Publications by authors named "Daniel L Villeneuve"

147 Publications

Identifying Chemicals and Mixtures of Potential Biological Concern Detected in Passive Samplers from Great Lakes Tributaries Using High-Throughput Data and Biological Pathways.

Environ Toxicol Chem 2021 Aug 8;40(8):2165-2182. Epub 2021 Jul 8.

Idaho Water Science Center, US Geological Survey, Boise, Idaho.

Waterborne contaminants were monitored in 69 tributaries of the Laurentian Great Lakes in 2010 and 2014 using semipermeable membrane devices (SPMDs) and polar organic chemical integrative samplers (POCIS). A risk-based screening approach was used to prioritize chemicals and chemical mixtures, identify sites at greatest risk for biological impacts, and identify potential hazards to monitor at those sites. Analyses included 185 chemicals (143 detected) including polycyclic aromatic hydrocarbons (PAHs), legacy and current-use pesticides, fire retardants, pharmaceuticals, and fragrances. Hazard quotients were calculated by dividing detected concentrations by biological effect concentrations reported in the ECOTOX Knowledgebase (toxicity quotients) or ToxCast database (exposure-activity ratios [EARs]). Mixture effects were estimated by summation of EAR values for chemicals that influence ToxCast assays with common gene targets. Nineteen chemicals-atrazine, N,N-diethyltoluamide, di(2-ethylhexyl)phthalate, dl-menthol, galaxolide, p-tert-octylphenol, 3 organochlorine pesticides, 3 PAHs, 4 pharmaceuticals, and 3 phosphate flame retardants-had toxicity quotients >0.1 or EARs for individual chemicals >10 at 10% or more of the sites monitored. An additional 4 chemicals (tributyl phosphate, triethyl citrate, benz[a]anthracene, and benzo[b]fluoranthene) were present in mixtures with EARs >10 . To evaluate potential apical effects and biological endpoints to monitor in exposed wildlife, in vitro bioactivity data were compared to adverse outcome pathway gene ontology information. Endpoints and effects associated with endocrine disruption, alterations in xenobiotic metabolism, and potentially neuronal development would be relevant to monitor at the priority sites. The EAR threshold exceedance for many chemical classes was correlated with urban land cover and wastewater effluent influence, whereas herbicides and fire retardants were also correlated to agricultural land cover. Environ Toxicol Chem 2021;40:2165-2182. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
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http://dx.doi.org/10.1002/etc.5118DOI Listing
August 2021

Assessing effects of aromatase inhibition on fishes with group-synchronous oocyte development using western mosquitofish (Gambusia affinis) as a model.

Aquat Toxicol 2021 Jan 5;232:105741. Epub 2021 Jan 5.

Great Lakes Toxicology and Ecology Division, U.S. Environmental Protection Agency, 6201 Congdon Boulevard, Duluth, MN, 55804, United States.

Exposure to certain anthropogenic chemicals can inhibit the activity to cytochrome P450 aromatase (CYP19) in fishes leading to decreased plasma 17β-estradiol (E2), plasma vitellogenin (VTG), and egg production. Reproductive dysfunction resulting from exposure to aromatase inhibitors has been extensively investigated in several laboratory model species of fish. These model species have ovaries that undergo asynchronous oocyte development, but many fishes have ovaries with group-synchronous oocyte development. Fishes with group-synchronous oocyte development have dynamic reproductive cycles which typically occur annually and are often triggered by complex environmental cues. This has resulted in a lack of test data and uncertainty regarding sensitivities to and adverse effects of aromatase inhibition. The present study used the western mosquitofish (Gambusia affinis) as a laboratory model to investigate adverse effects of chemical aromatase inhibition on group-synchronous oocyte development. Adult female western mosquitofish were exposed to either 0, 2, or 30 μg/L of the model nonsteroidal aromatase inhibiting chemical, fadrozole, for a complete reproductive cycle. Fish were sampled at four time-points representing pre-vitellogenic resting, early vitellogenesis, late vitellogenesis/early ovarian recrudescence, and late ovarian recrudescence. Temporal changes in numerous reproductive parameters were measured, including gonadosomatic index (GSI), plasma sex steroids, and expression of selected genes in the brain, liver, and gonad that are important for reproduction. In contrast to fish from the control treatment, fish exposed to 2 and 30 μg/L of fadrozole had persistent elevated expression of cyp19 in the ovary, depressed expression of vtg in the liver, and a low GSI. These responses suggest that completion of a group-synchronous reproductive cycle was unsuccessful during the assay in fish from either fadrozole treatment. These adverse effects data show that exposure to aromatase inhibitors has the potential to cause reproductive dysfunction in a wide range of fishes with both asynchronous and group-synchronous reproductive strategies.
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http://dx.doi.org/10.1016/j.aquatox.2020.105741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255332PMC
January 2021

Effects-Based Monitoring of Bioactive Chemicals Discharged to the Colorado River before and after a Municipal Wastewater Treatment Plant Replacement.

Environ Sci Technol 2021 01 29;55(2):974-984. Epub 2020 Dec 29.

U.S. Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, Minnesota 55804, United States.

Monitoring of the Colorado River near the Moab, Utah, wastewater treatment plant (WWTP) outflow has detected pharmaceuticals, hormones, and estrogen-receptor (ER)-, glucocorticoid receptor (GR)-, and peroxisome proliferator-activated receptor-gamma (PPARγ)-mediated biological activities. The aim of the present multi-year study was to assess effects of a WWTP replacement on bioactive chemical (BC) concentrations. Water samples were collected bimonthly, pre- and post-replacement, at 11 sites along the Colorado River upstream and downstream of the WWTP and analyzed for bioactivities (e.g., agonism of ER, GR, and PPARγ) and BC concentrations; fathead minnows were cage deployed pre- and post-replacement at sites with varying proximities to the WWTP. Before the WWTP replacement, ER (24 ng 17β-estradiol equivalents/L)-, GR (60 ng dexamethasone equivalents/L)-, and PPARγ-mediated activities were detected at the WWTP outflow but diminished downstream. In March 2018, the WWTP effluent was acutely toxic to the fish, likely due to elevated ammonia concentrations. Following the WWTP replacement, ER, GR, and PPARγ bioactivities were reduced by approximately 60-79%, no toxicity was observed in caged fish, and there were marked decreases in concentrations of many BCs. Results suggest that replacement of the Moab WWTP achieved a significant reduction in BC concentrations to the Colorado River.
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http://dx.doi.org/10.1021/acs.est.0c05269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8135223PMC
January 2021

Case Study in 21st Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Short-Term In Vivo Responses in Adult Female Fish.

