Antioxidant responses and NRF2 in synergistic developmental toxicity of PAHs in zebrafish

Author Posting. © The Authors, 2009. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Toxicological Sciences 109 (2009): 217-227, doi:10.1093/toxsci/kfp038.Early piscine life-stages are sensitive to polycyclic aromatic hydrocarbon (PAH) exposure, which can cause pericardial effusion and craniofacial malformations. We previously reported that certain combinations of PAHs cause synergistic developmental toxicity, as observed with co-exposure to the aryl hydrocarbon receptor (AHR) agonist β-naphthoflavone (BNF) and cytochrome P4501A inhibitor α-naphthoflavone (ANF). Herein, we hypothesized that oxidative stress is a component of this toxicity. We examined induction of antioxidant genes in zebrafish embryos (Danio rerio) exposed to BNF or ANF individually, a BNF+ANF combination, and a pro-oxidant positive control, tert-butylhydroperoxide (tBOOH). We measured total glutathione, and attempted to modulate deformities using the glutathione synthesis inhibitor buthionine sulfoxamine (BSO) and increase glutathione pools with N-acetyl cysteine (NAC). In addition, we used a morpholino to knockdown expression of the antioxidant response element transcription factor NRF2 to determine if this would alter gene expression or increase deformity severity. BNF+ANF co-exposure significantly increased expressions of superoxide dismutase1 and2, glutathione peroxidase 1, pi class glutathione-s-transferase, and glutamate cysteine-ligase to a greater extent than tBOOH, BNF, or ANF alone. BSO pretreatment decreased some glutathione levels, but did not worsen deformities, nor did NAC diminish toxicity. Knockdown of NRF2 increased mortality following tBOOH challenge, prevented significant upregulation of antioxidant genes following both tBOOH and BNF+ANF exposures, and exacerbated BNF+ANF‐related deformities. Collectively, these findings demonstrate that antioxidant responses are a component of PAH synergistic developmental toxicity, and that NRF2 is protective against prooxidant and PAH challenges during development.This work was supported by the National Institute for Environmental Health Sciencessupported Duke University Superfund Basic Research Program (P42 ES10356), National Institute for Environmental Health Sciences‐supported Duke University Integrated Toxicology & Environmental Health Program (TS ES07031), United States Environmental Protection Agency STAR fellowship (to A.T.‐L.), Duke University RJR‐Leon Golberg Memorial Postdoctoral Training Program in Toxicology (to A.T.‐L.), and the Postdoctoral Scholar Program at the Woods Hole Oceanographic Institution, with funding provided by the J. Seward Johnson Fund and The Walter A. and Hope Noyes Smith Chair (to A.T‐L)

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing \u3ci\u3eFundulus heteroclitus\u3c/i\u3e embryos

Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC50 and LD50 of TCDD for Fundulus embryos were ~19.7 ± 9.5 pg TCDD/µl (water bath) and 0.25 ± 0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing \u3ci\u3eFundulus heteroclitus\u3c/i\u3e embryos

Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC50 and LD50 of TCDD for Fundulus embryos were ~19.7 ± 9.5 pg TCDD/µl (water bath) and 0.25 ± 0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction

Development of the morpholino gene knockdown technique in Fundulus heteroclitus : a tool for studying molecular mechanisms in an established environmental model

Author Posting. © Elsevier B.V., 2008. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Aquatic Toxicology 87 (2008): 289-295, doi:10.1016/j.aquatox.2008.02.010.A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in β-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.This work was funded in part by the National Institute of Environmental Health Sciences through the Duke Superfund Basic Research Center (P42ES010356), the Boston University Superfund Basic Research Program (P42ES007381), and the Duke Integrated Toxicology and Environmental Health Program (ES-T32-0007031). Additional support was provided by a U.S. Environmental Protection Agency STAR fellowship awarded to C.R.F

Synergistic Embryotoxicity of Polycyclic Aromatic Hydrocarbon Aryl Hydrocarbon Receptor Agonists with Cytochrome P4501A Inhibitors in Fundulus heteroclitus

