Edward M. Mager

Deepwater Horizon crude oil impacts the developing hearts of large predatory pelagic fish

Significance The 2010 Deepwater Horizon (MC252) disaster in the northern Gulf of Mexico released more than 4 million barrels of crude oil. Oil rose from the ocean floor to the surface where many large pelagic fish spawn. Here we describe the impacts of field-collected oil samples on the rapidly developing embryos of warm-water predators, including bluefin and yellowfin tunas and an amberjack. For each species, environmentally relevant MC252 oil exposures caused serious defects in heart development. Moreover, abnormalities in cardiac function were highly consistent, indicating a broadly conserved developmental crude oil cardiotoxicity. Losses of early life stages were therefore likely for Gulf populations of tunas, amberjack, swordfish, billfish, and other large predators that spawned in oiled surface habitats. The Deepwater Horizon disaster released more than 636 million L of crude oil into the northern Gulf of Mexico. The spill oiled upper surface water spawning habitats for many commercially and ecologically important pelagic fish species. Consequently, the developing spawn (embryos and larvae) of tunas, swordfish, and other large predators were potentially exposed to crude oil-derived polycyclic aromatic hydrocarbons (PAHs). Fish embryos are generally very sensitive to PAH-induced cardiotoxicity, and adverse changes in heart physiology and morphology can cause both acute and delayed mortality. Cardiac function is particularly important for fast-swimming pelagic predators with high aerobic demand. Offspring for these species develop rapidly at relatively high temperatures, and their vulnerability to crude oil toxicity is unknown. We assessed the impacts of field-collected Deepwater Horizon (MC252) oil samples on embryos of three pelagic fish: bluefin tuna, yellowfin tuna, and an amberjack. We show that environmentally realistic exposures (1–15 µg/L total PAH) cause specific dose-dependent defects in cardiac function in all three species, with circulatory disruption culminating in pericardial edema and other secondary malformations. Each species displayed an irregular atrial arrhythmia following oil exposure, indicating a highly conserved response to oil toxicity. A considerable portion of Gulf water samples collected during the spill had PAH concentrations exceeding toxicity thresholds observed here, indicating the potential for losses of pelagic fish larvae. Vulnerability assessments in other ocean habitats, including the Arctic, should focus on the developing heart of resident fish species as an exceptionally sensitive and consistent indicator of crude oil impacts.

Acute embryonic or juvenile exposure to Deepwater Horizon crude oil impairs the swimming performance of mahi-mahi (Coryphaena hippurus).

The Deepwater Horizon incident likely resulted in exposure of commercially and ecologically important fish species to crude oil during the sensitive early life stages. We show that brief exposure of a water-accommodated fraction of oil from the spill to mahi-mahi as juveniles, or as embryos/larvae that were then raised for ∼25 days to juveniles, reduces their swimming performance. These physiological deficits, likely attributable to polycyclic aromatic hydrocarbons (PAHs), occurred at environmentally realistic exposure concentrations. Specifically, a 48 h exposure of 1.2 ± 0.6 μg L(-1) ΣPAHs (geometric mean ± SEM) to embryos/larvae that were then raised to juvenile stage or a 24 h exposure of 30 ± 7 μg L(-1) ΣPAHs (geometric mean ± SEM) directly to juveniles resulted in 37% and 22% decreases in critical swimming velocities (Ucrit), respectively. Oil-exposed larvae from the 48 h exposure showed a 4.5-fold increase in the incidence of pericardial and yolk sac edema relative to controls. However, this larval cardiotoxicity did not manifest in a reduced aerobic scope in the surviving juveniles. Instead, respirometric analyses point to a reduction in swimming efficiency as a potential alternative or contributing mechanism for the observed decreases in Ucrit.

High rates of HCO3- secretion and Cl- absorption against adverse gradients in the marine teleost intestine: the involvement of an electrogenic anion exchanger and H+-pump metabolon?

