ToxSci Advance Access originally published online on February 20, 2009
Toxicological Sciences 2009 109(2):217-227; doi:10.1093/toxsci/kfp038
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Antioxidant Responses and NRF2 in Synergistic Developmental Toxicity of PAHs in Zebrafish
* Integrated Toxicology and Environmental Health Program, Nicholas School of the Environment, Duke University, Durham, NC 27708
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710
2 To whom correspondence should be addressed at Duke University, Research Drive, Levine Science Research Center, Room A346, Durham, NC 27708-0328. Fax: (919)-668-1799. E-mail: richd{at}duke.edu.
Received December 16, 2008; accepted February 4, 2009
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Abstract |
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Early piscine life stages are sensitive to polycyclic aromatic hydrocarbon (PAH) exposure, which can cause pericardial effusion and craniofacial malformations. We previously reported that certain combinations of PAHs cause synergistic developmental toxicity, as observed with coexposure to the aryl hydrocarbon receptor agonist β-naphthoflavone (BNF) and cytochrome P4501A inhibitor 
-naphthoflavone (ANF). Herein, we hypothesized that oxidative stress is a component of this toxicity. We examined induction of antioxidant genes in zebrafish embryos (Danio rerio) exposed to BNF or ANF individually, a BNF + ANF combination, and a prooxidant positive control, tert-butylhydroperoxide (tBOOH). We measured total glutathione (GSH) and attempted to modulate deformities using the GSH synthesis inhibitor L-buthionine (S,R)-sulfoximine (BSO) and increase GSH pools with N-acetyl cysteine (NAC). In addition, we used a morpholino to knockdown expression of the antioxidant response element transcription factor NRF2 to determine if this would alter gene expression or increase deformity severity. BNF + ANF coexposure significantly increased expressions of superoxide dismutase 1 and 2, glutathione peroxidase 1, pi class glutathione-s-transferase, and glutamate cysteine-ligase to a greater extent than tBOOH, BNF, or ANF alone. BSO pretreatment decreased some GSH levels, but did not worsen deformities, nor did NAC diminish toxicity. Knockdown of NRF2 increased mortality following tBOOH challenge, prevented significant upregulation of antioxidant genes following both tBOOH and BNF + ANF exposures, and exacerbated BNF + ANF–related deformities. Collectively, these findings demonstrate that antioxidant responses are a component of PAH synergistic developmental toxicity and that NRF2 is protective against prooxidant and PAH challenges during development.
Key Words: PAH; NRF2; redox; ROS; embryonic development; glutathione.
1 Present address: Biology Department, Woods Hole Oceanographic Institute, Woods Hole, MA 02543