Environ Toxicol Chem 2021 Apr 10;40(4):1155-1170. Epub 2021 Mar 10.

US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, Minnesota.

The present study evaluated whether in vitro measures of aromatase inhibition as inputs into a quantitative adverse outcome pathway (qAOP) construct could effectively predict in vivo effects on 17β-estradiol (E2) and vitellogenin (VTG) concentrations in female fathead minnows. Five chemicals identified as aromatase inhibitors in mammalian-based ToxCast assays were screened for their ability to inhibit fathead minnow aromatase in vitro. Female fathead minnows were then exposed to 3 of those chemicals: letrozole, epoxiconazole, and imazalil in concentration-response (5 concentrations plus control) for 24 h. Consistent with AOP-based expectations, all 3 chemicals caused significant reductions in plasma E2 and hepatic VTG transcription. Characteristic compensatory upregulation of aromatase and follicle-stimulating hormone receptor (fshr) transcripts in the ovary were observed for letrozole but not for the other 2 compounds. Considering the overall patterns of concentration-response and temporal concordance among endpoints, data from the in vivo experiments strengthen confidence in the qualitative relationships outlined by the AOP. Quantitatively, the qAOP model provided predictions that fell within the standard error of measured data for letrozole but not for imazalil and epoxiconazole. However, the inclusion of measured plasma concentrations of the test chemicals as inputs improved model predictions, with all predictions falling within the range of measured values. Results highlight both the utility and limitations of the qAOP and its potential use in 21st century ecotoxicology. Environ Toxicol Chem 2021;40:1155-1170. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.
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http://dx.doi.org/10.1002/etc.4968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127875PMC
April 2021

Harmonized Cross-Species Assessment of Endocrine and Metabolic Disruptors by Ecotox FACTORIAL Assay.

Environ Sci Technol 2020 10 23;54(19):12142-12153. Epub 2020 Sep 23.

Attagene, Research Triangle Park, Morrisville, North Carolina 27709, United States.

Environmental pollution is a threat to humans and wildlife species. Of particular concern are endocrine disrupting chemicals (EDCs). An important target of EDCs is nuclear receptors (NRs) that control endocrine and metabolic responses through transcriptional regulation. Owing in part to structural differences of NRs, adverse effects of EDCs vary significantly among species. Here, we describe a multiplexed reporter assay (the Ecotox FACTORIAL) enabling parallel assessment of compounds' effects on estrogen, androgen, thyroid, and PPARγ receptors of representative mammals, birds, reptiles, amphibians, and fish. The Ecotox FACTORIAL is a single-well assay comprising a set of species-specific, one-hybrid GAL4-NR reporter constructs transiently transfected into test cells. To harmonize cross-species assessments, we used a combination of two approaches. First, we used the same type of test cells for all reporters; second, we implemented a parallel detection of reporter RNAs. The assay demonstrated excellent quality, reproducibility, and insignificant intra-assay variability. Importantly, the EC50 values for NR ligands were consistent with those reported for conventional assays. Using the assay allowed ranking the hazard potential of environmental pollutants (e.g., bisphenols, polycyclic aromatic hydrocarbons, and synthetic progestins) across species. Furthermore, the assay permitted detecting taxa-specific effects of surface water samples. Therefore, the Ecotox FACTORIAL enables harmonized assessment of the endocrine and metabolic disrupting activity of chemicals and surface water in humans as well as in wildlife species.
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http://dx.doi.org/10.1021/acs.est.0c03375DOI Listing
October 2020

Toward an AOP Network-Based Tiered Testing Strategy for the Assessment of Thyroid Hormone Disruption.

Environ Sci Technol 2020 07 9;54(14):8491-8499. Epub 2020 Jul 9.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.

A growing number of environmental pollutants are known to adversely affect the thyroid hormone system, and major gaps have been identified in the tools available for the identification, and the hazard and risk assessment of these thyroid hormone disrupting chemicals. We provide an example of how the adverse outcome pathway (AOP) framework and associated data generation can address current testing challenges in the context of fish early life stage tests, and fish tests in general. We demonstrate how a suite of assays covering biological processes involved in the underlying toxicological pathways can be implemented in a tiered screening and testing approach for thyroid hormone disruption, using the levels of assessment of the OECD's Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals as a guide.
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http://dx.doi.org/10.1021/acs.est.9b07205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477622PMC
July 2020

Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish.

Environ Sci Technol 2020 05 29;54(10):6213-6223. Epub 2020 Apr 29.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.

A set of adverse outcome pathways (AOPs) linking inhibition of thyroperoxidase and deiodinase to impaired swim bladder inflation in fish has recently been developed. These AOPs help to establish links between these thyroid hormone (TH) disrupting molecular events and adverse outcomes relevant to aquatic ecological risk assessment. Until now, very little data on the effects of TH disruption on inflation of the anterior chamber (AC) of the swim bladder were available. The present study used zebrafish exposure experiments with three model compounds with distinct thyroperoxidase and deiodinase inhibition potencies (methimazole, iopanoic acid, and propylthiouracil) to evaluate this linkage. Exposure to all three chemicals decreased whole body triiodothyronine (T3) concentrations, either through inhibition of thyroxine (T4) synthesis or through inhibition of Dio mediated conversion of T4 to T3. A quantitative relationship between reduced T3 and reduced AC inflation was established, a critical key event relationship linking impaired swim bladder inflation to TH disruption. Reduced inflation of the AC was directly linked to reductions in swimming distance compared to controls as well as to chemical-exposed fish whose ACs inflated. Together the data provide compelling support for AOPs linking TH disruption to impaired AC inflation in fish.
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http://dx.doi.org/10.1021/acs.est.9b07204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477623PMC
May 2020

A method for CRISPR/Cas9 mutation of genes in fathead minnow (Pimephales promelas).

