Heart development in two populations of Atlantic killifish (Fundulus heteroclitus) following exposure to a polycyclic aromatic hydrocarbon mixture

Abstract

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.

Keywords: Fundulus; Heart development; Histology; Morphometry; PAHs.

Fig. 1

Percent of individuals with severity scores 0–2 in each population (King’s Creek, KC; Atlantic Wood, AW) for each ERSE concentration 0% (control) to 5%. Severity scores of 0 are in white, 1 are grey, and 2 are black. Width of bars reflects the number of individuals in each group, with wider bars representing more individuals.

Fig. 2

Ventricular wall thickness for each population (King’s Creek, KC; Atlantic Wood, AW) for each ERSE concentration 0% (control) through 5%. KC 5% has significantly thinner walls than all other groups (*p<0.05) except KC 3%. KC is the dashed line and AW the solid line lines represent means ± SEM.

Fig. 3

Pericardial differences for each population (King’s Creek, KC; Atlantic Wood, AW) for each ERSE concentration 0% (control) through 5%. A) Heart chamber area (μm²) divided by pericardial (PC) area (μm²). Values closer to 1 represent normal where visceral pericardium is tight to the heart, and values approaching 0 represent increasingly severe pericardial edema. B) Amount of eosinophilic material in pericardial cavity per unit area calculated by dividing area of stained material (μm²; PC Contents) by PC area (μm²). Values closer to 1 represent pericardium filled with stained material, and as values approach 0, they represent less material per unit of pericardial cavity area. Different letters represent significant differences (p<0.05). KC is dashed line and AW the solid lines; lines represent means ± SEM.

Fig. 4

Transverse sections of representative morphologies of the F. heteroclitus embryo heart (144 hpf). A-C show whole embryos and D-F corresponding magnified images of hearts. A,D) Normal heart from Atlantic wood embryo exposed to 2% ERSE. Visceral pericardium is tight to the heart chambers, with a small amount of proteinaceous material between. B,E) Embryo from AW exposed to 5% ERSE with pericardial edema extending laterally and coming around the head. Edema moderately deformed heart (e.g., some elongation and thinning of chamber walls). Proteinaceous material is thin and wispy within pericardial cavity. C,F) KC embryo exposed to 5% ERSE shows severe pericardial edema evident in extended margins of visceral pericardium, which appears wavy because of fluid loss during processing. Little to no yolk visible as edema has pushed it caudally and proteinaceous material is thin and wispy within pericardial cavity. Tube heart visible as elongated ventricle (elongated atrium in another section, not pictured). Ventricular walls are thin and basophilic with no visible trabeculae. Arrowheads: Atrium – black, ventricle – white, parietal pericardium – yellow, sinus venosus – green, proteinaceous material in pericardial cavity – red; y – yolk, c – chorion, L – lipid droplet, PE – pericardial edema. A-C scale bars are 10μm and D-E are 5μm.

Fig. 5

Alterations in heart chamber walls. A-B) Control atrium and ventricle from AW 0% embryo. A) Atrial walls are 1–2 cell layers thick and smooth on the luminal and visceral surfaces. While somewhat flattened, atrial myocytes have rounded, centrally located nuclei. B) Ventricular wall are 3–5 cells layers thick and are trabeculated. Ventricular myocytes are stratified cuboidal with round, centrally located nuclei. C) Altered ventricular myocytes from embryo in AW 5% showing cytosolic expansion appearing as perinuclear clearing. Affected cells are throughout the ventricular wall. D) High magnification of tube heart from embryo in Fig. 4C–F (KC 5%) showing ventricular myocytes to be small and basophilic. Ventricle walls are very thin, only 1–2 cell layer thick, and without trabeculae. y – yolk, c – chorion, L – lipid droplet, PE – pericardial edema. Arrowheads: atrium – black, ventricle – white, parietal pericardium – yellow, visceral pericardium – blue, cytosolic expansion of ventricular myocytes –green, trabeculae – orange arrowhead. Scale bars are 20μm.