Di Giulio Richard T.

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Di Giulio
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Richard T.
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  • Preprint
    Development of the morpholino gene knockdown technique in Fundulus heteroclitus : a tool for studying molecular mechanisms in an established environmental model
    ( 2008-02-13) Matson, Cole W. ; Clark, Bryan W. ; Jenny, Matthew J. ; Fleming, Carrie R. ; Hahn, Mark E. ; Di Giulio, Richard T.
    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.
  • Preprint
    Fundulus as the premier teleost model in environmental biology : opportunities for new insights using genomics
    ( 2007-09-01) Burnett, Karen G. ; Bain, Lisa J. ; Baldwin, William S. ; Callard, Gloria V. ; Cohen, Sarah ; Di Giulio, Richard T. ; Evans, David H. ; Gomez-Chiarri, Marta ; Hahn, Mark E. ; Hoover, Cindi A. ; Karchner, Sibel I. ; Katoh, Fumi ; MacLatchy, Deborah L. ; Marshall, William S. ; Meyer, Joel N. ; Nacci, Diane E. ; Oleksiak, Marjorie F. ; Rees, Bernard B. ; Singer, Thomas D. ; Stegeman, John J. ; Towle, David W. ; Van Veld, Peter A. ; Vogelbein, Wolfgang K. ; Whitehead, Andrew ; Winn, Richard N. ; Crawford, Douglas L.
    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.
  • Preprint
    Antioxidant responses and NRF2 in synergistic developmental toxicity of PAHs in zebrafish
    ( 2008-12) Timme-Laragy, Alicia R. ; Van Tiem, Lindsey A. ; Linney, Elwood A. ; Di Giulio, Richard T.
    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.