Aquat Toxicol 2020 May 2;222:105464. Epub 2020 Mar 2.

Great Lakes Toxicology and Ecology Division, US Environmental Protection Agency, 6201 Congdon Blvd., Duluth, MN, 55804, USA.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing allows for the disruption or modification of genes in a multitude of model organisms. In the present study, we describe and employ the method for use in the fathead minnow (Pimephales promelas), in part, to assist in the development and validation of adverse outcome pathways (AOPs). The gene coding for an enzyme responsible for melanin production, tyrosinase (tyr), was the initial target chosen for development and assessment of the method since its disruption results in abnormal pigmentation, a phenotype obvious within 3-4 d after injection of fathead minnow embryos. Three tyrosinase-targeting guide strands were generated using the fathead minnow sequence in tandem with the CRISPOR guide strand selection tool. The strands targeted two areas: one stretch of sequence in a conserved region that demonstrated homology to EGF-like or laminin-like domains as determined by Protein Basic Local Alignment Search Tool in concert with the Conserved Domain Database, and a second area in the N-terminal region of the tyrosinase domain. To generate one cell embryos, in vitro fertilization was performed, allowing for microinjection of hundreds of developmentally-synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Altered retinal pigmentation was observed in a portion of the tyr guide strand injected population within 3 d post fertilization (dpf). By 14 dpf, fish without skin and swim bladder pigmentation were observed. Among the three guide strands injected, the guide targeting the EGF/laminin-like domain was most effective in generating mutants. CRISPR greatly advances our ability to directly investigate gene function in fathead minnow, allowing for advanced approaches to AOP validation and development.
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http://dx.doi.org/10.1016/j.aquatox.2020.105464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280908PMC
May 2020

Adverse Outcome Pathway Network-Based Assessment of the Interactive Effects of an Androgen Receptor Agonist and an Aromatase Inhibitor on Fish Endocrine Function.

Environ Toxicol Chem 2020 04 21;39(4):913-922. Epub 2020 Feb 21.

Great Lakes Toxicology and Ecology Division, US Environmental Protection Agency, Duluth, Minnesota, USA.

Predictive approaches to assessing the toxicity of contaminant mixtures have been largely limited to chemicals that exert effects through the same biological molecular initiating event. However, by understanding specific pathways through which chemicals exert effects, it may be possible to identify shared "downstream" nodes as the basis for forecasting interactive effects of chemicals with different molecular initiating events. Adverse outcome pathway (AOP) networks conceptually support this type of analysis. We assessed the utility of a simple AOP network for predicting the effects of mixtures of an aromatase inhibitor (fadrozole) and an androgen receptor agonist (17β-trenbolone) on aspects of reproductive endocrine function in female fathead minnows. The fish were exposed to multiple concentrations of fadrozole and 17β-trenbolone individually or in combination for 48 or 96 h. Effects on 2 shared nodes in the AOP network, plasma 17β-estradiol (E2) concentration and vitellogenin (VTG) production (measured as hepatic vtg transcripts) responded as anticipated to fadrozole alone but were minimally impacted by 17β-trenbolone alone. Overall, there were indications that 17β-trenbolone enhanced decreases in E2 and vtg in fadrozole-exposed fish, as anticipated, but the results often were not statistically significant. Failure to consistently observe hypothesized interactions between fadrozole and 17β-trenbolone could be due to several factors, including lack of impact of 17β-trenbolone, inherent biological variability in the endpoints assessed, and/or an incomplete understanding of interactions (including feedback) between different pathways within the hypothalamic-pituitary-gonadal axis. Environ Toxicol Chem 2020;39:913-922. © 2020 SETAC.
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http://dx.doi.org/10.1002/etc.4668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357796PMC
April 2020

De Facto Water Reuse: Bioassay suite approach delivers depth and breadth in endocrine active compound detection.

Sci Total Environ 2020 Jan 4;699:134297. Epub 2019 Sep 4.

U.S. Environmental Protection Agency, National Health and Environmental Effects Laboratory, Research Triangle Park, NC, United States of America. Electronic address:

Although endocrine disrupting compounds have been detected in wastewater and surface waters worldwide using a variety of in vitro effects-based screening tools, e.g. bioassays, few have examined potential attenuation of environmental contaminants by both natural (sorption, degradation, etc.) and anthropogenic (water treatment practices) processes. This study used several bioassays and quantitative chemical analyses to assess residence-time weighted samples at six sites along a river in the northeastern United States beginning upstream from a wastewater treatment plant outfall and proceeding downstream along the stream reach to a drinking water treatment plant. Known steroidal estrogens were quantified and changes in signaling pathway molecular initiating events (activation of estrogen, androgen, glucocorticoid, peroxisome proliferator-activated, pregnane X receptor, and aryl hydrocarbon receptor signaling networks) were identified in water extracts. In initial multi-endpoint assays geographic and receptor-specific endocrine activity patterns in transcription factor signatures and nuclear receptor activation were discovered. In subsequent single endpoint receptor-specific bioassays, estrogen (16 of 18 samples; 0.01 to 28 ng estradiol equivalents [E2Eqs]/L) glucocorticoid (3 of 18 samples; 1.8 to 21 ng dexamethasone equivalents [DexEqs]/L), and androgen (2 of 18 samples; 0.95 to 2.1 ng dihydrotestosterone equivalents [DHTEqs]/L) receptor transcriptional activation occurred above respective assay method detection limits (0.04 ng E2Eqs/L, 1.2 ng DexEqs/L, and 0.77 ng DHTEqs/L) in multiple sampling events. Estrogen activity, the most often detected, correlated well with measured concentrations of known steroidal estrogens (r = 0.890). Overall, activity indicative of multiple types of endocrine active compounds was highest in wastewater effluent samples, while activity downstream was progressively lower, and negligible in unfinished treated drinking water. Not only was estrogenic and glucocorticoid activity confirmed in the effluent by utilizing multiple methods concurrently, but other activated signaling networks that historically received less attention (i.e. peroxisome proliferator-activated receptor) were also detected.
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http://dx.doi.org/10.1016/j.scitotenv.2019.134297DOI Listing
January 2020

Prioritizing chemicals of ecological concern in Great Lakes tributaries using high-throughput screening data and adverse outcome pathways.

