Metabolomic changes following GenX and PFBS exposure in developing zebrafish.

Short chain per- and polyfluoroalkyl substances (PFAS), including hexafluoropropylene oxide dimer acid (GenX) and perfluorobutane sulfonate (PFBS), are replacement chemicals for environmentally persistent, long-chain PFAS. Although GenX and PFBS have been detected in surface and ground water worldwide, few studies provide information on the metabolic alterations or risks associated with their exposures. In this study, larval zebrafish were used to investigate the toxicity of early-life exposure to GenX or PFBS. Zebrafish were chronically exposed from 4 h post-fertilization (hpf) to 6 days post-fertilization (dpf) to 150 µM GenX or 95.0 µM PFBS. Ultra-high-performance liquid chromatography paired with high-resolution mass spectrometry was used to quantify uptake of GenX and PFBS into zebrafish larvae and perform targeted and untargeted metabolomics. Our results indicate that PFBS was 20.4 % more readily absorbed into the zebrafish larvae compared to GenX. Additionally, PFBS exposure significantly altered 13 targeted metabolites and 21 metabolic pathways, while GenX exposure significantly altered 1 targeted metabolite and 17 metabolic pathways. Exposure to GenX, and to an even greater extent PFBS, resulted in a number of altered metabolic pathways in the amino acid metabolism, with other significant alterations in the carbohydrate, lipid, cofactors and vitamins, nucleotide, and xenobiotics metabolisms. Our results indicate that GenX and PFBS impact the zebrafish metabolome, with implications of global metabolic dysregulation, particularly in metabolic pathways relating to growth and development.

Loss of developmentally derived Irf8+ macrophages promotes hyperinnervation and arrhythmia in the adult zebrafish heart.

Recent developments in cardiac macrophage biology have broadened our understanding of the critical functions of macrophages in the heart. As a result, there is further interest in understanding the independent contributions of distinct subsets of macrophage to cardiac development and function. Here, we demonstrate that genetic loss of interferon regulatory factor 8 (Irf8)-positive embryonic-derived macrophages significantly disrupts cardiac conduction, chamber function, and innervation in adult zebrafish. At 4 months post-fertilization (mpf), homozygous irf8st96/st96 mutants have significantly shortened atrial action potential duration and significant differential expression of genes involved in cardiac contraction. Functional in vivo assessments via electro- and echocardiograms at 12 mpf reveal that irf8 mutants are arrhythmogenic and exhibit diastolic dysfunction and ventricular stiffening. To identify the molecular drivers of the functional disturbances in irf8 null zebrafish, we perform single cell RNA sequencing and immunohistochemistry, which reveal increased leukocyte infiltration, epicardial activation, mesenchymal gene expression, and fibrosis. Irf8 null hearts are also hyperinnervated and have aberrant axonal patterning, a phenotype not previously assessed in the context of cardiac macrophage loss. Gene ontology analysis supports a novel role for activated epicardial-derived cells (EPDCs) in promoting neurogenesis and neuronal remodeling in vivo. Together, these data uncover significant cardiac abnormalities following embryonic macrophage loss and expand our knowledge of critical macrophage functions in heart physiology and governing homeostatic heart health.

Evaluation of Neural Regulation and Microglial Responses to Brain Injury in Larval Zebrafish Exposed to Perfluorooctane Sulfonate.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are biopersistent, global pollutants. Although some in vitro and epidemiological studies have explored the neurotoxic potential of perfluorooctane sulfonate (PFOS), a prevalent PFAS congener, it is unknown how developmental PFOS exposure affects neuronal signaling, microglia development, and microglial-neuron communication. OBJECTIVES: We sought to determine the extent to which PFOS exposure disrupts brain health, neuronal activity, and microglia-neuron communication during development. In addition, although PFOS impairs humoral immunity, its impact on innate immune cells, including resident microglia, is unclear. As such, we investigated whether microglia are cellular targets of PFOS, and, if so, whether disrupted microglial development or function could contribute to or is influenced by PFOS-induced neural dysfunction. METHODS: Zebrafish were chronically exposed to either a control solution [0.1% dimethyl sulfoxide (DMSO)], 7µM PFOS, 14µM PFOS, 28µM PFOS, or 64µM perfluorooctanoic acid (PFOA). We used in vivo imaging and gene expression analysis to assess microglial populations in the developing brain and to determine shifts in the microglia state. We functionally challenged microglia state using a brain injury model and, to assess the neuronal signaling environment, performed functional neuroimaging experiments using the photoconvertible calcium indicator calcium-modulated photoactivatable ratiometric integrator (CaMPARI). These studies were paired with optogenetic manipulations of neurons and microglia, an untargeted metabolome-wide association study (MWAS), and behavioral assays. RESULTS: Developmental PFOS exposure resulted in a shift away from the homeostatic microglia state, as determined by functional and morphological differences in exposed larvae, as well as up-regulation of the microglia activation gene p2ry12. PFOS-induced effects on microglia state exacerbated microglia responses to brain injury in the absence of increased cell death or inflammation. PFOS exposure also heightened neural activity, and optogenetic silencing of neurons or microglia independently was sufficient to normalize microglial responses to injury. An untargeted MWAS of larval brains revealed PFOS-exposed larvae had neurochemical signatures of excitatory-inhibitory imbalance. Behaviorally, PFOS-exposed larvae also exhibited anxiety-like thigmotaxis. To test whether the neuronal and microglial phenotypes were specific to PFOS, we exposed embryos to PFOA, a known immunotoxic PFAS. PFOA did not alter thigmotaxis, neuronal activity, or microglial responses, further supporting a role for neuronal activity as a critical modifier of microglial function following PFOS exposure. DISCUSSION: Together, this study provides, to our knowledge, the first detailed account of the effects of PFOS exposure on neural cell types in the developing brain in vivo and adds neuronal hyperactivity as an important end point to assess when studying the impact of toxicant exposures on microglia function. https://doi.org/10.1289/EHP12861.