The environmental release of nanoparticles is attracting increasing attention. Graphene oxide (GO) embedded in epoxy resin (ER) is a popular composite that has been used in various fields, but the environmental release of GO-ER composites and the effects on organisms in the environment remain unknown. The present work found that GO-ER composites in water for 2–7 days resulted in the release of 0.3–2.1% GO-ER at nanoscale (2–3 nm thickness and approximately 70–130 nm lateral length). Interestingly, pristine GO quenched 30–45% hydroxyl and 12% nitroxide free radicals, whereas this capacity was not observed for the released particles from GO-ER. At environmentally relevant concentrations (μg/L), released GO-ER particles, but not GO or ER matrix, promoted algal reproduction by 34% and chlorophyll biosynthesis by 65–127% at 96 h. Released GO-ER entered algal cells and induced a slight increase in reactive oxygen species but did not elicit notable cell structure damage. The upregulated amino acids and phenylalanine metabolism, and the downregulated fatty acid biosynthesis contributed to algal growth promoted by released GO-ER. Previous studies of pristine nanoparticles were unable to reflect the environmental effects of released nanoparticles into the environment, and our research on the exposure-toxicological continuum adds important contributions to this field.

Developmental toxicity is a critical issue in nanotoxicity. However, very little is known about the effects of graphene oxide (GO, a widely used carbon material) at predicted environmental concentrations on biological development or the specific molecular mechanisms. The present study established that the development of zebrafish embryos exposed to trace concentrations (1–100 μg/L) of GO was impaired because of DNA modification, protein carbonylation and excessive generation of reactive oxygen species (ROS), especially the superoxide radical. Noticeably, there was a nonmonotonic response of zebrafish developmental toxicity to GO at μg/L to mg/L levels. Transcriptomics analysis revealed that disturbing collagen- and matrix metalloproteinase (MMP)-related genes affected the skeletal and cardiac development of zebrafish. Moreover, metabolomics analysis showed that the inhibition of amino acid metabolism and the ratios of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) contributed to the above developmental toxicity. The present work verifies the developmental toxicity of GO at trace concentrations and illustrates for the first time the specific molecular mechanisms thereof. Because of the potential developmental toxicity of GO at trace concentrations, government administrators and nanomaterial producers should consider its potential risks prior to the widespread environmental exposure to GO.

Alcohol overconsumption as a worldwide issue results in alcoholic liver disease (ALD), such as steatosis, alcoholic hepatitis, and cirrhosis. The treatment of ALD has been widely investigated but remains challenging. In this work, the protective effects of graphene oxide quantum dots (GOQDs) as novel nanozymes against alcohol overconsumption are discovered, and the specific mechanisms underlying these effects are elucidated via omics analysis. GOQDs dramatically alleviate the reduction of cell viability induced by ethanol and can act as nanozymes to accelerate ethanol metabolism and avoid the accumulation of toxic intermediates in cells. Mitochondrial damage and the excessive generation of free radicals were mitigated by GOQDs. The mechanisms underlying the cellular protective effects were also related to alterations in metabolic and protein signals, especially those involved in lipid metabolism. The moderately increased autophagy induced by GOQDs explained the removal of accumulated lipids and the subsequent elimination of excessive GOQDs. These findings suggest that GOQDs have an antagonistic capacity against the adverse effects caused by ethanol and provide new insights into the direct applications of GOQDs. In addition to traditional antioxidation, this work also establishes metabolomics and proteomics techniques as effective tools to discover the multiple functions of nanozymes.Keywords: artificial enzyme; ethanol; graphene; hepatocyte; nanozyme; oxidative stress;

