The NCBI Comparative Genome Viewer (CGV) is an interactive visualization tool for the analysis of whole-genome eukaryotic alignments.

We report a new visualization tool for analysis of whole-genome assembly-assembly alignments, the Comparative Genome Viewer (CGV) (https://ncbi.nlm.nih.gov/genome/cgv/). CGV visualizes pairwise same-species and cross-species alignments provided by National Center for Biotechnology Information (NCBI) using assembly alignment algorithms developed by us and others. Researchers can examine large structural differences spanning chromosomes, such as inversions or translocations. Users can also navigate to regions of interest, where they can detect and analyze smaller-scale deletions and rearrangements within specific chromosome or gene regions. RefSeq or user-provided gene annotation is displayed where available. CGV currently provides approximately 800 alignments from over 350 animal, plant, and fungal species. CGV and related NCBI viewers are undergoing active development to further meet needs of the research community in comparative genome visualization.

TERT-independent telomere elongation and shelterin dysregulation after pulmonary exposure to stainless-steel welding fume in-vivo.

Telomeres are inert DNA sequences (TTAGGG) at the end of chromosomes that protect genetic information and maintain DNA integrity. Emerging evidence has demonstrated that telomere alteration can be closely related to occupational exposure and the development of various disease conditions, including cancer. However, the functions and underlying molecular mechanisms of telomere alteration and shelterin dysregulation after welding fume exposures have not been broadly defined. In this study, we analyzed telomere length and shelterin complex proteins in peripheral blood mononuclear cells (PBMCs) and in lung tissue recovered from male Sprague-Dawley rats following exposure by intratracheal instillation (ITI) to 2 mg/rat of manual metal arc-stainless steel (MMA-SS) welding fume particulate or saline (vehicle control). PBMCs and lung tissue were harvested at 30 d after instillation. Our study identified telomere elongation and shelterin dysregulation in PBMCs and lung tissue after welding fume exposure. Mechanistically, telomere elongation was independent of telomerase reverse transcriptase (TERT) activation. Collectively, our findings demonstrated that welding fume-induced telomere elongation was (a) TERT-independent and (b) associated with shelterin complex dysregulation. It is possible that an alteration of telomere length and its regulatory proteins may be utilized as predictive biomarkers for various disease conditions after welding fume exposure. This needs further investigation.

Interactive visualization of whole eukaryote genome alignments using NCBI's Comparative Genome Viewer (CGV).

We report a new visualization tool for analysis of whole genome assembly-assembly alignments, the Comparative Genome Viewer (CGV) (https://ncbi.nlm.nih.gov/genome/cgv/). CGV visualizes pairwise same-species and cross-species alignments provided by NCBI using assembly alignment algorithms developed by us and others. Researchers can examine the alignments between the two assemblies using two alternate views: a chromosome ideogram-based view or a 2D genome dotplot. Whole genome alignment views expose large structural differences spanning chromosomes, such as inversions or translocations. Users can also navigate to regions of interest, where they can detect and analyze smaller-scale deletions and rearrangements within specific chromosome or gene regions. RefSeq or user-provided gene annotation is displayed in the ideogram view where available. CGV currently provides approximately 700 alignments from over 300 animal, plant, and fungal species. CGV and related NCBI viewers are undergoing active development to further meet needs of the research community in comparative genome visualization.

Selective involvement of UGGT variant: UGGT2 in protecting mouse embryonic fibroblasts from saturated lipid-induced ER stress.

Secretory proteins and lipids are biosynthesized in the endoplasmic reticulum (ER). The "protein quality control" system (PQC) monitors glycoprotein folding and supports the elimination of terminally misfolded polypeptides. A key component of the PQC system is Uridine diphosphate glucose:glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 re-glucosylates unfolded glycoproteins, to enable the re-entry in the protein-folding cycle and impede the aggregation of misfolded glycoproteins. In contrast, a complementary "lipid quality control" (LQC) system that maintains lipid homeostasis remains elusive. Here, we demonstrate that cytotoxic phosphatidic acid derivatives with saturated fatty acyl chains are one of the physiological substrates of UGGT2, an isoform of UGGT1. UGGT2 produces lipid raft-resident phosphatidylglucoside regulating autophagy. Under the disruption of lipid metabolism and hypoxic conditions, UGGT2 inhibits PERK-ATF4-CHOP-mediated apoptosis in mouse embryonic fibroblasts. Moreover, the susceptibility of UGGT2 KO mice to high-fat diet-induced obesity is elevated. We propose that UGGT2 is an ER-localized LQC component that mitigates saturated lipid-associated ER stress via lipid glucosylation.

