Rapid and efficient testing of the toxicity of graphene-related materials in primary human lung cells

Graphene and its derivative materials are manufactured by numerous companies and research laboratories, during which processes they can come into contact with their handlers' physiological barriers—for instance, their respiratory system. Despite their potential toxicity, these materials have even been used in face masks to prevent COVID-19 transmission. The increasingly widespread use of these materials requires the design and implementation of appropriate, versatile, and accurate toxicological screening methods to guarantee their safety. Murine models are adequate, though limited when exploring different doses and lengths of exposure—as this increases the number of animals required, contrary to the Three R's principle in animal experimentation. This article proposes an in vitro model using primary, non-transformed normal human bronchial epithelial (NHBE) cells as an alternative to the most widely used model to date, the human lung tumor cell line A549. The model has been tested with three graphene derivatives—graphene oxide (GO), few-layer graphene (FLG), and small FLG (sFLG). We observed a cytotoxic effect (necrosis and apoptosis) at early (6- and 24-h) exposures, which intensified after seven days of contact between cells and the graphene-related materials (GRMs)—with cell death reaching 90% after a 5 µg/mL dose. A549 cells are more resistant to necrosis and apoptosis, yielding values less than half of NHBE cells at low concentrations of GRMs (between 0.05 and 5 µg/mL). Indeed, GRM-induced cell death in NHBE cells is comparable to that induced by toxic compounds such as diesel exhaust particles on the same cell line. We propose NHBE as a suitable model to test GRM-induced toxicity, allowing refinement of the dose concentrations and exposure timings for better-designed in vivo mouse assays

Assessment of Genotoxicity Induced by Subchronic Exposure to Graphene in HaCaT Human Skin Cell Line

The applications of graphene-based materials (GBMs) and even their processing involve prolonged contact with cellular barriers such as human skin. Even though the potential cytotoxicity of graphene has been studied in recent years, the impact of long-term graphene exposure has rarely been explored. We tested in the HaCaT epithelial cells, in vitro, the effect of subchronic treatments with subletal doses of four different, well-characterized GBMs, two commercial graphene oxides (GO) and two few-layer graphenes (FLG). Cells were exposed weekly to low doses of the GBMs for 14 days, 30 days, 3 months, and 6 months. GBMs-cells uptake was assessed by confocal microscopy. Cell death and cell cycle were determined by fluorescence microscopy and cytometry, respectively. DNA damage was measured by comet assay and g-H2AX staining, followed by determination of p-p53 and p-ATR by immunolabeling. Subchronic exposure to different GBMs at non-cytotoxic doses has potential genotoxic effects on HaCaT epithelial cells, that can be recovered depending on the GBM and exposure time. Specifically, GO-induced genotoxicity can be detected after 14 and 30 days from treatment. At this time, FLG appears less genotoxic than GO, and cells can recover more easily when genotoxic pressure disappears after some days removal of the GBM. Long-term exposure, 3 and 6 months, to different GBMs induces permanent, non-reversible, genotoxic damage that is comparable than the exerted by arsenite. This should be considered for the production and future applications of GBMs in scenarios where low concentrations of the material interact chronically with epithelial barriers

Subchronic Graphene Exposure Reshapes Skin Cell Metabolism

In recent years, the toxicity of graphene-related materials (GRMs) has been evaluated in diverse models to guarantee their safety. In most applications, sublethal doses of GRMs contact human barriers such as skin in a subchronic way. Herein, the subchronic effect (30 day exposure) of three GRMs (GO 1, GO 2, and FLG) with different oxidation degrees and sizes was studied. The effects of these materials on human skin cells, HaCaTs, were assayed through high-throughput metabolic-based readout and other cell-based assays. A differential effect was found between the different GRMs. GO 2 induced a metabolic remodeling in epithelial cells, increasing the level of tricarboxylic acid components, mirrored by increased cell proliferation and changes in cell phenotype. The oxidation degree, size, and method of manufacture of GRMs dictated harmful effects on cell metabolism and behavior generated by nontoxic exposures. Therefore, a “safe by design” procedure is necessary when working with these nanomaterial

