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

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.Keywords: Killifish, Vitellogenin, Choriogenin, Estrogen, Biomarkers, Endocrine disruptionKeywords: Killifish, Vitellogenin, Choriogenin, Estrogen, Biomarkers, Endocrine disruptio

An integrated biomarker approach for assessing exposure and effects of endocrine disruptors and other contaminants in killifish (Fundulus heteroclitus) from the New York-New Jersey harbor estuary:

A multi-tiered approach was used to evaluate killifish (Fundulus heteroclitus) health by examining a suite of biomarkers in killifish inhabiting the heavily industrialized Newark Bay and a reference population in Tuckerton, NJ. The general hypothesis of this study was that Newark Bay killifish would exhibit biomarker responses indicative of impaired health when compared to a reference population from Tuckerton, NJ. The biomarkers investigated included classical endpoints (histopathology, morphometrics, gonad maturation), hepatic mRNA expression (CYP1A, metallothionein, vitellogenin I), gonadal aromatase mRNA expression, hepatic protein levels (CYP1A and vitellogenin I) and chemical exposure analyses (bile PAHs). Newark Bay fish had significantly higher levels of bile PAHs compared to reference fish. Females had significantly higher concentrations of naphthalene, pyrene and benzo[a]pyrene (3, 6 and 4 fold higher, respectively, p<0.05). Males had significantly higher concentrations of pyrene (7-fold higher, p<0.05), and higher concentrations of naphthalene (4-fold higher, p=0.06) and benzo[a]pyrene (9-fold higher, p=0.07). Histological lesions of the liver and pancreas in Newark Bay fish were similar to reference fish. Newark Bay fish had significantly higher expression of hepatic CYP1A for both males and females (7 and 3 fold higher, respectively, p<0.05) and CYP1A protein levels for both males and females (5 and 8 fold higher, p<0.05). Endocrine disruption in male gonads is demonstrated by a decreased gonad weight, altered testis development and upregulated aromatase expression (3-fold, p<0.05), which indicates exposure to endocrine active compounds. Efforts to strip spawn Newark Bay females produced few viable eggs, while at the same time Tuckerton females produced large numbers of viable eggs. There was a significant increase in the percent of pre-vitellogenic follicles (43% at Tuckerton, 64% at Newark Bay) and a significantly decreased percent of follicles at the mid-vitellogenic and mature stages (25% at Tuckerton, 3% at Newark Bay) in Newark Bay females (p<0.05). Vitellogenin mRNA and protein (egg yolk-protein) was significantly decreased in Newark Bay females (6-fold lower mRNA, 27-fold lower protein, p<0.05) while gonadal aromatase (produces 17β-estradiol) was significantly increased (210-fold higher, p<0.05). Killifish in the NY-NJ Harbor Estuary are exposed to high amounts of PAHs and aryl hydrocarbon agonists and their reproductive health is impaired. Impaired reproductive health is possibly due to disruption of steroid signaling by aryl hydrocarbon receptor-estrogen receptor crosstalk leading to decreased vitellogenin production.M.S.Includes bibliographical references (p. 91-100)by Sean M. Buge

AHR2 required for normal behavioral responses and proper development of the skeletal and reproductive systems in zebrafish

<div><p>The aryl hydrocarbon receptor (AHR) is a conserved ligand-activated transcription factor required for proper vertebrate development and homeostasis. The inappropriate activation of AHR by ubiquitous pollutants can lead to adverse effects on wildlife and human health. The zebrafish is a powerful model system that provides a vertebrate data stream that anchors hypothesis at the genetic and cellular levels to observations at the morphological and behavioral level, in a high-throughput format. In order to investigate the endogenous functions of AHR, we generated an AHR2 (homolog of human AHR)-null zebrafish line (<i>ahr2</i>^osu1) using the clustered, regulatory interspaced, short palindromic repeats (CRISPR)-Cas9 precision genome editing method. In zebrafish, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) mediated toxicity requires AHR2. The AHR2-null line was resistant to TCDD-induced toxicity, indicating the line can be used to investigate the biological and toxicological functions of AHR2. The AHR2-null zebrafish exhibited decreased survival and fecundity compared to the wild type line. At 36 weeks, histological evaluations of the AHR2-null ovaries revealed a reduction of mature follicles when compared to wild type ovaries, suggesting AHR2 regulates follicle growth in zebrafish. AHR2-null adults had malformed cranial skeletal bones and severely damaged fins. Our data suggests AHR2 regulates some aspect(s) of neuromuscular and/or sensory system development, with impaired behavioral responses observed in larval and adult AHR2-null zebrafish. This study increases our understanding of the endogenous functions of AHR, which may help foster a better understanding of the target organs and molecular mechanisms involved in AHR-mediated toxicities.</p></div

Schematic diagram of the mutation sequence and predicted AHR2 protein.

<p><b>(A)</b> The AHR2 exon 1 DNA sequence in <i>ahr2</i>⁺ (top) and <i>ahr2</i>^osu1 (bottom) zebrafish. The <i>ahr2</i>^osu1 mutant has an 11 bp deletion. The target sequence is in green and the PAM site is in red (CGG). <b>(B)</b> The translated mutant sequence results in a frameshift mutation and is predicted to result in a premature stop codon at amino acid residue 23. <b>(C)</b> Schematic diagram of the <i>ahr2</i>⁺ (top) and predicted <i>ahr2</i>⁺ (bottom) protein. The predicted truncated protein does not contain the bHLH, PAS, or transactivation domains. NLS = nuclear localization signal, NES1 = nuclear export signal 1, NES2 = nuclear export signal 2.</p

<i>ahr2</i>^osu1 mutants exhibit reduced <i>ahr2</i> mRNA expression levels and reduced expression of known AHR2 target genes.

