Deviant development of pancreatic beta cells from embryonic exposure to PCB-126 in zebrafish

Abstract

Exposures to co-planar PCBs and dioxins have been associated with diabetes in epidemiologic studies. Individuals may be predisposed to diseases such as diabetes as a result of exposure to environmental contaminants during early life, resulting in dysmorphic pancreatic islets or metabolically fragile β-cells. We tested the hypothesis that embryonic exposure to a model Ahr-ligand, PCB-126 would cause structural and/or functional alterations to the developing primary pancreatic islet in the zebrafish (Danio rerio). To assess β-cell development, transgenic zebrafish embryos (Tg(ins:GFP) and Tg(ins:mcherry) were exposed to nominal concentrations of 2 or 5 nM PCB-126 or DMSO from 24–48 h post fertilization (hpf), and imaged via time-lapse microscopy from 80–102 hpf. We identified defects including hypomorphic islets, altered islet migration, islet fragmentation, and formation of ectopic β-cells. As we recently showed the transcription factor Nrf2a is protective in PCB-126 embryotoxicity, we then assessed the transcriptional function of the islets in wildtype and nrf2a^fh318/fh318 mutant embryos. We measured gene expression of preproinsulin a, somatostatin2, pdx1, ghrelin, and glucagon. Expression of preproinsulin a increased with PCB treatment in wildtype embryos, while expression of all measured pancreas genes was altered by the nrf2a mutant genotype, suggesting misregulation of the glucose homeostasis axis in those embryos, independent of PCB treatment. This study shows that embryonic exposure to PCB-126 can result in deviant development of the pancreatic islet and suggests that Nrf2a plays a role in regulating glucose homeostasis during development.

Introduction

Embryonic exposures to contaminants can result in subtle structural and/or metabolic changes that may underlie susceptibility to diseases that emerge at later life stages, such as diabetes. In recent decades, rates of both Type 1 and Type 2 diabetes have increased; numerous experts agree that this rapid increase among genetically stable populations indicates that there must be a strong environmental component driving this increase (Gale, 2002, Pitkaniemi et al., 2004, Hectors et al., 2011). Part of the explanation for this may be due to early-life exposures to environmental contaminants that result in aberrant development of the endocrine pancreas, but given the expense and encumbrances of rodent developmental toxicity assays (e.g. multiple doses, difficulty isolating sensitive developmental windows, maternal and fetal sacrifice to examine effects on the pancreas), this has not been adequately tested in any model system. Thus, we know very little about how embryonic chemical exposures affect pancreas development.

Polychlorinated biphenyls (PCBs) are legacy contaminants formerly used mainly in electrical and hydraulic equipment, but also as plasticizers in rubber, paint, caulk, and plastics. Despite being banned in the late 1970s, they remain widespread and persistent environmental contaminants. There are 209 PCB congeners; arguably one of the most widely studied is 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), that has been shown to be associated with Type 2 diabetes in human epidemiology studies (Carpenter, 2006, Kuo et al., 2013). This coplanar PCB is a potent ligand for the Aryl hydrocarbon receptor (AHR²). The AHR is a member of the basic helix-loop-helix] Per-Arnt-Sim (bHLH/PAS) family of transcription factors, and is a constitutively expressed, cytosolic protein. The canonical activation of the Ahr pathway is initiated by ligand binding. Ahr then translocates to the nucleus, where it forms a heterodimer with the aryl hydrocarbon nuclear translocator (Arnt). This heterodimer binds to xenobiotic response elements (XREs) in promoters and initiates the transcription of a variety of genes.

There is strong evidence that an even more potent ligand for the AHR than PCB-126, dioxin (2,3,7,8-Tetrachlorodibenzo-p-dioxin; TCDD), is associated with an increased risk of diabetes (Bertazzi et al., 1998, Henriksen et al., 1997). Further, a Belgian population study found that elevated PCB serum levels and dioxin levels were associated with diabetes (Fierens et al., 2003), and pregnant women with diabetes were found to have a clear dose response relationship between serum PCB levels and diabetes (serum levels 2.5–> 5 ppb) (Longnecker et al., 2001). Despite these strong associations among adults, the biological basis for this association has not been established. Moreover, to the best of our knowledge, the impacts of embryonic PCB exposures on endocrine pancreas development have not yet been explored.

The zebrafish embryo model has been instrumental in understanding the developmental toxicity of PCBs, dioxins, and PAHs, which exert toxicity via the Ahr pathway (Billiard et al., 2006, Carney et al., 2006, King-Heiden et al., 2012). One of the most sensitive developmental defects observed with exposure to PCB-126 is the failure of the swim bladder to inflate by 96 hpf (Jonsson et al., 2012). The swim bladder is derived from the same pool of progenitor cells as those of the pancreatic β-cells (Kimmel and Meyer, 2010); these cells may be an important but overlooked target tissue of PCB and dioxin embryotoxicity.

The Ahr participates in crosstalk with the redox-sensitive transcription factor, nuclear factor erythroid related factor 2-like-2 (Nfe2l2 or Nrf2³) (Miao et al., 2005). Nrf2 is a “master regulator” of the adaptive response to oxidative stress (Ohtsuji et al., 2008). This basic-region leucine zipper transcription factor is constitutively and ubiquitously expressed, and repressed by Kelch-like ECH-associated protein 1 (Keap1) in the cytosol, targeting it for ubiquitination (Cullinan et al., 2004). Nrf2 can be activated by PERK signaling from the endoplasmic reticulum, phosphorylation, and electrophilic or ROS interactions (Bryan et al., 2013, Niture et al., 2014). Upon activation, Nrf2 accumulates in the nucleus where it dimerizes with small Maf proteins to upregulate transcription of cytoprotective genes through binding to antioxidant response elements (AREs) (Ma et al., 2004). AREs are frequently located in promoter regions of genes involved in the oxidative stress response and Phase II detoxification, but also in genes with other functions such as cell cycle regulation and lipid metabolism (Malhotra et al., 2010). There is also ample evidence for an emerging role of NRF2 in diabetes (Uruno et al., 2013, Uruno et al., 2015, Jimenez-Osorio et al., 2014, Bhakkiyalakshmi et al., 2015).

Early stages of development cannot be directly observed in mammalian, in utero systems. Zebrafish overcome this limitation because their embryos develop externally and are transparent, allowing in vivo observation of development in real time. The embryonic development of the pancreas is highly conserved between fish and mammals, as are the signaling pathways that guide development and gluco-regulatory functions (Ham and Stoffers, 2011, Tehrani and Lin, 2011). Zebrafish pancreas, and specifically β-cells, have been extensively characterized, and human relevance has been firmly established (diIorio et al., 2002, Tehrani and Lin, 2011, Wang et al., 2011). The primary goal of this exploratory study was to determine if exposure to PCB-126 alters the development and/or function of the pancreatic β cells. Also, as we recently identified a protective role for Nrf2a in PCB-126 embryotoxicity (Rousseau et al., 2015), we also investigated whether Nrf2a plays a role in regulating transcription of genes in the developing endocrine pancreas.