Analysis of carbon nanotube levels in organic matter: an inter-laboratory comparison to determine best practice.

Carbon nanotubes (CNTs) are increasingly being used in industrial applications, but their toxicological data in animals and humans are still sparse. To assess the toxicological dose-response of CNTs and to evaluate their pulmonary biopersistence, their quantification in tissues, especially lungs, is crucial. There are currently no reference methods or reference materials for low levels of CNTs in organic matter. Among existing analytical methods, few have been fully and properly validated. To remedy this, we undertook an inter-laboratory comparison on samples of freeze-dried pig lung, ground and doped with CNTs. Eight laboratories were enrolled to analyze 3 types of CNTs at 2 concentration levels each in this organic matrix. Associated with the different analysis techniques used (specific to each laboratory), sample preparation may or may not have involved prior digestion of the matrix, depending on the analysis technique and the material being analyzed. Overall, even challenging, laboratories' ability to quantify CNT levels in organic matter is demonstrated. However, CNT quantification is often overestimated. Trueness analysis identified effective methods, but systematic errors persisted for some. Choosing the assigned value proved complex. Indirect analysis methods, despite added steps, outperform direct methods. The study emphasizes the need for reference materials, enhanced precision, and organized comparisons.

Disruption of phosphofructokinase activity and aerobic glycolysis in human bronchial epithelial cells by atmospheric ultrafine particulate matter.

Exposure to ambient ultrafine particulate matter (UPM) causes respiratory disorders; however, the underlying molecular mechanisms remain unclear. In this study, we synthesized simulated UPM (sUPM) with controlled physicochemical properties using the spark-discharge method. Subsequently, we investigated the biological effects of sUPM using BEAS-2B human bronchial epithelial cells (HBECs) and a mouse intratracheal instillation model. High throughput RNA-sequencing and bioinformatics analyses revealed that dysregulation of the glycolytic metabolism is involved in the inhibited proliferation and survival of HBECs by sUPM treatment. Furthermore, signaling pathway and enzymatic analyses showed that the treatment of BEAS-2B cells with sUPM induces the inactivation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB, also known as AKT), resulting in the downregulation of phosphofructokinase 2 (PFK2) S483 phosphorylation, PFK enzyme activity, and aerobic glycolysis in HBECs in an oxidative stress-independent manner. Additionally, intratracheal instillation of sUPM reduced the phosphorylation of ERK, AKT, and PFK2, decreased proliferation, and increased the apoptosis of bronchial epithelial cells in mice. The findings of this study imply that UPM induces pulmonary toxicity by disrupting aerobic glycolytic metabolism in lung epithelial cells, which can provide novel insights into the toxicity mechanisms of UPM and strategies to prevent their toxic effects.

Effect of sp3/sp2 carbon ratio and hydrodynamic size on the biodistribution kinetics of nanodiamonds in mice via intravenous injection.

BACKGROUND: Nanodiamonds (NDs) have gained a rapidly growing interest in biomedical applications; however, little is known regarding their biokinetics owing to difficulties in measurements and limited synthesis/purification technologies. In this study, we investigated the distribution kinetics of detonation-synthesized NDs in mice via intravenous injection to evaluate the parameters that determine the behavior of the particles. We prepared two distinctive NDs that controlled the sp3/sp2 carbon ratio and particle size by coating them with serum proteins. The four control samples were intravenously injected into mice, and tissue distribution and clearance were evaluated at 30 min and 1, 7, and 28 days post-injection. RESULTS: The sp3/sp2 carbon ratio showed no correlation with the organ distribution of the NDs. However, hydrodynamic size showed an excellent correlation with organ distribution levels: a negative correlation in the liver and positive correlations in the spleen and lungs. Furthermore, the deposition levels of NDs in the lung suggest that particles smaller than 300 nm could avoid lung deposition. Finally, a similar organ distribution pattern was observed in mice injected with carbon black nanoparticles controlled hydrodynamic size. CONCLUSIONS: In conclusion, the tissue distribution of NDs is modulated not by the sp3/sp2 carbon ratio but by the hydrodynamic size, which can provide helpful information for targeting the tissue of NDs. Furthermore, the organ distribution pattern of the NDs may not be specific to NDs but also can apply to other nanoparticles, such as carbon black.

Size- and oxidative potential-dependent toxicity of environmentally relevant expanded polystyrene styrofoam microplastics to macrophages.

