Abstract
The toxic effects of cadmium and other heavy metals have been well established, and many of these and other environmental pollutants are known to be embryotoxic or teratogenic. However, it has proven difficult to identify individual cells that respond to toxicants among the wide range of cell populations in an intact animal, particularly during early development when cells are continually changing their molecular and physiologic characteristics as they differentiate. Here we report the establishment of an in vivo system that uses hsp70 gene activation as a measure of cadmium toxicity in living early larvae of transgenic zebrafish carrying a stably integrated hsp70-enhanced green fluorescent protein (eGFP) reporter gene. We demonstrate that eGFP expression in this strain of fish acts as an accurate and reproducible indicator of cell-specific induction of hsp70 gene expression. Furthermore, the transgene responds in a dose-dependent manner at concentrations similar to those observed for morphologic indicators of early-life-stage toxicity and is sensitive enough to detect cadmium at doses below the median combined adverse effect concentration and the median lethal concentration. The stable nature of this transgenic line should allow for extremely rapid and reproducible toxicologic profiling of embryos and larvae throughout development.
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- Adám A., Bártfai R., Lele Z., Krone P. H., Orbán L. Heat-inducible expression of a reporter gene detected by transient assay in zebrafish. Exp Cell Res. 2000 Apr 10;256(1):282–290. doi: 10.1006/excr.2000.4805. [DOI] [PubMed] [Google Scholar]
- Aït-Aïssa S., Porcher J., Arrigo A., Lambré C. Activation of the hsp70 promoter by environmental inorganic and organic chemicals: relationships with cytotoxicity and lipophilicity. Toxicology. 2000 Apr 14;145(2-3):147–157. doi: 10.1016/s0300-483x(00)00145-1. [DOI] [PubMed] [Google Scholar]
- Bierkens J. G. Applications and pitfalls of stress-proteins in biomonitoring. Toxicology. 2000 Nov 16;153(1-3):61–72. doi: 10.1016/s0300-483x(00)00304-8. [DOI] [PubMed] [Google Scholar]
- Bigsby R., Chapin R. E., Daston G. P., Davis B. J., Gorski J., Gray L. E., Howdeshell K. L., Zoeller R. T., vom Saal F. S. Evaluating the effects of endocrine disruptors on endocrine function during development. Environ Health Perspect. 1999 Aug;107 (Suppl 4):613–618. doi: 10.1289/ehp.99107s4613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blechinger Scott R., Evans Tyler G., Tang Ping Tao, Kuwada John Y., Warren James T., Jr, Krone Patrick H. The heat-inducible zebrafish hsp70 gene is expressed during normal lens development under non-stress conditions. Mech Dev. 2002 Mar;112(1-2):213–215. doi: 10.1016/s0925-4773(01)00652-9. [DOI] [PubMed] [Google Scholar]
- Braeckman B., Smagghe G., Brutsaert N., Cornelis R., Raes H. Cadmium uptake and defense mechanism in insect cells. Environ Res. 1999 Apr;80(3):231–243. doi: 10.1006/enrs.1998.3897. [DOI] [PubMed] [Google Scholar]
- Candido E. P., Jones D. Transgenic Caenorhabditis elegans strains as biosensors. Trends Biotechnol. 1996 Apr;14(4):125–129. doi: 10.1016/0167-7799(96)10016-0. [DOI] [PubMed] [Google Scholar]
- Carvan M. J., 3rd, Dalton T. P., Stuart G. W., Nebert D. W. Transgenic zebrafish as sentinels for aquatic pollution. Ann N Y Acad Sci. 2000;919:133–147. doi: 10.1111/j.1749-6632.2000.tb06875.x. [DOI] [PubMed] [Google Scholar]
- Dave G. The influence of pH on the toxicity of aluminum, cadmium, and iron to eggs and larvae of the zebrafish, Brachydanio rerio. Ecotoxicol Environ Saf. 1985 Oct;10(2):253–267. doi: 10.1016/0147-6513(85)90072-7. [DOI] [PubMed] [Google Scholar]
- De Smet H., Blust R. Stress responses and changes in protein metabolism in carp Cyprinus carpio during cadmium exposure. Ecotoxicol Environ Saf. 2001 Mar;48(3):255–262. doi: 10.1006/eesa.2000.2011. [DOI] [PubMed] [Google Scholar]
- Glynn A. W. Cadmium and zinc kinetics in fish: studies on water-borne 109Cd and 65Zn turnover and intracellular distribution in minnows, Phoxinus phoxinus. Pharmacol Toxicol. 1991 Jun;68(6):485–491. doi: 10.1111/j.1600-0773.1991.tb01274.x. [DOI] [PubMed] [Google Scholar]
