Chronic exposure of killifish to a highly polluted environment desensitizes estrogen-responsive reproductive and biomarker genes

Abstract

Reproductive and endocrine disruption is commonly reported in aquatic species exposed to complex contaminant mixtures. We previously reported that Atlantic killifish (Fundulus heteroclitus) from the chronically contaminated Newark Bay, NJ, exhibit multiple endocrine disrupting effects, including inhibition of vitellogenesis (yolk protein synthesis) in females and false negative vitellogenin biomarker responses in males. Here, we characterized the effects on estrogen signaling and the transcriptional regulation of estrogen–responsive genes in this model population. First, a dose–response study tested the hypothesis that reproductive biomarkers (vtg1, vtg2, chg H, chg Hm, chg L) in Newark Bay killifish are relatively less sensitive to 17β–estradiol at the transcriptional level, relative to a reference (Tuckerton, NJ) population. The second study assessed expression for various metabolism (cyp1a, cyp3a30, mdr) and estrogen receptor (ER α, ER βa, ER βb) genes under basal and estrogen treatment conditions in both populations. Hepatic metabolism of 17β–estradiol was also evaluated in vitro as an integrated endpoint for adverse effects on metabolism. In the third study, gene methylation was evaluated for promoters of vtg1 (8 CpGs) and vtg2 (10 CpGs) in both populations, and vtg1 promoter sequences were examined for single nucleotide polymorphism (SNPs). Overall, these studies show that multi–chemical exposures at Newark Bay have desensitized all reproductive biomarkers tested to estrogen. For example, at 10 ng/g 17β–estradiol, inhibition of gene induction ranged from 62% to 97% for all genes tested in the Newark Bay population, relative to induction levels in the reference population. The basis for this recalcitrant phenotype could not be explained by a change in 17β– estradiol metabolism, nuclear estrogen receptor expression, promoter methylation (gene silencing) or SNPs, all of which were unaltered and normal in the Newark Bay population. The decreased transcriptional sensitivity of estrogen–responsive genes is suggestive of a broad effect on estrogen receptor pathway signaling, and provides insight into the mechanisms of the endocrine disrupting effects in the Newark Bay population.

Feminizing effects of ethinylestradiol in roach (Rutilus rutilus) populations with different estrogenic pollution exposure histories

“…We found no differences in response in either sex, despite differences in the exposure histories of these populations and genetic differences between the Grantham population and the two Thames populations (Hamilton et al, 2020). To our knowledge the only other study comparing sensitivity of fish populations to estrogens has been conducted on killifish where fish taken from a polluted site were desensitized to estrogen exposure compared to exposed fish from a reference location (Bugel et al, 2014), which differs from our findings for responses in the roach. A possible explanation for these differences is our use of offspring of fish caught from the wild, rather than using the adult fish taken directly from the wild as in the killifish study.…”

“…In contrast little is known of whether fish populations differ in their sensitivity to endocrine disrupting chemicals (EDCs). The only exception we could find in the literature to this was for adult killifish (Fundulus heteroclitus) from chemically impacted Newark Bay (USA) that were found to be desensitized when exposed to 17β-estradiol, compared to fish from a reference population, as assessed using responses of vitellogenin and choriogenin genes (Bugel et al, 2014). In that study, the mechanism of desensitization was not established, although later studies using genome wide comparisons identified evidence for selection at estrogen-responsive genes in killifish populations at other polluted sites (Reid et al, 2016).…”

Cloning of multiple ERα mRNA variants in killifish (Fundulus heteroclitus), and differential expression by tissue type, stage of reproduction, and estrogen exposure in fish from polluted and unpolluted environments

“…Although induction of AroB is strictly ERβb mediated, estrogen upregulation of ERα and Vtg mRNAs are ERα-mediated, at least in zebrafish (Griffin et al, 2013). Also, the normal or exaggerated estrogen responses we record in adult male and female killifish are in contrast to the hypo-responsiveness seen when ERα and AroB mRNAs are measured in NBH embryos (Greytak et al, 2010), and when a variety of estrogen markers are measured in killifish from other polluted sites (Bugel et al, 2014; Bugel et al, 2011). …”

When evolution is the solution to pollution: Key principles, and lessons from rapid repeated adaptation of killifish (Fundulus heteroclitus) populations

“…In addition, Atlantic Wood offspring were more susceptible to mortality associated with infectious disease (Frederick, Van Veld, & Rice, 2007), to toxicity associated with photoactivation of PAHs, and to acute hypoxia (Meyer & Di Giulio, 2003), although they were less susceptible to the model pro‐oxidant tert ‐butyl hydroperoxide than reference fish (Meyer, Smith, Winston, & Di Giulio, 2003). Altered estrogen signaling has been observed in DLC‐tolerant killifish from both Newark and NBH (Bugel et al., 2011, 2014; Greytak & Callard, 2007; Greytak et al., 2010). NBH killifish are more sensitive to the pro‐oxidant chemical tert ‐butylhydroquinone as compared to fish from a reference site (Harbeitner, Hahn, & Timme‐Laragy, 2013); however, the opposite effect was seen in ER fish, which were more tolerant of the oxidant tert ‐butylhydroperoxide than reference fish (Meyer et al., 2003).…”

“…Many pollutants in urban estuaries may act as endocrine disruptors (Oberdorster & Cheek, 2001) through interference with estrogen receptor signaling (Shanle & Xu, 2011) and altered estrogen signaling has been observed in tolerant populations of killifish (Bugel, Bonventre, White, Tanguay, & Cooper, 2014;Bugel, White, & Cooper, 2011;Greytak & Callard, 2007;Greytak, Tarrant, Nacci, Hahn, & Callard, 2010 (Soto et al, 1995) in BP fish, prompting adaptation of the ESR2b gene.…”