Coupling Genome-wide Transcriptomics and Developmental Toxicity Profiles in Zebrafish to Characterize Polycyclic Aromatic Hydrocarbon (PAH) Hazard.

Polycyclic Aromatic Hydrocarbons (PAHs) are diverse environmental pollutants associated with adverse human health effects. Many studies focus on the carcinogenic effects of a limited number of PAHs and there is an increasing need to understand mechanisms of developmental toxicity of more varied yet environmentally relevant PAHs. A previous study characterized the developmental toxicity of 123 PAHs in zebrafish. Based on phenotypic responses ranging from complete inactivity to acute mortality, we classified these PAHs into eight bins, selected 16 representative PAHs, and exposed developing zebrafish to the concentration of each PAH that induced 80% phenotypic effect. We conducted RNA sequencing at 48 h post fertilization to identify gene expression changes as a result of PAH exposure. Using the Context Likelihood of Relatedness algorithm, we inferred a network that links the PAHs based on coordinated gene responses to PAH exposure. The 16 PAHs formed two broad clusters: Cluster A was transcriptionally more similar to the controls, while Cluster B consisted of PAHs that were generally more developmentally toxic, significantly elevated cyp1a transcript levels, and induced Ahr2-dependent Cyp1a protein expression in the skin confirmed by gene-silencing studies. We found that cyp1a transcript levels were associated with transcriptomic response, but not with PAH developmental toxicity. While all cluster B PAHs predominantly activated Ahr2, they also each enriched unique pathways like ion transport signaling, which likely points to differing molecular events between the PAHs downstream of Ahr2. Thus, using a systems biology approach, we have begun to evaluate, classify, and define mechanisms of PAH toxicity.

Systematic developmental neurotoxicity assessment of a representative PAH Superfund mixture using zebrafish.

Superfund sites often consist of complex mixtures of polycyclic aromatic hydrocarbons (PAHs). It is widely recognized that PAHs pose risks to human and environmental health, but the risks posed by exposure to PAH mixtures are unclear. We constructed an environmentally relevant PAH mixture with the top 10 most prevalent PAHs (SM10) from a Superfund site derived from environmental passive sampling data. Using the zebrafish model, we measured body burden at 48 hours post fertilization (hpf) and evaluated the developmental and neurotoxicity of SM10 and the 10 individual constituents at 24 hours post fertilization (hpf) and 5 days post fertilization (dpf). Zebrafish embryos were exposed from 6 to 120 hpf to (1) the SM10 mixture, (2) a variety of individual PAHs: pyrene, fluoranthene, retene, benzo[a]anthracene, chrysene, naphthalene, acenaphthene, phenanthrene, fluorene, and 2-methylnaphthalene. We demonstrated that SM10 and only 3 of the individual PAHs were developmentally toxic. Subsequently, we constructed and exposed developing zebrafish to two sub-mixtures: SM3 (comprised of 3 of the developmentally toxicity PAHs) and SM7 (7 non-developmentally toxic PAHs). We found that the SM3 toxicity profile was similar to SM10, and SM7 unexpectedly elicited developmental toxicity unlike that seen with its individual components. The results demonstrated that the overall developmental toxicity in the mixtures could be explained using the general concentration addition model. To determine if exposures activated the AHR pathway, spatial expression of CYP1A was evaluated in the 10 individual PAHs and the 3 mixtures at 5 dpf. Results showed activation of AHR in the liver and vasculature for the mixtures and some individual PAHs. Embryos exposed to SM10 during development and raised in chemical-free water into adulthood exhibited decreased learning and responses to startle stimulus indicating that developmental SM10 exposures affect neurobehavior. Collectively, these results exemplify the utility of zebrafish to investigate the developmental and neurotoxicity of complex mixtures.

Comparative developmental toxicity of a comprehensive suite of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that occur in complex mixtures. Several PAHs are known or suspected mutagens and/or carcinogens, but developmental toxicity data is lacking for PAHs, particularly their oxygenated and nitrated derivatives. Such data are necessary to understand and predict the toxicity of environmental mixtures. 123 PAHs were assessed for morphological and neurobehavioral effects for a range of concentrations between 0.1 and 50 µM, using a high throughput early-life stage zebrafish assay, including 33 parent, 22 nitrated, 17 oxygenated, 19 hydroxylated, 14 methylated, 16 heterocyclic, and 2 aminated PAHs. Additionally, each PAH was evaluated for AHR activation, by assessing CYP1A protein expression using whole animal immunohistochemistry (IHC). Responses to PAHs varied in a structurally dependent manner. High-molecular weight PAHs were significantly more developmentally toxic than the low-molecular weight PAHs, and CYP1A expression was detected in five distinct tissues, including vasculature, liver, skin, neuromasts and yolk.