Widespread contamination of aquatic systems with polycyclic aromatic hydrocarbons (PAHs) has led to concern about effects of PAHs on aquatic life. Some PAHs have been shown to cause deformities in early life stages of fish that resemble those elicited by planar halogenated aromatic hydrocarbons (pHAHs) that are agonists for the aryl hydrocarbon receptor (AHR). Previous studies have suggested that activity of cytochrome P4501A, a member of the AHR gene battery, is important to the toxicity of pHAHs, and inhibition of CYP1A can reduce the early-life-stage toxicity of pHAHs. In light of the effects of CYP1A inhibition on pHAH-derived toxicity, we explored the impact of both model and environmentally relevant CYP1A inhibitors on PAH-derived embryotoxicity. We exposed Fundulus heteroclitus embryos to two PAH-type AHR agonists, β-naphthoflavone and benzo(a)pyrene, and one pHAH-type AHR agonist, 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), alone and in combination with several CYP1A inhibitors. In agreement with previous studies, coexposure of embryos to PCB-126 with the AHR antagonist and CYP1A inhibitor α-naphthoflavone decreased frequency and severity of deformities compared with embryos exposed to PCB-126 alone. In contrast, embryos coexposed to the PAHs with each of the CYP1A inhibitors tested were deformed with increased severity and frequency compared with embryos dosed with PAH alone. The mechanism by which inhibition of CYP1A increased embryotoxicity of the PAHs tested is not understood, but these results may be helpful in elucidating mechanisms by which PAHs are embryotoxic. Additionally, these results call into question additive models of PAH embryotoxicity for environmental PAH mixtures that contain both AHR agonists and CYP1A inhibitors

Effects of benzo[a]pyrene on mitochondrial and nuclear DNA damage in Atlantic killifish (Fundulus heteroclitus) from a creosote-contaminated and reference site

Benzo[a]pyrene (BaP) is a known genotoxicant that affects both mitochondrial and nuclear DNA (mtDNA, nDNA). Here, we examined mtDNA and nDNA damage in the Atlantic killifish (Fundulus heteroclitus) from a highly contaminated Superfund site (Elizabeth River, VA, USA) and from a reference site (King’s Creek, VA, USA) that were dosed with 10 mg/kg BaP. Using the long amplicon quantitative PCR technique, we observed similar increases in mitochondrial and nuclear DNA damage in King’s Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish compared to King’s Creek fish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Measurements of oxidative DNA damage (8-oxo-deoxyguanine by LC-MS/MS) showed no differences among treatment groups, suggesting that the majority of DNA damage is from covalent binding of BaP metabolites to DNA. This study shows for the first time that mitochondria can be an important target of BaP toxicity in fish, indicating that BaP exposures could have important energetic consequences. Results also suggest that multi-generational exposures in the wild may lead to adaptations that dampen DNA damage arising from BaP exposure

Genotoxicity in Atlantic killifish (Fundulus heteroclitus) from a PAH-contaminated Superfund site on the Elizabeth River, Virginia

The Atlantic Wood Industries Superfund site (AWI) on the Elizabeth River in Portsmouth, VA is heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) from a wood treatment facility. Atlantic killifish, or mummichog (Fundulus heteroclitus), at this Superfund site are exposed to very high concentrations of several carcinogens. In this study, we measured PAH concentrations in both fish tissues and sediments. Concurrently, we assessed different aspects of genotoxicity in the killifish exposed in situ. Both sediment and tissue PAH levels were significantly higher in AWI samples, relative to a reference site, but the chemistry profile was different between sediments and tissues. Killifish at AWI exhibited higher levels of DNA damage compared to reference fish, as measured via the flow cytometric method (FCM), and the damage was consistent with sediment PAH concentrations. Covalent binding of benzo[a]pyrene (BaP) metabolites to DNA, as measured via LC-MS/MS adduct detection methods, were also elevated and could be partially responsible for the DNA damage. Using similar LC-MS/MS methods, we found no evidence that oxidative DNA adducts had a role in observed genotoxicity

\u3ci\u3eFundulus\u3c/i\u3e as the premier teleost model in environmental biology: Opportunities for new insights using genomics

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms

Highlights from the Pierre Auger Observatory

The Pierre Auger Observatory is the world's largest cosmic ray observatory. Our current exposure reaches nearly 40,000 km$^2$ str and provides us with an unprecedented quality data set. The performance and stability of the detectors and their enhancements are described. Data analyses have led to a number of major breakthroughs. Among these we discuss the energy spectrum and the searches for large-scale anisotropies. We present analyses of our X$_{max}$ data and show how it can be interpreted in terms of mass composition. We also describe some new analyses that extract mass sensitive parameters from the 100% duty cycle SD data. A coherent interpretation of all these recent results opens new directions. The consequences regarding the cosmic ray composition and the properties of UHECR sources are briefly discussed.Comment: 9 pages, 12 figures, talk given at the 33rd International Cosmic Ray Conference, Rio de Janeiro 201