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Tanguay, Robyn L.

Publications and source records attributed to Tanguay, Robyn L..

10 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Aryl hydrocarbon receptor-dependent toxicity by retene requires metabolic competence

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds frequently detected in the environment with widely varying toxicities. Many PAHs activate the aryl hydrocarbon receptor (AHR), inducing the expression of a battery of genes, including xenobiotic metabolizing enzymes like cytochrome P450s (CYPs); however, not all PAHs act via this mechanism. We screened several parent and substituted PAHs in in vitro AHR activation assays to classify their unique activity. Retene (1-methyl-7-isopropylphenanthrene) displays Ahr2-dependent teratogenicity in zebrafish, but did not activate human AHR or zebrafish Ahr2, suggesting a retene metabolite activates Ahr2 in zebrafish to induce developmental toxicity. To investigate the role of metabolism in retene toxicity, studies were performed to determine the functional role of cyp1a, cyp1b1, and the microbiome in retene toxicity, identify the zebrafish window of susceptibility, and measure retene uptake, loss, and metabolite formation in vivo. Cyp1a-null fish were generated using CRISPR-Cas9. Cyp1a-null fish showed increased sensitivity to retene toxicity, whereas Cyp1b1-null fish were less susceptible, and microbiome elimination had no significant effect. Zebrafish required exposure to retene between 24 and 48 hours post fertilization (hpf) to exhibit toxicity. After static exposure, retene concentrations in zebrafish embryos increased until 24 hpf, peaked between 24 and 36 hpf, and decreased rapidly thereafter. We detected retene metabolites at 36 and 48 hpf, indicating metabolic onset preceding toxicity. This study highlights the value of combining molecular and systems biology approaches with mechanistic and predictive toxicology to interrogate the role of biotransformation in AHR-dependent toxicity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Diverse PFAS produce unique transcriptomic changes linked to developmental toxicity in zebrafish

Per- and polyfluoroalkyl substances (PFAS) are a widespread and persistent class of contaminants posing significant environmental and human health concerns. Comprehensive understanding of the modes of action underlying toxicity among structurally diverse PFAS is mostly lacking. To address this need, we recently reported on our application of developing zebrafish to evaluate a large library of PFAS for developmental toxicity. In the present study, we prioritized 15 bioactive PFAS that induced significant morphological effects and performed RNA-sequencing to characterize early transcriptional responses at a single timepoint (48 h post fertilization) after early developmental exposures (8 h post fertilization). Internal concentrations of 5 of the 15 PFAS were measured from pooled whole fish samples across multiple timepoints between 24–120 h post fertilization, and additional temporal transcriptomics at several timepoints (48–96 h post fertilization) were conducted for Nafion byproduct 2. A broad range of differentially expressed gene counts were identified across the PFAS exposures. Most PFAS that elicited robust transcriptomic changes affected biological processes of the brain and nervous system development. While PFAS disrupted unique processes, we also found that similarities in some functional head groups of PFAS were associated with the disruption in expression of similar gene sets. Body burdens after early developmental exposures to select sulfonic acid PFAS, including Nafion byproduct 2, increased from the 24–96 h post fertilization sampling timepoints and were greater than those of sulfonamide PFAS of similar chain lengths. In parallel, the Nafion byproduct 2-induced transcriptional responses increased between 48 and 96 h post fertilization. PFAS characteristics based on toxicity, transcriptomic effects, and modes of action will contribute to further prioritization of PFAS structures for testing and informed hazard assessment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

3′ RNA-seq is superior to standard RNA-seq in cases of sparse data but inferior at identifying toxicity pathways in a model organism

The application of RNA-sequencing has led to numerous breakthroughs related to investigating gene expression levels in complex biological systems. Among these are knowledge of how organisms, such as the vertebrate model organism zebrafish (Danio rerio), respond to toxicant exposure. Recently, the development of 3' RNA-seq has allowed for the determination of gene expression levels with a fraction of the required reads compared to standard RNA-seq. While 3' RNA-seq has many advantages, a comparison to standard RNA-seq has not been performed in the context of whole organism toxicity and sparse data. Here, we examined samples from zebrafish exposed to perfluorobutane sulfonamide (FBSA) with either 3' or standard RNA-seq to determine the advantages of each with regards to the identification of functionally enriched pathways. We found that 3' and standard RNA-seq showed specific advantages when focusing on annotated or unannotated regions of the genome. We also found that standard RNA-seq identified more differentially expressed genes (DEGs), but that this advantage disappeared under conditions of sparse data. We also found that standard RNA-seq had a significant advantage in identifying functionally enriched pathways via analysis of DEG lists but that this advantage was minimal when identifying pathways via gene set enrichment analysis of all genes. These results show that each approach has experimental conditions where they may be advantageous. Our observations can help guide others in the choice of 3' RNA-seq vs standard RNA sequencing to query gene expression levels in a range of biological systems.

