[Immunogenic and toxic effects of graphene oxide nanoparticles in mouse skeletal muscles and human red blood cells].

OBJECTIVE: To investigate immunogenic and toxic effects of graphene oxide (GO) nanoparticles in mouse skeletal muscles and in human blood in vitro. METHODS: GO nanoparticles prepared using a probe sonicator were supended in deionized H2O or PBS, and particle size and surface charge of the nanoparticles were measured with dynamic light scattering (DLS). Different concentrations (0.5, 1.0 and 2.0 mg/mL) of GO suspension or PBS were injected at multiple sites in the gastrocnemius muscle (GN) of C57BL/6 mice, and inflammatory response and immune cell infiltrations were detected with HE and immunofluorescence staining. We also examined the effects of GO nanoparticles on human red blood cell (RBC) morphology, hemolysis and blood coagulation using scanning electron microscope (SEM), spectrophotometry, and thromboelastography (TEG). RESULTS: GO nanoparticles suspended in PBS exhibited better colloidal dispersity, stability and surface charge effects than those in deionized H2O. In mouse GNs, injection of GO suspensions dose- and time-dependently resulted in sustained muscular inflammation and myofiber degeneration at the injection sites, which lasted till 8 weeks after the injection; immunofluorescence staining revealed obvious infiltration of monocytes, macrophages, dendritic cells and CD4+ T cells around the injection sites in mouse GNs. In human RBCs, incubation with GO suspensions at 0.2, 2.0 and 20 mg/mL, but not at 0.002 or 0.02 mg/mL, caused significant alterations of cell morphology and hemolysis. TEG analysis showed significant abnormalities of blood coagulation parameters following treatment with high concentrations of GO. CONCLUSION: GO nanoparticles can induce sustained inflammatory and immunological responses in mouse GNs and cause RBC hemolysis and blood coagulation impairment, suggesting its muscular toxicity and hematotoxicity at high concentrations.

Comparison of developmental toxicity of graphene oxide and graphdiyne to zebrafish larvae.

Graphdiyne (GDY) is a new member of family of carbon-based 2D nanomaterials (NMs), but the environmental toxicity is less investigated compared with other 2D NMs, such as graphene oxide (GO). In this study, we compared with developmental toxicity of GO and GDY to zebrafish larvae. It was shown that exposure of zebrafish embryos from 5 h post fertilization to GO and GDY for up to 5 days decreased hatching rate and induced morphological deformity. Behavioral tests indicated that GO and GDY treatment led to hyperactivity of larvae. However, blood flow velocity was not significantly affected by GO or GDY. RNA-sequencing data revealed that both types of NMs altered gene expression profiles as well as gene ontology terms and KEGG pathways related with metabolism. We further confirmed that the NMs altered the expression of genes related with lipid droplets and autophagy, which may be account for the delayed development of zebrafish larvae. At the same mass concentrations, GO induced comparable or even larger toxic effects compared with GDY, indicating that GDY might be more biocompatible compared with GO. These results may provide novel understanding about the environmental toxicity of GO and GDY in vivo.

Molecular dynamics simulations suggest the potential toxicity of fluorinated graphene to HP35 protein via unfolding the α-helix structure.

Fluorinated graphene, a two-dimensional nanomaterial composed of three atomic layers, a central carbon layer sandwiched between two layers of fluorine atoms, has attracted considerable attention across various fields, particularly for its potential use in biomedical applications. Nonetheless, scant effort has been devoted to assessing the potential toxicological implications of this nanomaterial. In this study, we scrutinize the potential impact of fluorinated graphene on a protein model, HP35 by utilizing extensive molecular dynamics (MD) simulation methods. Our MD results elucidate that upon adsorption to the nanomaterial, HP35 undergoes a denaturation process initiated by the unraveling of the second helix of the protein and the loss of the proteins hydrophobic core. In detail, substantial alterations in various structural features of HP35 ensue, including alterations in hydrogen bonding, Q value, and RMSD. Subsequent analyses underscore that hydrophobic and van der Waals interactions (predominant), alongside electrostatic energy (subordinate), exert influence over the adsorption of HP35 on the fluorinated graphene surface. Mechanistic scrutiny attests that the unrestrained lateral mobility of HP35 on the fluorinated graphene nanomaterial primarily causes the exposure of HP35's hydrophobic core, resulting in the eventual structural denaturation of HP35. A trend in the features of 2D nanostructures is proposed that may facilitate the denaturation process. Our findings not only substantiate the potential toxicity of fluorinated graphene but also unveil the underlying molecular mechanism, which thereby holds significance for the prospective utilization of such nanomaterials in the field of biomedicine.

