The Zebrafish (Danio rerio) Embryo Model as a Tool to Assess Drinking Water Treatment Efficacy for Freshwater Impacted by Crude Oil Spill.

Traditional approaches toward evaluating oil spill mitigation effectiveness in drinking water supplies using analytical chemistry can overlook residual hydrocarbons and treatment byproducts of unknown toxicity. Zebrafish (Danio rerio) were used to address this limitation by evaluating the reduction in toxicity to fish exposed to laboratory solutions of dissolved crude oil constituents treated with 3 mg/L ozone (O3 ) with or without a peroxone-based advanced oxidation process using 0.5 M H2 O2 /M O3 or 1 M H2 O2 /M O3 . Crude oil water mixtures (OWMs) were generated using three mixing protocols-orbital (OWM-Orb), rapid (OWM-Rap), and impeller (OWM-Imp) and contained dissolved total aromatic concentrations of 106-1019 µg/L. In a first experiment, embryos were exposed at 24 h post fertilization (hpf) to OWM-Orb or OWM-Rap diluted to 25%-50% of full-strength samples and in a second experiment, to untreated or treated OWM-Imp mixtures at 50% dilutions. Toxicity profiles included body length, pericardial area, and swim bladder inflation, and these varied depending on the OWM preparation, with OWM-Rap resulting in the most toxicity, followed by OWM-Imp and then OWM-Orb. Zebrafish exposed to a 50% dilution of OWM-Imp resulted in 6% shorter body length, 83% increased pericardial area, and no swim bladder inflation, but exposure to a 50% dilution of OWM-Imp treated with O3 alone or with 0.5 M H2 O2 /M O3 resulted in normal zebrafish development and average total aromatic destruction of 54%-57%. Additional aromatic removal occurred with O3 + 1 M H2 O2 /M O3 but without further attenuation of toxicity to zebrafish. This study demonstrates using zebrafish as an additional evaluation component for modeling the effectiveness of freshwater oil spill treatment methods. Environ Toxicol Chem 2022;41:2822-2834. © 2022 SETAC.

Dibenzyl trisulfide binds to and competitively inhibits the cytochrome P450 1A1 active site without impacting the expression of the aryl hydrocarbon receptor.

The toxicological manifestation of many pollutants relies upon their binding to the aryl hydrocarbon receptor (AHR), and it follows a cascade of reactions culminating in an elevated expression of cytochrome P450 (CYP) 1 enzymes. CYP1A1 and CYP1B1 are associated with enhanced carcinogenesis when chronically exposed to certain polyaromatic hydrocarbons, and their inhibition may lead to chemoprevention. We evaluated dibenzyl trisulfide (DTS), expressed in the ethnomedical plant, Petiveria alliacea, for such potential chemoprevention. Using recombinant human CYP1A1 and CYP1B1 bactosomes on a fluorogenic assay, we first demonstrated that DTS moderately inhibited both enzymes with half maximal inhibitory concentration (IC50) values of 1.3 ± 0.3 and 1.7 ± 0.3 µM, respectively. Against CYP1A1, DTS was a reversible, competitive inhibitor with an apparent inhibitory constant (Ki) of 4.55 ± 0.37 µM. In silico molecular modeling showed that DTS binds with an affinity of -39.8 kJ·mol-1, situated inside the binding pocket, approximately 4.3 Å away from the heme group, exhibiting interactions with phenylalanine residue 123 (Phe-123), Phe-224, and Phe-258. Lastly, zebrafish (Danio rerio) embryos were exposed to 0.08-0.8 µM DTS from 24 to 96 h post fertilization (hpf) with the in vivo ethoxyresorufin-O-deethylase (EROD) assay, and, at 96 hpf, DTS significantly suppressed EROD CYP1A activity in a dose-dependent manner, with up to 60% suppression in the highest 0.8 µM exposure group. DTS had no impact on gene transcription levels for cyp1a and aryl hydrocarbon receptor 2 (ahr2). In co-exposure experiments, DTS suppressed CYP1A activity induced by both B[a]P and PCB-126, although these reductions were not significant. Taken together, these results demonstrate that DTS is a direct, reversible, competitive inhibitor of the carcinogen-activating CYP1A enzyme, binding in the active site pocket close to the heme site, and shows potential in chemoprevention.

Using Monochlorobimane to Visualize Glutathione Utilization in the Developing Zebrafish (Danio rerio) Embryo.