A joint NCBI and EMBL-EBI transcript set for clinical genomics and research.

Comprehensive genome annotation is essential to understand the impact of clinically relevant variants. However, the absence of a standard for clinical reporting and browser display complicates the process of consistent interpretation and reporting. To address these challenges, Ensembl/GENCODE1 and RefSeq2 launched a joint initiative, the Matched Annotation from NCBI and EMBL-EBI (MANE) collaboration, to converge on human gene and transcript annotation and to jointly define a high-value set of transcripts and corresponding proteins. Here, we describe the MANE transcript sets for use as universal standards for variant reporting and browser display. The MANE Select set identifies a representative transcript for each human protein-coding gene, whereas the MANE Plus Clinical set provides additional transcripts at loci where the Select transcripts alone are not sufficient to report all currently known clinical variants. Each MANE transcript represents an exact match between the exonic sequences of an Ensembl/GENCODE transcript and its counterpart in RefSeq such that the identifiers can be used synonymously. We have now released MANE Select transcripts for 97% of human protein-coding genes, including all American College of Medical Genetics and Genomics Secondary Findings list v3.0 (ref. 3) genes. MANE transcripts are accessible from major genome browsers and key resources. Widespread adoption of these transcript sets will increase the consistency of reporting, facilitate the exchange of data regardless of the annotation source and help to streamline clinical interpretation.

RefSeq Functional Elements as experimentally assayed nongenic reference standards and functional interactions in human and mouse.

Eukaryotic genomes contain many nongenic elements that function in gene regulation, chromosome organization, recombination, repair, or replication, and mutation of those elements can affect genome function and cause disease. Although numerous epigenomic studies provide high coverage of gene regulatory regions, those data are not usually exposed in traditional genome annotation and can be difficult to access and interpret without field-specific expertise. The National Center for Biotechnology Information (NCBI) therefore provides RefSeq Functional Elements (RefSeqFEs), which represent experimentally validated human and mouse nongenic elements derived from the literature. The curated data set is comprised of richly annotated sequence records, descriptive records in the NCBI Gene database, reference genome feature annotation, and activity-based interactions between nongenic regions, target genes, and each other. The data set provides succinct functional details and transparent experimental evidence, leverages data from multiple experimental sources, is readily accessible and adaptable, and uses a flexible data model. The data have multiple uses for basic functional discovery, bioinformatics studies, genetic variant interpretation; as known positive controls for epigenomic data evaluation; and as reference standards for functional interactions. Comparisons to other gene regulatory data sets show that the RefSeqFE data set includes a wider range of feature types representing more areas of biology, but it is comparatively smaller and subject to data selection biases. RefSeqFEs thus provide an alternative and complementary resource for experimentally assayed functional elements, with future data set growth expected.

Oxidized carbon black nanoparticles induce endothelial damage through C-X-C chemokine receptor 3-mediated pathway.

Oxidation of engineered nanomaterials during application in various industrial sectors can alter their toxicity. Oxidized nanomaterials also have widespread industrial and biomedical applications. In this study, we evaluated the cardiopulmonary hazard posed by these nanomaterials using oxidized carbon black (CB) nanoparticles (CBox) as a model particle. Particle surface chemistry was characterized by X-ray photo electron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR). Colloidal characterization and in vitro dosimetry modeling (particle kinetics, fate and transport modeling) were performed. Lung inflammation was assessed following oropharyngeal aspiration of CB or oxidized CBox particles (20 µg per mouse) in C57BL/6J mice. Toxicity and functional assays were also performed on murine macrophage (RAW 264.7) and endothelial cell lines (C166) with and without pharmacological inhibitors. Oxidant generation was assessed by electron paramagnetic resonance spectroscopy (EPR) and via flow cytometry. Endothelial toxicity was evaluated by quantifying pro-inflammatory mRNA expression, monolayer permeability, and wound closure. XPS and FTIR spectra indicated surface modifications, the appearance of new functionalities, and greater oxidative potential (both acellular and in vitro) of CBox particles. Treatment with CBox demonstrated greater in vivo inflammatory potentials (lavage neutrophil counts, secreted cytokine, and lung tissue mRNA expression) and air-blood barrier disruption (lavage proteins). Oxidant-dependent pro-inflammatory signaling in macrophages led to the production of CXCR3 ligands (CXCL9,10,11). Conditioned medium from CBox-treated macrophages induced significant elevation in endothelial cell pro-inflammatory mRNA expression, enhanced monolayer permeability and impairment of scratch healing in CXCR3 dependent manner. In summary, this study mechanistically demonstrated an increased biological potency of CBox particles and established the role of macrophage-released chemical mediators in endothelial damage.