Expanded polystyrene (EPS), also known as Styrofoam, is a widespread global pollutant, and its lightweight floating property increases its chances of weathering by abrasion and ultraviolet (UV) irradiation, resulting in microplastics. Herein, we investigated the effects of particle size ((1 µm versus 10 µm), UV irradiation (pristine versus UV oxidation), and origin (secondary versus primary) on the toxicity of Styrofoam microplastics. The target cells used in this study were selected based on human exposure-relevant cell lines: differentiated THP-1 cells for macrophages, Caco-2 for enterocytes, HepG2 for hepatocytes, and A549 for alveolar epithelial cells. In the differentiated THP-1 cells, the levels of cytotoxicity and inflammatory cytokines showed size- (1 µm > 10 µm), UV oxidation- (UV > pristine), and origin- (secondary > primary) dependency. Furthermore, the intrinsic oxidative potential of the test particles was positively correlated with cellular oxidative levels and toxicity endpoints, suggesting that the toxicity of Styrofoam microplastics also follows the oxidative stress paradigm. Additionally, all microplastics induced the activation of the pyrin domain-containing protein 3 (NLRP3) inflammasome and the release of interleukin-1ß (IL-1ß). These results imply that weathering process can aggravate the toxicity of Styrofoam microplastics due to the increased oxidative potential and decreased particle size.

Zinc oxide nanoparticles trigger autophagy-mediated cell death through activating lysosomal TRPML1 in normal kidney cells.

Zinc oxide nanoparticles (ZnO NPs) have been widely used in various materials including sunscreens, cosmetics, over-the-counter topical skin products, and pigments. As traces of the used ZnO NPs have been found in the kidney, it is crucial to uncover their potential risks. The aim of this study is to elucidate detrimental effects of ZnO NPs and the molecular mechanism behind their renal toxicity. Cytotoxic effects were measured by MTT assay after HK2 cells were exposed to ZnO NPs for 24 h and IC50 value was determined. ROS and intracellular Zn2+ levels were detected by flow cytometry, and localization of Zn2+ and lysosome was determined by confocal microscopy. Occurrence of autophagy and detection of autophagic flux were determined by Western blot and confocal microscopy, respectively. We performed unpaired student t test for two groups, and one-way ANOVA with Tukey's post hoc for over three groups. ZnO NPs induced cell death in human renal proximal tubule epithelial cells, HK2. Cytosolic Zn2+ caused autophagy-mediated cell death rather than apoptosis. Cytosolic Zn2+ processed in lysosome was released by TRPML1, and inhibition of TRPML1 significantly decreased autophagic flux and cell death. The findings of this study suggest that ZnO NPs strongly induce autophagy-mediated cell death in human kidney cells. Controlling TRPML1 can be potentially used to prevent the kidney from ZnO NPs-induced toxicity.

Differential particle and ion kinetics of silver nanoparticles in the lungs and biotransformation to insoluble silver sulfide.

The measurement of nanoparticles (NPs) in a biological matrix is essential in various toxicity studies. However, the current knowledge has limitations in differentiating particulate and ionic forms and further identification of their biotransformation. Herein, we evaluate the biotransformation and differential lung clearance kinetics of particulate and ionic forms using PEGylated silver NPs (AgNP-PEGs; 47.51 nm) and PEGylated gold NPs (AuNP-PEGs; 11.76 nm). At 0, 3, and 6 h and 1, 3, 7, and 14 days after a single pharyngeal aspiration in mice at 25 µg/mouse, half of the lung is digested by proteinase K (PK) to separate particulates and ions, and the other half is subjected to the acid digestion method for comparison. The quantitative and qualitative evaluation of lung clearance kinetics suggests that AgNP-PEGs are quickly dissolved and transformed into insoluble silver sulfide (Ag2S), which shows a fast-clearing early phase (0 -6 h; particle T1/2: 4.8 h) and slow-clearing late phase (1 -14 days; particle T1/2: 13.20 days). In contrast, AuNP-PEGs were scarcely cleared or biotransformed in the lungs for 14 days. The lung clearance kinetics of AgNPs and biotransformation shown in this study can be informed by the PK digestion method and cannot be obtained using the acid digestion method.

Mutual regulation between phosphofructokinase 1 platelet isoform and VEGF promotes glioblastoma tumor growth.