Blood is the main biological fluid of humans and most animals. Understanding the effects of the biotransformation of nanomaterials in blood plasma on their interactions with cells is fundamental for the evaluation of their health risks and applications. However, there is a lack of related information. The present work found that free radicals and biological molecules in human blood plasma simultaneously drive the formation of a biological corona on biodegraded graphene oxide (GO) nanosheets. Importantly, the above biotransformation affected the interactions of GO with cells. For example, the biotransformed GO induced lower levels of reactive oxygen species and cell ultrastructure damage than did the pristine GO. In addition to the well-known protein corona, the small-molecule corona on the biodegraded GO also plays a critical role in the reduction of GO cytotoxicity through quenching excessive free radical generation. Metabolomic analysis revealed that the biotransformation reduced the oxidative stress induced by GO mainly via upregulation of the fatty acid metabolism and downregulation of the galactose metabolism. Overall, the present work clearly shows that the biotransformation of GO in blood plasma influences the nanotoxicity of GO. Compared with pristine nanomaterials, biotransformed materials are more biologically relevant to assessments of their environmental health risks.

Metabolomics is a new tool used in nanotoxicology to provide comprehensive insight into overall stress responses, while the relationships between global metabolic disturbance and the alterations of biological endpoints remain unclear from effect onset to cessation. Herein, single-cell Chlorella vulgaris was exposed to graphene oxide (GO). The inhibition of cell division and chlorophyll a biosynthesis and the enhancement of reactive oxygen species (ROS) and cell plasmolysis were observed under GO exposure; these adverse effects returned to normal levels when the cells were placed in fresh medium, suggesting that the tested effects were recoverable. Using the data collected from biological effect onset to cessation, the metabolic endpoints (X variables) and biological endpoints (Y variables) were integrated using the orthogonal partial least squares discriminant analysis. It was found that fatty acid metabolism greatly contributed to chlorophyll a and ROS levels, while carbohydrate metabolism had a dominant influence on GO-induced cell plasmolysis. This study proposes the reversibility of GO nanotoxicity and provides deeper insight into nanotoxicological mechanisms via integrating metabolomics with biological endpoints than using metabolic analysis alone.

With the widening application of graphene oxide nanosheets (GONS), their safety has attracted much attention. Secretions from aquatic organisms are ubiquitous in natural water, but the effects of secretions on the characteristics and toxicity of GONS remain largely unknown. To help fill this knowledge gap, we characterized the GONS with biological secretions (GOBS) and the associated changes in apparent toxicity. Small organic molecules, proteins, nucleotides and mucopolysaccharides from secretions in zebrafish culture water bound to GONS. Compared with GONS, GOBS showed special nanoplate topography with thicknesses of approximately 10 nm and lateral lengths ranging from 19.5 to 282 nm. GOBS with smaller lateral sizes exhibited more negative surface charges and lower aggregation state than GONS. Furthermore, GOBS triggered higher toxicity than GONS, such as death, malformation, upregulation of β-galactosidase and loss in mitochondrial membrane potential of zebrafish embryos. The well-dispersive GOBS covered embryos, inhibiting oxygen and ion exchange; these phenomena were the specific mechanisms of the adverse effects. In future work, the acquired natural coatings on nanomaterials should be paid much attention in nanotoxicology, especially for the relationships among topography, aggregation state, and toxicity.

In response to the comment by Forstner and coworkers with regard to our paper ‘Graphene oxide regulates the bacterial community and exhibits property changes in soil’, we clarify the experimental design and data analysis, and provide additional information for discussion.

Antibiotics and antibiotic resistance genes (ARGs) in the natural environment have become substantial threats to the ecosystem and public health. Effective strategies to control antibiotics and ARG contaminations are emergent. A novel carbon nanomaterial, graphene oxide (GO), has attracted a substantial amount of attention in environmental fields. This study discovered the inhibition effects of GO on sulfamethoxazole (SMZ) uptake for bacteria and ARG transfer among microorganisms. GO promoted the penetration of SMZ from intracellular to extracellular environments by increasing the cell membrane permeability. In addition, the formation of a GO-SMZ complex reduced the uptake of SMZ in bacteria. Moreover, GO decreased the abundance of the sulI and intI genes by approximately 2–3 orders of magnitude, but the global bacterial activity was not obviously inhibited. A class I integron transfer experiment showed that the transfer frequency was up to 55-fold higher in the control than that of the GO-treated groups. Genetic methylation levels were not significant while sulI gene replication was inhibited. The biological properties of ARGs were altered due to the GO-ARG noncovalent combination, which was confirmed using multiple spectral analyses. This work suggests that GO can potentially be applied for controlling ARG contamination via inhibiting antibiotic uptake and ARG propagation.Keywords: antibiotic resistance genes; graphene oxide; nanotechnology; pollution control; sulfamethoxazole;