Iron-lanthanum supported on graphite sheets for As(III) removal from aqueous solution: kinetics, thermodynamic and ecotoxicity assessment.

Graphene-based material is widely used to remove arsenic from water due to its layered structure with high surface area. Here, we have successfully synthesized Fe-La bimetallic modified graphite sheet materials to more efficiently remove As(III) from aqueous solution. The results showed that Fe-La-graphite sheets (FL-graphite sheets) have a larger specific surface area (194.28 m2·g-1) than graphite sheets (2.80 m2·g-1). The adsorption capacity of FL-graphite sheets for As(III) was 51.69 mg·g-1, which was higher than that of graphite sheets (21.91 mg·g-1), La-graphite sheets (26.06 mg·g-1), and Fe-graphite sheets (40.26 mg·g-1). The FL-graphite sheets conformed to the Freundlich and Dubinin-Radushkevich isotherm, and the maximum adsorption capacity was 53.62 mg·g-1. The removal process obeys intra-particle diffusion and pore diffusion for As(III). The results of batch adsorption experiments and characterization analyses demonstrated that oxidation, ligand exchange, and inner sphere complexation mechanisms involved in the adsorption of FL-graphite sheets to As(III) in comparison with graphite sheets. In addition, electrostatic attraction mechanism was found vital in the adsorption. Ecotoxicity assessment revealed that FL-graphite sheets have little influence on rice germination and growth, but reduced the toxicity of As(III) to rice. Therefore, the FL-graphite sheets have good practical application value in purifying As(III) polluted water with litter ecotoxicity.

Potential toxicity of graphene (oxide) quantum dots via directly covering the active site of anterior gradient homolog 2 protein.

Graphene quantum dots (GQDs) have attracted significant attention in biomedicine, while extensive investigations have revealed a reverse regarding the potential biotoxicity of GQDs. In order to supplementing the understanding of the toxicity profile of GQDs, this study employs a molecular dynamics (MD) simulation approach to systematically investigate the potential toxicity of both GQDs and Graphene Oxide Quantum Dots (GOQDs) on the Anterior Gradient Homolog 2 (AGR2) protein, a key protein capable of protecting the intestine. We construct two typical simulation systems, in which an AGR2 protein is encircled by either GQDs or GOQDs. The MD results demonstrate that both GQDs and GOQDs can directly make contact with and even cover the active site (specifically, the Cys81 amino acid) of the AGR2 protein. This suggests that GQDs and GOQDs have the capability to inhibit or interfere with the normal biological interaction of the AGR2 active site with its target protein. Thus, GQDs and GOQDs exhibit potential detrimental effects on the AGR2 protein. Detailed analyses reveal that GQDs adhere to the Cys81 residue due to van der Waals (vdW) interaction forces, whereas GOQDs attach to the Cys81 residue through a combination of vdW (primary) and Coulomb (secondary) interactions. Furthermore, GQDs aggregation typically adsorb onto the AGR2 active site, while GOQDs adsorb to the active site of AGR2 one by one. Consequently, these findings shed new light on the potential adverse impact of GQDs and GOQDs on the AGR2 protein via directly covering the active site of AGR2, providing valuable molecular insights for the toxicity profile of GQD nanomaterials.

Nano-graphene oxide particles induce inheritable anomalies through altered gene expressions involved in oocyte maturation.