Glioblastoma (GBM) is a highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, whether PFKP and VEGF are reciprocally regulated during GBM tumor growth remains unknown. Here, we show that PFKP can promote EGFR activation-induced VEGF expression in HIF-1α-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in both AKT/SP1-mediated transcriptional expression of HIF-1α and in the AKT-mediated ß-catenin S552 phosphorylation, to fully enhance VEGF transcription, subsequently promoting blood vessel formation and brain tumor growth. Levels of PFKP Y64 phosphorylation in human GBM specimens are positively correlated with HIF-1α expression, ß-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth and also suggest that targeting the PFKP/VEGF regulatory loop might show therapeutic potential for treating GBM patients.

Biokinetics of fluorophore-conjugated polystyrene microplastics in marine mussels.

Biokinetic information on microplastics in bivalves is required to reduce the human exposure, but little is known about the time-course and size effect on tissue absorption and clearance. The biokinetics of fluorophore-labeled polystyrene microbeads with diameters 10 µm (PL10) and 90 µm (PL90) in Mytilus galloprovincialis marine mussels was investigated in the present study. It was found that both PL10 and PL90 showed a biphasic tissue distribution pattern in digestive and non-digestive tissues, highlighting the significant tissue distribution starting from 48 h post-treatment. The differential size effect on tissue distribution was observed only in the gills, which suggests that PL10 accumulates more than PL90. The depuration kinetics show that particles of both sizes can be cleared in any tissue, but non-digestive tissue requires a longer duration for depuration than digestive tissue. The differential size effect on depuration was observed for both digestive and non-digestive tissues, suggesting that PL10 needed a longer duration for depuration than PL90. More than seven days were needed for depuration of microplastics in mussels, which is an exceptionally longer period compared to conventional depuration of bivalves. The most significant improvement of this study is providing the biokinetics of two different-sized microplastics in mussels and the differential time for purging microplastics from mussels.

Nanodiamond as a Cytokine Sponge in Infectious Diseases.

Cytokine release syndrome (CRS) is a systemic inflammatory response resulting in overexpression of cytokines in serum and tissues, which leads to multiple-organ failure. Due to rapid aggravation of symptoms, timely intervention is paramount; however, current therapies are limited in their capacity to address CRS. Here, we find that the intravenous injection of highly purified detonation-synthesized nanodiamonds (DND) can act as a therapeutic agent for treating CRS by adsorbing inflammatory cytokines. Highly purified DNDs successfully inactivated various key cytokines in plasma from CRS patients with pneumonia, septic shock, and coronavirus disease 2019 pandemic (COVID-19). The intravenous injection of the DND samples in a mouse sepsis model by cecal ligation and puncture significantly improved survival rates and prevented tissue damage by reducing the circulating inflammatory cytokines. The results of this study suggest that the clinical application of highly purified DND can provide survival benefits for CRS patients by adsorbing inflammatory cytokines.

Eugenitol ameliorates memory impairments in 5XFAD mice by reducing Aß plaques and neuroinflammation.

Alzheimer's disease (AD) is caused by various pathological mechanisms; therefore, it is necessary to develop drugs that simultaneously act on multiple targets. In this study, we investigated the effects of eugenitol, which has anti-amyloid ß (Aß) and anti-neuroinflammatory effects, in an AD mouse model. We found that eugenitol potently inhibited Aß plaque and oligomer formation. Moreover, eugenitol dissociated the preformed Aß plaques and reduced Aß-induced nero2a cell death. An in silico docking simulation study showed that eugenitol may interact with Aß1-42 monomers and fibrils. Eugenitol showed radical scavenging effects and potently reduced the release of proinflammatory cytokines from lipopolysaccharide-treated BV2 cells. Systemic administration of eugenitol blocked Aß aggregate-induced memory impairment in the Morris water maze test in a dose-dependent manner. In 5XFAD mice, prolonged administration of eugenitol ameliorated memory and hippocampal long-term potentiation impairment. Moreover, eugenitol significantly reduced Aß deposits and neuroinflammation in the hippocampus of 5XFAD mice. These results suggest that eugenitol, which has anti-Aß aggregation, Aß fibril dissociation, and anti-inflammatory effects, potently modulates AD-like pathologies in 5XFAD mice, and could be a promising candidate for AD therapy.

Effects of Perilla frutescens var. acuta in amyloid ß toxicity and Alzheimer's disease-like pathology in 5XFAD mice.