Aerobic Bioremediation of PAH Contaminated Soil Results in Increased Genotoxicity and Developmental Toxicity.

The formation of more polar and toxic polycyclic aromatic hydrocarbon (PAH) transformation products is one of the concerns associated with the bioremediation of PAH-contaminated soils. Soil contaminated with coal tar (prebioremediation) from a former manufactured gas plant (MGP) site was treated in a laboratory scale bioreactor (postbioremediation) and extracted using pressurized liquid extraction. The soil extracts were fractionated, based on polarity, and analyzed for 88 PAHs (unsubstituted, oxygenated, nitrated, and heterocyclic PAHs). The PAH concentrations in the soil tested, postbioremediation, were lower than their regulatory maximum allowable concentrations (MACs), with the exception of the higher molecular weight PAHs (BaA, BkF, BbF, BaP, and IcdP), most of which did not undergo significant biodegradation. The soil extract fractions were tested for genotoxicity using the DT40 chicken lymphocyte bioassay and developmental toxicity using the embryonic zebrafish (Danio rerio) bioassay. A statistically significant increase in genotoxicity was measured in the unfractionated soil extract, as well as in four polar soil extract fractions, postbioremediation (p < 0.05). In addition, a statistically significant increase in developmental toxicity was measured in one polar soil extract fraction, postbioremediation (p < 0.05). A series of morphological abnormalities, including peculiar caudal fin malformations and hyperpigmentation in the tail, were measured in several soil extract fractions in embryonic zebrafish, both pre- and postbioremediation. The increased toxicity measured postbioremediation is not likely due to the 88 PAHs measured in this study (including quinones), because most were not present in the toxic polar fractions and/or because their concentrations did not increase postbioremediation. However, the increased toxicity measured postbioremediation is likely due to hydroxylated and carboxylated transformation products of the 3- and 4-ring PAHs (PHE, 1MPHE, 2MPHE, PRY, BaA, and FLA) that were most degraded.

Aryl Hydrocarbon Receptor Mediates Larval Zebrafish Fin Duplication Following Exposure to Benzofluoranthenes.

The aryl hydrocarbon receptor (AHR) mediates developmental toxicity of several xenobiotic classes including polycyclic aromatic hydrocarbons. Using embryonic zebrafish, we previously identified 4 polycyclic aromatic hydrocarbons that caused a novel phenotype among AHR ligands-growth of a lateral, duplicate caudal fin fold. The window of sensitivity to the most potent inducer of this phenotype, benzo[k]fluoranthene (BkF), was prior to 36 h postfertilization (hpf), although the phenotype was not manifest until 60 hpf. AHR dependency via Ahr2 was demonstrated using morpholino knockdown. Hepatocyte ablation demonstrated that hepatic metabolism of BkF was not required for the phenotype, nor was it responsible for the window of sensitivity. RNA sequencing performed on caudal trunk tissue from BkF-exposed animals collected at 48, 60, 72, and 96 hpf showed upregulation of genes associated with AHR activation, appendage development, and tissue patterning. Genes encoding fibroblast growth factor and bone morphogenic protein ligands, along with retinaldehyde dehydrogenase, were prominently upregulated. Gene Ontology term analysis revealed that upregulated genes were enriched for mesoderm development and fin regeneration, whereas downregulated genes were enriched for Wnt signaling and neuronal development. MetaCore (Clarivate Analytics) systems analysis of orthologous human genes predicted that R-SMADs, AP-1, and LEF1 regulated the expression of an enriched number of gene targets across all time points. Our results demonstrate a novel aspect of AHR activity with implications for developmental processes conserved across vertebrate species.

Multidimensional in vivo hazard assessment using zebrafish.

There are tens of thousands of man-made chemicals in the environment; the inherent safety of most of these chemicals is not known. Relevant biological platforms and new computational tools are needed to prioritize testing of chemicals with limited human health hazard information. We describe an experimental design for high-throughput characterization of multidimensional in vivo effects with the power to evaluate trends relating to commonly cited chemical predictors. We evaluated all 1060 unique U.S. EPA ToxCast phase 1 and 2 compounds using the embryonic zebrafish and found that 487 induced significant adverse biological responses. The utilization of 18 simultaneously measured endpoints means that the entire system serves as a robust biological sensor for chemical hazard. The experimental design enabled us to describe global patterns of variation across tested compounds, evaluate the concordance of the available in vitro and in vivo phase 1 data with this study, highlight specific mechanisms/value-added/novel biology related to notochord development, and demonstrate that the developmental zebrafish detects adverse responses that would be missed by less comprehensive testing strategies.