Polybrominated Diphenyl Ethers and Gut Microbiome Modulate Metabolic Syndrome-Related Aqueous Metabolites in Mice.

Polybrominated diphenyl ethers (PBDEs) are persistent environmental toxicants associated with increased risk for metabolic syndrome. Intermediary metabolism is influenced by the intestinal microbiome. To test the hypothesis that PBDEs reduce host-beneficial intermediary metabolites in an intestinal microbiome-dependent manner, 9-week old male conventional (CV) and germ-free (GF) C57BL/6 mice were orally gavaged once daily with vehicle, BDE-47, or BDE-99 (100 µmol/kg) for 4 days. Intestinal microbiome (16S rDNA sequencing), liver transcriptome (RNA-Seq), and intermediary metabolites in serum, liver, as well as small and large intestinal contents (SIC and LIC; LC-MS) were examined. Changes in intermediary metabolite abundances in serum, liver, and SIC, were observed under basal conditions (CV vs. GF mice) and by PBDE exposure. PBDEs altered the largest number of metabolites in the LIC; most were regulated by PBDEs in GF conditions. Importantly, intestinal microbiome was necessary for PBDE-mediated decreases in branched-chain and aromatic amino acid metabolites, including 3-indolepropionic acid, a tryptophan metabolite recently shown to be protective against inflammation and diabetes. Gene-metabolite networks revealed a positive association between the hepatic glycan synthesis gene α-1,6-mannosyltransferase (Alg12) mRNA and mannose, which are important for protein glycosylation. Glycome changes have been observed in patients with metabolic syndrome. In LIC of CV mice, 23 bacterial taxa were regulated by PBDEs. Correlations of certain taxa with distinct serum metabolites further highlight a modulatory role of the microbiome in mediating PBDE effects. In summary, PBDEs impact intermediary metabolism in an intestinal microbiome-dependent manner, suggesting that dysbiosis may contribute to PBDE-mediated toxicities that include metabolic syndrome.

Quantum dots and mouse strain influence house dust mite-induced allergic airway disease.

Quantum dot nanoparticles (QDs) are engineered nanomaterials (ENMs) that have utility in many industries due to unique optical properties not available in small molecules or bulk materials. QD-induced acute lung inflammation and toxicity in rodent models raise concerns about potential human health risks. Recent studies have also shown that some ENMs can exacerbate allergic airway disease (AAD). In this study, C57BL/6J and A/J mice were exposed to saline, house dust mite (HDM), or a combination of HDM and QDs on day 1 of the sensitization protocol. Mice were then challenged on days 8, 9 and 10 with HDM or saline only. Significant differences in cellular and molecular markers of AAD induced by both HDM and HDM + QD were observed between C57BL/6J and A/J mice. Among A/J mice, HDM + QD co-exposure, but not HDM exposure alone, significantly increased levels of bronchoalveolar lavage fluid (BALF). IL-33 compared to saline controls. BALF total protein levels in both mouse strains were also only significantly increased by HDM + QD co-exposure. In addition, A/J mice had significantly more lung type 2 innate lymphoid cells (ILC2s) cells than C57BL/6J mice. A/J lung ILC2s were inversely correlated with lung glutathione and MHC-IIhigh resident macrophages, and positively correlated with MHC-IIlow resident macrophages. The results from this study suggest that 1) QDs influence HDM-induced AAD by potentiating and/or enhancing select cytokine production; 2) that genetic background modulates the impact of QDs on HDM sensitization; and 3) that potential ILC2 contributions to HDM induced AAD are also likely to be modulated by genetic background.

Genetic determinants of susceptibility to silver nanoparticle-induced acute lung inflammation in mice.

