Manganese dioxide nanosheets induce mitochondrial toxicity in fish gill epithelial cells.

The development and production of engineered 2D nanomaterials are expanding exponentially, increasing the risk of their release into the aquatic environment. A recent study showed 2D MnO2 nanosheets, under development for energy and biomedical applications, dissolve upon interaction with biological reducing agents, resulting in depletion of intracellular glutathione levels within fish gill cells. However, little is known concerning their toxicity and interactions with subcellular organelles. To address this gap, we examined cellular uptake, cytotoxicity and mitochondrial effects of 2D MnO2 nanosheets using an in vitro fish gill cell line to represent a target tissue of rainbow trout, a freshwater indicator species. The data demonstrate cellular uptake of MnO2 nanosheets into lysosomes and potential mechanisms of dissolution within the lysosomal compartment. MnO2 nanosheets induced severe mitochondrial dysfunction at sub-cytotoxic doses. Quantitative, single cell fluorescent imaging revealed mitochondrial fission and impaired mitochondrial membrane potential following MnO2 nanosheet exposure. Seahorse analyses for cellular respiration revealed that MnO2 nanosheets inhibited basal respiration, maximal respiration and the spare respiratory capacity of gill cells, indicating mitochondrial dysfunction and reduced cellular respiratory activity. MnO2 nanosheet exposure also inhibited ATP production, further supporting the suppression of mitochondrial function and cellular respiration. Together, these observations indicate that 2D MnO2 nanosheets impair the ability of gill cells to respond to energy demands or prolonged stress. Finally, our data demonstrate significant differences in the toxicity of the 2D MnO2 nanosheets and their microparticle counterparts. This exemplifies the importance of considering the unique physical characteristics of 2D nanomaterials when conducting safety assessments.

The Key Characteristics of Carcinogens: Relationship to the Hallmarks of Cancer, Relevant Biomarkers, and Assays to Measure Them.

The key characteristics (KC) of human carcinogens provide a uniform approach to evaluating mechanistic evidence in cancer hazard identification. Refinements to the approach were requested by organizations and individuals applying the KCs. We assembled an expert committee with knowledge of carcinogenesis and experience in applying the KCs in cancer hazard identification. We leveraged this expertise and examined the literature to more clearly describe each KC, identify current and emerging assays and in vivo biomarkers that can be used to measure them, and make recommendations for future assay development. We found that the KCs are clearly distinct from the Hallmarks of Cancer, that interrelationships among the KCs can be leveraged to strengthen the KC approach (and an understanding of environmental carcinogenesis), and that the KC approach is applicable to the systematic evaluation of a broad range of potential cancer hazards in vivo and in vitro We identified gaps in coverage of the KCs by current assays. Future efforts should expand the breadth, specificity, and sensitivity of validated assays and biomarkers that can measure the 10 KCs. Refinement of the KC approach will enhance and accelerate carcinogen identification, a first step in cancer prevention.See all articles in this CEBP Focus section, "Environmental Carcinogenesis: Pathways to Prevention."

Chemical and Colloidal Dynamics of MnO2 Nanosheets in Biological Media Relevant for Nanosafety Assessment.

Many layered crystal phases can be exfoliated or assembled into ultrathin 2D nanosheets with novel properties not achievable by particulate or fibrous nanoforms. Among these 2D materials are manganese dioxide (MnO2 ) nanosheets, which have applications in batteries, catalysts, and biomedical probes. A novel feature of MnO2 is its sensitivity to chemical reduction leading to dissolution and Mn2+ release. Biodissolution is critical for nanosafety assessment of 2D materials, but the timing and location of MnO2 biodissolution in environmental or occupational exposure scenarios are poorly understood. This work investigates the chemical and colloidal dynamics of MnO2 nanosheets in biological media for environmental and human health risk assessment. MnO2 nanosheets are insoluble in most aqueous phases, but react with strong and weak reducing agents in biological fluid environments. In vitro, reductive dissolution can be slow enough in cell culture media for MnO2 internalization by cells in the form of intact nanosheets, which localize in vacuoles, react to deplete intracellular glutathione, and induce cytotoxicity that is likely mediated by intracellular Mn2+ release. The results are used to classify MnO2 nanosheets within a new hazard screening framework for 2D materials, and the implications of MnO2 transformations for nanotoxicity testing and nanosafety assessment are discussed.

