The use of early life stage fathead minnows (Pimephales promelas) as a toxicological model for the study of endocrine disrupting compounds

Sonia Mae Johns, Purdue University

Abstract

The fathead minnow (Pimephales promelas) is one of the most widely used test species for studying the effects of endocrine disrupting compounds (EDCs) on fish. However, most of these studies have focused on the adult life stages of the species despite the fact that early life stages have been identified as being more sensitive to contaminants. In order to address this gap in the research, the first month of development in the fathead minnow was monitored for growth and the expression of nine genes important for growth, development, and sexual reproduction were characterized using quantitative polymerase chain reaction in order to provide a baseline of information for future research concerning the early life stages of this species. There was a significant up-regulation of growth hormone (gh) mRNA and no significant changes in the expression of insulin-like growth factor 1 (igf1) over the developmental period studied. Thyroid hormone receptors A and B expression stayed relatively constant while there was a large variation in cytochrome P45019A mRNA expression during the first week of development returning to 0 dpf expression levels thereafter. Estrogen receptor 2B was up-regulated during the first three weeks post-fertilization, returning to pre-hatch values by 28 dpf. Expression of hydroxysteroid dehydrogenase 3B and steroidogenic acute regulatory (star) protein increased after the third or fourth week post-fertilization, respectively. Vitellogenin (vtg) exhibited a large degree of variation within time points, especially after day 15, and a significant up-regulation of this gene was observed at 7 and 10 dpf, likely due to differences between gene expressions in the sexes. Knowledge of the normal changes in gene expression during embryo and larval development will allow for better experimental design and selection of suitable biomarkers when testing the potential toxicological effects of EDCs in this model fish species. To further validate the utility of the early life stage fathead minnow for EDC toxicity testing, embryos were exposed to a potent estrogen (17α-ethinyl estradiol, EE2, 2, 10, and 50 ng/L); a weak estrogen (mycotoxin zearalenone, ZEAR, same concentrations as above); an antiestrogen (ZM 189, 154; 40, 250, and 1000 ng/L); and to mixtures of EE2 and ZM (EE 2 50 ng/L combined with ZM 189, 154; 40, 250, and 1000 ng/L) for seven days. Following exposure, expression of gh, igf1, vtg, star, and lutenizing hormone (lh) were examined using Q-PCR. Exposure to all concentrations of ZEAR and to the lowest concentration of ZM resulted in increased body sizes. High concentrations of EE2 decreased body sizes. There was a significant increase in the frequency of abnormalities (mostly edema) in larvae exposed to all concentrations of EE2, and high ZEAR, and EE2 + ZM mixture groups. Expression of growth hormone was up-regulated by most of the conditions tested. Exposure to 50 ng/L ZEAR caused an induction of ifg1, whereas exposure to 40 ng/L ZM caused a down-regulation of this gene. Expression of star protein gene was significantly up-regulated after exposure to all concentrations of EE2 and lh expression increased significantly in response to all treatments tested. As expected, EE2 induced vtg expression; however, ZEAR also induced expression of this gene to similar levels compared to EE2. Overall, exposure to EE2 + ZM mixture resulted in a different expression pattern compared to single exposures. The results of this study suggest that novel pathways of xenoestrogen toxicity may exist in larval fathead minnows. Our study implies that a 7-day exposure to EDCs is adequate to cause physiological changes as well as changes in the expression of genes important for reproduction and growth in early life stages of this fish species.

Degree

M.S.

Advisors

Sepulveda, Purdue University.

Subject Area

Molecular biology|Ecology

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