Silver nanoparticles (AgNPs) are employed in a variety of consumer products; however, in vivo rodent studies indicate that AgNPs can cause lung inflammation and toxicity in a strain- and particle type-dependent manner, but mechanisms of susceptibility remain unclear. The aim of this study was to assess the variation in AgNP-induced lung inflammation and toxicity across multiple inbred mouse strains and to use genome-wide association (GWA) mapping to identify potential candidate susceptibility genes. Mice received doses of 0.25 mg/kg of either 20-nm citrate-coated AgNPs or citrate buffer using oropharyngeal aspiration. Neutrophils in bronchoalveolar lavage fluid (BALF) served as markers of inflammation. We found significant strain- and treatment-dependent variation in neutrophils in BALF. GWA mapping identified 10 significant single-nucleotide polymorphisms (false discovery rate, 15%) in 4 quantitative trait loci on mouse chromosomes 1, 4, 15, and 18, and Nedd4l (neural precursor cell expressed developmentally downregulated gene 4-like; chromosome 18), Ano6 (anocatmin 6; chromosome 15), and Rnf220 (Ring finger protein 220; chromosome 4) were considered candidate genes. Quantitative RT-PCR revealed significant inverse associations between mRNA levels of these genes and neutrophil influx. Nedd4l, Ano6, and Rnf220 are candidate susceptibility genes for AgNP-induced lung inflammation that warrant additional exploration in future studies.-Scoville, D. K., Botta, D., Galdanes, K., Schmuck, S. C., White, C. C., Stapleton, P. L., Bammler, T. K., MacDonald, J. W., Altemeier, W. A., Hernandez, M., Kleeberger, S. R., Chen, L.-C., Gordon, T., Kavanagh, T. J. Genetic determinants of susceptibility to silver nanoparticle-induced acute lung inflammation in mice.

Quantum dot induced acute changes in lung mechanics are mouse strain dependent.

INTRODUCTION: Concerns have been raised regarding occupational exposure to engineered nanomaterials (ENMs). Potential impacts on lung function from inhalation exposures are of concern as the lung is a sensitive ENM target in animals. Epidemiological data suggest that occupational exposure to ENMs may impact respiratory and cardiovascular health. Quantum dots (QDs) are ENMs with outstanding semiconductor and fluorescent properties with uses in biomedicine and electronics. QDs are known to induce inflammation and cytotoxicity in rodents and high dose exposures impact lung function 2 weeks after exposure. However, effects of mouse strain and the temporality of QD effects on lung function at more occupationally relevant doses have not been well-established. OBJECTIVE: We evaluated the impact of QD exposure on respiratory mechanics in C57BL/6J and A/J mice. Previous work found a greater initial inflammatory response to QD exposure in A/J mice compared to C57BL/6J mice. Thus, we hypothesized that A/J mice would be more sensitive to QD-induced effects on lung mechanics. METHODS: C57BL/6J and A/J mice were exposed to 6 µg/kg Cd equivalents of amphiphilic polymer-coated Cd/Se core, ZnS shell QDs via oropharyngeal aspiration. Lung mechanics were measured using forced oscillation, and inflammation was characterized by neutrophils and cytokines in bronchoalveolar lavage fluid. RESULTS: Both strains showed signs of QD-induced acute lung inflammation. However, lung mechanics were impacted by QD exposure in A/J mice only. CONCLUSIONS: Our findings suggest that susceptibility to QDs and similar ENM-induced changes in lung function may depend at least in part on genetic background.

Susceptibility to quantum dot induced lung inflammation differs widely among the Collaborative Cross founder mouse strains.

