Kazuma Higashisaka

Silica and titanium dioxide nanoparticles cause pregnancy complications in mice

The increasing use of nanomaterials has raised concerns about their potential risks to human health. Recent studies have shown that nanoparticles can cross the placenta barrier in pregnant mice and cause neurotoxicity in their offspring, but a more detailed understanding of the effects of nanoparticles on pregnant animals remains elusive. Here, we show that silica and titanium dioxide nanoparticles with diameters of 70 nm and 35 nm, respectively, can cause pregnancy complications when injected intravenously into pregnant mice. The silica and titanium dioxide nanoparticles were found in the placenta, fetal liver and fetal brain. Mice treated with these nanoparticles had smaller uteri and smaller fetuses than untreated controls. Fullerene molecules and larger (300 and 1,000 nm) silica particles did not induce these complications. These detrimental effects are linked to structural and functional abnormalities in the placenta on the maternal side, and are abolished when the surfaces of the silica nanoparticles are modified with carboxyl and amine groups.

Carbon Nanotubes Elicit DNA Damage and Inflammatory Response Relative to Their Size and Shape

Carbon nanotubes (CNTs) have been one of the most extensively researched and developed nanomaterials. However, little concern has been placed on their safety. The biological effects of CNTs are believed to differ relative to size and shape. Thus, the relationship between the characteristics of CNTs and their safety needs to be evaluated. In this study, we examined the biological effects of different-sized multi-walled CNTs (MWCNTs) and single-walled CNTs (SWCNTs). Long and thick MWCNTs induced the strongest DNA damage while similar SWCNTs caused little effect. Comparison of inflammatory responses of various types of CNTs found that peritoneal CNT administration of long and thick MWCNTs increased the total cell number in abdominal lavage fluid in mice. These results indicate that long and thick MWCNT, but not short and thin MWCNT, cause DNA damage and severe inflammatory effects. These findings might provide useful information for constructing novel CNTs with safety.

Acute phase proteins as biomarkers for predicting the exposure and toxicity of nanomaterials

Recently, nanomaterials have become an integral part of our daily lives. However, there is increasing concern about the potential risk to human health. Here, we attempted to identify biomarkers for predicting the exposure and toxicity of nanomaterials by using a proteomics based approach. We evaluated the changes of protein expression in plasma after treatment with silica nanoparticles. Our analyses identified haptoglobin, one of the acute phase proteins, as a candidate biomarker. The results of ELISA showed that the level of haptoglobin was significantly elevated in plasma of mice exposed to silica nanoparticles with a diameter of 70 nm (nSP70) compared to normal mice and those exposed to silica particles with a diameter of 1000 nm. Furthermore, the other acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA) were also elevated in plasma of nSP70 treated mice. In addition, the level of these acute phase proteins was elevated in the plasma of mice after intranasal treatment with nSP30. Our results suggest that haptoglobin, CRP and SAA are highly sensitive biomarkers for assessing the risk of exposure to silica nanoparticles. We believe this study will contribute to the development of global risk assessment techniques for nanomaterials.

Intranasal exposure to amorphous nanosilica particles could activate intrinsic coagulation cascade and platelets in mice.