Glutathione (GSH) plays fundamental roles in cellular redox buffering and is a common detoxification pathway for excretion of xenobiotics. This is especially crucial during vertebrate embryogenesis, when an organism is at one of its most vulnerable life stages. Importantly, GSH content and redox potential can dictate cell fate decisions, which can have profound consequences if altered by early life xenobiotic exposures. Owing to technical limitations, the best available method to detect and quantify changes in GSH has been high-pressure liquid chromatography, a terminal method that prevents suborganism-level resolution of these changes in developing embryos. Here, we describe a protocol that leverages the transparent nature of zebrafish embryos and the compatibility of monochlorobimane with the zebrafish GSH-S-transferase enzymes, to allow for the visualization of changes in GSH via S-glutathionylation in a live, developing embryo. This method can find broad application in developmental biology and toxicology. © 2021 Wiley Periodicals LLC.

Modulating glutathione thiol status alters pancreatic ß-cell morphogenesis in the developing zebrafish (Danio rerio) embryo.

Emerging evidence suggests that redox-active chemicals perturb pancreatic islet development. To better understand potential mechanisms for this, we used zebrafish (Danio rerio) embryos to investigate roles of glutathione (GSH; predominant cellular redox buffer) and the transcription factor Nrf2a (Nfe2l2a; zebrafish Nrf2 co-ortholog) in islet morphogenesis. We delineated critical windows of susceptibility to redox disruption of ß-cell morphogenesis, interrogating embryos at 24, 48 and 72 h post fertilization (hpf) and visualized Nrf2a expression in the pancreas using whole-mount immunohistochemistry at 96 hpf. Chemical GSH modulation at 48 hpf induced significant islet morphology changes at 96 hpf. Pro-oxidant exposures to tert-butylhydroperoxide (77.6 µM; 10-min at 48 hpf) or tert-butylhydroquinone (1 µM; 48-56 hpf) decreased ß-cell cluster area at 96 hpf. Conversely, exposures to antioxidant N-acetylcysteine (bolsters GSH pools; 100 µM; 48-72 hpf) or sulforaphane (activates Nrf2a; 20 µM; 48-72 hpf) significantly increased islet areas. Nrf2a was also stabilized in ß-cells: 10-min exposures to 77.6 µM tert-butylhydroperoxide significantly increased Nrf2a protein compared to control islet cells that largely lack stabilized Nrf2a; 10-min exposures to higher (776 µM) tert-butylhydroperoxide concentration stabilized Nrf2a throughout the pancreas. Using biotinylated-GSH to visualize in situ protein glutathionylation, islet cells displayed high protein glutathionylation, indicating oxidized GSH pools. The 10-min high (776 µM) tert-butylhydroperoxide exposure (induced Nrf2a globally) decreased global protein glutathionylation at 96 hpf. Mutant fish expressing inactive Nrf2a were protected against tert-butylhydroperoxide-induced abnormal islet morphology. Our data indicate that disrupted redox homeostasis and Nrf2a stabilization during pancreatic ß-cell development impact morphogenesis, with implications for disease states at later life stages. Our work identifies a potential molecular target (Nrf2) that mediates abnormal ß-cell morphology in response to redox disruptions. Moreover, our findings imply that developmental exposure to exogenous stressors at distinct windows of susceptibility could diminish the reserve redox capacity of ß-cells, rendering them vulnerable to later-life stresses and disease.

Perfluorobutanesulfonic Acid (PFBS) Induces Fat Accumulation in HepG2 Human Hepatoma.

Per- and poly-fluoroalkyl substances, especially perfluorooctanesulfonic acid, have been extensively used for over 50 years. A growing body of evidence has emerged demonstrating the potential adverse effects of these substances, including its effect on the development of non-alcoholic fatty liver disease, as one of the most prevalent chronic liver diseases. Nonetheless, there is no report of effects of perfluorobutanesulfonic acid, the major replacement for perfluorooctanesulfonic acid, on non-alcoholic fatty liver disease. Therefore, the effects of perfluorobutanesulfonic acid exposure on fat accumulation in a human hepatoma cell line were examined. Cells were exposed to perfluorobutanesulfonic acid with or without 300 µmol/L fatty acid mixture (oleic acid:palmitic acid = 2:1) conjugated by bovine serum albumin as an inducer of steatosis for 48 hours. Perfluorobutanesulfonic acid at 200 µmol/L significantly increased the triglyceride level in the presence of fatty acid compared to the control, but not without fatty acid, which was abolished by a specific peroxisome proliferator-activated receptor gamma antagonist. Perfluorobutanesulfonic acid upregulated key genes controlling lipogenesis and fatty acid uptake. Perfluorobutanesulfonic acid treatment also promoted the production of reactive oxygen species, an endoplasmic reticulum stress marker and cytosolic calcium. In conclusion, perfluorobutanesulfonic acid increased fat accumulation, in part, via peroxisome proliferator-activated receptor gamma-mediated pathway in hepatoma cells.