Quantum dots (QDs) are engineered semiconductor nanoparticles with unique physicochemical properties that make them potentially useful in clinical, research and industrial settings. However, a growing body of evidence indicates that like other engineered nanomaterials, QDs have the potential to be respiratory hazards, especially in the context of the manufacture of QDs and products containing them, as well as exposures to consumers using these products. The overall goal of this study was to investigate the role of mouse strain in determining susceptibility to QD-induced pulmonary inflammation and toxicity. Male mice from 8 genetically diverse inbred strains (the Collaborative Cross founder strains) were exposed to CdSe-ZnS core-shell QDs stabilized with an amphiphilic polymer. QD treatment resulted in significant increases in the percentage of neutrophils and levels of cytokines present in bronchoalveolar lavage fluid (BALF) obtained from NOD/ShiLtJ and NZO/HlLtJ mice relative to their saline (Sal) treated controls. Cadmium measurements in lung tissue indicated strain-dependent differences in disposition of QDs in the lung. Total glutathione levels in lung tissue were significantly correlated with percent neutrophils in BALF as well as with lung tissue Cd levels. Our findings indicate that QD-induced acute lung inflammation is mouse strain dependent, that it is heritable, and that the choice of mouse strain is an important consideration in planning QD toxicity studies. These data also suggest that formal genetic analyses using additional strains or recombinant inbred strains from these mice could be useful for discovering potential QD-induced inflammation susceptibility loci.

Spatial transcriptomics of murine bone marrow megakaryocytes at single-cell resolution.

Background: While megakaryocytes are known for making platelets, recent single-cell RNA sequencing data have revealed subpopulations of megakaryocytes with predicted immunoregulatory and bone marrow niche-supporting roles. Although these studies uncovered interesting information regarding the transcriptional variation of megakaryocytes, the generation, localization, and regulation of these subsets have not yet been studied and therefore remain incompletely understood. Considering the complex organization of the bone marrow, we reasoned that the application of spatial transcriptomic approaches could help dissect megakaryocyte heterogeneity within a spatiotemporal context. Objectives: The aim of this study was to combine spatial context and transcriptomics to assess the heterogeneity of murine bone marrow megakaryocytes in situ at a single-cell level. Methods: Bone marrow sections were obtained from femurs of C57BL/6J mice. Using the murine whole transcriptome array on the Nanostring GeoMx digital spatial profiling platform, we profiled 44 individual megakaryocytes (CD41+ by immunofluorescence) in situ throughout the bone marrow, both adjacent and nonadjacent to the endothelium (directly in contact with vascular endothelial-cadherin-positive cells). Results: Principal component analysis revealed no association between transcriptomic profile and adjacency to the vasculature. However, there was a significant effect of proximal vs distal regions of the bone. Two and 3 genes were found overexpressed in the proximal and distal sides, respectively. Of note, proplatelet basic protein and platelet factor 4, 2 genes associated with platelet production, had higher expression in proximal megakaryocytes. Conclusion: This study indicates a possible effect of spatial location on megakaryocyte heterogeneity and substantiate further interest in investigating megakaryocyte subpopulations in the context of their spatial orientation.

Spatial architecture of high-grade glioma reveals tumor heterogeneity within distinct domains.

Background: High-grade gliomas (HGGs) are aggressive primary brain cancers with poor response to standard regimens, driven by immense heterogeneity. In isocitrate dehydrogenase (IDH) wild-type HGG (glioblastoma, GBM), increased intratumoral heterogeneity is associated with more aggressive disease. Methods: Spatial technologies can dissect complex heterogeneity within the tumor ecosystem by preserving cellular organization in situ. We employed GeoMx digital spatial profiling, CosMx spatial molecular imaging, Xenium in situ mapping and Visium spatial gene expression in experimental and validation patient cohorts to interrogate the transcriptional landscape in HGG. Results: Here, we construct a high-resolution molecular map of heterogeneity in GBM and IDH-mutant patient samples to investigate the cellular communities that compose HGG. We uncovered striking diversity in the tumor landscape and degree of spatial heterogeneity within the cellular composition of the tumors. The immune distribution was diverse between samples, however, consistently correlated spatially with distinct tumor cell phenotypes, validated across tumor cohorts. Reconstructing the tumor architecture revealed two distinct niches, one composed of tumor cells that most closely resemble normal glial cells, associated with microglia, and the other niche populated by monocytes and mesenchymal tumor cells. Conclusions: This primary study reveals high levels of intratumoral heterogeneity in HGGs, associated with a diverse immune landscape within spatially localized regions.

