Molecular and developmental effects of exposure to pyrene in the early life-stages of Cyprinodon variegatus

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Polycyclic aromatic hydrocarbons (PAHs) have been connected to developmental toxicity in the early life-stages of many species by their ability to bind to the aryl hydrocarbon receptor (AHR), which dimerizes with ARNT (AHR nuclear translocator) to induce transcription of genes such as CYP1A1. ARNT also dimerizes with HIF (hypoxia-inducible factor α) to induce transcription of genes such as VEGF (vascular endothelial growth factor), suggesting that PAHs may interfere with transcription of VEGF by competing for ARNT. Herein, we address the molecular and developmental effects of exposures to the weak AHR agonist pyrene on the early life-stages of the sheepshead minnow Cyprinodon variegatus. Embryos were exposed under flow-through conditions to 0, 20, 60, or 150 ppb pyrene up to 432 hours post-fertilization (hpf). RNA was extracted at 5 time points (12, 24, 48, 96, and 432 hpf) and changes in CYP1A1 and VEGF expression were assessed by real-time RT-PCR. Since few genes have been documented for the sheepshead minnow, we first cloned and sequenced CYP1A1, VEGF and internal standard 18S rRNA. Pyrene significantly induced the AHR-regulated gene, CYP1A1, in a time- and dose-dependent manner, while pyrene failed to alter the HIF-regulated gene, VEGF. However, VEGF was found to change during various stages of normal development in this study. Although a normal hatch time (5 dpf) was observed for all treatments, pyrene-treated embryos showed dose-dependent abnormalities such as severe dorsal body curvature, mild pericardial and yolk-sac edema, and increased mortality. Taken together, these data indicate that embryonic exposure of sheepshead minnows to pyrene disrupts normal development and alters expression of an AHR/ARNT-regulated gene. In addition, embryonic exposure to pyrene failed to provide evidence of possible AHR-HIF pathway cross-talk since developmental expression of VEGF was unaltered.