Intrinsic and Selective Activity of Functionalized Carbon Nanotube/Nanocellulose Platforms Against Colon Cancer Cells

[Abstract] Given their large surface area and versatile chemical reactivity, single-walled carbon nanotubes (SWCNTs) are regarded as the basis of new pharmacological complexes. In this study, SWCNTs are chemically functionalized with fluorescein, folic acid, and capecitabine, a drug that is commonly used against colorectal cancer. These functionalized SWCNTs are dispersed in water by taking advantage of their synergistic interaction with type-II nanocrystalline cellulose (II-NCC), and the resulting colloidal system is tested in vitro on both normal (differentiated) and cancerous (proliferative) human colon cells (Caco-2). The functionalized SWCNT/II-NCC hybrids show a higher activity than the reference (capecitabine) against the Caco-2 cancer cell line. However, this effect appears to be intrinsically associated with the SWCNT/II-NCC complex, particularly boosted by fluorescein, as the presence of capecitabine is not required. In addition, confocal microscopy fluorescence imaging using cell cultures highlights the enormous potential of this nanohybrid platform for colon cancer theranostics.This research was funded by the regional government of Aragón, DGA (Grupos Reconocidos DGA-T03_17R, DGA-T03_20R and DGA-A20_20R), together with associated EU Regional Development Funds, and also the Spanish MINEICO through a “Juan de la Cierva Incorporación” contract, and their associated research funds (ref. IJCI-2016–27789). A.C. thanks the Xunta de Galicia for an “Atracción de Talento” research grant (no. ED431H 2020/17)Gobierno de Aragón; DGA-T03_17RGobierno de Aragón; DGA-T03_20RGobierno de Aragón; DGA-A20_20RXunta de Galicia; ED431H 2020/1

Easy and Versatile Synthesis of Bulk Quantities of Highly Enriched 13C-Graphene Materials for Biological and Safety Applications

The preparation of bulk quantities of 13C-labeled graphene materials is relevant for basic investigations and for practical applications. In addition, 13C-labeled graphene materials can be very useful in biological and environmental studies, as they may allow the detection of graphene or its derivatives in cells or organs. In this paper, we describe the synthesis of 13C-labeled graphene materials (few-layer graphene, FLG, and graphene oxide, GO) on a tens of mg scale, starting from 13C-labeled methane to afford carbon fibers, followed by liquid-phase exfoliation (FLG) or oxidation (GO). The materials have been characterized by several analytical and microscopic techniques, including Raman and nuclear magnetic resonance spectroscopies, thermogravimetric analysis, X-ray photoelectron spectroscopy, and X-ray powder diffraction. As a proof of concept, the distribution of the title compounds in cells has been investigated. In fact, the analysis of the 13C/12C ratio with isotope ratio mass spectrometry (IRMS) allows the detection and quantification of very small amounts of material in cells or biological compartments with high selectivity, even when the material has been degraded. During the treatment of 13C-labeled FLG with HepG2 cells, 4.1% of the applied dose was found in the mitochondrial fraction, while 4.9% ended up in the nuclear fraction. The rest of the dose did not enter into the cell and remained in the plasma membrane or in the culture media.La preparación de cantidades a granel de materiales de grafeno marcados con 13C es relevante para investigaciones básicas y para aplicaciones prácticas. Además, los materiales de grafeno marcados con 13C pueden ser muy útiles en estudios biológicos y medioambientales, ya que pueden permitir la detección de grafeno o sus derivados en células u órganos. En este trabajo describimos la síntesis de materiales de grafeno marcados con 13C (grafeno de pocas capas, FLG, y óxido de grafeno, GO) a escala de decenas de mg, partiendo de metano marcado con 13C para obtener fibras de carbono, seguidas de exfoliación en fase líquida (FLG) u oxidación (GO). Los materiales se han caracterizado mediante varias técnicas analíticas y microscópicas, como las espectroscopias Raman y de resonancia magnética nuclear, el análisis termogravimétrico, la espectroscopia de fotoelectrones de rayos X y la difracción de rayos X en polvo. Como prueba de concepto, se ha investigado la distribución de los compuestos del título en las células. De hecho, el análisis de la relación 13C/12C con espectrometría de masas de relación isotópica (IRMS) permite la detección y cuantificación de cantidades muy pequeñas de material en células o compartimentos biológicos con alta selectividad, incluso cuando el material se ha degradado. Durante el tratamiento de FLG marcado con 13C con células HepG2, el 4,1% de la dosis aplicada se encontró en la fracción mitocondrial, mientras que el 4,9% acabó en la fracción nuclear. El resto de la dosis no entró en la célula y permaneció en la membrana plasmática o en el medio de cultivo

