, Volume 20, Issue 6, pp 1300–1314 | Cite as

The role of CYP1A inhibition in the embryotoxic interactions between hypoxia and polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures in zebrafish (Danio rerio)

  • Carrie R. Fleming
  • Richard T. Di Giulio


Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with elevated concentrations in waters that may also experience hypoxia. Previous research has shown interactions between hypoxia and some PAHs (fluoranthene, α-naphthoflavone) but no interaction with others (benzo[a]pyrene (BaP), β-naphthoflavone). Here we examine how hypoxia (7.4% oxygen, ~35% of normoxia) affects the embryotoxicity of PAHs that act through different mechanisms and the role that CYP1A inhibition may play in these interactions. About 500 μg/l BaP and 1–200 μg/l benzo[k]fluoranthene (BkF) interacted synergistically with hypoxia to induce pericardial edema in developing zebrafish (Danio rerio). Hypoxia protected from the embryotoxicity of pyrene (PY) and had no effect on the toxicity of polychlorinated biphenyl-126. Despite previous reports of other CYP1A inhibitors interacting with hypoxia, up to 2,000 μg/l dibenzothiophene, 2-aminoanthracene (AA), and carbazole (CB) all failed to induce embryotoxicity under normoxic or hypoxic conditions. The toxicity of PAH mixtures—including binary mixtures of BaP/AA and BaP/CB and two environmentally relevant, complex mixtures—were exacerbated severely by hypoxia to induce or worsen pericardial edema and cause mortality. The interactions between hypoxia and BkF and PY were closely mimicked by morpholino knockdown of CYP1A, indicating a potential role for metabolism of these compounds in their toxicity. Our results indicate that various PAHs may exhibit synergistic, antagonistic or additive toxicity with hypoxia. The enhanced toxicity of environmental mixtures of PAHs under hypoxia suggests that risk assessments that do not take into account potential interactions with hypoxia may underestimate the threat of PAHs to fish in contaminated sites.


Polycyclic aromatic hydrocarbons Hypoxia Zebrafish Multiple stressors CYP1A 



We would like to thank Kyle Erwin in the laboratory of Margaret Kirby for the generous gift of the PCB-126 and β-actin primers used in these experiments. We would also like to thank Bryan Clark and Lindsey Van Tiem for technical assistance in the collection and preparation of the Elizabeth River Sediment Extract and Cole Matson for statistical advice. Funding was provided by the National Institute of Environmental Health Sciences-supported Duke University Superfund Research Center (P42-ES-10356), the Integrated Toxicology and Environmental Health Program (T32-ES-007031) and an Environmental Protection Agency STAR grant to C. Fleming.

Supplementary material

10646_2011_686_MOESM1_ESM.tif (493 kb)
Supplemental Fig. 1: Sample images of pericardial edema. Bars represent the normalized 2D area of the pericardium of each accompanying image. Images are not all from the same exposure, but are presented as a visual reference for the degrees of pericardial edema caused by various exposures in our experiments (TIFF 493 kb)
10646_2011_686_MOESM2_ESM.tif (15.4 mb)
Supplemental Fig. 2: Induction of CYP1A mRNA by BkF or PY in zebrafish larvae. BkF and PY both induced CYP1A mRNA over control levels (main effect of BkF: p<0.0001; main effect of PY: p<0.0001). Data presented are means of fold change compared to normoxia DMSO values ± standard error. Treatments that do not share a letter are significantly different from one another in pairwise comparisons (p<0.05) (TIFF 15752 kb)
10646_2011_686_MOESM3_ESM.tif (1.3 mb)
Supplemental Fig. 3: The effects of control-mo injection on zebrafish larvae exposed to BkF or PY and hypoxia or normoxia. BkF (A, B, C) (A) BkF-induced EROD activity was decreased by hypoxia and the control-mo did not alter these effects (BkF*oxygen interaction: p<0.0001; morpholino main effect: p=0.8; BkF*oxygen*morpholino interaction: p=0.5). (B) Hypoxia interacted with BkF to induce pericardial edema at otherwise non-toxic concentrations and control-mo had no effect on this interaction (BkF*oxygen interaction: p<0.0001; morpholino main effect: p=0.9; BkF*oxygen*morpholino interaction: p=0.7). (C) Hypoxia interacted with BkF to induce mortality at otherwise non-lethal concentrations, and control morpholino had no effect on this interaction (BkF*oxygen interaction: p<0.0008; morpholino main effect: p=0.7, BkF*oxygen*morpholino interaction: p=0.9). PY (D) 1000 μg/L PY induced pericardial edema under normoxic conditions; concurrent treatment with hypoxia prevented this effect and control-mo had no effect (PY*oxygen interaction: p<0.002; morpholino main effect: p=0.8; PY*oxygen*morpholino interaction: p=0.9). Treatments that do not share a letter are significantly different from one another in pairwise comparisons (p<0.05). Error bars are ± standard error (TIFF 1314 kb)


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Integrated Toxicology and Environmental Health Program, Nicholas School for the Environment and Earth SciencesDuke UniversityDurhamUSA

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