Sci Total Environ 2019 Oct 5;686:995-1009. Epub 2019 Jun 5.

U.S. Geological Survey, Boise, ID 83702, United States.

Chemical monitoring data were collected in surface waters from 57 Great Lakes tributaries from 2010 to 13 to identify chemicals of potential biological relevance and sites at which these chemicals occur. Traditional water-quality benchmarks for aquatic life based on in vivo toxicity data were available for 34 of 67 evaluated chemicals. To expand evaluation of potential biological effects, measured chemical concentrations were compared to chemical-specific biological activities determined in high-throughput (ToxCast) in vitro assays. Resulting exposure-activity ratios (EARs) were used to prioritize the chemicals of greatest potential concern: 4‑nonylphenol, bisphenol A, metolachlor, atrazine, DEET, caffeine, tris(2‑butoxyethyl) phosphate, tributyl phosphate, triphenyl phosphate, benzo(a)pyrene, fluoranthene, and benzophenone. Water-quality benchmarks were unavailable for five of these chemicals, but for the remaining seven, EAR-based prioritization was consistent with that based on toxicity quotients calculated from benchmarks. Water-quality benchmarks identified three additional PAHs (anthracene, phenanthrene, and pyrene) not prioritized using EARs. Through this analysis, an EAR of 10 was identified as a reasonable threshold above which a chemical might be of potential concern. To better understand apical hazards potentially associated with biological activities captured in ToxCast assays, in vitro bioactivity data were matched with available adverse outcome pathway (AOP) information. The 49 ToxCast assays prioritized via EAR analysis aligned with 23 potentially-relevant AOPs present in the AOP-Wiki. Mixture effects at monitored sites were estimated by summation of EAR values for multiple chemicals by individual assay or individual AOP. Commonly predicted adverse outcomes included impacts on reproduction and mitochondrial function. The EAR approach provided a screening-level assessment for evidence-based prioritization of chemicals and sites with potential for adverse biological effects. The approach aids prioritization of future monitoring activities and provides testable hypotheses to help focus those efforts. This also expands the fraction of detected chemicals for which biologically-based benchmark concentrations are available to help contextualize chemical monitoring results.
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http://dx.doi.org/10.1016/j.scitotenv.2019.05.457DOI Listing
October 2019

Quantitative Response-Response Relationships Linking Aromatase Inhibition to Decreased Fecundity are Conserved Across Three Fishes with Asynchronous Oocyte Development.

Environ Sci Technol 2019 Sep 14;53(17):10470-10478. Epub 2019 Aug 14.

Mid-Continent Ecology Division , U.S. Environmental Protection Agency , Duluth , Minnesota 55804 United States.

Quantitative adverse outcome pathways (qAOPs) describe quantitative response-response relationships that can predict the probability or severity of an adverse outcome for a given magnitude of chemical interaction with a molecular initiating event. However, the taxonomic domain of applicability for these predictions is largely untested. The present study began defining this applicability for a previously described qAOP for aromatase inhibition leading to decreased fecundity developed using data from fathead minnow (). This qAOP includes quantitative response-response relationships describing plasma 17β-estradiol (E2) as a function of plasma fadrozole, plasma vitellogenin (VTG) as a function of plasma E2, and fecundity as a function of plasma VTG. These quantitative response-response relationships simulated plasma E2, plasma VTG, and fecundity measured in female zebrafish () exposed to fadrozole for 21 days but not these responses measured in female Japanese medaka (). However, Japanese medaka had different basal levels of plasma E2, plasma VTG, and fecundity. Normalizing basal levels of each measurement to equal those of female fathead minnow enabled the relationships to accurately simulate plasma E2, plasma VTG, and fecundity measured in female Japanese medaka. This suggests that these quantitative response-response relationships are conserved across these three fishes when considering relative change rather than absolute measurements. The present study represents an early step toward defining the appropriate taxonomic domain of applicability and extending the regulatory applications of this qAOP.
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http://dx.doi.org/10.1021/acs.est.9b02606DOI Listing
September 2019

Predictive Analysis Using Chemical-Gene Interaction Networks Consistent with Observed Endocrine Activity and Mutagenicity of U.S. Streams.

Environ Sci Technol 2019 Aug 22;53(15):8611-8620. Epub 2019 Jul 22.

South Atlantic Water Science Center , U.S. Geological Survey , Columbia , South Carolina 29210 , United States.

In a recent U.S. Geological Survey/U.S. Environmental Protection Agency study assessing more than 700 organic compounds in 38 streams, assays indicated generally low estrogen, androgen, and glucocorticoid receptor activities, with 13 surface waters with 17β-estradiol-equivalent (E2Eq) activities greater than a 1-ng/L estimated effects-based trigger value for estrogenic effects in male fish. Among the 36 samples assayed for mutagenicity in the bioassay (reported here), 25% had low mutagenic activity and 75% were not mutagenic. Endocrine and mutagenic activities of the water samples were well correlated with each other and with the total number and cumulative concentrations of detected chemical contaminants. To test the predictive utility of knowledge-base-leveraging approaches, site-specific predicted chemical-gene (pCGA) and predicted analogous pathway-linked (pPLA) association networks identified in the Comparative Toxicogenomics Database were compared with observed endocrine/mutagenic bioactivities. We evaluated pCGA/pPLA patterns among sites by cluster analysis and principal component analysis and grouped the pPLA into broad mode-of-action classes. Measured E2eq and mutagenic activities correlated well with predicted pathways. The pPLA analysis also revealed correlations with signaling, metabolic, and regulatory groups, suggesting that other effects pathways may be associated with chemical contaminants in these waters and indicating the need for broader bioassay coverage to assess potential adverse impacts.
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http://dx.doi.org/10.1021/acs.est.9b02990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770991PMC
August 2019

Differential Sensitivity to In Vitro Inhibition of Cytochrome P450 Aromatase (CYP19) Activity Among 18 Freshwater Fishes.

Toxicol Sci 2019 08;170(2):394-403

Mid-Continent Ecology Division.