SUMMARY Anion exchange contributes significantly to intestinal Cl– absorption in marine teleost fish and is thus vital for successful osmoregulation. This anion exchange process leads to high luminal HCO3– concentrations (up to ∼100 mmol l–1) and high pH and results in the formation of CaCO3 precipitates in the intestinal lumen. Recent advances in our understanding of the transport processes involved in intestinal anion exchange in marine teleost fish include the demonstration of a role for the H+-pump (V-ATPase) in apical H+ extrusion and the presence of an electrogenic (nHCO3–/Cl–) exchange protein (SLC26a6). The H+-V-ATPase defends against cellular acidification, which might otherwise occur as a consequence of the high rates of base secretion. In addition, apical H+ extrusion probably maintains lower HCO3– concentrations in the unstirred layer at the apical surface than in the bulk luminal fluids and thus facilitates continued anion exchange. Furthermore, H+-V-ATPase activity hyperpolarizes the apical membrane potential that provides the driving force for apical electrogenic nHCO3–/Cl– exchange, which appears to occur against both Cl– and HCO3– electrochemical gradients. We propose that a similar coupling between apical H+ extrusion and nHCO3–/Cl– exchange accounts for Cl– uptake in freshwater fish and amphibians against very steep Cl– gradients.

The effects of weathering and chemical dispersion on Deepwater Horizon crude oil toxicity to mahi-mahi (Coryphaena hippurus) early life stages

To better understand the impact of the Deepwater Horizon (DWH) incident on commercially and ecologically important pelagic fish species, a mahi-mahi spawning program was developed to assess the effect of embryonic exposure to DWH crude oil with particular emphasis on the effects of weathering and dispersant on the magnitude of toxicity. Acute lethality (96 h LC50) ranged from 45.8 (28.4-63.1) μg l(-1) ΣPAH for wellhead (source) oil to 8.8 (7.4-10.3) μg l(-1) ΣPAH for samples collected from the surface slick, reinforcing previous work that weathered oil is more toxic on a ΣPAH basis. Differences in toxicity appear related to the amount of dissolved 3 ringed PAHs. The dispersant Corexit 9500 did not influence acute lethality of oil preparations. Embryonic oil exposure resulted in cardiotoxicity after 48 h, as evident from pericardial edema and reduced atrial contractility. Whereas pericardial edema appeared to correlate well with acute lethality at 96 h, atrial contractility did not. However, sub-lethal cardiotoxicity may impact long-term performance and survival. Dispersant did not affect the occurrence of pericardial edema; however, there was an apparent reduction in atrial contractility at 48 h of exposure. Pericardial edema at 48 h and lethality at 96 h were equally sensitive endpoints in mahi-mahi.

Basolateral NBCe1 plays a rate-limiting role in transepithelial intestinal HCO3- secretion, contributing to marine fish osmoregulation.

SUMMARY Although endogenous CO2 hydration and serosal HCO3– are both known to contribute to the high rates of intestinal HCO3– secretion important to marine fish osmoregulation, the basolateral step by which transepithelial HCO3– secretion is accomplished has received little attention. Isolated intestine HCO3– secretion rates, transepithelial potential (TEP) and conductance were found to be dependent on serosal HCO3– concentration and sensitive to serosal DIDS. Elevated mucosal Cl– concentration had the unexpected effect of reducing HCO3– secretion rates, but did not affect electrophysiology. These characteristics indicate basolateral limitation of intestinal HCO3– secretion in seawater gulf toadfish, Opsanus beta. The isolated intestine has a high affinity for serosal HCO3– in the physiological range (Km=10.2 mmol l–1), indicating a potential to efficiently fine-tune systemic acid–base balance. We have confirmed high levels of intestinal tract expression of a basolateral Na+/HCO3– cotransporter of the electrogenic NBCe1 isoform in toadfish (tfNBCe1), which shows elevated expression following salinity challenge, indicating its importance in marine fish osmoregulation. When expressed in Xenopus oocytes, isolated tfNBCe1 has transport characteristics similar to those in the isolated tissue, including a similar affinity for HCO3– (Km=8.5 mmol l–1). Reported affinity constants of NBC1 for Na+ are generally much lower than physiological Na+ concentrations, suggesting that cotransporter activity is more likely to be modulated by HCO3– rather than Na+ availability in vivo. These similar functional characteristics of isolated tfNBCe1 and the intact tissue suggest a role of this cotransporter in the high HCO3– secretion rates of the marine fish intestine.