BackgroundNanomaterials with particle sizes <100 nm have been already applied in various applications such as cosmetics, medicines, and foods. Therefore, ensuring the safety of nanomaterials is becoming increasingly important. Here we examined the localization and biological responses of intranasally administered amorphous nanosilica particles in mice, focusing on the coagulation system.MethodsWe used nanosilica particles with diameters of 30, 70, or 100 nm (nSP30, nSP70, or nSP100 respectively), and conventional microscale silica particles with diameters of 300 or 1000 nm (mSP300 or mSP1000, respectively). BALB/c mice were intranasally exposed to nSP30, nSP70, nSP100, mSP300, or mSP1000 at concentrations of 500 μg/mouse for 7 days. After 24 hours of last administration, we performed the in vivo transmission electron microscopy analysis, hematological examination and coagulation tests.ResultsIn vivo transmission electron microscopy analysis showed that nanosilica particles with a diameter <100 nm were absorbed through the nasal cavity and were distributed into liver and brain. Hematological examination and coagulation tests showed that platelet counts decreased and that the activated partial thromboplastin time was prolonged in nSP30 or nSP70-treated groups of mice, indicating that nanosilica particles might have activated a coagulation cascade. In addition, in in vitro activation tests of human plasma, nanosilica particles had greater potential than did conventional microscale silica particles to activate coagulation factor XII. In nanosilica-particle-treated groups, the levels of soluble CD40 ligand, and von Willebrand factor which are involved in stimulating platelets tended to slightly increase with decreasing particle size.ConclusionsThese results suggest that intranasally administered nanosilica particles with diameters of 30 and 70 nm could induce abnormal activation of the coagulation system through the activation of an intrinsic coagulation cascade. This study provides information to advance the development of safe and effective nanosilica particles.

Liver-specific microRNAs as biomarkers of nanomaterial-induced liver damage

Although nanomaterials are being used in various fields, their safety is not yet sufficiently understood. We have been attempting to establish a nanomaterials safety-assessment system by using biomarkers to predict nanomaterial-induced adverse biological effects. Here, we focused on microRNAs (miRNAs) because of their tissue-specific expression and high degree of stability in the blood. We previously showed that high intravenous doses of silica nanoparticles of 70 nm diameter (nSP70) induced liver damage in mice. In this study, we compared the effectiveness of serum levels of liver-specific or -enriched miRNAs (miR-122, miR-192, and miR-194) with that of conventional hepatic biomarkers (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) as biomarkers for nSP70. After mice had been treated with nSP70, their serum miRNAs levels were measured by using quantitative RT-PCR. Serum levels of miR-122 in nSP70-treated mice were the highest among the three miRNAs. The sensitivity of miR-122 for liver damage was at least as good as those of ALT and AST. Like ALT and AST, miR-122 may be a useful biomarker of nSP70. We believe that these findings will help in the establishment of a nanomaterials safety-assessment system.

Metal nanoparticles in the presence of lipopolysaccharides trigger the onset of metal allergy in mice

Many people suffer from metal allergy, and the recently demonstrated presence of naturally occurring metal nanoparticles in our environment could present a new candidate for inducing metal allergy. Here, we show that mice pretreated with silver nanoparticles (nAg) and lipopolysaccharides, but not with the silver ions that are thought to cause allergies, developed allergic inflammation in response to the silver. nAg-induced acquired immune responses depended on CD4(+) T cells and elicited IL-17A-mediated inflammation, similar to that observed in human metal allergy. Nickel nanoparticles also caused sensitization in the mice, whereas gold and silica nanoparticles, which are minimally ionizable, did not. Quantitative analysis of the silver distribution suggested that small nAg (≤10 nm) transferred to the draining lymph node and released ions more readily than large nAg (>10 nm). These results suggest that metal nanoparticles served as ion carriers to enable metal sensitization. Our data demonstrate a potentially new trigger for metal allergy.

Protein corona changes mediated by surface modification of amorphous silica nanoparticles suppress acute toxicity and activation of intrinsic coagulation cascade in mice.

Recently, nanomaterial-mediated biological effects have been shown to be governed by the interaction of nanomaterials with some kinds of proteins in biological fluids, and the physical characteristics of the nanomaterials determine the extent and type of their interactions with proteins. Here, we examined the relationships between the surface properties of amorphous silica nanoparticles with diameters of 70 nm (nSP70), their interactions with some proteins in biological fluids, and their toxicity in mice after intravenous administration. The surface modification of nSP70 with amino groups (nSP70-N) prevented acute lethality and abnormal activation of the coagulation cascade found in the nSP70-treated group of mice. Since our previous study showed that coagulation factor XII played a role in the nSP70-mediated abnormal activation of the coagulation cascade, we examined the interaction of nSP70 and nSP70-N with coagulation factor XII. Coagulation factor XII bonded to the surface of nSP70 to a greater extent than that observed for nSP70-N, and consequently more activation of coagulation factor XII was observed for nSP70 than for nSP70-N. Collectively, our results suggest that controlling the interaction of nSP70 with blood coagulation factor XII by modifying the surface properties would help to inhibit the nSP70-mediated abnormal activation of the blood coagulation cascade.