There is significant concern regarding potential impairment of fish reproduction associated with endocrine disrupting chemicals. Aromatase (CYP19) is a steroidogenic enzyme involved in the conversion of androgens to estrogens. Inhibition of aromatase by chemicals can result in reduced concentrations of estrogens leading to adverse reproductive effects. These effects have been extensively investigated in a small number of laboratory model fishes, such as fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio). But, differences in sensitivity among species are largely unknown. Therefore, this study took a first step toward understanding potential differences in sensitivity to aromatase inhibitors among fishes. Specifically, a standard in vitro aromatase inhibition assay using subcellular fractions of whole tissue homogenates was used to evaluate the potential sensitivity of 18 phylogenetically diverse species of freshwater fish to the nonsteroidal aromatase inhibitor fadrozole. Sensitivity to fadrozole ranged by more than 52-fold among these species. Five species were further investigated for sensitivity to up to 4 additional nonsteroidal aromatase inhibitors, letrozole, imazalil, prochloraz, and propiconazole. Potencies of each of these chemicals relative to fadrozole ranged by up to 2 orders of magnitude among the 5 species. Fathead minnow, Japanese medaka, and zebrafish were among the least sensitive to all the investigated chemicals; therefore, ecological risks of aromatase inhibitors derived from these species might not be adequately protective of more sensitive native fishes. This information could guide more objective ecological risk assessments of native fishes to chemicals that inhibit aromatase.
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http://dx.doi.org/10.1093/toxsci/kfz115DOI Listing
August 2019

The Next Generation Blueprint of Computational Toxicology at the U.S. Environmental Protection Agency.

Toxicol Sci 2019 06;169(2):317-332

National Center for Computational Toxicology, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.

The U.S. Environmental Protection Agency (EPA) is faced with the challenge of efficiently and credibly evaluating chemical safety often with limited or no available toxicity data. The expanding number of chemicals found in commerce and the environment, coupled with time and resource requirements for traditional toxicity testing and exposure characterization, continue to underscore the need for new approaches. In 2005, EPA charted a new course to address this challenge by embracing computational toxicology (CompTox) and investing in the technologies and capabilities to push the field forward. The return on this investment has been demonstrated through results and applications across a range of human and environmental health problems, as well as initial application to regulatory decision-making within programs such as the EPA's Endocrine Disruptor Screening Program. The CompTox initiative at EPA is more than a decade old. This manuscript presents a blueprint to guide the strategic and operational direction over the next 5 years. The primary goal is to obtain broader acceptance of the CompTox approaches for application to higher tier regulatory decisions, such as chemical assessments. To achieve this goal, the blueprint expands and refines the use of high-throughput and computational modeling approaches to transform the components in chemical risk assessment, while systematically addressing key challenges that have hindered progress. In addition, the blueprint outlines additional investments in cross-cutting efforts to characterize uncertainty and variability, develop software and information technology tools, provide outreach and training, and establish scientific confidence for application to different public health and environmental regulatory decisions.
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http://dx.doi.org/10.1093/toxsci/kfz058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542711PMC
June 2019

Extracting and Benchmarking Emerging Adverse Outcome Pathway Knowledge.

Toxicol Sci 2019 04;168(2):349-364

Ecotoxicology and Wildlife Health Division, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Raven Road, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6 Canada.

As the community of toxicological researchers, risk assessors, and risk managers adopt the adverse outcome pathway (AOP) framework for organizing toxicological knowledge, the number and diversity of AOPs in the online AOP knowledgebase (KB) continues to grow. To track and investigate this growth, AOPs in the AOP-KB were assembled into a single network. Summary measures on the current state of the AOP-KB and the overall connectivity and structural features of the resulting network were calculated. Our results show that networking the 187 user-defined AOPs currently described in the AOP-KB resulted in the emergence of 9405 unique, previously undescribed, linear AOPs (LAOPs). To investigate patterns in this emerging knowledge, we assembled the AOP-KB network retrospectively by sequentially adding each of the 187 user-defined AOPs and found that the creation of new AOPs that borrowed components from previously existing AOPs in the KB most described emergence of new LAOPs. However, the introduction of nonadjacent key event relationships and cycles among KEs also play key roles in emergent LAOPs. We provide examples of how to identify application-specific critical paths from this large number of LAOPs. Our research shows that the global AOP network may have considerable value as a source of emergent toxicological knowledge. These findings are not only helpful for understanding the nature of this emergent information but can also be used to manage and guide future development of the AOP-KB, and how to tailor this wealth of information to specific applications.
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http://dx.doi.org/10.1093/toxsci/kfz006DOI Listing
April 2019

High-throughput screening and environmental risk assessment: State of the science and emerging applications.

Environ Toxicol Chem 2019 01 20;38(1):12-26. Epub 2018 Dec 20.

Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, Mississippi.

In 2007 the United States National Research Council (NRC) published a vision for toxicity testing in the 21st century that emphasized the use of in vitro high-throughput screening (HTS) methods and predictive models as an alternative to in vivo animal testing. In the present study we examine the state of the science of HTS and the progress that has been made in implementing and expanding on the NRC vision, as well as challenges to implementation that remain. Overall, significant progress has been made with regard to the availability of HTS data, aggregation of chemical property and toxicity information into online databases, and the development of various models and frameworks to support extrapolation of HTS data. However, HTS data and associated predictive models have not yet been widely applied in risk assessment. Major barriers include the disconnect between the endpoints measured in HTS assays and the assessment endpoints considered in risk assessments as well as the rapid pace at which new tools and models are evolving in contrast with the slow pace at which regulatory structures change. Nonetheless, there are opportunities for environmental scientists and policymakers alike to take an impactful role in the ongoing development and implementation of the NRC vision. Six specific areas for scientific coordination and/or policy engagement are identified. Environ Toxicol Chem 2019;38:12-26. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.
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http://dx.doi.org/10.1002/etc.4315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698360PMC
January 2019

Potential Toxicity of Complex Mixtures in Surface Waters from a Nationwide Survey of United States Streams: Identifying in Vitro Bioactivities and Causative Chemicals.

Environ Sci Technol 2019 01 21;53(2):973-983. Epub 2018 Dec 21.

U.S. EPA, Mid-Continent Ecology Division , 6201 Congdon Boulevard , Duluth , Minnesota 55804 , United States.