Life After Being a Pathology Department Chair III: Reflections on the "Afterlife".

The Association of Pathology Chairs Senior Fellows Group provided reflections on activities that have kept them engaged and inspired after stepping down as chair. They offered advice to current chairs who were considering leaving their positions and also to individuals contemplating becoming pathology chairs. A majority (35/41) responded: 60% maintained teaching/mentoring activities; 43% engaged in hobbies; 40% took other administrative positions including deans, medical center chief executive officers, and residency program directors; 31% continued research; 28% wrote books; 20% performed community service; 14% led professional organizations; 14% developed specialized programs; 11% engaged in clinical service; and 11% performed entrepreneurial activities. Most individuals had several of these activities. One-third indicated that those considering becoming chair should be able to place faculty and department needs before their own. One-fourth emphasized the need to know why one wants to become chair, the need to develop clear goals, and the need to know what one wants to accomplish as chair before applying for and accepting the position. More than half (57%) indicated that before stepping down as chair, one should have a clear plan and/or professional goals that can be served by stepping down. Some even suggested that this be in place before applying for the chair. Almost two-thirds (63%) indicated they had no regrets stepping down as chair. These findings may be valuable to those contemplating stepping down from or stepping into any department chair position or other academic leadership role.

A novel human 3D lung microtissue model for nanoparticle-induced cell-matrix alterations.

BACKGROUND: Multi-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm. RESULTS: As inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins. CONCLUSION: 3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing.

The asbestos-carbon nanotube analogy: An update.

Nanotechnology is an emerging industry based on commercialization of materials with one or more dimensions of 100 nm or less. Engineered nanomaterials are currently incorporated into thin films, porous materials, liquid suspensions, or filler/matrix nanocomposites with future applications predicted in energy and catalysis, microelectronics, environmental sensing and remediation, and nanomedicine. Carbon nanotubes are one-dimensional fibrous nanomaterials that physically resemble asbestos fibers. Toxicologic studies in rodents demonstrated that some types of carbon nanotubes can induce mesothelioma, and the World Health Organization evaluated long, rigid multiwall carbon nanotubes as possibly carcinogenic for humans in 2014. This review summarizes key physicochemical similarities and differences between asbestos fibers and carbon nanotubes. The "fiber pathogenicity paradigm" has been extended to include carbon nanotubes as well as other high-aspect-ratio fibrous nanomaterials including metallic nanowires. This paradigm identifies width, length, and biopersistence of high-aspect-ratio fibrous nanomaterials as critical determinants of lung disease, including mesothelioma, following inhalation. Based on recent theoretical modeling studies, a fourth factor, mechanical bending stiffness, will be considered as predictive of potential carcinogenicity. Novel three-dimensional lung tissue platforms provide an opportunity for in vitro screening of a wide range of high aspect ratio fibrous nanomaterials for potential lung toxicity prior to commercialization.

Biodissolution and Cellular Response to MoO3 Nanoribbons and a New Framework for Early Hazard Screening for 2D Materials.

Two-dimensional (2D) materials are a broad class of synthetic ultra-thin sheet-like solids whose rapid pace of development motivates systematic study of their biological effects and safe design. A challenge for this effort is the large number of new materials and their chemical diversity. Recent work suggests that many 2D materials will be thermodynamically unstable and thus non-persistent in biological environments. Such information could inform and accelerate safety assessment, but experimental data to confirm the thermodynamic predictions is lacking. Here we propose a framework for early hazard screening of nanosheet materials based on biodissolution studies in reactive media, specially chosen for each material to match chemically feasible degradation pathways. Simple dissolution and in vitro tests allow grouping of nanosheet materials into four classes: A, potentially biopersistent; B: slowly degradable (>24-48 hours); C, biosoluble with potentially hazardous degradation products; and D, biosoluble with low-hazard degradation products. The proposed framework is demonstrated through an experimental case study on MoO3 nanoribbons, which have a dual 2D / 1D morphology and have been reported to be stable in aqueous stock solutions. The nanoribbons are shown to undergo rapid dissolution in biological simulant fluids and in cell culture, where they elicit no adverse responses up to 100µg ml-1 dose. These results place MoO3 nanoribbons in Class D, and assigns them a low priority for further nanotoxicology testing. We anticipate use of this framework could accelerate the risk assessment for the large set of new powdered 2D nanosheet materials, and promote their safe design and commercialization.