Oxidant-induced epithelial alarmin pathway mediates lung inflammation and functional decline following ultrafine carbon and ozone inhalation co-exposure.

Environmental inhalation exposures are inherently mixed (gases and particles), yet regulations are still based on single toxicant exposures. While the impacts of individual components of environmental pollution have received substantial attention, the impact of inhalation co-exposures is poorly understood. Here, we mechanistically investigated pulmonary inflammation and lung function decline after inhalation co-exposure and individual exposures to ozone (O3) and ultrafine carbon black (CB). Environmentally/occupationally relevant lung deposition levels in mice were achieved after inhalation of stable aerosols with similar aerodynamic and mass median distributions. X-ray photoemission spectroscopy detected increased surface oxygen contents on particles in co-exposure aerosols. Compared with individual exposures, co-exposure aerosols produced greater acellular and cellular oxidants detected by electron paramagnetic resonance (EPR) spectroscopy, and in vivo immune-spin trapping (IST), as well as synergistically increased lavage neutrophils, lavage proteins and inflammation related gene/protein expression. Co-exposure induced a significantly greater respiratory function decline compared to individual exposure. A synthetic catalase-superoxide dismutase mimetic (EUK-134) significantly blunted lung inflammation and respiratory function decline confirming the role of oxidant imbalance. We identified a significant induction of epithelial alarmin (thymic stromal lymphopoietin-TSLP)-dependent interleukin-13 pathway after co-exposure, associated with increased mucin and interferon gene expression. We provided evidence of interactive outcomes after air pollution constituent co-exposure and identified a key mechanistic pathway that can potentially explain epidemiological observation of lung function decline after an acute peak of air pollution. Developing and studying the co-exposure scenario in a standardized and controlled fashion will enable a better mechanistic understanding of how environmental exposures result in adverse outcomes.

The pulmonary toxicity of carboxylated or aminated multi-walled carbon nanotubes in mice is determined by the prior purification method.

BACKGROUND: Inhalation of multi-walled carbon nanotubes (MWCNTs) poses a potential risk to human health. In order to safeguard workers and consumers, the toxic properties of MWCNTs need to be identified. Functionalization has been shown to either decrease or increase MWCNT-related pulmonary injury, depending on the type of modification. We, therefore, investigated both acute and chronic pulmonary toxicity of a library of MWCNTs derived from a common pristine parent compound (NC7000). METHODS: MWCNTs were thermally or chemically purified and subsequently surface functionalized by carboxylation or amination. To evaluate pulmonary toxicity, male C57BL6 mice were dosed via oropharyngeal aspiration with either 1.6 or 4 mg/kg of each MWCNT type. Mitsui-7 MWCNT was used as a positive control. Necropsy was performed at days 3 and 60 post-exposure to collect bronchoalveolar lavage fluid (BALF) and lungs. RESULTS: At day 3 all MWCNTs increased the number of neutrophils in BALF. Chemical purification had a greater effect on pro-inflammatory cytokines (IL-1ß, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-ß1). At day 60, thermally purified, carboxylated MWCNTs had the strongest effect on lymphocyte numbers in BALF. Thermally purified MWCNTs caused the greatest increase in LDH and total protein in BALF. Furthermore, the thermally purified and carboxyl- or amine-functionalized MWCNTs caused the greatest number of granulomatous lesions in the lungs. The physicochemical characteristics mainly associated with increased toxicity of the thermally purified derivatives were decreased surface defects and decreased amorphous content as indicated by Raman spectroscopy. CONCLUSIONS: These data demonstrate that the purification method is an important determinant of lung toxicity induced by carboxyl- and amine-functionalized MWCNTs.

A possible relationship between telomere length and markers of neurodegeneration in rat brain after welding fume inhalation exposure.

