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The sea urchin, Paracentrotus lividus, embryo as a “bioethical” model for neurodevelopmental toxicity testing

Effects of diazinon on the intracellular distribution of OTX2-like proteins

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Abstract

Presently, a large effort is being made worldwide to increase the sustainability of industrial development, while preserving not only the quality of the environment but also that of animal and human life. In this work, sea urchin early developmental stages were used as a model to test the effects of the organophosphate pesticide (diazinon) on the regulation of gene expression by immunohistochemical localization of the human regulatory protein against the human OTX2. Egg exposure to diazinon did not affect fertilization; however, at concentrations 10−5–10−6 M, it did cause developmental anomalies, among which was the dose-dependent alteration of the intracellular distribution of a regulatory protein that is immunologically related to the human OTX2. The severe anomalies and developmental delay observed after treatment at 10−5 M concentration are indicators of systemic toxicity, while the results after treatment at 10−6 M suggest a specific action of the neurotoxic compound. In this second case, exposure to diazinon caused partial delivery of the protein into the nuclei, a defective translocation that particularly affected the blastula and gastrula stages. Therefore, the possibility that neurotoxic agents such as organophosphates may damage embryonic development is taken into account. Specifically, the compounds are known to alter cytoplasmic dynamics, which play a crucial role in regulating the distribution of intracellular structures and molecules, as well as transcription factors. Speculatively, basing our assumptions on Fura2 experiments, we submit the hypothesis that this effect may be due to altered calcium dynamics, which in turn alter cytoskeleton dynamics: the asters, in fact, appear strongly positive to the OTX2 immunoreaction, in both control and exposed samples. Coimmunoprecipitation experiments seem to supply evidence to the hypothesis.

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Abbreviations

ACh:

acetylcholine

AChE:

acetylcholinesterase, EC: 3.1.1.7

BChE:

butyrylcholinesterase, EC: 3.1.1.8

[Ca+]I:

intracellular calcium concentration

dzn:

diazinon

IR:

immunoreactivity

OP:

organophosphate compounds (esthers of the pyrophosphoric acid) in the literature, these compounds are also erroneously called organophosphorous compounds

Otx:

regulatory protein of the Otx gene

OTX2:

human regulatory protein of the orthodenticle homolog 2 (Drosophila), paired homeobox, bicoid family

PMCs:

primary mesenchyme cells, represented by micromeres performing the first ingression into the blastocoele, at the beginning of gastrulation

PMSF:

Phenylmethylsulfonyl fluoride

Siamois:

the gene responsible for the specification of dorsal mesoderm

WGA:

wheat germ agglutinin

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Acknowledgments

We warmly thank Dr. Mario Mori (University of Genova) for continuous supply of living P. lividus specimens in optimal reproductive conditions and Dr. William H. Klein (M. D. Anderson Cancer Center, Houston, TX) for the gift of the anti-SpOtx-β antibody. We also thank Prof. Michael J. Whitaker and Dr. Patrick K. Harrison for their hospitality in the Laboratory of Newcastle upon Tyne University and help in performing the Fura2 experiments.

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Authors and Affiliations

  1. Dipartimento di Biologia Sperimentale, Ambientale ed Applicata (DI.BI.S.A.A.), University of Genova, Genoa, Italy

    M. G. Aluigi, C. Angelini & C. Falugi

  2. IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy

    G. Corte

  3. Dipartimento di Biologia, Università di Genova, Viale Benedetto XV, n°5, 16132, Genoa, Italy

    C. Falugi

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  1. M. G. Aluigi
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  2. C. Angelini
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  3. G. Corte
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  4. C. Falugi
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Correspondence to C. Falugi.

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Aluigi, M.G., Angelini, C., Corte, G. et al. The sea urchin, Paracentrotus lividus, embryo as a “bioethical” model for neurodevelopmental toxicity testing. Cell Biol Toxicol 24, 587–601 (2008). https://doi.org/10.1007/s10565-008-9061-2

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