Although accumulation of amyloid ß (Aß) plaque is a major hallmark of Alzheimer's disease (AD), various pathologies have been suggested therapeutic targets. Therefore, therapies-targeting multiple pathologies would be required for effective managements of AD. Accordingly, natural products, which has multiple active ingredients, have been receiving a lot of attention. In this study, we tested whether standardized ethanol extract of leaves of Perilla frutescens var. acuta (L.) Britt. (Lamiaceae) (ELPF) could modulate various pathologies in AD using 5XFAD mice. ELPF blocked Aß aggregation and disassembled pre-formed Aß aggregates. ELPF blocked Aß aggregates-induced LTP impairment and ELPF-disassembled Aß aggregates failed to impair hippocampal LTP. Systemic administration of ELPF blocked Aß aggregates-induced memory impairment in a passive avoidance test. ELPF-disassembled Aß aggregates failed to impair passive avoidance memory. Prolonged administration of ELPF ameliorated memory impairments in 5XFAD mice. In the hippocampus of 5XFAD mice, ELPF administration significantly reduced Aß deposits and neuroinflammation. These results demonstrate that ELPF could be a promising therapeutic candidate for AD.

Hydrangea macrophylla and Thunberginol C Attenuate Stress-Induced Anxiety in Mice.

Stress is an important neurological input for successful life. However, chronic stress and stress hormones could be a cause of various neurological disorders including anxiety disorders. Therefore, there have been many efforts to find effective materials for curing stress-induced neurological disorders. In this study, we examined the effect of Hydrangea macrophylla (HM) on corticosterone-induced neurotoxicity, stress-induced anxiety in mice and suggested a possible active ingredient of HM. HM protected cortical neurons against neurotoxicity of corticosterone (CORT), a stress hormone. HM also blocked CORT-induced hippocampal synaptic deficit via regulating Akt signaling. Oral administration of HM improved chronic restraint stress-induced anxiety in Elevated Plus maze test along with reduction of plasma corticosterone and TNF-α levels. Moreover, HM reduced stress-induced neuroinflammation and oxidative stress. Thunberginol C, an active ingredient of HM, also prevented CORT-induced neuronal cell death and restraint stress-induced anxiety. Moreover, thunberginol C reduced plasma TNF-α level and neuroinflammation and oxidative stress. Collectively, HM could be a good candidate for preventing stress-induced neurological disorders and thunberginol C may be an active ingredient of HM for this purpose.

Exposure to Nickel Oxide Nanoparticles Induces Acute and Chronic Inflammatory Responses in Rat Lungs and Perturbs the Lung Microbiome.

Nickel oxide nanoparticles (NiO NPs) are highly redox active nanoparticles. They can cause acute and chronic inflammation in rat lungs. Unlike the gut microbiome, the association between the lung microbiome's role and pulmonary inflammatory response to inhaled nanoparticles remains largely unexplored. We aimed to explore the interaction between the lung microbiome and inflammatory responses in rats exposed to NiO NPs. Thirty female Wistar rats were randomly categorized into control and low- (50 cm2/rat), and high- (150 cm2/rat) dose NiO NPs exposure groups. NiO NPs were intratracheally instilled, and cytological, biochemical, proinflammatory cytokine, and lung microbiome analyses of bronchoalveolar lavage fluid were performed at 1 day and 4 weeks after instillation. NiO NPs caused a neutrophilic and lymphocytic inflammatory response in rat lung. We demonstrated that exposure to NiO NPs can alter the lung microbial composition in rats. In particular, we found that more Burkholderiales are present in the NiO NPs exposure groups than in the control group at 1 day after instillation. Dysbiosis in the lung microbiome is thought to be associated with acute lung inflammation. We also suggested that Burkholderiales may be a key biomarker associated with lung neutrophilic inflammation after NiO NPs exposure.

Role of extracellular signal-regulated kinase in rubrofusarin-enhanced cognitive functions and neurite outgrowth.