3’ RNA-seq↗
DOE OSTI2023

The Superfund Research Program Analytics Portal: linking environmental chemical exposure to biological phenotypes

The OSU/PNNL Superfund Research Program represents a longstanding collaboration to quantify Polycyclic Aromatic Hydrocarbons (PAHs) at various superfund sites in the Pacific Northwest and assess their potential impact on human health. To link the chemical measurements to biological activity, we describe the use of the zebrafish as a high-throughput developmental model of human exposure that provides quantitative measurements of the biological ramifications of exposure to toxicants. Toward this end, we have linked over 150 PAHs found at Superfund sites to the effect of these same chemicals in zebrafish, creating a rich dataset that links environmental exposure to biological response. To quantify this response, we have implemented a dose-response modelling pipeline to calculate benchmark dose parameters which enable potency comparison across over 500 chemicals and 10 zebrafish-specific phenotypes. Our portal provides public access to this dataset via an interactive web site designed to support exploration and re-use of these data by the scientific community at http://srp.pnnl.gov.

Gosline, Sara JC↗
DOE OSTI2023

Coupling Environmental Whole Mixture Toxicity Screening with Unbiased RNA-Seq Reveals Site-Specific Biological Responses in Zebrafish

Passive sampling device (PSD) extracts paired with developmental toxicity assays in Danio Rerio (zebrafish) are excellent sensors for whole mixture toxicity associated with the bioavailable non-polar organics at environmental sites. We expand this concept by incorporating RNA-Seq in 48-h post fertilization zebrafish statically exposed to PSD extracts from two Portland Harbor Superfund Site locations: river mile 6.5W (RM 6.5W) and river mile 7W (RM 7W). RM 6.5W contained higher concentrations of polycyclic aromatic hydrocarbons (PAHs), but the diagnostic ratios of both extracts indicated similar PAH sourcing and composition. Developmental screens determined RM 6.5W to be more toxic with the most sensitive endpoint being a “wavy” notochord malformation. Differential gene expression from exposure to both extracts was largely parallel, although more pronounced for RM 6.5W. When compared to the gene expression associated with individual chemical exposures, PSD extracts produced some gene signatures parallel to PAHs but were more closely matched by oxygenated-PAHs. Additionally, differential expression, reminiscent of the wavy notochord phenotype, was not accounted for by either class of chemical, indicating the potential of other contaminants driving mixture toxicity. These techniques offer a compelling method for non-targeted hazard characterization of whole mixtures in an in vivo vertebrate system without requiring complete chemical characterization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Concentration-response gene expression analysis in zebrafish reveals phenotypically-anchored transcriptional responses to retene

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants and are associated with human disease. Canonically, many PAHs induce toxicity via activation of the aryl hydrocarbon receptor (AHR) pathway. While the interaction between PAHs and the AHR is well-established, understanding which AHR-regulated transcriptional effects directly result in observable phenotypes and which are adaptive or benign is important to better understand PAH toxicity. Retene is a frequently detected PAH in environmental sampling and has been associated with AHR2-dependent developmental toxicity in zebrafish, though its mechanism of toxicity has not been fully elucidated. To interrogate transcriptional changes causally associated with retene toxicity, we conducted whole-animal RNA sequencing at 48 hours post-fertilization after exposure to eight retene concentrations. The concentrations were selected to produce effects ranging from no phenotype to mortality and malformations in 100% of animals at 5 days post-fertilization. We identified a concentration-response relationship between retene teratogenicity and differential gene expression in both number of DEGs and magnitude of expression change. Elevated expression of cyp1a at retene concentrations below the threshold for teratogenicity suggested that while cyp1a expression is a sensitive biomarker of AHR activation, it may be too sensitive to serve as a biomarker of AHR-dependent teratogenicity. Genes differentially expressed at only non-teratogenic concentrations were enriched for transforming growth factor-ß (TGF-ß) signaling pathway disruption while DEGs identified at only teratogenic concentrations were significantly enriched for response to xenobiotic stimulus and reduction-oxidation reaction activity. DEGs which spanned both non-teratogenic and teratogenic concentrations showed similar disrupted biological processes to those unique to teratogenic concentrations, indicating these processes were disrupted at low exposure concentrations. Gene co-expression network analysis identified several gene modules, including those associated with PAHs and AHR2 activation. One, Module 7, was strongly enriched for AHR2-associated genes and contained the strongest responses to retene. Benchmark concentration (BMC) of Module 7 genes identified a median BMC of 7.5 µM, nearly the highest retene concentration with no associated teratogenicity, supporting the hypothesis that Module 7 genes are largely responsible for retene toxicity.