While chemical analysis of contaminant mixtures remains an essential component of environmental monitoring, bioactivity-based assessments using in vitro systems increasingly are used in the detection of biological effects. Historically, in vitro assessments focused on a few biological pathways, for example, aryl hydrocarbon receptor (AhR) or estrogen receptor (ER) activities. High-throughput screening (HTS) technologies have greatly increased the number of biological targets and processes that can be rapidly assessed. Here we screened extracts of surface waters from a nationwide survey of United States streams for bioactivities associated with 69 different end points using two multiplexed HTS assays. Bioactivity of extracts from 38 streams was evaluated and compared with concentrations of over 700 analytes to identify chemicals contributing to observed effects. Eleven primary biological end points were detected. Pregnane X receptor (PXR) and AhR-mediated activities were the most commonly detected. Measured chemicals did not completely account for AhR and PXR responses. Surface waters with AhR and PXR effects were associated with low intensity, developed land cover. Likewise, elevated bioactivities frequently associated with wastewater discharges included endocrine-related end points ER and glucocorticoid receptor. These results underscore the value of bioassay-based monitoring of environmental mixtures for detecting biological effects that could not be ascertained solely through chemical analyses.
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http://dx.doi.org/10.1021/acs.est.8b05304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467772PMC
January 2019

Evidence for Cross Species Extrapolation of Mammalian-Based High-Throughput Screening Assay Results.

Environ Sci Technol 2018 12 13;52(23):13960-13971. Epub 2018 Nov 13.

Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division , US Environmental Protection Agency , 6201 Congdon Blvd. , Duluth , Minnesota 55804 , United States.

High-throughput screening (HTS) and computational technologies have emerged as important tools for chemical hazard identification. The US Environmental Protection Agency (EPA) launched the Toxicity ForeCaster (ToxCast) Program, which has screened thousands of chemicals in hundreds of mammalian-based HTS assays for biological activity. The data are being used to prioritize toxicity testing on those chemicals likely to lead to adverse effects. To use HTS assays in predicting hazard to both humans and wildlife, it is necessary to understand how broadly these data may be extrapolated across species. The US EPA Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS; https://seqapass.epa.gov/seqapass/ ) tool was used to assess conservation of the 484 protein targets represented in the suite of ToxCast assays and other HTS assays. To demonstrate the utility of the SeqAPASS data for guiding extrapolation, case studies were developed which focused on targets of interest to the US Endocrine Disruptor Screening Program and the Organisation for Economic Cooperation and Development. These case studies provide a line of evidence for conservation of endocrine targets across vertebrate species, with few exceptions, and demonstrate the utility of SeqAPASS for defining the taxonomic domain of applicability for HTS results and identifying organisms for suitable follow-up toxicity tests.
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http://dx.doi.org/10.1021/acs.est.8b04587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8283686PMC
December 2018

Representing the Process of Inflammation as Key Events in Adverse Outcome Pathways.

Toxicol Sci 2018 06;163(2):346-352

Department of Physiology, University of Lausanne, CH 1005 Lausanne, Switzerland.

Inflammation is an important biological process involved in many target organ toxicities. However, there has been little consensus on how to represent inflammatory processes using the adverse outcome pathway (AOP) framework. In particular, there were concerns that inflammation was not being represented in a way that it would be recognized as a highly connected, central node within the global AOP network. The consideration of salient features common to the inflammatory process across tissues was used as a basis to propose 3 hub key events (KEs) for use in AOP network development. Each event, "tissue resident cell activation", "increased pro-inflammatory mediators", and "leukocyte recruitment/activation," is viewed as a hallmark of inflammation, independent of tissue, and can be independently measured. Using these proposed hub KEs, it was possible to link together a series of AOPs that previously had no shared KEs. Significant challenges remain with regard to accurate prediction of inflammation-related toxicological outcomes even if a broader and more connected network of inflammation-centered AOPs is developed. Nonetheless, the current proposal addresses one of the major hurdles associated with representation of inflammation in AOPs and may aid fit-for-purpose evaluations of other AOPs operating in a network context.
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http://dx.doi.org/10.1093/toxsci/kfy047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309953PMC
June 2018

The advantages of linear concentration-response curves for in vitro bioassays with environmental samples.

Environ Toxicol Chem 2018 09 11;37(9):2273-2280. Epub 2018 Jul 11.

Mid-Continent Ecology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Duluth, Minnesota.

In vitro assays and high-throughput screening (HTS) tools are increasingly being employed as replacements for animal testing, but most concentration-response curves are still evaluated with models developed for animal testing. We argue that application of in vitro assays, particularly reporter gene assays, to environmental samples can benefit from a different approach to concentration-response modeling. First, cytotoxicity often occurs at higher concentrations, especially for weakly acting compounds and in complex environmental mixtures with many components. In these cases, specific effects can be masked by cytotoxicity. Second, for many HTS assays, low effect levels can be precisely quantified because of the low variability of controls in cell-based assays and the opportunity to run many concentrations and replicates when using high-density well-plate formats (e.g., 384 or more wells per plate). Hence, we recommend focusing concentration-response modeling on the lower portion of the concentration-response curve, which is approximately linear. Effect concentrations derived from low-effect level linear concentration-response models facilitate simple derivation of relative effect potencies and the correct application of mixture toxicity models in the calculation of bioanalytical equivalent concentrations. Environ Toxicol Chem 2018;37:2273-2280. © 2018 SETAC.
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http://dx.doi.org/10.1002/etc.4178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150494PMC
September 2018

Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish.

Gen Comp Endocrinol 2018 09 4;266:87-100. Epub 2018 May 4.