Neurotrophins and Tonsillar Hypertrophy in Children With Obstructive Sleep Apnea

Enlarged adenotonsillar tissue (AT) is a major determinant of obstructive sleep apnea (OSA) severity in children; however, mechanisms of AT proliferation are poorly understood. We hypothesized that early exposure to respiratory syncytial virus (RSV) may modify AT proliferation through up-regulation of nerve growth factor (NGF)-neurokinin 1 (NK1) receptor dependent pathways. AT harvested from 34 children with OSA and 25 children with recurrent tonsillitis (RI) were examined for mRNA expression of multiple growth factors and their receptors. In addition, NK1 receptor expression and location, and substance P tissue concentrations were compared in AT from OSA and RI children. NGF mRNA and its high-affinity tyrosine kinase receptor (trkA) expression were selectively increased in OSA (p < 0.001). NK1 receptor mRNA and protein expression were also enhanced in OSA (p < 0.01), and substance P concentrations in OSA patients were higher than in RI (p < 0.0001). AT from OSA children exhibit distinct differences in the expression of NGF and trkA receptors, NK1 receptors, and substance P. The homology between these changes and those observed in the lower airways following RSV infection suggests that RSV may have induced neuro-immunomodulatory changes within AT, predisposing them to increased proliferation, and ultimately contribute to emergence of OSA.

Corresponding morphological and molecular indicators of crude oil toxicity to the developing hearts of mahi mahi.

Crude oils from distinct geological sources worldwide are toxic to developing fish hearts. When oil spills occur in fish spawning habitats, natural resource injury assessments often rely on conventional morphometric analyses of heart form and function. The extent to which visible indicators correspond to molecular markers for cardiovascular stress is unknown for pelagic predators from the Gulf of Mexico. Here we exposed mahi (Coryphaena hippurus) embryos to field-collected crude oil samples from the 2010 Deepwater Horizon disaster. We compared visible heart defects (edema, abnormal looping, reduced contractility) to changes in expression of cardiac-specific genes that are diagnostic of heart failure in humans or associated with loss-of-function zebrafish cardiac mutants. Mahi exposed to crude oil during embryogenesis displayed typical symptoms of cardiogenic syndrome as larvae. Contractility, looping, and circulatory defects were evident, but larval mahi did not exhibit downstream craniofacial and body axis abnormalities. A gradation of oil exposures yielded concentration-responsive changes in morphometric and molecular responses, with relative sensitivity being influenced by age. Our findings suggest that 1) morphometric analyses of cardiac function are more sensitive to proximal effects of crude oil-derived chemicals on the developing heart, and 2) molecular indicators reveal a longer-term adverse shift in cardiogenesis trajectory.

Toxicogenomics of water chemistry influence on chronic lead exposure to the fathead minnow (Pimephales promelas)

Establishment of water quality criteria (WQC), intended to protect aquatic life, continues to rely principally on water hardness (i.e. Ca(2+)) for lead (Pb) despite growing evidence that other chemical parameters also strongly influence toxicity. To more clearly define the water chemistry parameters mediating Pb toxicity, we evaluated the effects of hardness as CaSO(4) and dissolved organic carbon (DOC) as humic acid during chronic (150 days) exposures to the fathead minnow. Measured Pb concentrations ranged from 157+/-5 nM (33+/-1 microg/L) Pb in base water to 177+/-7 (37+/-1 microg/L) and 187+/-7 nM (39+/-1 microg/L) Pb in CaSO(4)- or HA-supplemented water, respectively. Fish were collected at 2, 4, 10, 30, 63, 90 and 150 days of exposure. Traditional toxicological endpoints were examined alongside gene transcription analyses to help clarify the underlying mechanisms of Pb toxicity and to identify candidate molecular markers that might ultimately serve as robust indicators of exposure and effect. Addition of CaSO(4) did not prevent whole body Pb accumulation whereas DOC afforded strong protection (about half the amount accumulated by fish in base water) suggesting that current, hardness-based WQC are likely inaccurate for predicting chronic Pb effects in aquatic systems. Custom-made microarrays were co-hybridized with base water samples+/-Pb up to the 30 days time point. Quantitative PCR was employed to verify gene transcription responses and to extend analysis to the CaSO(4) and HA treatments and the 150 days time point. Identification of four genes by microarray analysis revealed clear Pb-induced responses over time: glucose-6-phosphate dehydrogenase, glutathione-S-transferase, ferritin and beta-globin. Results obtained by qPCR were in strong agreement with microarray data by regression analysis (r(2)=0.82, slope=1.28). The associated pathways implicated herein for these genes provide further evidence supporting roles for anemia and neurological disorders in chronic Pb toxicity. Effects of water chemistry on Pb accumulation and gene transcription responses were in close parallel, though alterations in ionoregulatory and morphological endpoints were not observed. Whereas DOC was protective against Pb accumulation and mRNA expression changes, Ca(2+) was not. Additionally, several hypothesis-driven genes (ECaC, DMT-1, and ALA-D) were examined by qPCR but revealed either no change or small Pb-induced responses lacking any clear influence attributable to water chemistry. These findings should help pave the way toward development of a new chronic Pb BLM and a Pb-responsive gene transcript profile for fathead minnows, both of which would greatly aid future environmental monitoring and regulatory strategies for Pb.