Expression of Eph receptor A10 is correlated with lymph node metastasis and stage progression in breast cancer patients.

Eph receptor A10 (EphA10) is a valuable breast cancer marker that is highly expressed in breast cancer tissues by comparison with normal breast tissues, as we previously reported. However, the role of EphA10 expression in breast cancer is not well understood. Here, we have analyzed the expression of EphA10 at the mRNA‐ and protein‐level in clinical breast cancer tissues and then evaluated the relationship with clinicopathological parameters for each sample. EphA10 mRNA expression was quantified by real‐time polymerase chain reaction using complimentary DNA (cDNA) samples derived from breast cancer patients. Lymph node (LN) metastasis and stage progression were significantly correlated with EphA10 expression at the mRNA level (P = 0.0091 and P = 0.034, respectively). Furthermore, immunohistochemistry (IHC) staining of breast cancer tissue microarrays (TMAs) revealed that EphA10 expression at the protein level was also associated with LN metastasis and stage progression (P = 0.016 and P = 0.011, respectively). These results indicate that EphA10 expression might play a role in tumor progression and metastasis. Our findings will help elucidate the role of EphA10 in clinical breast cancer progression.

Ephrin receptor A10 is a promising drug target potentially useful for breast cancers including triple negative breast cancers.

Ephrin receptor A10 (EphA10) is a relatively uncharacterized protein which is expressed in many breast cancers but not expressed in normal breast tissues. Here, we examined the potential of EphA10 as a drug target in breast cancer. Immunohistochemical staining of clinical tissue sections revealed that EphA10 was expressed in various breast cancer subtypes, including triple negative breast cancers (TNBCs), with no expression observed in normal tissues apart from testis. Ligand-dependent proliferation was observed in EphA10-transfected MDA-MB-435 cells (MDA-MB-435(EphA10)) and native TNBC cells (MDA-MB-436). However, this phenomenon was not observed in parental MDA-MB-435 cells which express a low level of EphA10. Finally, tumor growth was significantly suppressed by administration of an anti-EphA10 monoclonal antibody in a xenograft mouse model. These results suggest that inhibition of EphA10 signaling may be a novel therapeutic option for management of breast cancer, including TNBCs which are currently not treated with molecularly targeted agents.

Aminopeptidase P3, a new member of the TNF-TNFR2 signaling complex, induces phosphorylation of JNK1 and JNK2.

ABSTRACT Tumor necrosis factor (TNF) is an important mediator that triggers onset of autoimmune diseases and exerts its biological effects by interacting through two receptors, TNFR1 (also known as TNFRSF1A) and TNFR2 (also known as TNFRSF1B). TNFR2 signaling has significant potential to exert pro-survival and protective roles in several diseases. Unlike TNFR1 signaling, however, the mechanism of TNFR2 signal transduction is poorly understood, and few of its adaptor molecules are known. The present study utilized a proteomics approach to search for adaptor molecules in the TNFR2 signaling complex and identified aminopeptidase P3 (APP3, also known as XPNPEP3) to be a key molecule. One of its two isoforms, mitochondrial APP3 (APP3m) but not cytosolic APP3 (APP3c), was recruited to TNFR2 and shown to regulate TNF–TNFR2-dependent phosphorylation of JNK1 (also known as MAPK8) and JNK2 (also known as MAPK9). Furthermore, APP3m was released from mitochondria upon TNF stimulation in the absence of mitochondrial outer membrane permeabilization (MOMP). The observation of increased cell death upon downregulation of APP3m also suggested that APP3m exerts an anti-apoptotic function. These findings reveal that APP3m is a new member of the TNF–TNFR2 signaling complex and characterize an APP3-mediated TNFR2 signal transduction mechanism that induces activation of JNK1 and JNK2.