Inhalation of welding fume (WF) can result in the deposition of toxic metals, such as manganese (Mn), in the brain and may cause neurological changes in exposed workers. Alterations in telomere length are indicative of cellular aging and, possibly, neurodegeneration. Here, we investigated the effect of WF inhalation on telomere length and markers of neurodegeneration in whole brain tissue in rats. Male Fischer-344 (F-344) rats were exposed by inhalation to stainless steel WF (20 mg/m3 x 3 h/d x 4 d/wk x 5 wk) or filtered air (control). Telomere length, DNA-methylation, gene expression of Trf1, Trf2, ATM, and APP, protein expression of p-Tau, α-synuclein, and presenilin 1 and 2 were assessed in whole brain tissue at 12 wk after WF exposure ended. Results suggest that WF inhalation increased telomere length without affecting telomerase in whole brain. Moreover, we observed that components of the shelterin complex, Trf1 and Trf2, play an important role in telomere end protection, and their regulation may be responsible for the increase in telomere length. In addition, expression of different neurodegeneration markers, such as p-Tau, presenilin 1-2 and α-synuclein proteins, were increased in brain tissue from the WF-exposed rats as compared to control. These findings suggest a possible correlation between epigenetic modifications, telomere length alteration, and neurodegeneration because of the presence of factors in serum after WF exposure that may cause extra-pulmonary effects as well as the translocation of potentially neurotoxic metals associated with WF to the central nervous system (CNS). Further studies are needed to investigate the brain region specificity and temporal response of these effects.

Grouping of carbonaceous nanomaterials based on association of patterns of inflammatory markers in BAL fluid with adverse outcomes in lungs.

Carbonaceous nanomaterials (CNMs) are universally being used to make commodities, as they present unique opportunities for development and innovation in the fields of engineering, biotechnology, etc. As technology advances to incorporate CNMs in industry, the potential exposures associated with these particles also increase. CNMs have been found to be associated with substantial pulmonary toxicity, including inflammation, fibrosis, and/or granuloma formation in animal models. This study attempts to categorize the toxicity profiles of various carbon allotropes, in particular, carbon black, different multi-walled carbon nanotubes, graphene-based materials, and their derivatives. Statistical and machine learning-based approaches were used to identify groups of CNMs with similar pulmonary toxicity responses from a panel of proteins measured in bronchoalveolar lavage (BAL) fluid samples and with similar pathological outcomes in the lungs. Thus, grouped particles, based on their pulmonary toxicity profiles, were used to select a small set of proteins that could potentially identify and discriminate between the toxicity profiles associated within each group. Specifically, MDC/CCL22 and MIP-3ß/CCL19 were identified as common protein markers associated with both toxicologically distinct groups of CNMs. In addition, the persistent expression of other selected protein markers in BAL fluid from each group suggested their ability to predict toxicity in the lungs, i.e. fibrosis and microgranuloma formation. The advantages of such approaches can have positive implications for further research in toxicity profiling.

All that is silver is not toxic: silver ion and particle kinetics reveals the role of silver ion aging and dosimetry on the toxicity of silver nanoparticles.

BACKGROUND: When suspended in cell culture medium, nano-objects composed of soluble metals such as silver can dissolve resulting in ion formation, altered particle properties (e.g. mass, morphology, etc.), and modulated cellular dose. Cultured cells are exposed not just to nanoparticles but to a complex, dynamic mixture of altered nanoparticles, unbound ions, and ion-ligand complexes. Here, three different cell types (RAW 264.7 macrophages and bone marrow derived macrophages from wild-type C57BL/6 J mice and Scavenger Receptor A deficient (SR-A(-/-)) mice) were exposed to 20 and 110 nm silver nanoparticles, and RAW 264.7 cells were exposed to freshly mixed silver ions, aged silver ions (ions incubated in cell culture medium), and ions formed from nanoparticle dissolution. The In Vitro Sedimentation, Diffusion, Dissolution, and Dosimetry Model (ISD3) was used to predict dose metrics for each exposure scenario. RESULTS: Silver nanoparticles, freshly mixed ions, and ions from nanoparticle dissolution were toxic, while aged ions were not toxic. Macrophages from SR-A(-/-) mice did not take up 20 nm silver nanoparticles as well as wild-types but demonstrated no differences in silver levels after exposure to 110 nm nanoparticles. Dose response modeling with ISD3 predicted dose metrics suggest that amount of ions in cells and area under the curve (AUC) of ion amount in cells are the most predictive of cell viability after nanoparticle and combined nanoparticle/dissolution-formed-ions exposures, respectively. CONCLUSIONS: Results of this study suggest that the unbound silver cation is the ultimate toxicant, and ions formed extracellularly drive toxicity after exposure to nanoparticles. Applying computational modeling (ISD3) to better understand dose metrics for soluble nanoparticles allows for better interpretation of in vitro hazard assessments.

Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Sparse Supervised Classification Methods Predict and Characterize Nanomaterial Exposures: Independent Markers of MWCNT Exposures.

Recent experimental evidence indicates significant pulmonary toxicity of multiwalled carbon nanotubes (MWCNTs), such as inflammation, interstitial fibrosis, granuloma formation, and carcinogenicity. Although numerous studies explored the adverse potential of various CNTs, their comparability is often limited. This is due to differences in administered dose, physicochemical characteristics, exposure methods, and end points monitored. Here, we addressed the problem through sparse classification method, a supervised machine learning approach that can reduce the noise contained in redundant variables for discriminating among MWCNT-exposed and MWCNT-unexposed groups. A panel of proteins measured from bronchoalveolar lavage fluid (BAL) samples was used to predict exposure to various MWCNT and determine markers that are attributable to MWCNT exposure and toxicity in mice. Using sparse support vector machine-based classification technique, we identified a small subset of proteins clearly distinguishing each exposure. Macrophage-derived chemokine (MDC/CCL22), in particular, was associated with various MWCNT exposures and was independent of exposure method employed, that is, oropharyngeal aspiration versus inhalation exposure. Sustained expression of some of the selected protein markers identified also suggests their potential role in MWCNT-induced toxicity and proposes hypotheses for future mechanistic studies. Such approaches can be used more broadly for nanomaterial risk profiling studies to evaluate decisions related to dose/time-response relationships that could delineate experimental variables from exposure markers.

Consensus coding sequence (CCDS) database: a standardized set of human and mouse protein-coding regions supported by expert curation.

The Consensus Coding Sequence (CCDS) project provides a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assembly in genome annotations produced independently by NCBI and the Ensembl group at EMBL-EBI. This dataset is the product of an international collaboration that includes NCBI, Ensembl, HUGO Gene Nomenclature Committee, Mouse Genome Informatics and University of California, Santa Cruz. Identically annotated coding regions, which are generated using an automated pipeline and pass multiple quality assurance checks, are assigned a stable and tracked identifier (CCDS ID). Additionally, coordinated manual review by expert curators from the CCDS collaboration helps in maintaining the integrity and high quality of the dataset. The CCDS data are available through an interactive web page (https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi) and an FTP site (ftp://ftp.ncbi.nlm.nih.gov/pub/CCDS/). In this paper, we outline the ongoing work, growth and stability of the CCDS dataset and provide updates on new collaboration members and new features added to the CCDS user interface. We also present expert curation scenarios, with specific examples highlighting the importance of an accurate reference genome assembly and the crucial role played by input from the research community.

Acute in vitro and in vivo toxicity of a commercial grade boron nitride nanotube mixture.

Boron nitride nanotubes (BNNTs) are an emerging engineered nanomaterial attracting significant attention due to superior electrical, chemical and thermal properties. Currently, the toxicity profile of this material is largely unknown. Commercial grade BNNTs are composed of a mixture (BNNT-M) of ∼50-60% BNNTs, and ∼40-50% impurities of boron and hexagonal boron nitride. We performed acute in vitro and in vivo studies with commercial grade BNNT-M, dispersed by sonication in vehicle, in comparison to the extensively studied multiwalled carbon nanotube-7 (MWCNT-7). THP-1 wild-type and NLRP3-deficient human monocytic cells were exposed to 0-100 µg/ml and C57BL/6 J male mice were treated with 40 µg of BNNT-M for in vitro and in vivo studies, respectively. In vitro, BNNT-M induced a dose-dependent increase in cytotoxicity and oxidative stress. This was confirmed in vivo following acute exposure increase in bronchoalveolar lavage levels of lactate dehydrogenase, pulmonary polymorphonuclear cell influx, loss in mitochondrial membrane potential and augmented levels of 4-hydroxynonenal. Uptake of this material caused lysosomal destabilization, pyroptosis and inflammasome activation, corroborated by an increase in cathepsin B, caspase 1, increased protein levels of IL-1ß and IL-18 both in vitro and in vivo. Attenuation of these effects in NLRP3-deficient THP-1 cells confirmed NLRP3-dependent inflammasome activation by BNNT-M. BNNT-M induced a similar profile of inflammatory pulmonary protein production when compared to MWCNT-7. Functionally, pretreatment with BNNT-M caused suppression in bacterial uptake by THP-1 cells, an effect that was mirrored in challenged alveolar macrophages collected from exposed mice and attenuated with NLRP3 deficiency. Analysis of cytokines secreted by LPS-challenged alveolar macrophages collected after in vivo exposure to dispersions of BNNT-M showed a differential macrophage response. The observed results demonstrated acute inflammation and toxicity in vitro and in vivo following exposure to sonicated BNNT-M was in part due to NLRP3 inflammasome activation.