- Halloran M. C., Sato-Maeda M., Warren J. T., Su F., Lele Z., Krone P. H., Kuwada J. Y., Shoji W. Laser-induced gene expression in specific cells of transgenic zebrafish. Development. 2000 May;127(9):1953–1960. doi: 10.1242/dev.127.9.1953. [DOI] [PubMed] [Google Scholar]
- Jowett T., Yan Y. L. Double fluorescent in situ hybridization to zebrafish embryos. Trends Genet. 1996 Oct;12(10):387–389. doi: 10.1016/s0168-9525(96)90091-8. [DOI] [PubMed] [Google Scholar]
- Kimmel C. B., Ballard W. W., Kimmel S. R., Ullmann B., Schilling T. F. Stages of embryonic development of the zebrafish. Dev Dyn. 1995 Jul;203(3):253–310. doi: 10.1002/aja.1002030302. [DOI] [PubMed] [Google Scholar]
- Krone P. H., Lele Z., Sass J. B. Heat shock genes and the heat shock response in zebrafish embryos. Biochem Cell Biol. 1997;75(5):487–497. [PubMed] [Google Scholar]
- Lele Z., Engel S., Krone P. H. hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos. Dev Genet. 1997;21(2):123–133. doi: 10.1002/(SICI)1520-6408(1997)21:2<123::AID-DVG2>3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
- Mantovani A., Stazi A. V., Macrì C., Maranghi F., Ricciardi C. Problems in testing and risk assessment of endocrine disrupting chemicals with regard to developmental toxicology. Chemosphere. 1999 Oct;39(8):1293–1300. doi: 10.1016/s0045-6535(99)00197-6. [DOI] [PubMed] [Google Scholar]
- Mattingly C. J., McLachlan J. A., Toscano W. A., Jr Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AhR) function in developing zebrafish (Danio rerio). Environ Health Perspect. 2001 Aug;109(8):845–849. doi: 10.1289/ehp.01109845. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mosser D. D., Theodorakis N. G., Morimoto R. I. Coordinate changes in heat shock element-binding activity and HSP70 gene transcription rates in human cells. Mol Cell Biol. 1988 Nov;8(11):4736–4744. doi: 10.1128/mcb.8.11.4736. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Motoike T., Loughna S., Perens E., Roman B. L., Liao W., Chau T. C., Richardson C. D., Kawate T., Kuno J., Weinstein B. M. Universal GFP reporter for the study of vascular development. Genesis. 2000 Oct;28(2):75–81. doi: 10.1002/1526-968x(200010)28:2<75::aid-gene50>3.0.co;2-s. [DOI] [PubMed] [Google Scholar]
- Perz-Edwards A., Hardison N. L., Linney E. Retinoic acid-mediated gene expression in transgenic reporter zebrafish. Dev Biol. 2001 Jan 1;229(1):89–101. doi: 10.1006/dbio.2000.9979. [DOI] [PubMed] [Google Scholar]
- Rani A. U. Cadmium-induced bioaccumulation in the selected tissues of a freshwater teleost, Oreochromis mossambicus (Tilapia). Ann N Y Acad Sci. 2000;919:318–320. doi: 10.1111/j.1749-6632.2000.tb06895.x. [DOI] [PubMed] [Google Scholar]
- Salminen W. F., Jr, Voellmy R., Roberts S. M. Induction of hsp 70 in HepG2 cells in response to hepatotoxicants. Toxicol Appl Pharmacol. 1996 Nov;141(1):117–123. [PubMed] [Google Scholar]
- Tanguay R. L., Andreasen E., Heideman W., Peterson R. E. Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions. Biochim Biophys Acta. 2000 Nov 15;1494(1-2):117–128. doi: 10.1016/s0167-4781(00)00225-6. [DOI] [PubMed] [Google Scholar]
- Tully D. B., Collins B. J., Overstreet J. D., Smith C. S., Dinse G. E., Mumtaz M. M., Chapin R. E. Effects of arsenic, cadmium, chromium, and lead on gene expression regulated by a battery of 13 different promoters in recombinant HepG2 cells. Toxicol Appl Pharmacol. 2000 Oct 15;168(2):79–90. doi: 10.1006/taap.2000.9014. [DOI] [PubMed] [Google Scholar]
- Westerfield M., Wegner J., Jegalian B. G., DeRobertis E. M., Püschel A. W. Specific activation of mammalian Hox promoters in mosaic transgenic zebrafish. Genes Dev. 1992 Apr;6(4):591–598. doi: 10.1101/gad.6.4.591. [DOI] [PubMed] [Google Scholar]
- Willey J. B., Krone P. H. Effects of endosulfan and nonylphenol on the primordial germ cell population in pre-larval zebrafish embryos. Aquat Toxicol. 2001 Sep;54(1-2):113–123. doi: 10.1016/s0166-445x(00)00178-8. [DOI] [PubMed] [Google Scholar]