Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fish development

Abstract

Polycyclic aromatic hydrocarbons (PAHs), derived largely from fossil fuels and their combustion, are pervasive contaminants in rivers, lakes, and nearshore marine habitats. Studies after the Exxon Valdez oil spill demonstrated that fish embryos exposed to low levels of PAHs in weathered crude oil develop a syndrome of edema and craniofacial and body axis defects. Although mechanisms leading to these defects are poorly understood, it is widely held that PAH toxicity is linked to aryl hydrocarbon receptor (AhR) binding and cytochrome P450 1A (CYP1A) induction. Using zebrafish embryos, we show that the weathered crude oil syndrome is distinct from the well-characterized AhR-dependent effects of dioxin toxicity. Blockade of AhR pathway components with antisense morpholino oligonucleotides demonstrated that the key developmental defects induced by weathered crude oil exposure are mediated by low-molecular-weight tricyclic PAHs through AhR-independent disruption of cardiovascular function and morphogenesis. These findings have multiple implications for the assessment of PAH impacts on coastal habitats.

Figure 1

AhR2 knockdown prevents CYP1A induction by phenanthrene and dibenzothiophene, but not cardiac dysfunction. (A–F) Lateral light microscopic views of live embryos at 48 hpf (anterior at left) are paired with corresponding ventral confocal images (anterior at top) of CYP1A (green) and myosin heavy chain (red) immunofluorescence. (A, B) Embryo exposed to solvent (DMSO). (C, D) AhR2-MIS–injected embryo exposed to 28 μM phenanthrene. (E, F) AhR2 morphant exposed to 28 μM phenanthrene. Black arrowheads and arrows (C, E ) indicate pericardial and yolk sac edema, respectively. CYP1A immunofluorescence induced by phenanthrene (D) in the cranial division of the internal carotid artery (CrDI) and optic artery (OA) was blocked by AhR2-MO injection (F). Solid white arrowheads indicate cross-reactive immunofluorescence in the jaw cartilage, and unfilled white arrowheads indicate the ventricular myocardium. (G, H) Higher magnification confocal images showing CYP1A (green) and myocardial myosin heavy chain (red) immunofluorescence at 48 hpf in embryos with cardiac dysfunction after exposure to 28 μM dibenzothiophene (lateral views with anterior at left). In an uninjected embryo (G), the proximal portion of the mandibular arch (AA1, arrow) is CYP1A⁺, whereas the ventricular endothelium is CYP1A⁻ (asterisk). Only cross-reactive signal is seen in the jaw cartilage (arrowhead) in an AhR2 morphant (H). Bars = 100 μm (A–F) and 50 μm (G, H).

Figure 2

AhR2 morphants are resistant to pyrene toxicity. Control (uninjected or AhR2-MIS injected) and AhR2 morphant embryos were exposed to 5 μM pyrene through 100 hpf. Lateral (A, B) and dorsal (C, D) views showing edema (arrows) in uninjected larvae. Higher magnification light micrographs of the trunk region of uninjected (E, G) and AhR2 morphant (F, H) larvae showing cell death (E, granular appearance) in the neural tube (nt, neural tube; nc, notochord) and a reduction of erythrocytes (G, arrows) in the ventral aorta (VA) and caudal vein (CV) of uninjected larvae. (I–L) CYP1A immunofluorescence in the trunk (I, J) and head regions (K, L) of pyrene-exposed larvae. In AhR2-MIS–injected larvae (I, K) the vasculature (DLAV, dorsal longitudinal anastomotic vessel; Se, intersegmental vessels; AA, branchial arches) and liver are CYP1A⁺, whereas only weak signal is seen in the liver of the AhR morphant (J, L). In (E–L,) anterior is to the left and dorsal at top. Bars = 200 μm (A–D) and 50 μm (E–L).

