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.

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

Significantly differentially regulated biological processes in female <i>G. holbrooki</i> residing downstream of a paper mill as compared to a control site as determined by gene set enrichment and gene ontology (GO) analyses.

a<p> Percentage of genes within the GO category that were significantly differentially regulated between Econfina and Fenholloway groups.</p>b<p> Percentage of genes within the GO category that were not significantly differentially regulated between Econfina and Fenholloway groups.</p><p>Significantly differentially regulated biological processes in female <i>G. holbrooki</i> residing downstream of a paper mill as compared to a control site as determined by gene set enrichment and gene ontology (GO) analyses.</p

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

<p>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.</p

Focused hepatic gene expression patterns in female Eastern mosquitofish (<i>Gambusia holbrooki</i>) using qualitative polymerase chain reaction analysis.

<p>Gene expression of (A) vitellogenin (<i>vtg</i>), (B) zona pellucida glycoprotein 2 (<i>zp2</i>), (C) 17β-hydroxysteroid dehydrogenase 3 (<i>17βhsd3</i>) 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.</p

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

<p>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.</p

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.

<p>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.</p

Anal fin gene expression patterns of sonic hedgehog (<i>shh</i>) in female Eastern mosquitofish (<i>G. holbrooki</i>).

<p>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.</p

Blood Transcriptomics Analysis of Fish Exposed to Perfluoro Alkyls Substances: Assessment of a Non-Lethal Sampling Technique for Advancing Aquatic Toxicology Research

In contrast to mammals, the blood from other vertebrates such as fish contains nucleated red cells. Using a fathead minnow (Pimephales promelas) oligonucleotide microarray, we compared altered transcripts in the liver and whole blood after exposure to environmentally relevant concentrations of perfluorooctanesulfonic acid (PFOS) and a mixture of seven types of perfluoro alkyl substances (PFAS), including perfluorooctanoic acid (PFOA). We used quantitative polymerase chain reactions and cell-based assays to confirm the main effects and found that blood responded with a greater number of altered genes than the liver. The exposure to PFAS altered similar genes with central roles in a cellular pathway in both tissues, including estrogen receptor α and peroxisome proliferator activator β and ?, indicating that the genes previously associated with PFAS exposure are differentially expressed in blood and liver. The altered transcripts are involved with cholesterol metabolism and mitochondrial function. Our data confirmed that PFAS are weak xenoestrogens and exert effects on DNA integrity. Gene expression profiling from blood samples not related with the immune system, including very-low-density lipoprotein, vitellogenin, estrogen receptor, and thyroid hormone receptor, demonstrated that blood is a useful tissue for assessing endocrine disruption in non-mammalian vertebrates. We conclude that the use of blood for non-lethal sampling in genomics studies is informative and particularly useful for assessing the effects of pollution in endangered species. Further, using blood will reduce animal use and widen the experimental design options for studying the effects of contaminant exposure on wildlife.Fil: Rodríguez Jorquera, Ignacio A.. Universidad Austral de Chile; Chile. School Of Natural Resources And Environment; . University of Florida; Estados UnidosFil: Colli Dula, R. Cristina. INSTITUTO POLITÉCNICO NACIONAL (IPN); . University of Florida; Estados UnidosFil: Kroll, Kevin. University of Florida; Estados UnidosFil: Jayasinghe, B. Sumith. University of Florida; Estados UnidosFil: Parachu Marco, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Silva Sanchez, Cecilia. University of Florida; Estados UnidosFil: Toor, Gurpal S.. University of Maryland; Estados UnidosFil: Denslow, Nancy D.. University of Florida; Estados Unido

Interlaboratory comparison of in vitro bioassays for screening of endocrine active chemicals in recycled water.

In vitro bioassays have shown promise as water quality monitoring tools. In this study, four commercially available in vitro bioassays (GeneBLAzer(®) androgen receptor (AR), estrogen receptor-alpha (ER), glucocorticoid receptor (GR) and progesterone receptor (PR) assays) were adapted to screen for endocrine active chemicals in samples from two recycled water plants. The standardized protocols were used in an interlaboratory comparison exercise to evaluate the reproducibility of in vitro bioassay results. Key performance criteria were successfully achieved, including low background response, standardized calibration parameters and high intra-laboratory precision. Only two datasets were excluded due to poor calibration performance. Good interlaboratory reproducibility was observed for GR bioassay, with 16-26% variability among the laboratories. ER and PR bioactivity was measured near the bioassay limit of detection and showed more variability (21-54%), although interlaboratory agreement remained comparable to that of conventional analytical methods. AR bioassay showed no activity for any of the samples analyzed. Our results indicate that ER, GR and PR, were capable of screening for different water quality, i.e., the highest bioactivity was observed in the plant influent, which also contained the highest concentrations of endocrine active chemicals measured by LC-MS/MS. After advanced treatment (e.g., reverse osmosis), bioactivity and target chemical concentrations were both below limits of detection. Comparison of bioassay and chemical equivalent concentrations revealed that targeted chemicals accounted for ≤5% of bioassay activity, suggesting that detection limits by LC-MS/MS for some chemicals were insufficient and/or other bioactive compounds were present in these samples. Our study demonstrated that in vitro bioassays responses were reproducible, and can provide information to complement conventional analytical methods for a more comprehensive water quality assessment