Emerging evidence suggests that redox-active chemicals perturb pancreatic islet development. To better understand potential mechanisms for this, we used zebrafish (Danio rerio) embryos to investigate roles of glutathione (GSH; predominant cellular redox buffer) and the transcription factor Nrf2a (Nfe2l2a; zebrafish Nrf2 co-ortholog) in islet morphogenesis. We delineated critical windows of susceptibility to redox disruption of β-cell morphogenesis, interrogating embryos at 24, 48 and 72 h post fertilization (hpf) and visualized Nrf2a expression in the pancreas using whole-mount immunohistochemistry at 96 hpf. Chemical GSH modulation at 48 hpf induced significant islet morphology changes at 96 hpf. Pro-oxidant exposures to tert-butylhydroperoxide (77.6 μM; 10-min at 48 hpf) or tert-butylhydroquinone (1 μM; 48-56 hpf) decreased β-cell cluster area at 96 hpf. Conversely, exposures to antioxidant N-acetylcysteine (bolsters GSH pools; 100 μM; 48-72 hpf) or sulforaphane (activates Nrf2a; 20 μM; 48-72 hpf) significantly increased islet areas. Nrf2a was also stabilized in β-cells: 10-min exposures to 77.6 μM tert-butylhydroperoxide significantly increased Nrf2a protein compared to control islet cells that largely lack stabilized Nrf2a; 10-min exposures to higher (776 μM) tert-butylhydroperoxide concentration stabilized Nrf2a throughout the pancreas. Using biotinylated-GSH to visualize in situ protein glutathionylation, islet cells displayed high protein glutathionylation, indicating oxidized GSH pools. The 10-min high (776 μM) tert-butylhydroperoxide exposure (induced Nrf2a globally) decreased global protein glutathionylation at 96 hpf. Mutant fish expressing inactive Nrf2a were protected against tert-butylhydroperoxide-induced abnormal islet morphology. Our data indicate that disrupted redox homeostasis and Nrf2a stabilization during pancreatic β-cell development impact morphogenesis, with implications for disease states at later life stages. Our work identifies a potential molecular target (Nrf2) that mediates abnormal β-cell morphology in response to redox disruptions. Moreover, our findings imply that developmental exposure to exogenous stressors at distinct windows of susceptibility could diminish the reserve redox capacity of β-cells, rendering them vulnerable to later-life stresses and disease.

新的证据表明，氧化还原活性化学物质会扰乱胰岛的发育。为了更好地了解其潜在机制，我们使用斑马鱼（Danio rerio）胚胎来研究谷胱甘肽（GSH；主要细胞氧化还原缓冲液）和转录因子 Nrf2a（Nfe2l2a；斑马鱼 Nrf2 共直系同源物）在胰岛形态发生中的作用。我们描绘了对 β 细胞形态发生氧化还原破坏敏感的关键窗口，在受精后 24、48 和 72 小时 (hpf) 询问胚胎，并在 96 hpf 时使用整体免疫组织化学可视化胰腺中的 Nrf2a 表达。48 hpf 时的化学 GSH 调节在 96 hpf 时诱导显着的胰岛形态变化。促氧化剂暴露于叔丁基氢过氧化物（77.6 μM；48 hpf 10 分钟）或叔丁基氢醌（1 μM；48-56 hpf）可减少 96 hpf 时的 β 细胞簇面积。相反，暴露于抗氧化剂 N-乙酰半胱氨酸（增强 GSH 池；100 μM；48-72 hpf）或萝卜硫素（激活 Nrf2a；20 μM；48-72 hpf）会显着增加胰岛面积。Nrf2a 在 β 细胞中也得到稳定：与很大程度上缺乏稳定 Nrf2a 的对照胰岛细胞相比，暴露于 77.6 μM叔丁基过氧化氢10 分钟， Nrf2a 蛋白显着增加；暴露于较高浓度 (776 μM)叔丁基过氧化氢10 分钟可稳定整个胰腺的 Nrf2a。使用生物素化-GSH 观察原位蛋白质谷胱甘肽化，胰岛细胞显示出高蛋白质谷胱甘肽化，表明氧化的 GSH 池。10 分钟高（776 μM）叔丁基氢过氧化物暴露（全局诱导 Nrf2a）降低了 96 hpf 时的全局蛋白质谷胱甘肽化。表达失活 Nrf2a 的突变鱼受到保护，免受叔丁基过氧化氢诱导的异常胰岛形态的影响。我们的数据表明，胰腺 β 细胞发育过程中氧化还原稳态和 Nrf2a 稳定性的破坏会影响形态发生，并对生命后期的疾病状态产生影响。我们的工作确定了一个潜在的分子靶标 (Nrf2)，该靶标可介导异常的 β 细胞形态以响应氧化还原破坏。此外，我们的研究结果表明，在不同的易感期暴露于外源性压力源可能会降低β细胞的储备氧化还原能力，使它们在以后的生活中容易受到压力和疾病的影响。