No substantial changes in estrogen receptor and estrogen-related receptor orthologue gene transcription in Marisa cornuarietis exposed to estrogenic chemicals

Abstract

Estrogen receptor orthologues in molluscs may be targets for endocrine disruptors, although mechanistic evidence is lacking. Molluscs are reported to be highly susceptible to effects caused by very low concentrations of environmental estrogens which, if substantiated, would have a major impact on the risk assessment of many chemicals. The present paper describes the most thorough evaluation to-date of the susceptibility of Marisa cornuarietis ER and ERR gene transcription to modulation by vertebrate estrogens in vivo and in vitro. We investigated the effects of estradiol-17β and 4-tert-Octylphenol exposure on in vivo estrogen receptor (ER) and estrogen-related receptor (ERR) gene transcription in the reproductive and neural tissues of the gastropod snail M. cornuarietis over a 12-week period. There was no significant effect (p>0.05) of treatment on gene transcription levels between exposed and non-exposed snails. Absence of a direct interaction of estradiol-17β and 4-tert-Octylphenol with mollusc ER and ERR protein was also supported by in vitro studies in transfected HEK-293 cells. Additional in vitro studies with a selection of other potential ligands (including methyl-testosterone, 17α-ethinylestradiol, 4-hydroxytamoxifen, diethylstilbestrol, cyproterone acetate and ICI182780) showed no interaction when tested using this assay. In repeated in vitro tests, however, genistein (with mcER-like) and bisphenol-A (with mcERR) increased reporter gene expression at high concentrations only (>10(-6)M for Gen and >10(-5)M for BPA, respectively). Like vertebrate estrogen receptors, the mollusc ER protein bound to the consensus vertebrate estrogen-response element (ERE). Together, these data provide no substantial evidence that mcER-like and mcERR activation and transcript levels in tissues are modulated by the vertebrate estrogen estradiol-17β or 4-tert-Octylphenol in vivo, or that other ligands of vertebrate ERs and ERRs (with the possible exception of genistein and bisphenol A, respectively) would do otherwise.

Keywords: Estrogen; Estrogen receptor; Estrogen-related receptor; Exposure; Gene transcription; Mollusc.

Fig. 1

Bar and Whisker plots showing the mRNA expression levels of mcER-like and mcERR genes in the penis and sheath assessed by aQPCR at pre-exposure (pre), and after 1 week (wk1). 6 week (wk6), and 12 week (wk12) exposure to 17β-estradiol (E2; 10, 100 and 1000 ng/L), 4-tert-Octylphenol (OP; 5 and 25 μg/L) or the water (DW) and solvent controls (SC). Week 1 DW n = 5♂; SC n = 5♂; E2 10 n = 4♂, E2 100 n = 5♂; E2 1000 n = 5♂; OP 5 n = 8♂; OP 25 n = 6♂. Week 6 DW n = 5♂; SC n = 4♂; E2 10 n = 7 ♂, E2 100 n = 5 ♂; E2 1000 n = 6♂; OP 5 n = 4♂; OP 25 n = 6♂; Week 12 DW n = 6♂; SC n = 5♂; E2 10 n = 4♂, E2 100 n = 5♂; E2 1000 n = 6♂; OP 5 n = 6♂; OP 25 n = 4♂ The ends of the whisker are set at 1.5*IQR above the third quartile (Q3) and 1.5*IQR below the first quartile (Q1). If the Minimum or Maximum values are outside this range, then they are shown as outliers (*). Equivalent plots of the responses of mcER-like and mcERR genes in male and female ganglia, gonad-digestive complex and the albumin gland (female only) can be found in Supplementary information Figures S2–S6).

Fig. 2

Response of the VP16-mcERR(LBD)-Gal4 (A) and VP16-mcER-like(LBD)-Gal4 (B) construct transfected in HEK293 cells in the presence and absence of a chemicals (one dose from a single assay is presented). A recombinant plasmid was constructed by restriction cloning using the mcER-like or mcERR LBD, a GAL4 DNA binding domain and a VP16 transactivation domain in pSG5 expression vector (Stratagene) shown above the charts. This construct was co-transfected into HEK293 cells along with a luciferase reporter coupled to a GAL4 DNA response element and a renilla plasmid used to control for transfection efficiency. Following transfection, cells were treated for 24 h with a suite of chemicals at a range of concentrations from to 100 pM to 100 μM. Luciferase and renilla activites were measured by luminometer. Response at 100 nm is shown for 17β-estradiol (E2), Response at 1 μM is shown for all other chemicals. OP, 4-tert-Octylphenol; BPA, bisphenol A; GEN, genistein. Luciferase counts were normalised to renilla counts to control for transfection efficiency. The data presented here was normalised to the constitutive activity observed when the mcERR (A) or mcER-like (B) construct was transfected and left untreated. A construct containing mouse ERα LBD was used as a positive control. A construct with the LBD excised (vector) was used as a negative control. Error bars represent one standard deviation of the mean from replicate wells.

Fig. 3

Examples of dose responses of transfected HEK293 cells containing mouse ER (mERα), marisa ER-like (mcER) or marisa ERR (mcERR) exposed to various chemicals. A) Response of mERα to E2 (10⁻¹⁰ to 10⁻⁶ M) and BPA (10⁻¹⁰ to 10⁻⁵ M) and mcERR to BPA (10⁻¹⁰ to 10⁻⁴ M). B) Response of mERα, mcER and mcERR to genistein from 10⁻¹¹ to 10⁻⁴ M. The inclusion of the construct with the LBD excised plus chemical (Mock) and mERα with solvent carrier (control) were used as negative controls. Error bars denote the standard deviation of two replicate samples per treatment. Concentrations above 10⁻⁶ M showed signs of toxicity and are therefore not included.