University of Antwerp, Zebrafishlab, Veterinary Physiology and Biochemistry, Dept. Veterinary Sciences, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

The hypothalamic-pituitary-thyroid (HPT) axis is known to play a crucial role in the development of teleost fish. However, knowledge of endogenous transcription profiles of thyroid-related genes in developing teleosts remains fragmented. We selected two model teleost species, the fathead minnow (Pimephales promelas) and the zebrafish (Danio rerio), to compare the gene transcription ontogeny of the HPT axis. Control organisms were sampled at several time points during embryonic and larval development until 33 days post-fertilization. Total RNA was extracted from pooled, whole fish, and thyroid-related mRNA expression was evaluated using quantitative polymerase chain reaction. Gene transcripts examined included: thyrotropin-releasing hormone receptor (trhr), thyroid-stimulating hormone receptor (tshr), sodium-iodide symporter (nis), thyroid peroxidase (tpo), thyroglobulin (tg), transthyretin (ttr), deiodinases 1, 2, 3a, and 3b (dio1, dio2, dio3a and 3b), and thyroid hormone receptors alpha and beta (thrα and β). A loess regression method was successful in identifying maxima and minima of transcriptional expression during early development of both species. Overall, we observed great similarities between the species, including maternal transfer, at least to some extent, of almost all transcripts (confirmed in unfertilized eggs), increasing expression of most transcripts during hatching and embryo-larval transition, and indications of a fully functional HPT axis in larvae. These data will aid in the development of hypotheses on the role of certain genes and pathways during development. Furthermore, this provides a background reference dataset for designing and interpreting targeted transcriptional expression studies both for fundamental research and for applications such as toxicology.
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http://dx.doi.org/10.1016/j.ygcen.2018.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540109PMC
September 2018

Evaluation of targeted and untargeted effects-based monitoring tools to assess impacts of contaminants of emerging concern on fish in the South Platte River, CO.

Environ Pollut 2018 Aug 30;239:706-713. Epub 2018 Apr 30.

U.S. EPA, National Exposure Research Laboratory, 960 College Station Rd., Athens, GA 30605, USA. Electronic address:

Rivers in the arid Western United States face increasing influences from anthropogenic contaminants due to population growth, urbanization, and drought. To better understand and more effectively track the impacts of these contaminants, biologically-based monitoring tools are increasingly being used to complement routine chemical monitoring. This study was initiated to assess the ability of both targeted and untargeted biologically-based monitoring tools to discriminate impacts of two adjacent wastewater treatment plants (WWTPs) on Colorado's South Platte River. A cell-based estrogen assay (in vitro, targeted) determined that water samples collected downstream of the larger of the two WWTPs displayed considerable estrogenic activity in its two separate effluent streams. Hepatic vitellogenin mRNA expression (in vivo, targeted) and NMR-based metabolomic analyses (in vivo, untargeted) from caged male fathead minnows also suggested estrogenic activity downstream of the larger WWTP, but detected significant differences in responses from its two effluent streams. The metabolomic results suggested that these differences were associated with oxidative stress levels. Finally, partial least squares regression was used to explore linkages between the metabolomics responses and the chemical contaminants that were detected at the sites. This analysis, along with univariate statistical approaches, identified significant covariance between the biological endpoints and estrone concentrations, suggesting the importance of this contaminant and recommending increased focus on its presence in the environment. These results underscore the benefits of a combined targeted and untargeted biologically-based monitoring strategy when used alongside contaminant monitoring to more effectively assess ecological impacts of exposures to complex mixtures in surface waters.
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http://dx.doi.org/10.1016/j.envpol.2018.04.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6147041PMC
August 2018

An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish.

Aquat Toxicol 2018 Jul 21;200:1-12. Epub 2018 Apr 21.

Zebrafishlab, Veterinary Physiology and Biochemistry, Department of Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. Electronic address:

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.
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http://dx.doi.org/10.1016/j.aquatox.2018.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002951PMC
July 2018

Differentiating Pathway-Specific From Nonspecific Effects in High-Throughput Toxicity Data: A Foundation for Prioritizing Adverse Outcome Pathway Development.

Toxicol Sci 2018 06;163(2):500-515

U.S. EPA National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804.

The U.S. Environmental Protection Agency's ToxCast program has screened thousands of chemicals for biological activity, primarily using high-throughput in vitro bioassays. Adverse outcome pathways (AOPs) offer a means to link pathway-specific biological activities with potential apical effects relevant to risk assessors. Thus, efforts are underway to develop AOPs relevant to pathway-specific perturbations detected in ToxCast assays. Previous work identified a "cytotoxic burst" (CTB) phenomenon wherein large numbers of the ToxCast assays begin to respond at or near test chemical concentrations that elicit cytotoxicity, and a statistical approach to defining the bounds of the CTB was developed. To focus AOP development on the molecular targets corresponding to ToxCast assays indicating pathway-specific effects, we conducted a meta-analysis to identify which assays most frequently respond at concentrations below the CTB. A preliminary list of potentially important, target-specific assays was determined by ranking assays by the fraction of chemical hits below the CTB compared with the number of chemicals tested. Additional priority assays were identified using a diagnostic-odds-ratio approach which gives greater ranking to assays with high specificity but low responsivity. Combined, the two prioritization methods identified several novel targets (e.g., peripheral benzodiazepine and progesterone receptors) to prioritize for AOP development, and affirmed the importance of a number of existing AOPs aligned with ToxCast targets (e.g., thyroperoxidase, estrogen receptor, aromatase). The prioritization approaches did not appear to be influenced by inter-assay differences in chemical bioavailability. Furthermore, the outcomes were robust based on a variety of different parameters used to define the CTB.
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http://dx.doi.org/10.1093/toxsci/kfy049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6820004PMC
June 2018

Adverse outcome pathway networks II: Network analytics.

Environ Toxicol Chem 2018 06 7;37(6):1734-1748. Epub 2018 May 7.

Zebrafishlab, Veterinary Physiology and Biochemistry, University of Antwerp, Wilrijk, Belgium.

Toxicological responses to stressors are more complex than the simple one-biological-perturbation to one-adverse-outcome model portrayed by individual adverse outcome pathways (AOPs). Consequently, the AOP framework was designed to facilitate de facto development of AOP networks that can aid in the understanding and prediction of pleiotropic and interactive effects more common to environmentally realistic, complex exposure scenarios. The present study introduces nascent concepts related to the qualitative analysis of AOP networks. First, graph theory-based approaches for identifying important topological features are illustrated using 2 example AOP networks derived from existing AOP descriptions. Second, considerations for identifying the most significant path(s) through an AOP network from either a biological or risk assessment perspective are described. Finally, approaches for identifying interactions among AOPs that may result in additive, synergistic, or antagonistic responses (or previously undefined emergent patterns of response) are introduced. Along with a companion article (part I), these concepts set the stage for the development of tools and case studies that will facilitate more rigorous analysis of AOP networks, and the utility of AOP network-based predictions, for use in research and regulatory decision-making. The present study addresses one of the major themes identified through a Society of Environmental Toxicology and Chemistry Horizon Scanning effort focused on advancing the AOP framework. Environ Toxicol Chem 2018;37:1734-1748. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.
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http://dx.doi.org/10.1002/etc.4124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010347PMC
June 2018

Adverse outcome pathway networks I: Development and applications.