The inheritable impact of exposure to graphene oxide nanoparticles (GO NPs) on vertebrate germline during critical windows of gamete development remain undetermined to date. Here, we analyzed the transgenerational effects of exposure to nano-graphene oxide particles (nGO) synthesized in house with lateral dimensions 300-600 nm and surface charge of -36.8 mV on different developmental stages of germ cells (GCs): (1) during GCs undergoing early development and differentiation, and (2) during GCs undergoing gametogenesis and maturation in adulthood. Biocompatibility analyses in Japanese medaka embryos showed lethality above 1 µg/ml and also an aberrant increase in germ cell count of both males and females at doses below the lethal dose. However, no lethality or anomalies were evident in adults up to 45 µg/ml. Long term exposure of embryos and adults for 21 days resulted in reduced fecundity. This effect was transmitted to subsequent generations, F1 and F2. Importantly, the inheritable effects of nGO in adults were pronounced at a high dose of 10 µg/ml, while 1 µg/ml showed no impact on the germline indicating lower doses used in this study to be safe. Further, expressions of selected genes that adversely affected oocyte maturation were enhanced in F1 and F2 individuals. Interestingly, the inheritance patterns differed corresponding to the stage at which the fish received the exposure.

Comparative toxicological analysis of two pristine carbon nanomaterials (graphene oxide and aminated graphene oxide) and their corresponding degraded forms using human in vitro models.

Despite the wide application of graphene-based materials, the information of the toxicity associated to some specific derivatives such as aminated graphene oxide is scarce. Likewise, most of these studies analyse the pristine materials, while the available data regarding the harmful effects of degraded forms is very limited. In this work, the toxicity of graphene oxide (GO), aminated graphene oxide (GO-NH2), and their respective degraded forms (dGO and dGO-NH2) obtained after being submitted to high-intensity sonication was evaluated applying in vitro assays in different models of human exposure. Viability and ROS assays were performed on A549 and HT29 cells, while their skin irritation potential was tested on a reconstructed human epidermis model. The obtained results showed that GO-NH2 and dGO-NH2 substantially decrease cell viability in the lung and gastrointestinal models, being this reduction slightly higher in the cells exposed to the degraded forms. In contrast, this parameter was not affected by GO and dGO which, conversely, showed the ability to induce higher levels of ROS than the pristine and degraded aminated forms. Furthermore, none of the materials is skin irritant. Altogether, these results provide new insights about the potential harmful effects of the selected graphene-based nanomaterials in comparison with their degraded counterparts.

Graphene oxide quantum dots (GOQDs) induce behavioral disorders via the disturbance of kynurenine pathway in zebrafish larvae.

The emergence of graphene quantum dots (GQDs) expands the use of graphene derivatives in nanomedicine for its direct therapeutic applications in treating neurodegeneration, inflammation, metabolic dysfunction, and among others. Nevertheless, the biosafety assessment of GQDs remains deficient mostly because of the diverse surface characteristics of the nanoparticles. Our prior work demonstrated that GQDs can induce strong thigmotactic effects in zebrafish larvae over a wide range of concentrations, yet the underlying metabolic mechanisms remain largely unknown. In this study, we conducted a further exploration about graphene oxide quantum dots (GOQDs) for its potential neurotoxic effect on the behaviors of zebrafish larvae by combining neurotransmitter-targeted metabolomics with locomotion analysis. After continuous exposure to a concentration gradient of GOQDs (12.5 - 25 - 50 - 100 - 200 µg/mL) for 7 days, the thigmotactic activities of zebrafish larvae were observed across all exposure concentrations relative to the control group, while the basal locomotor activities, including distance moved and average velocity, were significantly changed by low concentrations of GOQDs. Targeted metabolomics was performed using zebrafish larvae at 7 days post-fertilization (dpf) that were exposed to 12.5 and 200 µg/mL, both of which were found to perturb the kynurenine pathway by regulating the levels of kynurenine, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid (QA). Furthermore, the thigmotaxis of larval fish induced by GOQDs during exposure could be counteracted by supplementing Ro-61-8048, an agonist acting on kynurenine 3-monooxygenase (KMO). In conclusion, our study establishes the involvement of the kynurenine pathway in GOQDs-induced thigmotaxis, which is independent of the transcriptional modulation of glutamate receptor families.