Impact of emerging, high-production-volume graphene-based materials on the bioavailability of benzo(a)pyrene to brine shrimp and fish liver cells.

With increasing commercialization of high volume, two-dimensional carbon nanomaterials comes a greater likelihood of environmental release. In aquatic environments, black carbon binds contaminants like aromatic hydrocarbons, leading to changes in their uptake, bioavailability, and toxicity. Engineered carbon nanomaterials can also adsorb pollutants onto their carbon surfaces, and nanomaterial physicochemical properties can influence this contaminant interaction. We used 2D graphene nanoplatelets and isometric carbon black nanoparticles to evaluate the influence of particle morphology and surface properties on adsorption and bioavailability of benzo(a)pyrene, a model aromatic hydrocarbon, to brine shrimp (Artemia franciscana) and a fish liver cell line (PLHC-1). Acellular adsorption studies show that while high surface area carbon black (P90) was most effective at a given concentration, 2D graphene nanoplatelets (G550) adsorbed more benzo(a)pyrene than carbon black with comparable surface area (M120). In both biological models, co-exposure to nanomaterials lead to reduced bioavailability, with G550 graphene nanoplatelets cause a greater reduction in bioavailability or response than the M120 carbon black nanoparticles. However, on a mass basis the high surface area P90 carbon black was most effective. The trends in bioavailability and adsorption were consistent across all biological and acellular studies, demonstrating the biological relevance of these results in different models of aquatic organisms. While adsorption is limited by surface area, 2D graphene nanoplatelets adsorb more benzo(a)pyrene than carbon black nanoparticles of similar surface area and charge, demonstrating that both surface area and shape play important roles in the adsorption and bioavailability of benzo(a)pyrene to carbon nanomaterials.

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures.

To identify the potential environmental impacts of aquatic pollutants, rapid and sensitive screening tools are needed to assess adaptive and toxic responses. This study characterizes a novel fish liver microtissue model, produced with the cell line PLHC-1, as an in vitro aquatic toxicity testing platform. These 3D microtissues remain viable and stable throughout the 8-day testing period and relative to 2D monolayers, show increased basal expression of the xenobiotic metabolizing enzyme cytochrome P450 1A (Cyp1a). To evaluate pulsed, low-dose exposures at environmentally relevant concentrations, microtissue responsiveness to the model toxicant benzo(a)pyrene was assessed after single and repeated exposures for determination of both immediate and persistent effects. Significant induction of Cyp1a gene and protein expression was detected after a single 24h exposure to as little as 1nM benzo(a)pyrene, and after a 24h recovery period, Cyp1a expression declined in a dose-dependent manner. However, cell death continued to increase during the recovery period and alterations in microtissue architecture occurred at higher concentrations. To evaluate a pulsed or repeated exposure scenario, microtissues were exposed to benzo(a)pyrene, allowed to recover, then exposed a second time for 24h. Following pre-exposure to benzo(a)pyrene, cyp1a expression remained equally inducible and the pattern and level of Cyp1a protein response to a second exposure were comparable. However, pre-exposure to 1µM or 5µM of benzo(a)pyrene resulted in increased cell death, greater disruption of microtissue architecture, and alterations in cell morphology. Together, this study demonstrates the capabilities of this PLHC-1 microtissue model for sensitive assessment of liver toxicants over time and following single and repeated exposures.

Evaluating the mechanistic evidence and key data gaps in assessing the potential carcinogenicity of carbon nanotubes and nanofibers in humans.