Environ Toxicol Chem 2018 06 7;37(6):1723-1733. Epub 2018 May 7.

Mid-Continent Ecology Division, US Environmental Protection Agency, Duluth, Minnesota, USA.

Based on the results of a Horizon Scanning exercise sponsored by the Society of Environmental Toxicology and Chemistry that focused on advancing the adverse outcome pathway (AOP) framework, the development of guidance related to AOP network development was identified as a critical need. This not only included questions focusing directly on AOP networks, but also on related topics such as mixture toxicity assessment and the implementation of feedback loops within the AOP framework. A set of two articles has been developed to begin exploring these concepts. In the present article (part I), we consider the derivation of AOP networks in the context of how it differs from the development of individual AOPs. We then propose the use of filters and layers to tailor AOP networks to suit the needs of a given research question or application. We briefly introduce a number of analytical approaches that may be used to characterize the structure of AOP networks. These analytical concepts are further described in a dedicated, complementary article (part II). Finally, we present a number of case studies that illustrate concepts underlying the development, analysis, and application of AOP networks. The concepts described in the present article and in its companion article (which focuses on AOP network analytics) are intended to serve as a starting point for further development of the AOP network concept, and also to catalyze AOP network development and application by the different stakeholder communities. Environ Toxicol Chem 2018;37:1723-1733. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.
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http://dx.doi.org/10.1002/etc.4125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004608PMC
June 2018

A Reduced Transcriptome Approach to Assess Environmental Toxicants Using Zebrafish Embryo Test.

Environ Sci Technol 2018 01 2;52(2):821-830. Epub 2018 Jan 2.

State Key Laboratory of Pollution Control & Resource Reuse, School of the Environment, Nanjing University , Nanjing, P. R. China , 210023.

Omics approaches can monitor responses and alterations of biological pathways at genome-scale, which are useful to predict potential adverse effects by environmental toxicants. However, high throughput application of transcriptomics in chemical assessment is limited due to the high cost and lack of "standardized" toxicogenomic methods. Here, a reduced zebrafish transcriptome (RZT) approach was developed to represent the whole transcriptome and to profile bioactivity of chemical and environmental mixtures in zebrafish embryo. RZT gene set of 1637 zebrafish Entrez genes was designed to cover a wide range of biological processes, and to faithfully capture gene-level and pathway-level changes by toxicants compared with the whole transcriptome. Concentration-response modeling was used to calculate the effect concentrations (ECs) of DEGs and corresponding molecular pathways. To validate the RZT approach, quantitative analysis of gene expression by RNA-ampliseq technology was used to identify differentially expressed genes (DEGs) at 32 hpf following exposure to seven serial dilutions of reference chemical BPA (10-10EμM) or each of four water samples ranging from wastewater to drinking water (relative enrichment factors 10-6.4 × 10). The RZT-ampliseq-embryo approach was both sensitive and able to identify a wide spectrum of biological activities associated with BPA exposure. Water quality was benchmarked based on the sensitivity distribution curve of biological pathways detected using RZT-ampliseq-embryo. Finally, the most sensitive biological pathways were identified, including those linked with adverse reproductive outcomes, genotoxicity and development outcomes. RZT-ampliseq-embryo approach provides an efficient and cost-effective tool to prioritize toxicants based on responsiveness of biological pathways.
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http://dx.doi.org/10.1021/acs.est.7b04073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839301PMC
January 2018

High-resolution mass spectrometry of skin mucus for monitoring physiological impacts and contaminant biotransformation products in fathead minnows exposed to wastewater effluent.

Environ Toxicol Chem 2018 03 8;37(3):788-796. Epub 2017 Dec 8.

National Exposure Research Laboratory, US Environmental Protection Agency, Athens, Georgia.

High-resolution mass spectrometry is advantageous for monitoring physiological impacts and contaminant biotransformation products in fish exposed to complex wastewater effluent. We evaluated this technique using skin mucus from male and female fathead minnows (Pimephales promelas) exposed to control water or treated wastewater effluent at 5, 20, and 100% levels for 21 d, using an on-site, flow-through system providing real-time exposure. Both sex-specific and non-sex-specific responses were observed in the mucus metabolome, the latter suggesting the induction of general compensatory pathways for xenobiotic exposures. Altogether, 85 statistically significant treatment-dependent metabolite changes were observed out of the 310 total endogenous metabolites that were detected (156 of the 310 were annotated). Partial least squares-regression models revealed strong covariances between the mucus metabolomes and up-regulated hepatic messenger ribonucleic acid (mRNA) transcripts reported previously for these same fish. These regression models suggest that mucus metabolomic changes reflected, in part, processes by which the fish biotransformed xenobiotics in the effluent. In keeping with this observation, we detected a phase II transformation product of bisphenol A in the skin mucus of male fish. Collectively, these findings demonstrate the utility of mucus as a minimally invasive matrix for simultaneously assessing exposures and effects of environmentally relevant mixtures of contaminants. Environ Toxicol Chem 2018;37:788-796. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.
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http://dx.doi.org/10.1002/etc.4003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061956PMC
March 2018

First-generation annotations for the fathead minnow (Pimephales promelas) genome.

Environ Toxicol Chem 2017 Dec 29;36(12):3436-3442. Epub 2017 Aug 29.

Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, US Environmental Protection Agency, Duluth, Minnesota.

Ab initio gene prediction and evidence alignment were used to produce the first annotations for the fathead minnow (Pimephales promelas) genome. We also describe a genome browser, hosted by the Society of Environmental Toxicology and Chemistry, that provides simplified access to the annotation data in context with the genomic sequence. The present study extends the utility of the fathead minnow genome and supports the continued development of this species as a model organism for predictive toxicology. Environ Toxicol Chem 2017;36:3436-3442. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.
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http://dx.doi.org/10.1002/etc.3929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733733PMC
December 2017
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