A comprehensive systematic review of CSF proteins and peptides that defne Alzheimer’s disease

Background: During the last two decades, over 100 proteomics studies have identifed a variety of potential bio‑ markers in CSF of Alzheimer’s (AD) patients. Although several reviews have proposed specifc biomarkers, to date, the statistical relevance of these proteins has not been investigated and no peptidomic analyses have been generated on the basis of specifc up- or down- regulation. Herein, we perform an analysis of all unbiased explorative proteom‑ ics studies of CSF biomarkers in AD to critically evaluate whether proteins and peptides identifed in each study are consistent in distribution; direction change; and signifcance, which would strengthen their potential use in studies of AD pathology and progression. Methods: We generated a database containing all CSF proteins whose levels are known to be signifcantly altered in human AD from 47 independent, validated, proteomics studies. Using this database, which contains 2022 AD and 2562 control human samples, we examined whether each protein is consistently present on the basis of reliable statistical studies; and if so, whether it is over- or under-represented in AD. Additionally, we performed a direct analysis of available mass spectrometric data of these proteins to generate an AD CSF peptide database with 3221 peptides for further analysis. Results: Of the 162 proteins that were identifed in 2 or more studies, we investigated their enrichment or depletion in AD CSF. This allowed us to identify 23 proteins which were increased and 50 proteins which were decreased in AD, some of which have never been revealed as consistent AD biomarkers (i.e. SPRC or MUC18). Regarding the analysis of the tryptic peptide database, we identifed 87 peptides corresponding to 13 proteins as the most highly consistently altered peptides in AD. Analysis of tryptic peptide fngerprinting revealed specifc peptides encoded by CH3L1, VGF, SCG2, PCSK1N, FBLN3 and APOC2 with the highest probability of detection in AD. Conclusions: Our study reveals a panel of 27 proteins and 21 peptides highly altered in AD with consistent statistical signifcance; this panel constitutes a potent tool for the classifcation and diagnosis of AD

Spatial and Temporal Protein Modules Signatures Associated with Alzheimer Disease in 3xTg-AD Mice Are Restored by Early Ubiquinol Supplementation