Memory-enhancing agents have long been required for various reasons such as for obtaining a good score in a test in the young and for retaining memory in the aged. Although many studies have found that several natural products may be good candidates for memory enhancement, there is still a need for better agents. The present study investigated whether rubrofusarin, an active ingredient in Cassiae semen, enhances learning and memory in normal mice. Passive avoidance and Morris water maze tests were performed to determine the memory-enhancing ability of rubrofusarin. To investigate synaptic function, hippocampal long-term potentiation (LTP) was measured. Western blotting was performed to determine protein levels. To investigate neurite outgrowth, DCX immunohistochemistry and cell culture were utilised. Rubrofusarin (1, 3, 10, 30 mg/kg) enhanced memory in passive avoidance and Morris water maze tests. Moreover, rubrofusarin ameliorated scopolamine-induced memory impairment. In the rubrofusarin-treated group, high-frequency stimulation induced higher LTP in the hippocampal Schaffer-collateral pathway compared to the control group. The rubrofusarin-treated group showed a higher number of DCX-positive immature neurons with an increase in the length of dendrites compared to the control group in the hippocampal dentate gyrus region. In vitro experiments showed that rubrofusarin facilitated neurite outgrowth in neuro2a cells through extracellular signal-regulated kinase (ERK). Finally, we found that extracellular signal-regulated kinase (ERK) is required for rubrofusarin-induced enhancement of neurite outgrowth, learning and memory. These results demonstrate that rubrofusarin enhances learning and memory and neurite outgrowth, and these might need activation of ERK pathway.

Development of chitosan decorated Fe3O4 nanospheres for potential enhancement of photocatalytic degradation of Congo red dye molecules.

Fe3O4 nanospheres (Nsps) and chitosan (Cts)/Fe3O4 Nsps were prepared using a one-pot hydrothermal method and subsequently used as photocatalysts against the degradation of Congo red (CR) dye molecules. The sphere-shaped Fe3O4 nanoparticles were heterogeneously decorated by the Cts matrix, which was confirmed by powder X-ray diffraction, scanning and transmission electron microscopies. The Cts/Fe3O4 Nsps demonstrated 98% efficient photocatalytic activity against CR dye molecules upon 60 min exposure to visible light compared to Fe3O4 Nsps (77% for 60 min). When compared to Fe3O4 Nsps, the visible light photocatalytic efficiency of Cts/Fe3O4 Nsps against CR dye molecules was significantly improved.

Six-well plate-based colony-forming efficacy assay and Co-Culture application to assess toxicity of metal oxide nanoparticles.

The development of a universal, label-free, and reliable in vitro toxicity testing method for nanoparticles is urgent because most nanoparticles can interfere with toxicity assays. In this regard, the colony-forming efficacy (CFE) assay has been suggested as a suitable in vitro toxicity assay for testing nanoparticles without such interference. Recently, the Organisation for Economic Co-operation and Development (OECD) developed a 60 × 15 mm Petri dish-based CFE assay for testing nanoparticles in MDCK-1 cells. However, further investigations are needed, including testing with other cell types, at a smaller scale for greater efficiency, and the application of the co-culture technique. In this study, we selected TiO2, CuO, CeO2, and SiO2 as test nanoparticles and successfully developed a 6-well plate-based CFE assay using HepG2 and A549 cells and a co-culture assay for combinations of HepG2 cells and THP-1 macrophages or A549 cells and THP-1 monocytes. The results suggest that the 6-wellplate-based CFE assay for HepG2 and A549 cells can be applied to nanoparticles, but the co-culture CFE assay has limitations in that it is not different from the single culture study, and it inhibits colony-formation by A549 cells in the presence of macrophages; this warrant further study.

Acute and subacute repeated oral toxicity study of fragmented microplastics in Sprague-Dawley rats.

Polypropylene (PP) is the second most highly produced plastic worldwide, and its microplastic forms are found in water and food matrices. However, the effects of PP microplastics on human health remain largely unknown. Here, we prepared 85.2 µm-sized weathered PP (w-PP) microplastics by sieving the microplastic particles after fragmentation and accelerated weathering processes. The prepared particles are irregular in shape and no chemical additives including phthalates and bisphenol A were not released in simulated body fluids. Then, the w-PP samples were gavaged to rats for acute and subacute toxicity testing in accordance to the Organization for Economic Co-operation and Development (OECD) test guidelines under good laboratory practice regulations. The highest dose for gavaging to rats was 25 mg/kg bw/day, which was the maximum feasible dose based on the dispersibility of microplastics. Both toxicity testings for w-PP microplastics showed no adverse effects and mutagenicity. Thus, the no observed adverse effect level (NOAEL) of w-PP microplastics is higher than 25 mg/kg bw/day. Furthermore, the w-PP microplastics did not show any skin or eye irritation potentials in the 3-dimensional reconstructed human skin or corneal culture model. The dose of 25 mg/kg of w-PP microplastics is roughly equal to 2.82 × 105 particles/kg, which suggests that human exposure to w-PP microplastics in a real-life situation may not have any adverse effects.