In an evaluation of carbon nanotubes (CNTs) for the IARC Monograph 111, the Mechanisms Subgroup was tasked with assessing the strength of evidence on the potential carcinogenicity of CNTs in humans. The mechanistic evidence was considered to be not strong enough to alter the evaluations based on the animal data. In this paper, we provide an extended, in-depth examination of the in vivo and in vitro experimental studies according to current hypotheses on the carcinogenicity of inhaled particles and fibers. We cite additional studies of CNTs that were not available at the time of the IARC meeting in October 2014, and extend our evaluation to include carbon nanofibers (CNFs). Finally, we identify key data gaps and suggest research needs to reduce uncertainty. The focus of this review is on the cancer risk to workers exposed to airborne CNT or CNF during the production and use of these materials. The findings of this review, in general, affirm those of the original evaluation on the inadequate or limited evidence of carcinogenicity for most types of CNTs and CNFs at this time, and possible carcinogenicity of one type of CNT (MWCNT-7). The key evidence gaps to be filled by research include: investigation of possible associations between in vitro and early-stage in vivo events that may be predictive of lung cancer or mesothelioma, and systematic analysis of dose-response relationships across materials, including evaluation of the influence of physico-chemical properties and experimental factors on the observation of nonmalignant and malignant endpoints.

Nanomechanical mechanism for lipid bilayer damage induced by carbon nanotubes confined in intracellular vesicles.

Understanding the behavior of low-dimensional nanomaterials confined in intracellular vesicles has been limited by the resolution of bioimaging techniques and the complex nature of the problem. Recent studies report that long, stiff carbon nanotubes are more cytotoxic than flexible varieties, but the mechanistic link between stiffness and cytotoxicity is not understood. Here we combine analytical modeling, molecular dynamics simulations, and in vitro intracellular imaging methods to reveal 1D carbon nanotube behavior within intracellular vesicles. We show that stiff nanotubes beyond a critical length are compressed by lysosomal membranes causing persistent tip contact with the inner membrane leaflet, leading to lipid extraction, lysosomal permeabilization, release of cathepsin B (a lysosomal protease) into the cytoplasm, and cell death. The precise material parameters needed to activate this unique mechanical pathway of nanomaterials interaction with intracellular vesicles were identified through coupled modeling, simulation, and experimental studies on carbon nanomaterials with wide variation in size, shape, and stiffness, leading to a generalized classification diagram for 1D nanocarbons that distinguishes pathogenic from biocompatible varieties based on a nanomechanical buckling criterion. For a wide variety of other 1D material classes (metal, oxide, polymer), this generalized classification diagram shows a critical threshold in length/width space that represents a transition from biologically soft to stiff, and thus identifies the important subset of all 1D materials with the potential to induce lysosomal permeability by the nanomechanical mechanism under investigation.

Chemical Dissolution Pathways of MoS2 Nanosheets in Biological and Environmental Media.

Material stability and dissolution in aqueous media are key issues to address in the development of a new nanomaterial intended for technological application. Dissolution phenomena affect biological and environmental persistence; fate, transport, and biokinetics; device and product stability; and toxicity pathways and mechanisms. This article shows that MoS2 nanosheets are thermodynamically and kinetically unstable to O2-oxidation under ambient conditions in a variety of aqueous media. The oxidation is accompanied by nanosheet degradation and release of soluble molybdenum and sulfur species, and generates protons that can colloidally destabilize the remaining sheets. The oxidation kinetics are pH-dependent, and a kinetic law is developed for use in biokinetic and environmental fate modeling. MoS2 nanosheets fabricated by chemical exfoliation with n-butyl-lithium are a mixture of 1T (primary) and 2H (secondary) phases and oxidize rapidly with a typical half-life of 1-30 days. Ultrasonically exfoliated sheets are in pure 2H phase, and oxidize much more slowly. Cytotoxicity experiments on MoS2 nanosheets and molybdate ion controls reveal the relative roles of the nanosheet and soluble fractions in the biological response. These results indicate that MoS2 nanosheets will not show long-term persistence in living systems and oxic natural waters, with important implications for biomedical applications and environmental risk.

Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials.

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

Biological and environmental interactions of emerging two-dimensional nanomaterials.

Two-dimensional materials have become a major focus in materials chemistry research worldwide with substantial efforts centered on synthesis, property characterization, and technological application. These high-aspect ratio sheet-like solids come in a wide array of chemical compositions, crystal phases, and physical forms, and are anticipated to enable a host of future technologies in areas that include electronics, sensors, coatings, barriers, energy storage and conversion, and biomedicine. A parallel effort has begun to understand the biological and environmental interactions of synthetic nanosheets, both to enable the biomedical developments and to ensure human health and safety for all application fields. This review covers the most recent literature on the biological responses to 2D materials and also draws from older literature on natural lamellar minerals to provide additional insight into the essential chemical behaviors. The article proposes a framework for more systematic investigation of biological behavior in the future, rooted in fundamental materials chemistry and physics. That framework considers three fundamental interaction modes: (i) chemical interactions and phase transformations, (ii) electronic and surface redox interactions, and (iii) physical and mechanical interactions that are unique to near-atomically-thin, high-aspect-ratio solids. Two-dimensional materials are shown to exhibit a wide range of behaviors, which reflect the diversity in their chemical compositions, and many are expected to undergo reactive dissolution processes that will be key to understanding their behaviors and interpreting biological response data. The review concludes with a series of recommendations for high-priority research subtopics at the "bio-nanosheet" interface that we hope will enable safe and successful development of technologies related to two-dimensional nanomaterials.

Into the depths: Techniques for in vitro three-dimensional microtissue visualization.

Three-dimensional (3-D) in vitro platforms have been shown to closely recapitulate human physiology when compared with conventional two-dimensional (2-D) in vitro or in vivo animal model systems. This confers a substantial advantage in evaluating disease mechanisms, pharmaceutical drug discovery, and toxicity testing. Despite the benefits of 3-D cell culture, limitations in visualization and imaging of 3-D microtissues present significant challenges. Here we optimized histology and microscopy techniques to overcome the constraints of 3-D imaging. For morphological assessment of 3-D microtissues of several cell types, different time points, and different sizes, a two-step glycol methacrylate embedding protocol for evaluating 3-D microtissues produced using agarose hydrogels improved resolution of nuclear and cellular histopathology characteristic of cell death and proliferation. Additional immunohistochemistry, immunofluorescence, and in situ immunostaining techniques were successfully adapted to these microtissues and enhanced by optical clearing. Utilizing the Clear(T2) protocol greatly increased fluorescence signal intensity, imaging depth, and clarity, allowing for more complete confocal fluorescence microscopy imaging of these 3-D microtissues compared with uncleared samples. The refined techniques presented here address the key challenges associated with 3-D imaging, providing new and alternative methods in evaluating disease pathogenesis, delineating toxicity pathways, and enhancing the versatility of 3-D in vitro testing systems in pharmacological and toxicological applications.

Nanomaterial categorization for assessing risk potential to facilitate regulatory decision-making.

For nanotechnology to meet its potential as a game-changing and sustainable technology, it is important to ensure that the engineered nanomaterials and nanoenabled products that gain entry to the marketplace are safe and effective. Tools and methods are needed for regulatory purposes to allow rapid material categorization according to human health and environmental risk potential, so that materials of high concern can be targeted for additional scrutiny, while material categories that pose the least risk can receive expedited review. Using carbon nanotubes as an example, we discuss how data from alternative testing strategies can be used to facilitate engineered nanomaterial categorization according to risk potential and how such an approach could facilitate regulatory decision-making in the future.

Antioxidant chemistry of graphene-based materials and its role in oxidation protection technology.