Computational Assessment of the Biocompatibility of Two-Dimensional g-C3N3 Toward Lipid Membranes.

One of the most recent additions to the family of two-dimensional (2D) materials, graphitic C3N3 (g-C3N3), has been considered a viable contender for biomedical applications, although its potential toxicity remains elusive. We perform all-atom molecular dynamics simulations to decipher the interactions between model lipid membranes and g-C3N3 as a first step toward exploring the cytotoxicity induced at the nanoscale. We show that g-C3N3 can easily insert into the cellular membranes following a multistage mechanism consisting of simultaneous desolvation of the 2D material along with enrichment of nanomaterial-lipid interactions. Free energy calculations indicate that g-C3N3 is more stable in a membrane-bound state compared to an aqueous solution; however, the insertion of the material does not disturb the structural integrity of lipid membranes. After being inserted into a membrane, g-C3N3 is unlikely to be released into the cellular environment and is incapable of extracting lipid molecules from the membrane. The nature of interaction between the 2D material and membranes is found to be independent of the nanomaterial size. Also, the performance of g-C3N3 toward biomolecular delivery is shown to be significantly improved compared to the state-of-the-art 2D materials graphene and hexagonal boron nitride (h-BN). It is revealed that, the affinity of g-C3N3 toward lipid membranes is weaker compared to the nanotoxic graphene and h-BN, while being marginally higher than h2D-C2N, which in turn, increases the biocompatibility of the material, thereby brightening its future as a noncytotoxic material for forthcoming biomedical applications.

Graphene-Based Engineered Living Materials.

With the rise of engineered living materials (ELMs) as innovative, sustainable and smart systems for diverse engineering and biological applications, global interest in advancing ELMs is on the rise. Graphene-based nanostructures can serve as effective tools to fabricate ELMs. By using graphene-based materials as building units and microorganisms as the designers of the end materials, next-generation ELMs can be engineered with the structural properties of graphene-based materials and the inherent properties of the microorganisms. However, some challenges need to be addressed to fully take advantage of graphene-based nanostructures for the design of next-generation ELMs. This work covers the latest advances in the fabrication and application of graphene-based ELMs. Fabrication strategies of graphene-based ELMs are first categorized, followed by a systematic investigation of the advantages and disadvantages within each category. Next, the potential applications of graphene-based ELMs are covered. Moreover, the challenges associated with fabrication of next-generation graphene-based ELMs are identified and discussed. Based on a comprehensive overview of the literature, the primary challenge limiting the integration of graphene-based nanostructures in ELMs is nanotoxicity arising from synthetic and structural parameters. Finally, we present possible design principles to potentially address these challenges.

EPS-corona formation on graphene family nanomaterials (GO, rGO and graphene) and its role in mitigating their toxic effects in the marine alga Chlorella sp.

GFNs have widespread applications but can harm marine systems due to excessive use and improper disposal. Algae-secreted EPS can mitigate nanomaterial harm, but their impact on GFN toxicity is understudied. Hence, in the present study, we investigated the toxicity of three GFNs, graphene oxide (GO), reduced graphene oxide (rGO), and graphene, in pristine and EPS-adsorbed forms in the marine alga Chlorella sp. At an environmentally relevant concentration of 1 mgL-1, all three GFNs induced considerable oxidative stress and impeded growth and photosynthetic activity of the algae. The order of the toxic potential followed GO > rGO > graphene. The various facets of adsorption of EPS (1:1 mixture of loosely bound, and tightly bound EPS) on GFNs were investigated through microscopy, surface chemical analyses, fluorescence quenching studies, and isotherm and kinetics studies. Amongst the pristine GFNs treated with algal cells, GO was found to exert the maximum negative effects on algal growth. Upon adsorption of EPS over the GFNs, a significant decline in growth inhibition was observed compared to the respective pristine forms which strongly correlated with reduced oxidative stress and enhanced photosynthetic parameters in the cells. The formation of a layer of eco-corona after interaction of GFNs with EPS possibly caused a barrier effect which in turn diminished their toxic potential. The findings from the present investigation offer valuable insights into the environmental toxicity of GFNs and show that the eco-corona formation may lessen the risk posed by these materials in the marine environment.