Persistence of improved glucose homeostasis in Gclm null mice with age and cadmium treatment.

Antioxidant signaling/communication is among the most important cellular defense and survival pathways, and the importance of redox signaling and homeostasis in aging has been well-documented. Intracellular levels of glutathione (GSH), a very important endogenous antioxidant, both govern and are governed by the Nrf2 pathway through expression of genes involved in its biosynthesis, including the subunits of the rate-limiting enzyme (glutamate cysteine ligase, GCL) in GSH production, GCLC and GCLM. Mice homozygous null for the Gclm gene are severely deficient in GSH compared to wild-type controls, expressing approximately 10% of normal GSH levels. To compensate for GSH deficiency, Gclm null mice have upregulated redox-regulated genes, and, surprisingly, are less susceptible to certain types of oxidative damage. Furthermore, young Gclm null mice display an interesting lean phenotype, resistance to high fat diet-induced diabetes and obesity, improved insulin and glucose tolerance, and decreased expression of genes involved in lipogenesis. However, the persistence of this phenotype has not been investigated into old age, which is important in light of studies which suggest aging attenuates antioxidant signaling, particularly in response to exogenous stimuli. In this work, we addressed whether aging compromises the favorable phenotype of increased antioxidant activity and improved glucose homeostasis observed in younger Gclm null mice. We present data showing that under basal conditions and in response to cadmium exposure (2 mg/kg, dosed once via intraperitoneal injection), the phenotype previously described in young (<6 months) Gclm null mice persists into old age (24+ months). We also provide evidence that transcriptional activation of the Nrf2, AMPK, and PPARγ pathways underlie the favorable metabolic phenotype observed previously in young Gclm null mice.

The effects of gene × environment interactions on silver nanoparticle toxicity in the respiratory system: An adverse outcome pathway.

The Adverse Outcome Pathway (AOP) framework is serving as a basis to integrate new data streams in order to enhance the power of predictive toxicology. AOP development for engineered nanomaterials (ENM), including silver nanoparticles (AgNP), is currently lagging behind other chemicals of regulatory interest due to our limited understanding of the mechanism by which underlying genetics or diseases directly modify host response to AgNP exposures. This also highlights the importance of considering the Aggregate Exposure Pathway (AEP) framework, which precedes the AOP framework and outlines source to target site exposure. The AEP and AOP frameworks interface at the target site, where a molecular initiating event (MIE) occurs and is followed by key events (KE) for adverse cellular and organ responses along a biological pathway and ends with the adverse organism response. The primary goal of this study is to use AgNP to interrogate the AEP-AOP framework by organizing and integrating in vitro dose-response data and in vivo exposure-response data from previous studies to evaluate the effects of interactions between host genetic and acquired factors, or gene × environment interactions (G × E), on AgNP toxicity in the respiratory system. Using this framework will help us to identify plausible key event relationships (KER) between MIE and adverse organism responses when KE are not measured using the same assay in order to derive future predictive models, guide research, and support development of tools for making risk-based, regulatory decisions on ENM. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

In vitro to in vivo benchmark dose comparisons to inform risk assessment of quantum dot nanomaterials.

Engineered nanomaterials are currently under review for their potential toxicity; however, their use in consumer/commercial products has continued to outpace risk assessments. In vitro methods may be utilized as tools to improve the efficiency of risk assessment approaches. We propose a framework to compare relationships between previously published in vitro and in vivo toxicity assessments of cadmium-selenium containing quantum dots (QDs) using benchmark dose (BMD) and dosimetric assessment methods. Although data were limited this approach was useful for identifying sensitive assays and strains. In vitro studies assessed effects of QDs in three pulmonary cell types across two mouse strains. Significant dose-response effects were modeled and a standardized method of BMD analysis was performed as a function of both exposure dose and dosimetric dose. In vivo studies assessed pulmonary effects of QD exposure across eight mouse strains. BMD analysis served as a basis for relative comparison with in vitro studies. We found consistent responses in common endpoints between in vitro and in vivo studies. Strain sensitivity was consistent between in vitro and in vivo studies, showing A/J mice more sensitive to QDs. Cell types were found to differentially take up QDs. Dosimetric adjustments identified similar sensitivity among cell types. Thus, BMD analysis can be used as an effective tool to compare the sensitivity of different strains, cell types, and assays to QDs. These methods allow for in vitro assays to be used to predict in vivo responses, improve the efficiency of in vivo studies, and allow for prioritization of nanomaterial assessments. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