Despite its robust proteopathic nature, the spatiotemporal signature of disrupted protein modules in sporadic Alzheimer's disease (AD) brains remains poorly understood. This considered oxidative stress contributes to AD progression and early intervention with coenzyme Q10 or its reduced form, ubiquinol, delays the progression of the disease. Using MALDI-MSI and functional bioinformatic analysis, we have developed a protocol to express how deregulated protein modules arise from hippocampus and cortex in the AD mice model 3xTG-AD in an age-dependent manner. This strategy allowed us to identify which modules can be efficiently restored to a non-pathological condition by early intervention with ubiquinol. Indeed, an early deregulation of proteostasis-related protein modules, oxidative stress and metabolism has been observed in the hippocampus of 6-month mice (early AD) and the mirrored in cortical regions of 12-month mice (middle/late AD). This observation has been validated by IHC using mouse and human brain sections, suggesting that these protein modules are also affected in humans. The emergence of disrupted protein modules with AD signature can be prevented by early dietary intervention with ubiquinol in the 3xTG-AD mice model.A pesar de su robusta naturaleza proteopática, la firma espaciotemporal de los módulos de proteínas interrumpidos en los cerebros de la enfermedad de Alzheimer (EA) esporádica sigue siendo poco conocida. Este considerado estrés oxidativo contribuye a la progresión de la EA y la intervención precoz con coenzima Q10 o su forma reducida, el ubiquinol, retrasa la progresión de la enfermedad. Usando MALDI-MSI y análisis bioinformático funcional, hemos desarrollado un protocolo para expresar cómo surgen módulos de proteína desregulados del hipocampo y la corteza en el modelo de ratones AD 3xTG-AD de una manera dependiente de la edad. Esta estrategia nos permitió identificar qué módulos se pueden restaurar de manera eficiente a una condición no patológica mediante una intervención temprana con ubiquinol. De hecho, una desregulación temprana de los módulos proteicos relacionados con la proteostasis, Se ha observado estrés oxidativo y metabolismo en el hipocampo de ratones de 6 meses (EA temprana) y se refleja en regiones corticales de ratones de 12 meses (EA media/tardía). Esta observación ha sido validada por IHC utilizando secciones de cerebro humano y de ratón, lo que sugiere que estos módulos de proteína también se ven afectados en humanos. La aparición de módulos de proteínas interrumpidos con la firma AD puede prevenirse mediante una intervención dietética temprana con ubiquinol en el modelo de ratones 3xTG-AD

CoQ10 reduces glioblastoma growth and infiltration through proteome remodeling and inhibition of angiogenesis and inflammation

Purpose: Most monotherapies available against glioblastoma multiforme (GBM) target individual hallmarks of this aggressive brain tumor with minimal success. In this article, we propose a therapeutic strategy using coenzyme Q10 (CoQ10) as a pleiotropic factor that crosses the blood-brain barrier and accumulates in cell membranes acting as an antioxidant, and in mitochondrial membranes as a regulator of cell bioenergetics and gene expression. Methods: Xenografts of U251 cells in nu/nu mice were used to assay tumor growth, hypoxia, angiogenesis, and inflammation. An orthotopic model was used to explore microglial infiltration, tumor growth, and invasion into the brain parenchyma. Cell proliferation, migration, invasion, proteome remodeling, and secretome were assayed in vitro. Conditioned media were used to assay angiogenesis, monocyte chemoattraction, and differentiation into macrophages in vitro. Results: CoQ10 treatment decreased tumor volume in xenografts and orthotopic models, although its effect on tumor cell proliferation was not direct. Tumors from mice treated with CoQ10 were less hypoxic and vascularized, having less infiltration from inflammatory cells. Treatment-induced downregulation of HIF-1α and NF-kB led to a complete remodeling of the tumor cells proteome and secretome, impacting angiogenesis, monocyte infiltration, and their differentiation into macrophages. Besides, tumor cell migration and invasion were drastically restricted by mechanisms involving modulation of the actin cytoskeleton and downregulation of matrix metalloproteases (MMPs). Conclusions: CoQ10 has a pleiotropic effect on GBM growth, targeting several hallmarks simultaneously. Thus, its integration into current treatments of this fatal disease should be considered. Keywords: Angiogenesis; Coenzyme Q10; Glioblastoma; Inflammation; Invasion.Propósito: La mayoría de las monoterapias disponibles contra el glioblastoma multiforme (GBM) se dirigen a las características individuales de este tumor cerebral agresivo con un éxito mínimo. En este artículo proponemos una estrategia terapéutica utilizando la coenzima Q 10 (CoQ 10 ) como factor pleiotrópico que atraviesa la barrera hematoencefálica y se acumula en las membranas celulares actuando como antioxidante, y en las membranas mitocondriales como regulador de la bioenergética celular y gen expresión. Métodos: Se utilizaron xenoinjertos de células U251 en ratones nu/nu para analizar el crecimiento tumoral, la hipoxia, la angiogénesis y la inflamación. Se utilizó un modelo ortotópico para explorar la infiltración microglial, el crecimiento tumoral y la invasión del parénquima cerebral. Se ensayaron in vitro la proliferación celular, la migración, la invasión, la remodelación del proteoma y el secretoma. Se usaron medios acondicionados para analizar la angiogénesis, la quimioatracción de monocitos y la diferenciación en macrófagos in vitro. Resultados: el tratamiento con CoQ 10 disminuyó el volumen tumoral en xenoinjertos y modelos ortotópicos, aunque su efecto sobre la proliferación de células tumorales no fue directo. Los tumores de ratones tratados con CoQ 10 eran menos hipóxicos y vascularizados, con menos infiltración de células inflamatorias. La regulación a la baja inducida por el tratamiento de HIF-1α y NF-kB condujo a una remodelación completa del proteoma y el secretoma de las células tumorales, lo que impactó en la angiogénesis, la infiltración de monocitos y su diferenciación en macrófagos. Además, la migración e invasión de células tumorales se vieron drásticamente restringidas por mecanismos que involucran la modulación del citoesqueleto de actina y la regulación a la baja de las metaloproteasas de matriz (MMP). Conclusiones: CoQ 10 tiene un efecto pleiotrópico en el crecimiento de GBM, apuntando a varios sellos simultáneamente. Por lo tanto, se debe considerar su integración en los tratamientos actuales de esta enfermedad mortal