ABCG1 and ABCG4 as key transporters in the development of pulmonary alveolar proteinosis by nanoparticles.

Pulmonary alveolar proteinosis (PAP) has been reported in rodents treated with nanoparticles (NPs). However, little is known about the type of NPs producing PAP and their toxicity mechanisms. Here, we assembled seven PAP-inducing NPs and TiO2 NPs as a negative control. At 1 and 6 months after a single intratracheal instillation in rats, pulmonary inflammation and the gene expression of ATP-binding cassette (ABC) transporters and related genes were evaluated in separated alveolar macrophages (AMs). One month after intratracheal instillation, seven NPs (Eu2O3, In2O3, Pr6O11, Sm2O3, Tb4O7, and NiO) caused PAP, but only In2O3 NPs caused persistent PAP at 6 months after treatment. The levels of phospholipids, indicators of PAP, showed good correlations with the gene expression profile of five transporters (ABCA1, ABCB4, ABCB8, ABCG1, and ABCG4), which effluxing phospholipids in AMs. Among them, ABCG1 and ABCG4 might be key transporters involved in PAP development because both showed a negative correlation with the magnitude of PAP, while others might be compensatory transporters for PAP recovery, as they showed a positive correlation. In conclusion, the identification of seven PAP-producing NPs implies that PAP may be an emerging occupational disease and that ABCG1 and ABCG4 may be therapeutic targets for PAP.

The reactive oxygen species as pathogenic factors of fragmented microplastics to macrophages.

The presence of microplastics in the various food web raised concerns on human health, but little is known about the target cells and mechanism of toxicity of microplastics. In this study, we evaluated the toxicity of microplastics using relevant cell lines to the oral route of exposure. Approximately 100 µm-sized fragment-type polypropylene (PP) and polystyrene (PS) particles were prepared by sieving after pulverization and further applied the accelerated weathering using ultraviolet and heat. Thus, the panel of microplastics includes fresh PP (f-PP), fresh PS (f-PS), weathered PP (w-PP), and weathered PS (w-PS). The spherical PS with a similar size was used as a reference particle. Treatment of all types of PP and PS did not show any toxic effects to the Caco-2 cells and HepG2 cells. However, the treatment of microplastics to THP-1 macrophages showed significant toxicity in the order of f-PS > f-PP > w-PS > w-PP. The weathering process significantly reduced the reactive oxygen species (ROS) generation potential of both microplastics because the weathered microplastics have an increased affinity to bind serum protein which acts as a ROS scavenger. The intrinsic ROS generation potential of microplastics showed a good correlation with the toxicity endpoints including cytotoxicity and pro-inflammatory cytokines in THP-1 macrophages. In conclusion, the results of this study suggest that the target cell type of microplastics via oral administration can be macrophages and the pathogenic factor to THP-1 macrophages is the intrinsic ROS generation potential of microplastics. Nevertheless, the toxic effect of microplastics tested in this study was much less than that of nano-sized particles.

Combination effect of nanoparticles on the acute pulmonary inflammogenic potential: additive effect and antagonistic effect.

The combination effect of co-exposed different types of nanomaterials is little known although humans are generally exposed to a mixture of nanomaterials from urban ultrafine particles or industrial nanomaterials. Herein, we evaluated the combined effect of nanoparticles (NPs) using three types of NPs in different inflammogenic categories: carbon black (CB), nickel oxide (NiO), and copper oxide (CuO). A single type of NPs or NPs in combination was intratracheally instilled into the lungs of rats and the bronchoalveolar lavage fluid (BALF) was analyzed at 24 h after instillation to evaluate the acute inflammogenic potential. The percentage of neutrophils in BALF was selected as a toxicity endpoint and the potential for reactive oxygen species (ROS) generation, dose-response of the combined effect, sequential treatment of CB and NiO, and uptake of NiO to alveolar macrophages after combined treatment of CB and NiO were evaluated for the mechanism of the combined effect. Co-exposure of CuO and NiO showed an additive effect on the percentage of neutrophils and ROS generation potential, which implies that the physicochemical properties of each NP are not influenced by the other type. While CB exerted an antagonistic effect on the percentage of neutrophils in combined treatment with CuO or NiO. The antagonistic effect of CB was due to the scavenging activity of the ROS generated by the CuO and NiO rather than the competition in cellular uptake to target cells (i.e. alveolar macrophages), which highlight the importance of the combined effect of NPs in the risk assessment.