<p><b>(A)</b> Comparative gene expression of <i>ahr2</i> and known transcripts downstream of AHR2 activation in <i>ahr2</i>⁺ and <i>ahr2</i>^osu1 zebrafish at 5 dpf (<i>n</i> = 4 biological replicates with 4 embryos per replicate). Data for each gene was tested for normality using the Shapiro-Wilk normality test and equal variance using the Levene’s test for homogeneity of variance. Data was statistically analyzed in R using a two-sample <i>t</i>-test or Welch’s two-sample <i>t</i>-test for data that either passed or failed equal variance testing, respectively. The Holm- Šídák multiple comparisons method was used with <i>α</i> = 0.05. <b>(B)</b> Comparative gene expression of <i>ahr2</i> and known transcripts downstream of AHR2 activation in 5 dpf wild-type and <i>ahr2</i>^osu1 zebrafish developmentally exposed to 0.1% DMSO or 1 ng/mL TCDD (<i>n</i> = 4 biological replicates with 4 embryos per replicate). Data for each transcript was tested for normality and equal variance as described above. Statistical significance was analyzed using a two-way ANOVA and a correction for multiple comparisons was performed using the Dunnett’s test. For all qPCR data, expression values were analyzed with the 2^-ΔΔCT Pfaffl method and normalized to β-actin. Error bars indicate SD of the mean. * = <i>p</i> < 0.05, ** = <i>p</i> < 0.01, and *** = <i>p</i> < 0.001 compared to either <b>(A)</b> <i>ahr2</i>⁺ or <b>(B)</b> <i>ahr2</i>⁺ vehicle control (DMSO).</p

Reproductive impacts on the ovary are evident in <i>ahr2</i>^osu1 zebrafish.

<p><b>(A)</b> A fecundity study was performed with group and pair spawning between weeks 24 and 34 with age-matched cohorts of <i>ahr2</i>⁺ and <i>ahr2</i>^osu1 zebrafish. Weeks 24–34 correspond to the optimal reproductive period. Between spawns, pairs were re-combined into groups so that each pair spawn were randomly selected groups. *p < 0.05 for group spawning (Fisher’s exact test) or pair-wise spawning (Student’s t-test) when compared to wild type. For group spawns, n = 10 males, 10 females. For pair spawns, n = 5, each with 2 males and 2 females. <b>(B)</b> Ovarian histopathological assessments were performed on <i>ahr2</i>⁺ and <i>ahr2</i>^osu1 zebrafish to quantify the representative follicle distribution. Photomicrographs for the older cohort (36 weeks) are shown for each genotype using the sections most representative of group averages. <b>(C)</b> Differential follicle analysis was performed for reproductively active adult zebrafish at 20 and 36 weeks (n = 4 per genotype for each age group). Statistical differences between genotypes and developmental stages were determined using two-way ANOVA with Tukey <i>post hoc</i> test for multiple comparisons (<i>p</i> < 0.05). Significance is indicated using compact letter display, and bars not in the same letter group are significantly different. Follicles were scored as Stage I (pre-vitellogenic primary growth), Stage II (early-vitellogenic cortical alveolus stage), Stage III (mid-vitellogenic), Stage IV (late-vitellogenic mature), and atretic.</p

<i>ahr2</i>^osu1 mutants are resistant to TCDD-induced developmental toxicity.

<p><b>(A)</b> Lateral view of representative bright field images of 5 dpf <i>ahr2</i>⁺ and <i>ahr2</i>^osu1 zebrafish developmentally exposed to embryo medium (EM), 0.1% DMSO, or 1 ng/mL TCDD. J = jaw, E = eye, PE = pericardial edema, and YE = yolk sac edema. Black bar in bottom right corner = 100 μm. <b>(B)</b> A 5 dpf zebrafish embryo-larval developmental toxicity assay for <i>ahr2</i>⁺ and <i>ahr2</i>^osu1 mutants developmentally exposed to DMSO or TCDD (<i>n</i> = 32). The wild-type embryos exposed to TCDD exhibited significant malformations for 11 endpoints examined, including yolk sac and pericardial edema, and craniofacial malformations. No significant malformations were observed in the AHR2^-null zebrafish exposed to TCDD. Morphological evaluations were completed in a binary notation (present/absent) and statistically compared using Fisher’s exact test at <i>p</i> < 0.05 for each endpoint.</p

Larval and adult behavior irregularities observed in adult <i>ahr2</i>^osu1 mutant zebrafish.

<p><b>(A)</b> Larval photomotor response (LPR) in wild types and <i>ahr2</i>^osu1 mutants at 5 dpf (<i>n</i> = 32) using the ViewPoint ZebraBox systems. The LPR assay consisted of 3 minutes of light and dark alternating periods, for four light-dark transitions, with the first transition representing an acclimation period. The black and white bar along the y-axis indicates the 3 minutes of light (white) and dark (black) alternating periods. Larval zebrafish at this developmental stage display increased locomotion during periods of darkness. The overall area under the curve was analyzed for the last 3 light-dark cycles compared to control morphants using a Kolmogorov-Smirnov test (<i>p</i> < 0.01). <b>(B-D)</b> Wild-type and <i>ahr2</i>^osu1 mutant adult behavioral response including startle stimulus <b>(B)</b>, predator response (C), and social cohesion (D) assays. Statistical differences between mutants and controls were determined by two-way ANOVA with repeated measures and Tukey HSD <i>post hoc</i> test (<i>p</i> < 0.05).</p