Magnetic graphene oxide nanocomposites induce cytotoxicity in ADSCs via GPX4 regulating ferroptosis.

Magnetic graphene oxide nanocomposites (MGO NPs) have been widely studied in biomedical applications. However, their cytotoxicity and underlying mechanisms remain unclear. In this study, the biosafety of MGO NPs was investigated, and the mechanism involved in ferroptosis was further explored. MGO can produce cytotoxicity in ADSCs, which is dependent on their concentration. Ferroptosis was involved in MGO NP-induced ADSC survival inhibition by increasing total ROS and lipid ROS accumulation as well as regulating the expression levels of ferroptosis-related genes and proteins. GPX4 played a critical role in the MGO NP-induced ADSC ferroptosis process, and overexpressing GPX4 suppressed ferroptosis to increase cell survival. This study provides a theoretical basis for the biosafety management of MGO NPs used in the field of biomedical treatment.

Photostability and phototoxicity of graphene quantum dots interacting with red blood cells.

Here we discuss fluorescent properties of graphene quantum dots (GQDs) interacting with the membranes of red blood cells. We report the results of spectroscopic, microscopic, and photon-counting measurements of the GQDs in different surroundings for uncovering specific features of the GQD fluorescence, and describe two observed phenomena important for implementation of the GQDs as fluorescent labels and agents for drug delivery. Firstly, the GQDs can suffer from photodegradation but also can be stabilized in the presence of antioxidants (reduced glutathione, N-acetylcysteine, or 1,4-hydroquinone). Secondly, GQDs can accumulate in red blood cell membranes without compromising the viability of the cells but also can induce hemolysis in the presence of visible light. We discuss mechanisms and regimes of the photodegradation, stabilization, interaction of the GQDs with red blood cell membranes, and hemolysis. Notably, photohemolysis for the case is dependent on the light dose and GQD concentration but not caused by the production of reactive oxygen species.

Applications of graphene-based nanomaterials in drug design: The good, the bad and the ugly.

INTRODUCTION: Graphene-based materials (GBMs) have unique physicochemical properties that make them extremely attractive as platforms for the design of new drugs. Indeed, their bidimensional (2D) morphology, high surface area, mechanical and optical properties, associated to different possibilities for functionalization of their surface, provides opportunities for their use as nanomedicines for drug delivery and/or phototherapies. AREAS COVERED: This opinion paper provides an overview of the current status of GBMs in drug design, with a focus on their therapeutic applications, potential environmental and health risks, and some controversial results. The authors discuss the chemical modifications of GBMs for the treatment of various diseases. The potential toxicity associated with some GBMs is also presented, along with a safe-by-design approach to minimize the risks. Finally, the authors address some issues associated to the use of GBMs in the biomedical field, such as contradictory antibacterial effects, fluorescence quenching and imprecise chemical functionalization. EXPERT OPINION: GBMs are a promising and exciting area of research in drug delivery. It is however important that responsible and safe use of these materials is ensured to fully exploit their advantages and overcome their drawbacks.

Abnormality of mussel in the early developmental stages induced by graphene and triphenyl phosphate: In silico toxicogenomic data-mining, in vivo, and toxicity pathway-oriented approach.