Time- and Oil-Dependent Transcriptomic and Physiological Responses to Deepwater Horizon Oil in Mahi-Mahi (Coryphaena hippurus) Embryos and Larvae

The Deepwater Horizon (DWH) oil spill contaminated the spawning habitats for numerous commercially and ecologically important fishes. Exposure to the water accommodated fraction (WAF) of oil from the spill has been shown to cause cardiac toxicity during early developmental stages across fishes. To better understand the molecular events and explore new pathways responsible for toxicity, RNA sequencing was performed in conjunction with physiological and morphological assessments to analyze the time-course (24, 48, and 96 h post fertilization (hpf)) of transcriptional and developmental responses in embryos/larvae of mahi-mahi exposed to WAF of weathered (slick) and source DWH oils. Slick oil exposure induced more pronounced changes in gene expression over time than source oil exposure. Predominant transcriptomic responses included alteration of EIF2 signaling, steroid biosynthesis, ribosome biogenesis and activation of the cytochrome P450 pathway. At 96 hpf, slick oil exposure resulted in significant perturbations in eye development and peripheral nervous system, suggesting novel targets in addition to the heart may be involved in the developmental toxicity of DHW oil. Comparisons of changes of cardiac genes with phenotypic responses were consistent with reduced heart rate and increased pericardial edema in larvae exposed to slick oil but not source oil.

The serotonin subtype 1A receptor regulates cortisol secretion in the Gulf toadfish, Opsanus beta.

It is well established that serotonin (5-HT; 5-hydroxytryptamine) plays a role in mammalian regulation of the hypothalamic-pituitary-adrenal (HPA) axis via the 5-HT receptor subtype 1A (5-HT(1A)). To date, there has not been a comprehensive investigation of the molecular, pharmacological and physiological aspects of the 5-HT(1A) receptor and its role in the activation of the hypothalamic-pituitary-interrenal (HPI) axis in teleost fish. The 5-HT(1A) receptor of the Gulf toadfish (Opsanus beta) was cloned and sequenced, showing 67.5% amino acid similarity to the human homologue. The 5-HT(1A) receptor was distributed throughout the brain, with the whole brain containing significantly higher levels of 5-HT(1A) mRNA compared to all other tissues and the midbrain/diencephalon region containing significantly higher levels of transcript than any other brain region. Substantial levels of transcript were also found in the pituitary, while very low levels were in the kidney that contains the interrenal cells. Xenopus oocytes injected with toadfish 5-HT(1A) receptor cRNA displayed significantly higher binding of [(3)H]5-HT that was abolished by the mammalian 5-HT(1A) receptor agonist, 8-OH-DPAT, indicating a conserved binding site of the toadfish 5-HT(1A) receptor and a high specificity for the agonist. Supporting this, binding of [(3)H]5-HT was not affected by the mammalian 5-HT(1B) receptor agonist, 5-nonyloxytryptamine, the 5-HT(7) receptor antagonist, SB269970, or the 5-HT(2) receptor agonist, alpha-methylserotonin. Confirming these molecular and pharmacological findings, intravenous injection of 8-OH-DPAT stimulated the HPI axis to cause a 2-fold increase in circulating levels of cortisol. The present study of the 5-HT(1A) receptor in a single teleost species illustrates the high conservation of this 5-HT receptor amongst vertebrates.