In Vivo Toxicity Assessment of Occupational Components of the Carbon Nanotube Life Cycle To Provide Context to Potential Health Effects.

Pulmonary toxicity studies on carbon nanotubes focus primarily on as-produced materials and rarely are guided by a life cycle perspective or integration with exposure assessment. Understanding toxicity beyond the as-produced, or pure native material, is critical, due to modifications needed to overcome barriers to commercialization of applications. In the first series of studies, the toxicity of as-produced carbon nanotubes and their polymer-coated counterparts was evaluated in reference to exposure assessment, material characterization, and stability of the polymer coating in biological fluids. The second series of studies examined the toxicity of aerosols generated from sanding polymer-coated carbon-nanotube-embedded or neat composites. Postproduction modification by polymer coating did not enhance pulmonary injury, inflammation, and pathology or in vitro genotoxicity of as-produced carbon nanotubes, and for a particular coating, toxicity was significantly attenuated. The aerosols generated from sanding composites embedded with polymer-coated carbon nanotubes contained no evidence of free nanotubes. The percent weight incorporation of polymer-coated carbon nanotubes, 0.15% or 3% by mass, and composite matrix utilized altered the particle size distribution and, in certain circumstances, influenced acute in vivo toxicity. Our study provides perspective that, while the number of workers and consumers increases along the life cycle, toxicity and/or potential for exposure to the as-produced material may greatly diminish.

Oxidative Stress, DNA Methylation, and Telomere Length Changes in Peripheral Blood Mononuclear Cells after Pulmonary Exposure to Metal-Rich Welding Nanoparticles.

Welding fume is a complex mixture of different potentially cytotoxic and genotoxic metals, such as chromium (Cr), manganese (Mn), nickel (Ni), and iron (Fe). Documented health effects have been observed in workers exposed to welding fume. The objective of the study was to use an animal model to identify potential biomarkers of epigenetic changes (e.g., changes in telomere length, DNA methylation) in isolated peripheral blood mononuclear cells (PBMCs) after exposure to different welding fumes. Male Sprague-Dawley rats were exposed by intratracheal instillation (ITI) of 2.0 mg/rat of gas metal arc-mild steel (GMA-MS) or manual metal arc-stainless steel (MMA-SS) welding fume. Vehicle controls received sterile saline by ITI. At 4 h, 14 h, 1 d, 3 d, 10 d, and 30 d, bronchoalveolar lavage (BAL) was performed to assess lung inflammation. Whole blood was collected, and PBMCs were isolated. Dihydroethidium (DHE) fluorescence and 4-hydroxylnonenal protein adduct (P-HNE) formation were measured in PBMCs to assess reactive oxygen species (ROS) production. DNA alterations in PBMCs were determined by evaluating changes in DNA methylation and telomere length. Metal composition of the two fumes was different: MMA-SS (41 % Fe, 29 % Cr, 17 % Mn, 3 % Ni) versus GMA-MS (85 % Fe, 14 % Mn). The more soluble and chemically complex MMA-SS sample induced a more persistent and greater inflammatory response compared to the other groups. Also, oxidative stress markers increased at 24 h in the PBMCs recovered from the MMA-SS group compared to other group. No significant differences were observed when comparing DNA methylation between the welding fume and control groups at any of the time points, whereas the MMA-SS sample significantly increased telomere length at 1 and 30 d after a single exposure compared to the other groups. These findings suggest that genotoxic metals in MMA-SS fume (e.g., Cr and Ni), that are absent in the GMA-MS fume, may enhance lung toxicity, as well as induce markers of oxidative stress and increase telomere length in PBMCs. Importantly, the measurement of telomere length in cells isolated from peripheral blood may serve as a potential biomarker of response in the assessment of toxicity associated with welding fumes.