Increasing number of complex mixtures of organic pollutants in coastal area (especially for nanomaterials and micro/nanoplastics associated chemicals) threaten aquatic ecosystems and their joint hazards are complex and demanding tasks. Mussels are the most sensitive marine faunal groups in the world, and their early developmental stages (embryo and larvae) are particularly susceptible to environmental contaminants, which can distinguish the probable mechanisms of mixture-induced growth toxicity. In this study, the potential critical target and biological processes affected by graphene and triphenyl phosphate (TPP) were developed by mining public toxicogenomic data. And their combined toxic effects were verified by toxicological assay at early developmental stages in filter-feeding mussels (embryo and larvae). It showed that interactions among graphene/TPP with 111 genes (ABCB1, TP53, SOD, CAT, HSP, etc.) affected phenotypes along conceptual framework linking these chemicals to developmental abnormality endpoints. The PPAR signaling pathway, monocarboxylic acid metabolic process, regulation of lipid metabolic process, response to oxidative stress, and gonad development were noted as the key molecular pathways that contributed to the developmental abnormality. Enriched phenotype analysis revealed biological processes (cell proliferation, cell apoptosis, inflammatory response, response to oxidative stress, and lipid metabolism) affected by the investigated mixture. Combined, our results supported that adverse effects induced by contaminants/ mixture could not only be mediated by single receptor signaling or be predicted by the simple additive effect of contaminants. The results offer a framework for better comprehending the developmental toxicity of environmental contaminants in mussels and other invertebrate species, which have considerable potential for hazard assessment of coastal mixture.

Three-dimensional graphene aerogel mitigated the toxic impact of chloramphenicol wastewater on microorganisms in an EGSB reactor.

Anaerobic treatment of chloramphenicol wastewater holds significant promise due to its potential for bioenergy generation. However, the high concentration of organic matter and residual toxic substances in the wastewater severely inhibit the activity of microorganisms. In this study, a three-dimensional graphene aerogel (GA), as a conductive material with high specific surface area (114.942 m2 g-1) and pore volume (0.352 cm3 g-1), was synthesized and its role in the efficiency and related mechanism for EGSB reactor to treat chloramphenicol wastewater was verified. The results indicated that synergy effects of GA for Chemical Oxygen Demand (COD) removal (increased by 8.17 %), chloramphenicol (CAP) removal (increased by 4.43 %) and methane production (increased by 70.29 %). Furthermore, GA increased the average particle size of anaerobic granular sludge (AGS) and promoted AGS to secrete more redox active substances. Microbial community analysis revealed that GA increased the relative abundance of functional bacteria and archaea, specifically Syntrophomonas, Geobacter, Methanothrix, and Methanolinea. These microbial species can participate in direct interspecific electron transfer (DIET). This research serves as a theoretical foundation for the application of GA in mitigating the toxic impact of refractory organic substances, such as antibiotics, on microorganisms during anaerobic treatment processes.

Environmental biotechnology and the involving biological process using graphene-based biocompatible material.

Biotechnology is a promising approach to environmental remediation but requires improvement in efficiency and convenience. The improvement of biotechnology has been illustrated with the help of biocompatible materials as biocarrier for environmental remediations. Recently, graphene-based materials (GBMs) have become promising materials in environmental biotechnology. To better illustrate the principle and mechanisms of GBM application in biotechnology, the comprehension of the biological response of microorganisms and enzymes when facing the GBMs is needed. The review illustrated distinct GBM-microbe/enzyme composites by providing the GBM-microbe/enzyme interaction and the determining factors. There are diverse GBM modifications for distinct biotechnology applications. Each of these methods and applications depends on the physicochemical properties of GBMs. The applications of these composites were mainly categorized as pollutant adsorption, anaerobic digestion, microbial fuel cells, and organics degradation. Where information was available, the strategies and mechanisms of GBMs in improving application efficacies were also demonstrated. In addition, the biological response, from microbial community changes, extracellular polymeric substances changes to biological pathway alteration, may become important in the application of these composites. Furthermore, we also discuss challenges facing the environmental application of GBMs, considering their fate and toxicity in the ecosystem, and offer potential solutions. This research significantly enhances our comprehension of the fundamental principles, underlying mechanisms, and biological pathways for the in-situ utilization of GBMs.

In vitro assessment of skin irritation and corrosion properties of graphene-related materials on a 3D epidermis.