The interactions between nanomaterials and cells are fundamental in biological responses to nanomaterials. However, the size-dependent synergistic effects of envelopment and internalization as well as the metabolic mechanisms of nanomaterials have remained unknown. The nanomaterials tested here were larger graphene oxide nanosheets (GONS) and small graphene oxide quantum dots (GOQD). GONS intensively entrapped single-celled Chlorella vulgaris, and envelopment by GONS reduced the cell permeability. In contrast, GOQD-induced remarkable shrinkage of the plasma membrane and then enhanced cell permeability through strong internalization effects such as plasmolysis, uptake of nanomaterials, an oxidative stress increase, and inhibition of cell division and chlorophyll biosynthesis. Metabolomics analysis showed that amino acid metabolism was sensitive to nanomaterial exposure. Shrinkage of the plasma membrane is proposed to be linked to increases in the isoleucine levels. The inhibition of cell division and chlorophyll a biosynthesis was associated with decreases in aspartic acid and serine, the precursors of chlorophyll a. The increases in mitochondrial membrane potential loss and oxidative stress were correlated with an increase in linolenic acid. The above metabolites can be used as indicators of the corresponding biological responses. These results enhance our systemic understanding of the size-dependent biological effects of nanomaterials.Keywords: graphene oxide; metabolomics; nanotoxicology; quantum dot; reactive oxygen species; single cell

Graphene oxide (GO) is a widely used carbonaceous nanomaterial. To date, the influence of natural organic matter (NOM) on GO toxicity in aquatic vertebrates has not been reported. During zebrafish embryogenesis, GO induced a significant hatching delay and cardiac edema. The intensive interactions of GO with the chorion induces damage to chorion protuberances, excessive generation of •OH, and changes in protein secondary structure. In contrast, humic acid (HA), a ubiquitous form of NOM, significantly relieved the above adverse effects. HA reduced the interactions between GO and the chorion and mitigated chorion damage by regulating the morphology, structures, and surface negative charges of GO. HA also altered the uptake and deposition of GO and decreased the aggregation of GO in embryonic yolk cells and deep layer cells. Furthermore, HA mitigated the mitochondrial damage and oxidative stress induced by GO. This work reveals a feasible antidotal mechanism for GO in the presence of NOM and avoids overestimating the risks of GO in the natural environment.

The extensive use of pristine graphene oxide (PGO) increases its environmental release. The interactions of PGO with soils, one of the ultimate repositories for discharged nanomaterial, remain unclear. In the present study, a pyrosequencing analysis based on the bacterial 16S rRNA gene showed that the bacterial community in a PGO-soil sample (PGOS) became richer and more diverse compared with a control soil sample (CS). PGO altered the structure of soil bacterial communities, with some nitrogen-fixing and dissimilatory iron reducing bacteria being selectively enriched, especially at the genus level. SGO (soil-modified graphene oxide) exhibited a greater thickness, a higher C/O ratio, a rougher texture, a lower transparency and a smaller size than PGO. Nitrogen-containing groups and the elements including Mg, Al, Si, K, Ca and Fe were detected in SGO. The changes in surface groups were consistent with the formation of organic molecules coating the SGO. SGO, which exhibited fewer negative charges, was more unstable than PGO. In addition, SGO presented higher chemical activity than PGO; for example, SGO exhibited more unpaired electrons and disordered structures. This work highlights the critical interactions of PGO and soil which deserve comprehensive consideration in assessing the risks of nanomaterials.