Toxin, Zebrafish, Retene, Transcriptomics, network↗
DOE OSTI2022

The Ahr2-Dependent wfikkn1 Gene Influences Zebrafish Transcriptome, Proteome, and Behavior

The aryl hydrocarbon receptor (AHR) is required for vertebrate development and is also activated by exogenous chemicals, including polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR activation is well-understood, but roles of downstream molecular signaling events are largely unknown. From previous transcriptomics in 48 h postfertilization (hpf) zebrafish exposed to several PAHs and TCDD, we found wfikkn1 was highly coexpressed with cyp1a (marker for AHR activation). Thus, we hypothesized wfikkn1’s role in AHR signaling, and showed that wfikkn1 expression was Ahr2 (zebrafish ortholog of human AHR)-dependent in developing zebrafish exposed to TCDD. To functionally characterize wfikkn1, we made a CRISPR-Cas9 mutant line with a 16-bp deletion in wfikkn1’s exon, and exposed wildtype and mutants to dimethyl sulfoxide or TCDD. 48-hpf mRNA sequencing revealed over 700 genes that were differentially expressed (p < .05, log 2 FC > 1) between each pair of treatment combinations, suggesting an important role for wfikkn1 in altering both the 48-hpf transcriptome and TCDD-induced expression changes. Mass spectrometry-based proteomics of 48-hpf wildtype and mutants revealed 325 significant differentially expressed proteins. Functional enrichment demonstrated wfikkn1 was involved in skeletal muscle development and played a role in neurological pathways after TCDD exposure. Mutant zebrafish appeared morphologically normal but had significant behavior deficiencies at all life stages, and absence of Wfikkn1 did not significantly alter TCDD-induced behavior effects at all life stages. In conclusion, wfikkn1 did not appear to be significantly involved in TCDD’s overt toxicity but is likely a necessary functional member of the AHR signaling cascade.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A Comparative Multi-System Approach to Characterizing Bioactivity of Commonly Occurring Chemicals

A 2019 retrospective study analyzed wristband personal samplers from fourteen different communities across three different continents for over 1530 organic chemicals. Investigators identified fourteen chemicals (G14) detected in over 50% of personal samplers. The G14 represent a group of chemicals that individuals are commonly exposed to, and are mainly associated with consumer products including plasticizers, fragrances, flame retardants, and pesticides. The high frequency of exposure to these chemicals raises questions of their potential adverse human health effects. Additionally, the possibility of exposure to mixtures of these chemicals is likely due to their co-occurrence; thus, the potential for mixtures to induce differential bioactivity warrants further investigation. This study describes a novel approach to broadly evaluate the hazards of personal chemical exposures by coupling data from personal sampling devices with high-throughput bioactivity screenings using in vitro and non-mammalian in vivo models. To account for species and sensitivity differences, screening was conducted using primary normal human bronchial epithelial (NHBE) cells and early life-stage zebrafish. Mixtures of the G14 and most potent G14 chemicals were created to assess potential mixture effects. Chemical bioactivity was dependent on the model system, with five and eleven chemicals deemed bioactive in NHBE and zebrafish, respectively, supporting the use of a multi-system approach for bioactivity testing and highlighting sensitivity differences between the models. In both NHBE and zebrafish, mixture effects were observed when screening mixtures of the most potent chemicals. Observations of BMC-based mixtures in NHBE (NHBE BMC Mix) and zebrafish (ZF BMC Mix) suggested antagonistic effects. In this study, consumer product-related chemicals were prioritized for bioactivity screening using personal exposure data. High-throughput high-content screening was utilized to assess the chemical bioactivity and mixture effects of the most potent chemicals.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Gene co-expression network analysis in zebrafish reveals chemical class specific modules

Zebrafish is a popular animal model used for high-throughput screening of chemical hazards, however, investigations of transcriptomic mechanisms of toxicity are still needed. Here, our goal was to identify genes and biological pathways that Aryl Hydrocarbon Receptor 2 (AHR2) Activators and flame retardant chemicals (FRCs) alter in developing zebrafish. Taking advantage of a compendium of phenotypically-anchored RNA sequencing data collected from 48-h post fertilization (hpf) zebrafish, we inferred a co-expression network that grouped genes based on their transcriptional response. Genes responding to the FRCs and AHR2 Activators localized to distinct regions of the network, with FRCs inducing a broader response related to neurobehavior. AHR2 Activators centered in one region related to chemical stress responses. We also discovered several highly co-expressed genes in this module, including cyp1a, and we subsequently show that these genes are definitively within the AHR2 signaling pathway. Systematic removal of the two chemical types from the data, and analysis of network changes identified neurogenesis associated with FRCs, and regulation of vascular development associated with both chemical classes. We also identified highly connected genes responding specifically to each class that are potential biomarkers of exposure. Overall, we created the first zebrafish chemical-specific gene co-expression network illuminating how chemicals alter the transcriptome relative to each other. In addition to our conclusions regarding FRCs and AHR2 Activators, our network can be leveraged by other studies investigating chemical mechanisms of toxicity.

59 BASIC BIOLOGICAL SCIENCES↗