Mapping glutathione utilization in the developing zebrafish (Danio rerio) embryo.

Glutathione (GSH), the most abundant vertebrate endogenous redox buffer, plays key roles in organogenesis and embryonic development, however, organ-specific GSH utilization during development remains understudied. Monochlorobimane (MCB), a dye conjugated with GSH by glutathione-s-transferase (GST) to form a fluorescent adduct, was used to visualize organ-specific GSH utilization in live developing zebrafish (Danio rerio) embryos. Embryos were incubated in 20 µM MCB for 1 h and imaged on an epifluorescence microscope. GSH conjugation with MCB was high during early organogenesis, decreasing as embryos aged. The heart had fluorescence 21-fold above autofluorescence at 24 hpf, dropping to 8.5-fold by 48 hpf; this increased again by 72 hpf to 23.5-fold, and stayed high till 96 hpf (18-fold). The brain had lower fluorescence (10-fold) at 24 and 48 hpf, steadily increasing to 30-fold by 96 hpf. The sensitivity and specificity of MCB staining was then tested with known GSH modulators. A 10-min treatment at 48 hpf with 750 µM tert-butylhydroperoxide, caused organ-specific reductions in staining, with the heart losing 30% fluorescence, and, the brain ventricle losing 47% fluorescence. A 24 h treatment from 24-48 hpf with 100 µM of N-Acetylcysteine (NAC) resulted in significantly increased fluorescence, with the brain ventricle and heart showing 312% and 240% increases respectively, these were abolished upon co-treatment with 5 µM BSO, an inhibitor of the enzyme that utilizes NAC to synthesize GSH. A 60 min 100 µM treatment with ethacrynic acid, a specific GST inhibitor, caused 30% reduction in fluorescence across all measured structures. MCB staining was then applied to test for GSH disruptions caused by the toxicants perfluorooctanesulfonic acid and mono-(2-ethyl-hexyl)phthalate; MCB fluorescence responded in a dose, structure and age-dependent manner. MCB staining is a robust, sensitive method to detect spatiotemporal changes in GSH utilization, and, can be applied to identify sensitive target tissues of toxicants.

Nrf2a modulates the embryonic antioxidant response to perfluorooctanesulfonic acid (PFOS) in the zebrafish, Danio rerio.

The glutathione redox system undergoes precise and dynamic changes during embryonic development, protecting against and mitigating oxidative insults. The antioxidant response is coordinately largely by the transcription factor Nuclear factor erythroid-2 (Nrf2), an endogenous sensor for cellular oxidative stress. We have previously demonstrated that impaired Nrf family signaling disrupts the glutathione redox system in the zebrafish embryo, and that impaired Nrf2 function increases embryonic sensitivity to environmental toxicants. Here, we investigated the persistent environmental toxicant and reported pro-oxidant perfluorooctanesulfonic acid (PFOS), and its impact on the embryonic glutathione-mediated redox environment. We further examined whether impaired Nrf2a function exacerbates PFOS-induced oxidative stress and embryotoxicity in the zebrafish, and the potential for Nrf2-PPAR crosstalk in the embryonic adaptive response. Wild-type and nrf2afh318-/- mutant embryos were exposed daily to 0 (0.01% v/v DMSO), 16, 32, or 64 µM PFOS beginning at 3 h post fertilization (hpf). Embryonic glutathione and cysteine redox environments were examined at 72 hpf. Gross embryonic toxicity, antioxidant gene expression, and apoptosis were examined at 96 hpf. Mortality, pericardial edema, and yolk sac utilization were increased in wild-type embryos exposed to PFOS. Embryonic glutathione and cysteine redox couples and gene expression of Nrf2 pathway targets were modulated by both exposure and genotype. Apoptosis was increased in PFOS-exposed wild-type embryos, though not in nrf2a mutants. In silico examination of putative transcription factor binding site suggested potential crosstalk between Nrf2 and PPAR signaling, since expression of PPARs and gene targets was modulated by both PFOS exposure and Nrf2a genotype. Overall, this work demonstrates that nrf2a modulates the embryonic response to PFOS, and that PPAR signaling may play a role in the embryonic adaptive response to PFOS.