In the present work, we identified root exudates that were stimulated by pristine graphene oxide (PGO), including small-molecule acids, alcoholates, alkanes, amino acids and secondary metabolites. These exudates acted as ligands and became immobilized on the PGO to form ligand-GO complexes (LGO). Compared with PGO, LGO exhibited increased thickness, a higher C:O ratio, and reduced transparency and size. Nitrogen- and sulfur-containing groups were observed in LGO. LGO, with its decreased negative charges, was less stable than PGO. In addition, LGO exhibited more unpaired electrons and disordered structures and greater UV absorption compared with PGO. The above alterations in the properties of PGO that occurred after modification of the PGO by exudates induced significant malformations (abnormal tail flexure and pericardial edema) and the loss of mitochondrial membrane polarization in zebrafish as a model organism. This work revealed that root exudates act as natural ligands and significantly alter the properties of PGO.

•Biologically and environmentally relevant exposure doses and terms•Effects of matrices, coronas, transformed nanomaterials and by-products•Reversibility and adaptation to nanotoxicity and transgenerational effects•High-throughput assay integrating with omics•Special mechanisms and control strategies of nanotoxicologyWith the wide research and application of nanomaterials in various fields, the safety of nanomaterials attracts much attention. An increasing number of reports in the literature have shown the adverse effects of nanomaterials, representing the quick development of nanotoxicology. However, many studies in nanotoxicology have not reflected the real nanomaterial safety, and the knowledge gaps between nanotoxicological research and nanomaterial safety remain large. Considering the remarkable influence of biological or environmental matrices (e.g., biological corona) on nanotoxicity, the situation of performing nanotoxicological experiments should be relevant to the environment and humans. Given the possibility of long-term and low-concentration exposure of nanomaterials, the reversibility of and adaptation to nanotoxicity, and the transgenerational effects should not be ignored. Different from common pollutants, the specific analysis methodology for nanotoxicology need development and exploration furthermore. High-throughput assay integrating with omics was highlighted in the present review to globally investigate nanotoxicity. In addition, the biological responses beyond individual levels, special mechanisms and control of nanotoxicity deserve more attention. The progress of nanotoxicology has been reviewed by previous articles. This review focuses on the blind spots in nanotoxicological research and provides insight into what we should do in future work to support the healthy development of nanotechnology and the evaluation of real nanomaterial safety.

•Control strategy of nanotoxicity remains in its infancy.•Structure modifications and unintended byproducts•Improving biocompatibility by surface, size and shape control•Comparison of chemical and biological strategies•Future concerns in life-cycle, occupational exposure and methodologyWith rapid development of nanotechnology and nanomaterials, nanosafety has attracted wide attention in all fields related to nanotechnology. As well known, a grand challenge in nanomaterial applications is their biocompatibility. It is urgent to explore effective strategies to control the unintentional effects. Although many novel methods for the synthesis of biocompatible and biodegradable nanomaterials are reported, the control strategy of nanotoxicity remains in its infancy. It is urgent to review the archived strategies for improving nanomaterial biocompatibility to clarify what we have done and where we should be. In this review, the achievements and challenges in nanomaterial structure/surface modifications and size/shape controls were analyzed. Moreover, the chemical and biological strategies to make nanomaterial more biocompatible and biodegradable were compared. Finally, the concerns that have not been studied well were prospected, involving unintended releases, life-cycle, occupational exposure and methodology.Download high-res image (144KB)Download full-size image

Studies of the environmental and health risks of graphene oxide (GO, a carbon nanosheet of broad concern) have focused on direct exposure. In contrast, the effects of GO on offspring through parental exposure at trace concentrations remain largely unknown, particularly in sensitive neurological systems. Thus, parental zebrafish were exposed to GO nanosheets at concentrations of 0.01–1 μg/L. GO translocated from the water to the brains of parental and offspring fish with a significant loss of claudin5a (a core component of the neuroepithelial barrier system). GO did not trigger obvious neurotoxicity in parental zebrafish, whereas remarkable neurotoxicity occurred in the offspring, which exhibited a loss of dopaminergic neurons and reductions in acetylcholinesterase activity. In the offspring, ER damage, autophagy promotion, ubiquitin downregulation and increased β-galactosidase activity were observed. Orthogonal partial least squares discriminant analysis revealed that the failures of carbohydrate and fatty acid metabolisms positively contributed to the loss of offspring dopaminergic neurons. The above results support the need for offspring to be examined in nanotoxicology, even for environmentally relevant concentrations.