Figure 3

Chrysene induces CYP1A through both AhR1 and AhR2. All images show CYP1A immunofluorescence at 72 hpf (A, B, G, H) or 48 hpf (C–F) after exposure to 9 μM chrysene from 6 hpf. (A–F) Lateral epifluorescent images with anterior to the left in uninjected (A), AhR2 morphant (B), AhR1-MIS–injected (C), AhR1 morphant (D), AhR1/AhR2 double morphant (E), and CYP1A morphant (F ) embryos. Epidermal CYP1A is seen as punctate fluorescence on the surface of the embryos. Immunofluorescent signal in the otic capsule and jaw cartilage was often observed in unexposed embryos. This signal was resistant to CYP1A morpholino (F) and is therefore likely to represent a cross-reactive protein. (G, H) Confocal images of uninjected chrysene-exposed embryos. (G) Three-dimensional confocal projection (180 μm series of optical sections) of CYP1A immunofluorescence, ventral view with anterior at top. Arrows indicate CYP1A⁺ blood vessels; AA1, mandibular arch; CrDI, cranial division of the internal carotid artery; OA, optic artery; ORA, opercular artery. (H) Confocal optical section through the cardiac chambers (anterior at top) with CYP1A (green) and myosin heavy chain (red) immunofluorescence. The asterisk (*) indicates CYP1A⁺ endothelial cells lining the ventricle. Bars = 250 μm (A–F) and 50 μm (G, H).

Figure 4

Defects resulting from embryonic exposure to OGE. PAH levels are shown in Table 2 (results for column 1) and Supplemental Figure 2 [Supplemental Material available online (http://ehp.niehs.nih.gov/docs/2005/8230/supplement.pdf)]. (A, B) Gross appearance at 64 hpf of CGE-exposed (A) or OGE-exposed (B) larvae. (C, D) Cardiac morphology at 64 hpf in CGE-exposed (C) and OGE-exposed (D) larvae. (E, F) Cardiac chamber–specific immunofluorescence (red, ventricle; green, atrium) in CGE-exposed (E) and OGE-exposed (F) larvae at 39 hpf; dashed white lines indicate the angles measured to assess looping. (G, H) High-magnification views of the midbrain–hindbrain junction in CGE-exposed (G) and OGE-exposed (H) larvae. Arrows indicate red tinge from extracellular hemoglobin; arrowheads mark extravascular erythrocytes, and the floor of the brain ventricles is marked with unfilled arrowheads. (I–L) High-magnification views of pectoral (I, J) and caudal (K, L) fins in CGE-exposed (I, K) and OGE-exposed (J, L) larvae. The finfolds of OGE-exposed larvae have irregular margins and blisters (arrows). Bars = 500 μm (A, B) and 50 μm (C–L).

Figure 5

AhR1/AhR2 or CYP1A morphants are more sensitive to weathered crude oil toxicity. (A–E) Confocal immunofluorescence images of CYP1A (green) and myosin heavy chain marking myocardium (red). CGE-exposed embryos showed no CYP1A immunofluorescence at 39 hpf (A), whereas OGE-exposed embryos showed intense immunofluorescence in the epidermis (B; 180 μm series of optical sections, vasculature of the head (C, optical section; PICA, primitive internal carotid artery; AA1, mandibular arch), and endocardium (asterisks in D, E) in both the atrium (D) and ventricle (E). (F) Cardiac function (good: strong forward flow; weak: forward flow with atrial regurgitation; or none: erythrocytes pooled in the yolk sac) at 39–40 hpf in CGE- and OGE-exposed embryos that were uninjected, control morpholino injected, AhR1/AhR2 double morphant, or CYP1A morphant [Supplemental Movie 3; Supplemental Material available online (http://ehp.niehs.nih.gov/docs/2005/8230/supplement.pdf)] Bars represent the percentage of embryos in the three classifications (numbers within each bar indicate n). The data represent sets of embryos that were exposed sequentially in the effluent of a single pair of columns (control and oiled) run over 8 days. PAH levels are shown in Table 2 (results for column 6) and Supplemental Figure 3 [Supplemental Material available online (http://ehp.niehs.nih.gov/docs/2005/8230/supplement.pdf)]. Because weathering increases with duration of column flow, AhR1/AhR2 double morphants (AhR1+2-MO) and corresponding controls (bars under OGE days 0–2) were exposed to less weathered oil than were the CYP1A morphants and controls (bars under OGE days 5–7; see also Table 2). The increased severity of cardiac dysfunction in control embryos exposed during days 5–7 is consistent with the higher tricyclic component. (G–K) CYP1A immunofluorescence (epifluorescent images) of representative OGE-exposed embryos from experiments shown in (F) and similar experiments with AhR2 single morphants, fixed at 42 hpf. (G) Uninjected control with intense epidermal CYP1A signal. (H) AhR2-MIS–injected control. (I) AhR2 morphant with loss of epidermal CYP1A signal and robust vascular immunofluorescence. (J) AhR1/AhR2 double morphant with markedly reduced overall immunofluorescence. (K) CYP1A morphant with background staining only. Bars= 100 μm (A, B), 50 μm (C–E), and 500 μm (G–K).