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.

A novel human 3D lung microtissue model for nanoparticle-induced cell-matrix alterations.

BACKGROUND: Multi-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm. RESULTS: As inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins. CONCLUSION: 3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing.

Impact of emerging, high-production-volume graphene-based materials on the bioavailability of benzo(a)pyrene to brine shrimp and fish liver cells.

With increasing commercialization of high volume, two-dimensional carbon nanomaterials comes a greater likelihood of environmental release. In aquatic environments, black carbon binds contaminants like aromatic hydrocarbons, leading to changes in their uptake, bioavailability, and toxicity. Engineered carbon nanomaterials can also adsorb pollutants onto their carbon surfaces, and nanomaterial physicochemical properties can influence this contaminant interaction. We used 2D graphene nanoplatelets and isometric carbon black nanoparticles to evaluate the influence of particle morphology and surface properties on adsorption and bioavailability of benzo(a)pyrene, a model aromatic hydrocarbon, to brine shrimp (Artemia franciscana) and a fish liver cell line (PLHC-1). Acellular adsorption studies show that while high surface area carbon black (P90) was most effective at a given concentration, 2D graphene nanoplatelets (G550) adsorbed more benzo(a)pyrene than carbon black with comparable surface area (M120). In both biological models, co-exposure to nanomaterials lead to reduced bioavailability, with G550 graphene nanoplatelets cause a greater reduction in bioavailability or response than the M120 carbon black nanoparticles. However, on a mass basis the high surface area P90 carbon black was most effective. The trends in bioavailability and adsorption were consistent across all biological and acellular studies, demonstrating the biological relevance of these results in different models of aquatic organisms. While adsorption is limited by surface area, 2D graphene nanoplatelets adsorb more benzo(a)pyrene than carbon black nanoparticles of similar surface area and charge, demonstrating that both surface area and shape play important roles in the adsorption and bioavailability of benzo(a)pyrene to carbon nanomaterials.

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures.

To identify the potential environmental impacts of aquatic pollutants, rapid and sensitive screening tools are needed to assess adaptive and toxic responses. This study characterizes a novel fish liver microtissue model, produced with the cell line PLHC-1, as an in vitro aquatic toxicity testing platform. These 3D microtissues remain viable and stable throughout the 8-day testing period and relative to 2D monolayers, show increased basal expression of the xenobiotic metabolizing enzyme cytochrome P450 1A (Cyp1a). To evaluate pulsed, low-dose exposures at environmentally relevant concentrations, microtissue responsiveness to the model toxicant benzo(a)pyrene was assessed after single and repeated exposures for determination of both immediate and persistent effects. Significant induction of Cyp1a gene and protein expression was detected after a single 24h exposure to as little as 1nM benzo(a)pyrene, and after a 24h recovery period, Cyp1a expression declined in a dose-dependent manner. However, cell death continued to increase during the recovery period and alterations in microtissue architecture occurred at higher concentrations. To evaluate a pulsed or repeated exposure scenario, microtissues were exposed to benzo(a)pyrene, allowed to recover, then exposed a second time for 24h. Following pre-exposure to benzo(a)pyrene, cyp1a expression remained equally inducible and the pattern and level of Cyp1a protein response to a second exposure were comparable. However, pre-exposure to 1µM or 5µM of benzo(a)pyrene resulted in increased cell death, greater disruption of microtissue architecture, and alterations in cell morphology. Together, this study demonstrates the capabilities of this PLHC-1 microtissue model for sensitive assessment of liver toxicants over time and following single and repeated exposures.

Into the depths: Techniques for in vitro three-dimensional microtissue visualization.

Three-dimensional (3-D) in vitro platforms have been shown to closely recapitulate human physiology when compared with conventional two-dimensional (2-D) in vitro or in vivo animal model systems. This confers a substantial advantage in evaluating disease mechanisms, pharmaceutical drug discovery, and toxicity testing. Despite the benefits of 3-D cell culture, limitations in visualization and imaging of 3-D microtissues present significant challenges. Here we optimized histology and microscopy techniques to overcome the constraints of 3-D imaging. For morphological assessment of 3-D microtissues of several cell types, different time points, and different sizes, a two-step glycol methacrylate embedding protocol for evaluating 3-D microtissues produced using agarose hydrogels improved resolution of nuclear and cellular histopathology characteristic of cell death and proliferation. Additional immunohistochemistry, immunofluorescence, and in situ immunostaining techniques were successfully adapted to these microtissues and enhanced by optical clearing. Utilizing the Clear(T2) protocol greatly increased fluorescence signal intensity, imaging depth, and clarity, allowing for more complete confocal fluorescence microscopy imaging of these 3-D microtissues compared with uncleared samples. The refined techniques presented here address the key challenges associated with 3-D imaging, providing new and alternative methods in evaluating disease pathogenesis, delineating toxicity pathways, and enhancing the versatility of 3-D in vitro testing systems in pharmacological and toxicological applications.

Effects of surface-engineered nanoparticle-based dispersants for marine oil spills on the model organism Artemia franciscana.

Fine particles are under active consideration as alternatives to chemical dispersants for large-scale petroleum spills. Fine carbon particles with engineered surface chemistry have been shown to stabilize oil-in-water emulsions, but the environmental impacts of large-scale particle introduction to the marine environment are unknown. Here we study the impact of surface-engineered carbon-black materials on brine shrimp (Artemia franciscana) as a model marine microcrustacean. Mortality was characterized at 50-1000 mg/L, and levels of heat shock protein 70 (hsp70) were characterized at sublethal particle concentrations (25-50 mg/L). Functionalized carbon black (CB) nanoparticles were found to be nontoxic at all concentrations, while hydrophobic (annealed) and as-produced CB induced adverse effects at high concentrations. CB was also shown to adsorb benzene, a model hydrocarbon representing the more soluble and toxic low-molecular weight aromatic fraction of petroleum, but the extent of adsorption was insufficient to mitigate benzene toxicity to Artemia in coexposure experiments. At lower benzene concentrations (25-75 mg/L), coexposure with annealed and as-produced CB increased hsp70 protein levels. This study suggests that surface functionalization for increased hydrophilicity can not only improve the performance of CB-based dispersants but also reduce their adverse environmental impacts on marine organisms.