Lisa Truong

The Aryl Hydrocarbon Receptor (AHR) is a versatile receptor that binds various compounds, triggering downstream transcriptional changes involved in the regulation of xenobiotic metabolism and cell fate. The Ahr Interacting Protein (AIP), also known as XAP2 and ARA9, is a molecular chaperone essential for the cytosolic retention and stabilization of the AHR. Past studies, and AIP-linked human pathologies, suggest an alternative developmental role of the AIP apart from the AHR and canonical xenobiotic response. A null mutation of the AIP in zebrafish larvae (designated as aip^wh86) resulted in craniofacial malformations, failure to inflate the swim bladder, and an enlarged and darkened liver, emerging at 5–6 days post-fertilization (dpf). Histopathological analysis at 5 dpf suggested no histological differences in the homozygous (HMZ) mutant compared to the wild type (WT). Whole-larvae RNA sequencing at 6 and 7 dpf showed significant dysregulation of key transcripts in the mutant larvae compared to WT, with pathway analysis revealing alterations in embryonic hematopoiesis and cardiac development. RT-qPCR validation showed that transcripts involved in the Hif-mediated pathway were overexpressed as early as 5 dpf. O-dianisidine staining at 6 dpf showed an increase in hemoglobin within the liver and cardiac regions of the mutant AIP larvae. This study demonstrates the complex and diverse role of AIP in vertebrate embryonic development and physiology.

Drinking water contamination poses ongoing public health concerns due to limited analytical and toxicological coverage of emerging and unknown chemical contaminants. Here, we integrate suspect screening and nontarget screening with in vivo toxicity testing to characterize organic chemical contaminants in tap drinking water across western Oregon, USA. Twelve public tap drinking water samples and one commercially bottled water sample were analyzed using complementary extraction and chromatographic workflows paired with high-resolution mass spectrometry analysis. Suspect screening identified 52 Level 1-2 annotated compounds, with prioritization leading to 13 chemical confirmations including both known drinking water contaminants (e.g., propiconazole, benzyl butyl phthalate, saccharin) and previously unreported drinking water contaminants (e.g., 18β-glycyrrhetinic acid). Zebrafish embryo and larvae toxicity screening demonstrated significant morphological toxicity following exposure to 18β-glycyrrhetinic acid and a benzyl butyl phthalate/monobenzyl phthalate mixture, while behavioral assays identified altered behavior following exposure to one individual contaminant and seven mixtures. This integrated analytical-toxicological framework demonstrates broad physicochemical screening, expands the inventory of confirmed drinking water contaminants, and establishes primary reference concentration values at the 20% effect level for one novel drinking water contaminant and several representative contaminant mixtures.

Polychlorinated biphenyls (PCBs) persist in the environment as complex mixtures and undergo extensive biotransformation, yet the developmental toxicity of PCB metabolites remains poorly defined. We evaluated developmental, neurobehavioral, and molecular effects of parent PCBs, hydroxylated, methoxylated, and sulfated metabolites, and environmentally relevant mixtures using embryonic zebrafish. This study employed a high-throughput screening approach using nominal exposure concentrations to enable rapid hazard identification and prioritization across a large chemical series. Morphological abnormalities and photomotor behavior were assessed across early development, followed by targeted cyp1a reporter analysis and transcriptomic profiling for a subset of potent exposures. Most chemicals induced morphological effects, with hydroxylated and sulfated metabolites producing effects more frequently and at lower benchmark concentrations than parent congeners. Behavioral alterations were more prevalent in embryonic photomotor response than larval photomotor response and generally co-occurred with morphological effects. Environmental mixtures elicited broad phenotypic profiles comparable to highly active individual compounds. Transcriptomic analyses revealed minimal responses for parent PCBs but robust, exposure-specific gene expression changes for select metabolites, particularly 5-OH-PCB11, and mixtures. Differentially expressed genes were enriched for xenobiotic metabolism, immune signaling, and neuroactive pathways, alongside consistent downregulation of circadian regulators. Together, these results demonstrate contributions of PCB metabolites and mixtures to toxicity.

Humans are exposed to chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) that cause toxicity through activation of the aryl hydrocarbon receptor (AHR). There is inter-individual variation in sensitivity to the effects of AHR ligands, but it is not fully explained by variation in the AHR. A clue to the genetic mechanisms underlying differential sensitivity to AHR agonists has emerged from studies of Atlantic killifish (Fundulus heteroclitus) populations with evolved tolerance to PCBs, TCDD, and PAHs. Genomic studies of these populations identified AHR-interacting protein (AIP/Ara9/XAP2) as the strongest candidate resistance gene. However, the precise role of AIP in the mechanism of resistance is unknown. To understand the role of AIP in the toxicity of dioxin-like compounds in vivo, we used CRISPR-Cas9 to generate AIP loss-of-function alleles in killifish and zebrafish (Danio rerio). Homozygous mutant killifish and zebrafish die during larval development—by 30 and 12 days post-fertilization, respectively—while heterozygous mutants develop, survive, and reproduce normally. During embryonic and early larval stages, homozygous mutant zebrafish exhibit reduced sensitivity to embryotoxic effects of exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126) and TCDD. Gene expression profiling of aip-deficient larvae revealed hundreds of differentially expressed genes. PCB126 induced similar sets of well-known AHR-regulated genes in mutant and wild-type larvae, although with reduced magnitude overall in AIP mutants. This study highlights the important role of AIP in fish larval development and demonstrates that AIP status can influence the response of vertebrate embryos to dioxin-like compounds in vivo.

