Abstract
Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals.
Highlights
Oxidative stress occurs when redox signaling and control are disrupted, either through generation of non-physiological levels of reactive oxygen species (ROS) or by alterations in the regulation of key thiol/disulfide couples [1]
Oxidative stress disrupts the interaction between NF-E2-related factor 2 (NRF2) and KEAP1, after which NRF2 enters the nucleus and forms a heterodimer with one of several small Maf proteins; NRF2-Maf dimers bind to anti-oxidant response elements (ARE) and activate transcription of genes such as glutathione S-transferases (GST), NAD(P)H-quinone oxidoreductase (NQO1), glutamate-cysteine ligase, heme oxygenase (HMOX), and superoxide dismutase (SOD) [18,23,24]
Expression and induction of oxidative stress response genes in zebrafish embryos To assess the ability of embryos to mount a response to oxidative stress, we performed an experiment in which separate groups of embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6-dpf were exposed for 6 hr to Dimethyl sulfoxide (DMSO), tBHQ (10 mM), or TCDD (2 nM) and gene expression was measured by quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) and microarray
Summary
Oxidative stress occurs when redox signaling and control are disrupted, either through generation of non-physiological levels of reactive oxygen species (ROS) or by alterations in the regulation of key thiol/disulfide couples [1]. The constitutive (basal) expression and inducibility of antioxidant defenses are known to affect the susceptibility of adult tissues and cells to effects of oxidative stress [17,18], and are likely to be important determinants of susceptibility at early life stages as well [19,20]. Oxidative stress disrupts the interaction between NRF2 and KEAP1, after which NRF2 enters the nucleus and forms a heterodimer with one of several small Maf proteins; NRF2-Maf dimers bind to anti-oxidant response elements (ARE) and activate transcription of genes such as glutathione S-transferases (GST), NAD(P)H-quinone oxidoreductase (NQO1), glutamate-cysteine ligase (catalytic subunit; GCLC), heme oxygenase (HMOX), and superoxide dismutase (SOD) [18,23,24]
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