Evaluation of pulmonary and systemic toxicity following lung exposure to graphite nanoplates: a member of the graphene-based nanomaterial family.

BACKGROUND: Graphene, a monolayer of carbon, is an engineered nanomaterial (ENM) with physical and chemical properties that may offer application advantages over other carbonaceous ENMs, such as carbon nanotubes (CNT). The goal of this study was to comparatively assess pulmonary and systemic toxicity of graphite nanoplates, a member of the graphene-based nanomaterial family, with respect to nanoplate size. METHODS: Three sizes of graphite nanoplates [20 µm lateral (Gr20), 5 µm lateral (Gr5), and <2 µm lateral (Gr1)] ranging from 8-25 nm in thickness were characterized for difference in surface area, structure,, zeta potential, and agglomeration in dispersion medium, the vehicle for in vivo studies. Mice were exposed by pharyngeal aspiration to these 3 sizes of graphite nanoplates at doses of 4 or 40 µg/mouse, or to carbon black (CB) as a carbonaceous control material. At 4 h, 1 day, 7 days, 1 month, and 2 months post-exposure, bronchoalveolar lavage was performed to collect fluid and cells for analysis of lung injury and inflammation. Particle clearance, histopathology and gene expression in lung tissue were evaluated. In addition, protein levels and gene expression were measured in blood, heart, aorta and liver to assess systemic responses. RESULTS: All Gr samples were found to be similarly composed of two graphite structures and agglomerated to varying degrees in DM in proportion to the lateral dimension. Surface area for Gr1 was approximately 7-fold greater than Gr5 and Gr20, but was less reactive reactive per m(2). At the low dose, none of the Gr materials induced toxicity. At the high dose, Gr20 and Gr5 exposure increased indices of lung inflammation and injury in lavage fluid and tissue gene expression to a greater degree and duration than Gr1 and CB. Gr5 and Gr20 showed no or minimal lung epithelial hypertrophy and hyperplasia, and no development of fibrosis by 2 months post-exposure. In addition, the aorta and liver inflammatory and acute phase genes were transiently elevated in Gr5 and Gr20, relative to Gr1. CONCLUSIONS: Pulmonary and systemic toxicity of graphite nanoplates may be dependent on lateral size and/or surface reactivity, with the graphite nanoplates > 5 µm laterally inducing greater toxicity which peaked at the early time points post-exposure relative to the 1-2 µm graphite nanoplate.

Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function during Nanoparticle-Induced Oxidative Stress.

Engineered nanoparticles (ENPs) are increasingly utilized for commercial and medical applications; thus, understanding their potential adverse effects is an important societal issue. Herein, we investigated protein S-glutathionylation (SSG) as an underlying regulatory mechanism by which ENPs may alter macrophage innate immune functions, using a quantitative redox proteomics approach for site-specific measurement of SSG modifications. Three high-volume production ENPs (SiO2, Fe3O4, and CoO) were selected as representatives which induce low, moderate, and high propensity, respectively, to stimulate cellular reactive oxygen species (ROS) and disrupt macrophage function. The SSG modifications identified highlighted a broad set of redox sensitive proteins and specific Cys residues which correlated well with the overall level of cellular redox stress and impairment of macrophage phagocytic function (CoO > Fe3O4 ≫ SiO2). Moreover, our data revealed pathway-specific differences in susceptibility to SSG between ENPs which induce moderate versus high levels of ROS. Pathways regulating protein translation and protein stability indicative of ER stress responses and proteins involved in phagocytosis were among the most sensitive to SSG in response to ENPs that induce subcytoxic levels of redox stress. At higher levels of redox stress, the pattern of SSG modifications displayed reduced specificity and a broader set pathways involving classical stress responses and mitochondrial energetics (e.g., glycolysis) associated with apoptotic mechanisms. An important role for SSG in regulation of macrophage innate immune function was also confirmed by RNA silencing of glutaredoxin, a major enzyme which reverses SSG modifications. Our results provide unique insights into the protein signatures and pathways that serve as ROS sensors and may facilitate cellular adaption to ENPs, versus intracellular targets of ENP-induced oxidative stress that are linked to irreversible cell outcomes.