Pancreatic beta cells are a sensitive target of embryonic exposure to butylparaben in zebrafish (Danio rerio).

BACKGROUND: Butylparaben (butyl p-hydroxybenzoic acid) is a common cosmetic and pharmaceutical preservative reported to induce oxidative stress and endocrine disruption. Embryonic development is sensitive to oxidative stress, with redox potentials playing critical roles in progenitor cell fate decisions. Because pancreatic beta cells have been reported to have low antioxidant gene expression, they may be sensitive targets of oxidative stress. We tested the hypotheses that butylparaben causes oxidative stress in the developing embryo, and that pancreatic beta cells are a sensitive target of butylparaben embryotoxicity. METHODS: Transgenic insulin:GFP zebrafish embryos (Danio rerio) were treated daily with 0, 250, 500, 1,000, and 3,000 nM butylparaben. Pancreatic islet and whole embryo development were examined though 7 days postfertilization, and gene expression was measured by quantitative real-time PCR. Glutathione (GSH) and cysteine redox content were measured at 28 hr postfertilization using HPLC. RESULTS: Butylparaben exposure caused intestinal effusion, pericardial edema, and accelerated yolk utilization. At 250 nM, beta cell area increased by as much as 55%, and increased incidence of two aberrant morphologies were observed-fragmentation of the islet cluster and ectopic beta cells. Butylparaben concentrations of 500 and 1,000 nM increased GSH by 10 and 40%, respectively. Butylparaben exposure downregulated transcription factor pdx1, as well as genes involved in GSH synthesis, while upregulating GSH-disulfide reductase (gsr). CONCLUSIONS: The endocrine pancreas is a sensitive target of embryonic exposure to butylparaben, which also causes developmental deformities and perturbs redox conditions in the embryo.

Redox stress and signaling during vertebrate embryonic development: Regulation and responses.

Vertebrate embryonic development requires specific signaling events that regulate cell proliferation and differentiation to occur at the correct place and the correct time in order to build a healthy embryo. Signaling pathways are sensitive to perturbations of the endogenous redox state, and are also susceptible to modulation by reactive species and antioxidant defenses, contributing to a spectrum of passive vs. active effects that can affect redox signaling and redox stress. Here we take a multi-level, integrative approach to discuss the importance of redox status for vertebrate developmental signaling pathways and cell fate decisions, with a focus on glutathione/glutathione disulfide, thioredoxin, and cysteine/cystine redox potentials and the implications for protein function in development. We present a tissue-specific example of the important role that reactive species play in pancreatic development and metabolic regulation. We discuss NFE2L2 (also known as NRF2) and related proteins, their roles in redox signaling, and their regulation of glutathione during development. Finally, we provide examples of xenobiotic compounds that disrupt redox signaling in the context of vertebrate embryonic development. Collectively, this review provides a systems-level perspective on the innate and inducible antioxidant defenses, as well as their roles in maintaining redox balance during chemical exposures that occur in critical windows of development.

Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo.

Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous nonprotein antioxidant defense molecule is the tripeptide glutathione (γ-glutamylcysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0-5 days postfertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione using HPLC and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0-120 h of zebrafish development (including mature oocytes, fertilization, midblastula transition, gastrulation, somitogenesis, pharyngula, prehatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12h postfertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12h, and then oscillated around -190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (-220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis.

Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish.

The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR(2)) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on ß-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC(50) values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos(3) failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.

Antioxidant responses and NRF2 in synergistic developmental toxicity of PAHs in zebrafish.

Early piscine life stages are sensitive to polycyclic aromatic hydrocarbon (PAH) exposure, which can cause pericardial effusion and craniofacial malformations. We previously reported that certain combinations of PAHs cause synergistic developmental toxicity, as observed with coexposure to the aryl hydrocarbon receptor agonist beta-naphthoflavone (BNF) and cytochrome P4501A inhibitor alpha-naphthoflavone (ANF). Herein, we hypothesized that oxidative stress is a component of this toxicity. We examined induction of antioxidant genes in zebrafish embryos (Danio rerio) exposed to BNF or ANF individually, a BNF + ANF combination, and a prooxidant positive control, tert-butylhydroperoxide (tBOOH). We measured total glutathione (GSH) and attempted to modulate deformities using the GSH synthesis inhibitor L-buthionine (S,R)-sulfoximine (BSO) and increase GSH pools with N-acetyl cysteine (NAC). In addition, we used a morpholino to knockdown expression of the antioxidant response element transcription factor NRF2 to determine if this would alter gene expression or increase deformity severity. BNF + ANF coexposure significantly increased expressions of superoxide dismutase 1 and 2, glutathione peroxidase 1, pi class glutathione-s-transferase, and glutamate cysteine-ligase to a greater extent than tBOOH, BNF, or ANF alone. BSO pretreatment decreased some GSH levels, but did not worsen deformities, nor did NAC diminish toxicity. Knockdown of NRF2 increased mortality following tBOOH challenge, prevented significant upregulation of antioxidant genes following both tBOOH and BNF + ANF exposures, and exacerbated BNF + ANF-related deformities. Collectively, these findings demonstrate that antioxidant responses are a component of PAH synergistic developmental toxicity and that NRF2 is protective against prooxidant and PAH challenges during development.

