Interactions between hypoxia and sewage-derived contaminants on gene expression in fish embryos

Fish embryos were used to evaluate the interaction among common environmental and chemical stressors found in urban coastal environments, namely hypoxia, aryl hydrocarbon receptor (AhR) agonists, and estrogenic compounds. At the molecular level, the systems responding to these stressors share common response factors, and evidence exists for cross-talk between them. Biomarkers of exposure to these stressors, cytochrome P4501a (Cyp1a), estrogen receptor alpha (ERα), brain cytochrome P450 aromatase (Cyp19a2 or AromB), and hypoxia inducible factor 1 alpha (Hif-1α) mRNA expression were examined using qRT-PCR simultaneously in embryos of two well studied species, the Atlantic killifish, Fundulus heteroclitus, and the zebrafish Danio rerio. Embryos of both species were exposed to the model Cyp1a inducer β-naphthoflavone (BNF) or 17-β estradiol (E2) under either normoxic or hypoxic (5% oxygen atmosphere) conditions and harvested prior to hatch at 9 days post fertilization (dpf) for the killifish, and 48h post fertilization (hpf) for the zebrafish. BNF significantly induced Cyp1a expression in embryos of both species with killifish embryos being more responsive (700-fold>control) than zebrafish embryos (7-100-fold>control). AromB was also significantly influenced by treatment, but to a lesser extent, with mean expression levels increased by less than two-fold over control values in response to E2, and in one case upregulated by BNF. ERα and Hif-1α were constitutively expressed in embryos of both species, but expression was unaffected by exposure to either BNF or E2. Hypoxic conditions downregulated AromB expression strongly in killifish but not in zebrafish embryos. The impact of hypoxia on expression of other genes in either species was inconsistent, although an interactive effect between hypoxia and BNF on several of the genes evaluated was observed. These data are the first to examine expression patterns of these important environmental response genes together in embryos of two important model fish species. The results support the use of Cyp1a expression as a biomarker of AhR agonists in fish embryos, and indicate that AromB may be more responsive than ERα to estrogenic chemicals at this stage in development. Hif-1α expression was not found to be a good biomarker of hypoxic exposure in either killifish or zebrafish embryos. The interaction observed between BNF and co-exposure to hypoxia warrants further investigation. Finally killifish embryos are generally more sensitive than zebrafish embryos at this stage of development supporting their use in environmental assessments.