The pulmonary inflammatory response to multiwalled carbon nanotubes is influenced by gender and glutathione synthesis.

Inhalation of multiwalled carbon nanotubes (MWCNTs) during their manufacture or incorporation into various commercial products may cause lung inflammation, fibrosis, and oxidative stress in exposed workers. Some workers may be more susceptible to these effects because of differences in their ability to synthesize the major antioxidant and immune system modulator glutathione (GSH). Accordingly, in this study we examined the influence of GSH synthesis and gender on MWCNT-induced lung inflammation in C57BL/6 mice. GSH synthesis was impaired through genetic manipulation of Gclm, the modifier subunit of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis. Twenty-four hours after aspirating 25µg of MWCNTs, all male mice developed neutrophilia in their lungs, regardless of Gclm genotype. However, female mice with moderate (Gclm heterozygous) and severe (Gclm null) GSH deficiencies developed significantly less neutrophilia. We found no indications of MWCNT-induced oxidative stress as reflected in the GSH content of lung tissue and epithelial lining fluid, 3-nitrotyrosine formation, or altered mRNA or protein expression of several redox-responsive enzymes. Our results indicate that GSH-deficient female mice are rendered uniquely susceptible to an attenuated neutrophil response. If the same effects occur in humans, GSH-deficient women manufacturing MWCNTs may be at greater risk for impaired neutrophil-dependent clearance of MWCNTs from the lung. In contrast, men may have effective neutrophil-dependent clearance, but may be at risk for lung neutrophilia regardless of their GSH levels.

In vivo approaches to assessing the toxicity of quantum dots.

The small size and heavy metal composition of quantum dots (QDs) combined with their growing consumer product and biomedical research applications have generated concern over their safety. In an occupational setting where QD-enabled products are being manufactured, inhalation is a likely route of exposure. Since current research indicates that QDs could cause inflammation and toxicity in the respiratory tract, it is important that a variety of methods be available to further characterize this potential respiratory hazard. This chapter focuses primarily on in vivo methods for modeling the inhalation and assessing the pulmonary toxicity of QDs.

A cellular genetics approach identifies gene-drug interactions and pinpoints drug toxicity pathway nodes.

New approaches to toxicity testing have incorporated high-throughput screening across a broad-range of in vitro assays to identify potential key events in response to chemical or drug treatment. To date, these approaches have primarily utilized repurposed drug discovery assays. In this study, we describe an approach that combines in vitro screening with genetic approaches for the experimental identification of genes and pathways involved in chemical or drug toxicity. Primary embryonic fibroblasts isolated from 32 genetically-characterized inbred mouse strains were treated in concentration-response format with 65 compounds, including pharmaceutical drugs, environmental chemicals, and compounds with known modes-of-action. Integrated cellular responses were measured at 24 and 72 h using high-content imaging and included cell loss, membrane permeability, mitochondrial function, and apoptosis. Genetic association analysis of cross-strain differences in the cellular responses resulted in a collection of candidate loci potentially underlying the variable strain response to each chemical. As a demonstration of the approach, one candidate gene involved in rotenone sensitivity, Cybb, was experimentally validated in vitro and in vivo. Pathway analysis on the combined list of candidate loci across all chemicals identified a number of over-connected nodes that may serve as core regulatory points in toxicity pathways.