Two-dimensional nanomaterials have potential as a new class of antioxidants that combine physical barrier function with ultrahigh surface area for free radical scavenging. This work presents the first measurements of the chemical reactivities of graphene-based materials toward a set of model free radicals and reactive oxygen species using electron paramagnetic resonance spectroscopy (EPR) and sacrificial dye protection assays. Graphene-based materials are shown to protect a variety of molecular targets from oxidation by these species, and to be highly effective as hydroxyl-radical scavengers. When the hydroxyl radical is produced photolytically, the overall antioxidant effect is a combination of preventative antioxidant activity (UV absorption) and ˙OH radical scavenging. Few-layer graphene is more active than monolayer graphene oxide, despite its lower surface area, which indicates that the primary scavenging sites are associated with the sp(2)-carbon network rather than oxygen-containing functional groups. To explain this trend, we propose that GO is a weak hydrogen donor, due to the non-phenolic nature of most OH groups on GO, which reside at basal sp(3)-carbon sites that do not allow for radical resonance stabilization following hydrogen donation. As an example application of graphene antioxidant behavior, we show that encapsulation of TiO2 nanoparticles in graphene nanosacks reduces undesired photo-oxidative damage to nearby organic target molecules, which suggests graphene encapsulation as a new approach to managing adverse environmental or health impacts of redox-active nanomaterials.

Effects of surface-engineered nanoparticle-based dispersants for marine oil spills on the model organism Artemia franciscana.

Fine particles are under active consideration as alternatives to chemical dispersants for large-scale petroleum spills. Fine carbon particles with engineered surface chemistry have been shown to stabilize oil-in-water emulsions, but the environmental impacts of large-scale particle introduction to the marine environment are unknown. Here we study the impact of surface-engineered carbon-black materials on brine shrimp (Artemia franciscana) as a model marine microcrustacean. Mortality was characterized at 50-1000 mg/L, and levels of heat shock protein 70 (hsp70) were characterized at sublethal particle concentrations (25-50 mg/L). Functionalized carbon black (CB) nanoparticles were found to be nontoxic at all concentrations, while hydrophobic (annealed) and as-produced CB induced adverse effects at high concentrations. CB was also shown to adsorb benzene, a model hydrocarbon representing the more soluble and toxic low-molecular weight aromatic fraction of petroleum, but the extent of adsorption was insufficient to mitigate benzene toxicity to Artemia in coexposure experiments. At lower benzene concentrations (25-75 mg/L), coexposure with annealed and as-produced CB increased hsp70 protein levels. This study suggests that surface functionalization for increased hydrophilicity can not only improve the performance of CB-based dispersants but also reduce their adverse environmental impacts on marine organisms.

Biological and environmental transformations of copper-based nanomaterials.

Copper-based nanoparticles are an important class of materials with applications as catalysts, conductive inks, and antimicrobial agents. Environmental and safety issues are particularly important for copper-based nanomaterials because of their potential large-scale use and their high redox activity and toxicity reported from in vitro studies. Elemental nanocopper oxidizes readily upon atmospheric exposure during storage and use, so copper oxides are highly relevant phases to consider in studies of environmental and health impacts. Here we show that copper oxide nanoparticles undergo profound chemical transformations under conditions relevant to living systems and the natural environment. Copper oxide nanoparticle (CuO-NP) dissolution occurs at lysosomal pH (4-5), but not at neutral pH in pure water. Despite the near-neutral pH of cell culture medium, CuO-NPs undergo significant dissolution in media over time scales relevant to toxicity testing because of ligand-assisted ion release, in which amino acid complexation is an important contributor. Electron paramagnetic resonance (EPR) spectroscopy shows that dissolved copper in association with CuO-NPs are the primary redox-active species. CuO-NPs also undergo sulfidation by a dissolution-reprecipitation mechanism, and the new sulfide surfaces act as catalysts for sulfide oxidation. Copper sulfide NPs are found to be much less cytotoxic than CuO-NPs, which is consistent with the very low solubility of CuS. Despite this low solubility of CuS, EPR studies show that sulfidated CuO continues to generate some ROS activity due to the release of free copper by H2O2 oxidation during the Fenton-chemistry-based EPR assay. While sulfidation can serve as a natural detoxification process for nanosilver and other chalcophile metals, our results suggest that sulfidation may not fully and permanently detoxify copper in biological or environmental compartments that contain reactive oxygen species.