oxygen consumption rates in Rep embryos relative to KC embryos. Furthermore, juvenile F1 Rep fish had significantly lower maximal metabolic rates and aerobic scopes than KC juveniles. These results suggest that populations of killifish that have adapted or evolved to withstand the toxicity associated with PAHs consequently have altered energetic metabolism or demands. Such consequences could result in an enhanced vulnerability to other environmental and anthropogenic stressors in PAH-resistant killifish.

SUBMITTER: Lindberg CD

PROVIDER: S-EPMC5398948 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Resistance to polycyclic aromatic hydrocarbon toxicity and associated bioenergetic consequences in a population of Fundulus heteroclitus.

Lindberg C D CD Jayasundara N N Kozal J S JS Leuthner T C TC Di Giulio R T RT

Ecotoxicology (London, England) 20170217 3

Several locations in the Elizabeth River, VA, USA are highly contaminated with polycyclic aromatic hydrocarbons (PAHs) due to the release of creosote mixtures from wood treatment facilities. Interestingly, some populations of Atlantic killifish (Fundulus heteroclitus) inhabiting the Elizabeth River (ER) are resistant to PAH-induced teratogenesis. However, evolutionary resistance to PAHs due to chronic PAH exposure is associated with reduced fitness and increased susceptibility to other environme ...[more]

PMID: 28213827

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Project description:Historic industrial pollution of the Elizabeth River, Virginia resulted in polycyclic aromatic hydrocarbon (PAH) contamination in sediments. Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood (AW) industrial site adapted to complex PAH mixture at this Superfund site. Their embryos have proved highly resistant to cardiac abnormalities indicative of PAH toxicity. In this study, embryos spawned from adults collected at AW and King's Creek (KC), a reference site, were exposed at 24 h post fertilization (hpf) to Elizabeth River Sediment Extract (ERSE), a complex PAH mixture, in a range of concentrations (0, 5.04, 50.45, 100.90, 151.35, or 252.25 µg/L total PAHs). Embryos were processed for histology at 144 hpf to enable evaluations of hearts at tissue and cellular levels. Morphometry and severity scoring were used to evaluate the extent of alterations. Unexposed embryos were similar in both populations. ERSE exposure resulted in multiple changes to hearts of KC embryos but not AW. Alterations were particularly evident in KC embryos exposed to concentrations above 1% ERSE (50.45 µg/L), which had thinner ventricular walls and larger pericardial edema. Individuals with moderate pericardial edema maintained arrangement and proximity of heart chambers, but changes were seen in ventricular myocytes. Severe pericardial edema was prevalent in exposed KC embryos and typically resulted in tube heart formation. Ventricles of tube hearts had very thin walls composed of small, basophilic cells and lacked trabeculae. Edematous pericardial fluid contained small amounts of proteinaceous material, as did controls, and was free of cells. This fluid was primarily unstained, suggesting water influx due to increased permeability. The use of histological approaches provided more specific detail for tissue and cellular effects in hearts of embryos exposed to PAHs and enabled understanding of potential links to later life effects of early life exposure.

Project description:Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Industries Superfund Site (Elizabeth River, Portsmouth, VA, USA) are resistant to the acute toxicity and cardiac teratogenesis caused by high levels of polycyclic aromatic hydrocarbons (PAHs) from creosote. The resistance is linked to down regulation of the aryl hydrocarbon receptor (AHR) pathway. We investigated the association between CYP1 activity, as a marker of potential AHR pathway suppression, and contaminant resistance in killifish subpopulations from sites throughout the estuary that varied significantly in PAH contamination level. Adult killifish and sediments were collected from seven sites across approximately 13.7 km in river length within the estuary and from a nearby reference site. Sediment PAH levels were determined using gas chromatography mass spectrometry. Embryos obtained via manual spawning were exposed to individual AHR agonists and PAH mixtures 24 h post fertilization (hpf); CYP1 activity was determined by in ovo ethoxyresorufin-o-deethylase (EROD) at 96 hpf, and cardiac deformity severity was scored at 144 hpf. The total PAH levels measured among the sites varied from approximately 200 to 125,000 ng/g dry sediment. Overall, the resistance to teratogenesis was strongest in the subpopulations from sites in or closest to the major PAH contamination sites, but even embryos from less-contaminated sites within the Elizabeth River demonstrated at least partial resistance to many challenges. Surprisingly, all of the subpopulations tested were highly resistant to PCB-126 (3,3',4,4',5-pentachlorobiphenyl). However, the degree of CYP1 activity response varied significantly among subpopulations and did not always correlate strongly with resistance to teratogenesis; some subpopulations resisted the cardiac teratogenesis caused by the challenges at doses that still elicited strong EROD induction. Our results suggest that there is variation in the adaptive phenotype exhibited by laboratory-spawned embryos from killifish subpopulations throughout the estuary. Furthermore, the results show that contaminants have affected killifish subpopulations throughout the estuary, even in sites with lower levels of PAHs.

Project description:There is increased emphasis on understanding cumulative risk from the combined effects of chemical and non-chemical stressors as it relates to public health. Recent animal studies have identified pulmonary inflammation as a possible modifier and risk factor for chemical toxicity in the lung after exposure to inhaled pollutants; however, little is known about specific interactions and potential mechanisms of action. In this study, primary human bronchial epithelial cells (HBEC) cultured in 3D at the air-liquid interface (ALI) are utilized as a physiologically relevant model to evaluate the effects of inflammation on toxicity of polycyclic aromatic hydrocarbons (PAHs), a class of contaminants generated from incomplete combustion of fossil fuels. Normal HBEC were differentiated in the presence of IL-13 for 14 days to induce a profibrotic phenotype similar to asthma. Fully differentiated normal and asthmatic phenotype HBEC were treated with benzo[a]pyrene (BAP; 1 – 40 ug/mL) or 1% DMSO/PBS vehicle at the ALI for 48 hrs. Cells were evaluated for cytotoxicity, barrier integrity, and transcriptional biomarkers of chemical metabolism and inflammation by quantitative PCR. Cells with the asthmatic phenotype treated with BAP show some significant (p<0.05) increase in cytotoxicity and significantly (p<0.05) decreased barrier integrity compared to normal cells. Asthmatic phenotype cells also showed increase sensitivity to BAP metabolism compared to cells with the normal phenotype. Additionally, RNA sequencing data showed that a large number of genes were uniquely significantly expressed in cells with the induced asthmatic phenotype exposed to BAP. Future studies will further explore mechanisms of toxicity from global transcriptomics and investigate the role of microRNA in mediating mechanisms of toxicity and inflammation. These data are the first to evaluate the role of combined environmental factors associated with inflammation from pre-existing disease and PAH exposure on pulmonary toxicity in a physiologically relevant human in vitro model.

Project description:Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluation of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuable non-traditional model, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for off-target effects in AHR paralogs. No mutations were observed in closely related AHR genes (AHR1a, AHR1b, AHR2a, AHRR) in the CRISPR-Cas9-injected embryos. Overall, our results demonstrate that targeted genome-editing methods are efficient in inducing mutations at specific loci in embryos of a non-traditional model species, without detectable off-target effects in paralogous genes.

Dataset Information

Resistance to polycyclic aromatic hydrocarbon toxicity and associated bioenergetic consequences in a population of Fundulus heteroclitus.

Publications

Resistance to polycyclic aromatic hydrocarbon toxicity and associated bioenergetic consequences in a population of Fundulus heteroclitus.

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