Dietary exposure of largemouth bass to OCPs changes expression of genes important for reproduction

Abstract

Dieldrin and p,p'-DDE are ubiquitous contaminants known to act as endocrine disruptors, causing impaired development and reproduction in fish and wildlife. In order to elucidate the mechanisms by which dieldrin and p,p'-DDE cause endocrine disruption in largemouth bass (Micropterus salmoides), fish were exposed subchronically through the diet to both contaminants. Following 120 days of exposure, p,p'-DDE decreased estradiol in females, but increased 11-ketotestosterone in both sexes. Dieldrin on the other hand, decreased estradiol and 11-ketotestosterone in both sexes. Both pesticides also altered steady state mRNA expression levels of a set of genes chosen to represent three possible mechanisms of endocrine disruption: (1) direct interaction with soluble sex steroid receptors, (2) biosynthesis of endogenous sex hormones, and (3) metabolism of endogenous hormones. p,p'-DDE acted as a weak estrogen, increasing the expression of vitellogenin and estrogen receptor alpha in the liver. p,p'-DDE also altered the expression of genes involved in the synthesis of endogenous hormones as well as their metabolism. Dieldrin, on the other hand, only altered expression of vitellogenin and not estrogen receptor alpha. Dieldrin also altered the expression of genes involved in hormone synthesis and metabolism, and it dramatically lowered plasma hormone levels. Both pesticides targeted expression of genes involved in all three modes of action, suggesting that they each have multiple modes of action.

Fig. 1

Measurements of hormone levels in largemouth bass plasma from males and females performed using a radioimmunoassay (RIA). The graphs show the concentration of E₂ and 11-KT in plasma for control fish, fish treated with p,p′-DDE (both doses) and fish treated with dieldrin (both doses). All values are shown as pg/ml hormone in plasma.

Fig. 2

Changes in gene expression for the p,p′-DDE treatment for genes in the receptor-related pathway in liver and gonad for females and males. The treatments are low (5.3 μg/g feed, white bars) and high (45.9 μg/g feed, gray bars) p,p′-DDE. The following genes were analyzed for the liver: vtg, ERα, ERβb, ERβa and AR; and for the gonad: ERα, ERβb, ERβa and AR. Results are plotted as log₂ ΔΔCt. Statistical significance (p = 0.5) between control and treated fish and among treatments are represented with letters (a, b). Highlighted area corresponds to log₂ between 0.58 and −0.58, corresponding to a 1.5-fold change in either direction, the limits for up and down regulation, respectively.

Fig. 3

Changes in gene expression for the dieldrin treatment for genes in the receptor-related pathway in liver and gonad for females and males, as described in Fig. 2. The treatments are low (0.4 μg/g feed, white bars), and high (0.81 μg/g feed, gray bars) dieldrin.

Fig. 4

Changes in gene expression for the p,p′-DDE treatment in gonads for the hormone synthesis pathway. StAR and CYP19 were measured for females and males as described for Fig. 2.

Fig. 5

Changes in gene expression for the dieldrin treatment in gonads for the hormone synthesis pathway. StAR and CYP19 were measured for females and males as described for Fig. 3.

Fig. 6

Changes in gene expression in the liver for the p,p′-DDE treatment for the hormone metabolism pathway. CYP3A68, CYP3A69 and CYP1A were measured for females and males as described for Fig. 2. down regulated in both sexes. In the gonad, both ERβa and ERβb were highly down regulated by dieldrin in both sexes, whereas StAR, as in p,p′-DDE, was up regulated (Fig. 8C and D).

Fig. 7

Changes in gene expression in the liver for the dieldrin treatment for the hormone metabolism pathway. CYP3A68, CYP3A69 and Cyp1A were measured for females and males treated as described for Fig. 3.

Fig. 8

Summary of gene expression patterns for p,p′-DDE and dieldrin. Data for the high dose of p,p′-DDE and the low dose of dieldrin from Figs. 2–7 was re-plotted in the same order as for females treated with p,p′-DDE, where the genes were arranged from the most up regulated to the most down regulated. described as weak estrogens and weak androgens by a variety of in vivo and in vitro assays, thus also implicating receptor-mediated modes of action (Kelce et al., 1995, 1997; Soto et al., 1994, 1995; Ramamoorthy et al., 1997; Arcaro et al., 1998; Sohoni and Sumpter, 1998; Maness et al., 1998; Andersen et al., 2002; Scippo et al., 2004).