The increasing use of graphene-related materials (GRMs) in many technological applications, ranging from electronics to biomedicine, needs a careful evaluation of their impact on human health. Skin contact can be considered one of the most relevant exposure routes to GRMs. Hence, this study is focused on two main adverse outcomes at the skin level, irritation and corrosion, assessed following two specific Test Guidelines (TGs) defined by the Organization for Economic Co-operation and Development (OECD) (439 and 431, respectively) that use an in vitro 3D reconstructed human epidermis (RhE) model. After the evaluation of their suitability to test a large panel of powdered GRMs, it was found that the latter were not irritants or corrosive. Only GRMs prepared with irritant surfactants, not sufficiently removed, reduced RhE viability at levels lower than those predicting skin irritation (≤50%, after 42 min exposure followed by 42 h recovery), but not at levels lower than those predicting corrosion (<50%, after 3 min exposure or <15% after 1 h exposure). As an additional readout, a hierarchical clustering analysis on a panel of inflammatory mediators (interleukins: IL-1α, IL-1ß, IL-6, and IL-18; tumor necrosis factor-α and prostaglandin E2) released by RhE exposed to these materials supported the lack of irritant and pro-inflammatory properties. Overall, these results demonstrate that both TGs are useful in assessing GRMs for their irritant or corrosion potential, and that the tested materials did not cause these adverse effects at the skin level. Only GRMs prepared using toxic surfactants, not adequately removed, turned out to be skin irritants.

Graphene oxide had adverse effects on sperm motility and morphology through oxidative stress.

Graphene oxide (GO) is a new type of graphene material, but its effects on the male reproductive system are unclear. Here, we investigated the effects of GO on human sperm in vitro. Sperms were incubated with various doses of GO (0, 10, 20, or 40 µg/mL) for different times (1, 3, or 6 h) at 37 °C, followed by analyses of the sperm motility, viability, abnormalities, and DNA fragmentations. GO exposure significantly decreased sperm motility and viability, increased sperm abnormalities, and DNA fragmentation. Moreover, GO exposure resulted in a significant reduction of sperm mitochondrial membrane potential (MMP), which was confirmed by the ultrastructural changes of chromatin and mitochondria caused by GO. These data revealed the adverse effects of GO on sperm. Further research showed that GO exposure led to a significant increase in malondialdehyde (MDA) and reactive oxygen species (ROS) in sperm cells and a significant decrease in total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px). In addition, western blot analysis showed that the levels of Nrf-2 and HO-1 protein expression in GO-treated sperm cells were significantly increased compared to the control. These results indicated that GO had adverse effects on human sperm through oxidative stress, which was associated with Nrf-2/HO-1 signaling pathway.

Toxicological assessment of pristine and degraded forms of graphene functionalized with MnOx nanoparticles using human in vitro models representing different exposure routes.

The development of novel advanced nanomaterials (NMs) with outstanding characteristics for their use in distinct applications needs to be accompanied by the generation of knowledge on their potential toxicological impact, in particular, that derived from different occupational risk exposure routes, such as inhalation, ingestion, and skin contact. The harmful effects of novel graphene-metal oxide composites on human health are not well understood, many toxicological properties have not been investigated yet. The present study has evaluated several toxicological effects associated with graphene decorated with manganese oxide nanoparticles (GNA15), in a comparative assessment with those induced by simple graphene (G2), on human models representing inhalation (A549 cell line), ingestion (HT29 cell line) and dermal routes (3D reconstructed skin). Pristine and degraded forms of these NMs were included in the study, showing to have different physicochemical and toxicological properties. The degraded version of GNA15 (GNA15d) and G2 (G2d) exhibited clear structural differences with their pristine counterparts, as well as a higher release of metal ions. The viability of respiratory and gastrointestinal models was reduced in a dose-dependent manner in the presence of both GNA15 and G2 pristine and degraded forms. Besides this, all NMs induced the production of reactive oxygen species (ROS) in both models. However, the degraded forms showed to induce a higher cytotoxicity effect. In addition, we found that none of the materials produced irritant effects on 3D reconstructed skin when present in aqueous suspensions. These results provide novel insights into the potentially harmful effects of novel multicomponent NMs in a comprehensive manner. Furthermore, the integrity of the NMs can play a role in their toxicity, which can vary depending on their composition and the exposure route.