The Protective Effect of Ubiquinone against the Amyloid Peptide in Endothelial Cells Is Isoprenoid Chain Length-Dependent

Vascular brain pathology constitutes a common feature in neurodegenerative diseases that could underlie their development. Indeed, vascular dysfunction acts synergistically with neurodegenerative changes to exacerbate the cognitive impairment found in Alzheimer’s disease. Different injuries such as hypertension, high glucose, atherosclerosis associated with oxidized low-density lipoprotein or inflammation induce NADPH oxidase activation, overproduction of reactive oxygen species, and apoptosis in endothelial cells. Since it has been shown that pretreatment of cultured endothelial cells with the lipophilic antioxidant coenzyme Q10 (CoQ10) displays a protective effect against the deleterious injuries caused by different agents, this study explores the cytoprotective role of different CoQs homologues against Aβ25–35-induced damage and demonstrates that only pretreatment with CoQ10 protects endothelial brain cells from Aβ25–35-induced damage. Herein, we show that CoQ10 constitutes the most effective ubiquinone in preventing NADPH oxidase activity and reducing both reactive oxygen species generation and the increase in free cytosolic Ca2+ induced by Aβ25–35, ultimately preventing apoptosis and necrosis. The specific cytoprotective effect of CoQ with a side chain of 10 isoprenoid units could be explained by the fact that CoQ10 is the only ubiquinone that significantly reduces the entry of Aβ25–35 into the mitochondria

Biological Significance of the Protein Changes Occurring in the Cerebrospinal Fluid of Alzheimer’s Disease Patients: Getting Clues from Proteomic Studies

The fact that cerebrospinal fluid (CSF) deeply irrigates the brain together with the relative simplicity of sample extraction from patients make this biological fluid the best target for biomarker discovery in neurodegenerative diseases. During the last decade, biomarker discovery has been especially fruitful for the identification new proteins that appear in the CSF of Alzheimer’s disease (AD) patients together with amyloid-β (Aβ42), total tau (T-tau), and phosphorylated tau (P-tau). Thus, several proteins have been already stablished as important biomarkers, due to an increase (i.e., CHI3L1) or a decrease (i.e., VGF) in AD patients’ CSF. Notwithstanding this, only a deep analysis of a database generated with all the changes observed in CSF across multiple proteomic studies, and especially those using state-of-the-art methodologies, may expose those components or metabolic pathways disrupted at different levels in AD. Deep comparative analysis of all the up- and down-regulated proteins across these studies revealed that 66% of the most consistent protein changes in CSF correspond to intracellular proteins. Interestingly, processes such as those associated to glucose metabolism or RXR signaling appeared inversely represented in CSF from AD patients in a significant manner. Herein, we discuss whether certain cellular processes constitute accurate indicators of AD progression by examining CSF. Furthermore, we uncover new CSF AD markers, such as ITAM, PTPRZ or CXL16, identified by this study