Phytocannabinoids from Cannabis sativa exhibit therapeutic potential, yet historical research restrictions have limited the ability to acquire key physicochemical data. This study employs biomimetic chromatography to characterize the partitioning behavior of eight neutral and seven acidic phytocannabinoids using octadecylsilica (C18), an immobilized artificial membrane (IAM), human serum albumin (HSA), and α-1-acid glycoprotein (AGP) columns. Chromatographic hydrophobic index (CHI) scaled to octanol–water partition coefficient (log D) values ranged from 4.16 to 6.06 for neutral compounds and 2.09–3.15 for acidic ones. Despite lower lipophilicity, acidic phytocannabinoids showed stronger phospholipid and HSA binding at pH 7.4, driven by electrostatic interactions. Predicted pharmacokinetic parameters indicate high lung and brain-tissue affinity, consistent with psychoactive and therapeutic effects, while human-ether-a-go-go-related gene inhibition suggests cardiotoxicity risk. These findings provide a comprehensive data set for predicting in vivo distribution and assessing safety, highlighting biomimetic chromatography as an effective approach for advancing cannabinoid pharmacology.

Polycyclic aromatic hydrocarbons (PAHs) are a contaminant class characterized by fused aromatic rings, formed through the incomplete combustion of organic materials and petrogenic sources. Despite the abundance and toxicity of alkyl-substituted PAHs, most research and regulation focus on unsubstituted parent PAHs. Alkyl substitution of Benzo[a]pyrene (BaP), one of the most well-studied parent PAHs, drastically alters its bioactivity in zebrafish. In larval zebrafish exposed from 6 h post-fertilization (hpf), BaP caused behavioral effects but no morphological effects up to 50 µM at 120 hpf. In contrast, 8-methylbenzo[a]pyrene caused a distinct fin duplication phenotype by 0.26 µM and additional morphological effects by 1 µM. Alkyl substitution in different positions (7-, 6-, 9-, and 10-MBaP) did not elicit morphological effects at similar concentrations. This study characterized the morphological effects of 8-MBaP in zebrafish and investigated its mechanism(s) of action. Using knock-out lines, we demonstrated that 8-MBaP toxicity is Ahr2 dependent and that Cyp1a served a protective role. To identify underlying transcriptomic changes, embryos were exposed to 3 concentrations of BaP, 6-MBaP, and 8-MBaP. Whole embryos/larvae were collected at 48 and 72 hpf, which was before and during phenotype onset, respectively. Collecting RNA and morphological effects across concentration, time, and chemicals facilitated the identification of concentration-dependent transcriptional responses linked to the downstream morphological phenotypes unique to BaP methylation at the eighth position. This study improves environmental and human health hazard assessment by identifying critical structural features and mechanisms of action contributing to the toxicity of PAH mixtures in the environment.

Zebrafish (Danio rerio) are an ideal system for understanding developmental toxicity as they display similar toxicity outcomes to other vertebrates. Further, many molecules have been tested for developmental toxicity in zebrafish, providing an opportunity for machine learning model development. We curated 1,345 small molecules from ToxCast, flame retardant compounds, per- and polyfluoroalkyl substances (PFAS), and industrial chemicals published by the Superfund Research Program (SRP). Following curation, we trained machine learning models on the zebrafish toxicity endpoints ANY_ = any effect including mortality, ANY_BUT_MORT = any effect excluding mortality, MORT = mortality, i.e. did the embryo die, EDEM = did an edema form, and CRAN = Craniofacial malformation. We demonstrated that these models were better than random when compared with shuffled data. We also fine-tuned the molecular SMILES encoder MolBART to predict on all zebrafish toxicity endpoints and found it generally matched the performance of classical machine learning models for ANY_BUT_MORT, CRAN, and EDEM endpoints. We present new toxicity data for Proteolysis Targeting Chimeras (PROTACs) in zebrafish and machine learning models for these data by fingerprinting different parts of the molecule individually, yielding predictive performance (AUROC 0.6 to 0.7). If we are to reduce animal testing with new approach methodologies like these zebrafish toxicity models they need to be able adapt to new molecular classes like PROTACs.

The demand for safe, effective, and multifunctional magnetic resonance imaging (MRI) contrast agents (CAs) continues to drive the search for gadolinium-free alternatives.