CYP1B1 knockdown does not alter synergistic developmental toxicity of polycyclic aromatic hydrocarbons in zebrafish (Danio rerio).

Polycyclic aromatic hydrocarbons (PAHs) are contaminants increasing in the environment largely due to burning of fossil fuels. Our previous work identified a synergistic toxicity interaction in zebrafish embryos occurring when PAHs that are agonists for the aryl hydrocarbon receptor (AHR) co-occur with PAHs that are CYP1A inhibitors. This toxicity is mediated by the AHR2, and morpholino knockdown of CYP1A exacerbated toxicity. This study tested two hypotheses: (1) in the absence of functional CYP1A, metabolism of PAHs is shunted towards CYP1B1, which has been shown in mammals to produce more reactive metabolites of PAHs; alternatively, (2) CYP1B1 serves a protective role similar to CYP1A. We used a morpholino approach to knockdown CYP1B1 alone and in co-knockdown with CYP1A to determine whether we could alter deformities caused by synergistic toxicity of PAHs. CYP1B1 knockdown was not different from non-injected controls; nor were CYP1B1+CYP1A co-knockdown deformities different from CYP1A knockdown alone. These data suggest that CYP1B1 is not a significant factor in causing synergistic toxicity of PAHs, nor, in contrast to CYP1A, in providing protection.

Synergistic induction of AHR regulated genes in developmental toxicity from co-exposure to two model PAHs in zebrafish.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants created by the incomplete combustion of carbon, and are increasing in the environment largely due to the burning of fossil fuels. PAHs occur as complex mixtures, and some combinations have been shown to cause synergistic developmental toxicity in fish embryos, characterized by pericardial edema and craniofacial malformations. Previous studies have indicated that in the zebrafish model, this toxicity is mediated by the aryl hydrocarbon receptor 2 (AHR2), and enhanced by inhibition of CYP1A activity. In this study, we further examined this interaction of the model PAH and AHR agonist beta-naphthoflavone (BNF) with and without the AHR partial agonist/antagonist and CYP1A inhibitor alpha-naphthoflavone (ANF) to determine (1) whether ANF was acting as an AHR antagonist, (2) what alterations BNF and ANF both alone and in combination had on mRNA expression of the AHR regulated genes cytochrome P450 (cyp) 1a, 1 b 1, and 1 c 1, and the AHR repressor (ahrr2) prior to versus during deformity onset, and (3) compare CYP1A enzyme activity with mRNA induction. Zebrafish embryos were exposed from 24-48 or 24-96 hpf to BNF, 1-100 microg/L, ANF, 1-150 microg/L, a BNF+ANF co-exposure (1 microg/L+100 microg/L), or a DMSO solvent control. RNA was extracted and examined by quantitative real-time PCR. Both BNF and ANF each individually resulted in a dose dependent increase CYP1A, CYP1B1, CYP1C1, and AHRR2 mRNA, confirming their activities as AHR agonists. In the BNF+ANF co-exposures prior to deformity onset, expression of these genes was synergistic, and expression levels of the AHR regulated genes resembled the higher doses of BNF alone. Gene induction during deformities was also significantly increased in the co-exposure, but to a lesser magnitude than prior to deformity onset. EROD measurements of CYP1A activity showed ANF inhibited activity induction by BNF in the co-exposure group; this finding is not predicted by mRNA expression, which is synergistically induced in this treatment. This suggests that inhibition of CYP1A activity may alter metabolism and/or increase the half-life of the AHR agonist(s), allowing for increased AHR activation. This study furthers a mechanistic understanding of interactions underlying PAH synergistic toxicity.