Over the past decade, the safety of nanomaterials has attracted attention due to their rapid development. The relevant health threat of these materials remains largely unknown, particularly at environmentally or biologically relevant ultra-trace concentrations. To address this, we first found that graphene oxide (GO, a carbon nanomaterial that receives extensive attention across various disciplines) at concentrations of 0.01 μg/L–1 μg/L induced Parkinson's disease-like symptoms in zebrafish larvae. In this model, zebrafish showed a loss of more than 90% of dopamine neurons, a 69–522% increase in Lewy bodies (α-synuclein and ubiquitin) and significantly disturbed locomotive activity. Moreover, it was also shown that GO was able to translocate from the water environment to the brain and localize to the nucleus of the diencephalon, thereby inducing structural and morphological damage in the mitochondria. Cell apoptosis and senescence were triggered via oxidative stress, as shown by the upregulation of caspase 8 and β-galactosidase. Using metabolomics, we found that the upregulation of amino acid and some fatty acids (e.g. dodecanoic acid, hexadecanoic acid, octadecenoic acid, nonanoic acid, arachidonic acid, eicosanoic acid, propanoic acid and benzenedicarboxylic acid) metabolism and the downregulation of some other fatty acids (e.g. butanoic acid, phthalic acid and docosenoic acid) are linked to these Parkinson's disease-like symptoms. These findings broaden our understanding of nanomaterial safety at ultra-trace concentrations.

•Nanotoxicity is affected by environmental factors.•Agglomeration and physicochemical features of NPs change in natural environment.•Physicochemical properties of NPs are interdependent for nanotoxicity.•Challenges and perspectives of nanotoxicity in natural environment•Nanotoxicity in environmental field remains in infancy.Nanomaterials develop rapidly and are applied in various fields. Generally, these nanomaterials are released into the environment and lead to potential nanotoxicity and environmental risks. Importantly, the environmental behaviors and properties of nanomaterials will inevitably be influenced by environmental factors. As a result, biological responses of the nanomaterials under the action of environmental factors are largely different from those of pristine nanomaterials. Therefore, this review discussed the influence of environmental factors (e.g., light irradiation, natural organic matter, ionic strength, coexisting contaminants, temperature, biomedium and chemical surface modifications) on nanotoxicity and the relevant knowledge gaps, and then explored the mechanisms thereof. Environmental factors affected nanotoxicity by regulating their physicochemical features, changing physiological damage, oxidative stress, and biomolecular responses caused by nanomaterials. In addition, the synergic effects of environmental factors and the dominant factors in natural environment should be taken into consideration when nanotoxicity is discussed. At the end of the review, the global perspectives of nanotoxicity with the influence of environmental factors are proposed.Download high-res image (112KB)Download full-size image

Blood is the main biological fluid of humans and most animals. Understanding the effects of the biotransformation of nanomaterials in blood plasma on their interactions with cells is fundamental for the evaluation of their health risks and applications. However, there is a lack of related information. The present work found that free radicals and biological molecules in human blood plasma simultaneously drive the formation of a biological corona on biodegraded graphene oxide (GO) nanosheets. Importantly, the above biotransformation affected the interactions of GO with cells. For example, the biotransformed GO induced lower levels of reactive oxygen species and cell ultrastructure damage than did the pristine GO. In addition to the well-known protein corona, the small-molecule corona on the biodegraded GO also plays a critical role in the reduction of GO cytotoxicity through quenching excessive free radical generation. Metabolomic analysis revealed that the biotransformation reduced the oxidative stress induced by GO mainly via upregulation of the fatty acid metabolism and downregulation of the galactose metabolism. Overall, the present work clearly shows that the biotransformation of GO in blood plasma influences the nanotoxicity of GO. Compared with pristine nanomaterials, biotransformed materials are more biologically relevant to assessments of their environmental health risks.