Exposure to paper mill effluent at a site in North Central Florida elicits molecular-level changes in gene expression indicative of progesterone and androgen exposure

Abstract

Endocrine disrupting compounds (EDCs) are chemicals that negatively impact endocrine system function, with effluent from paper mills one example of this class of chemicals. In Florida, female Eastern mosquitofish (Gambusia holbrooki) have been observed with male secondary sexual characteristics at three paper mill-impacted sites, indicative of EDC exposure, and are still found at one site on the Fenholloway River. The potential impacts that paper mill effluent exposure has on the G. holbrooki endocrine system and the stream ecosystem are unknown. The objective of this study was to use gene expression analysis to determine if exposure to an androgen receptor agonist was occurring and to couple this analysis with in vitro assays to evaluate the presence of androgen and progesterone receptor active chemicals in the Fenholloway River. Focused gene expression analyses of masculinized G. holbrooki from downstream of the Fenholloway River paper mill were indicative of androgen exposure, while genes related to reproduction indicated potential progesterone exposure. Hepatic microarray analysis revealed an increase in the expression of metabolic genes in Fenholloway River fish, with similarities in genes and biological processes compared to G. holbrooki exposed to androgens. Water samples collected downstream of the paper mill and at a reference site indicated that progesterone and androgen receptor active chemicals were present at both sites, which corroborates previous chemical analyses. Results indicate that G. holbrooki downstream of the Fenholloway River paper mill are impacted by a mixture of both androgens and progesterones. This research provides data on the mechanisms of how paper mill effluents in Florida are acting as endocrine disruptors.

Figure 1. Spatial representation of the two sites where female Eastern mosquitofish (Gambusia holbrooki) were collected for this study.

The paper mill-impacted sites include Fenholloway River sites 1 and 2 and the reference site in the Econfina River. G. holbrooki were collected from Fenholloway site 1 on July 12^th, 2012 and from Fenholloway site 2 on April 10^th, 2013. G. holbrooki were collected from the Econfina River on August 7^th and October 4^th, 2012. The map was created using ArcGIS v10.0 (Esri, Redlands, USA).

Figure 2. Anal fin elongation and bone segment formation in anal fin ray 3 of female Eastern mosquitofish (Gambusia holbrooki) collected from the Fenholloway and Econfina Rivers.

Data are repsresented as (A) Mean (± standard deviation) of all collection events and, (B) Distribution of anal fin elongation classes between both paper mill exposed (Fenholloway) and reference (Econfina) field sites. An asterisk indicates statistical significance between the two groups as determined using a Student's t-test with p<0.05. For anal fin elongation levels there was an N of 46 and 50 from the Fenholloway and Econfina rivers respectively and a subset N of 4 from both sites for the bone segment evaluation.

Figure 3. Focused hepatic gene expression patterns in female Eastern mosquitofish (Gambusia holbrooki) using qualitative polymerase chain reaction analysis.

Gene expression of (A) vitellogenin (vtg), (B) zona pellucida glycoprotein 2 (zp2), (C) 17β-hydroxysteroid dehydrogenase 3 (17βhsd3) was analyzed between paper mill exposed (Fenholloway) and reference (Econfina) collection sites. Each point represents the mean of each group and error bars represent standard deviation; time points with no standard deviation have an N of 1. A box indicates statistical significance between groups at the selected oocyte developmental stages as determined using a Student's t-test with p<0.05.

Figure 4. Anal fin gene expression patterns of sonic hedgehog (shh) in female Eastern mosquitofish (G. holbrooki).

An asterisk indicates statistical significance between the paper mill exposed (Fenholloway) and reference (Econfina) rivers as determined using a Student's t-test with p<0.05.

Figure 5. Gene expression profile comparisons between Fenholloway (PME) and reference site (Econ) female G. holbrooki and female G. holbrooki exposed to the androgen 17β-trenbolone (TB) or the vehicle control (C).

This set of genes were differentially expressed between the PME, Econfina, and TB groups over the lab controls with at least a 1.5-fold difference of expression over the lab control and was expressed in the same direction in those groups. Data were median-centered by gene and clustered using spearman correlation and centroid linkage. Yellow genes are more highly expressed than the gene average and blue genes are expressed at a lower level than the gene average.

Figure 6. Results of progesterone receptor (PR) and androgen receptor (AR) GeneBLAzer assays for concentrated water samples collected downstream of the Fenholloway River paper mill and the Econfina River conservation area.

The graph bars represent the mean of three replicates from each dilution for the PR (A) and AR (B) assays. Error bars represent standard deviation of the three replicates per assay. The dose-responses of levonorgestrel, progesterone, and 17β-trenbolone (C) were also evaluated by the AR assay.