Modulation of the Redox Environment During Sensitive Developmental Periods Impairs Pancreatic Organogenesis in the Zebrafish (Danio rerio)

The developing pancreas has been identified as a potentially sensitive target for oxidative damage caused by environmental toxicant exposure in the developing zebrafish, Danio rerio. The objectives of this study were to 1) investigate the effects of exposure to a common pro-oxidizing toxicant, Mono(2-ethylhexyl) phthalate (MEHP), on pancreatic development, and 2) elucidate redox-sensitive temporal windows of pancreatic organogenesis in the zebrafish. We also investigated the involvement of Nrf2 (Nfe2l2), a transcription factor involved in the oxidative stress response, in toxicant-mediated pancreatic toxicity. In aim one, zebrafish embryos were exposed to 0 or 200 µg/L MEHP beginning at 3 hours post fertilization (hpf) through 168 hpf, and imaged live under a fluorescence microscope to visualize pancreas development from 48-168 hpf. Glutathione (GSH) and cysteine (CyS) redox couples were quantified by HPLC at 72 hpf, and gene expression was investigated at 96 hpf. This study utilized wild type (AB) Tg(ins:GFP), Tg(gcga:GFP) (endocrine islet), and Tg(ptf1a:GFP) (exocrine pancreas) zebrafish strains. We observed that MEHP exposure significantly reduced endocrine islet area and exocrine pancreas length at all timepoints (48, 72, 96, 168 hpf). No significant changes were observed in the redox potential of GSH or Cys, however MEHP exposure significantly altered expression of GSH-related genes (gsr, gstp1), as well as pancreas-specific genes (insa, sst2, ptf1a). These data indicate that the developing pancreas is a sensitive target tissue of embryonic exposure to MEHP. In aim two, we exposed transgenic Tg(ins:GFP) and Tg(gcga:GFP) zebrafish embryos to water, dimethyl sulfoxide (DMSO), N-acetyl cysteine (NAC), sulforophane (SFN), tert-butylhydroperoxide (tBOOH), or tert-butylhydroquinone (tBHQ) at 24, 48, or 72 hours post fertilization (hpf), and assessed endocrine islet morphology at 96 hpf. We found both chemical-, stage-, and cell-type specific effects of redox modulation on the endocrine pancreas. Pro-oxidant exposures resulted in decreased ß-cell cluster area and an increased frequency of islet variants, while antioxidant exposures significantly increased ß-cell cluster area. These effects were most significant at the 48 hpf exposure timepoint. a-cell cluster area was only affected by prooxidant exposure at 48 hpf. These results indicate that ß-cells are uniquely sensitive to oxidative stress, specifically at 48 hpf