FCHO1560-571 peptide, a PKB kinase motif, inhibits tumor progression.

BACKGROUND: Cell division is regulated by protein kinase B (PKB)-mediated FCH domain only 1 (FCHO1) phosphorylation. METHODS: FCHO1560-571, a synthetic water-soluble peptide, was generated from the PKB substrate motif 560PPRRLRSRKVSC571 found in the human FCHO1 protein. RESULTS: In this study, we found that in vitro FCHO1560-571 inhibits cell proliferation via PKB/ERK/SMAD4 pathways in KRAS-mutated A549 lung cancer cells. In addition, FCHO1560-571, at effective doses of 15 and 30 mg/kg, significantly suppressed tumor growth and decreased the size and weight of tumors in A549-xenograft mice. CONCLUSION: These results suggest that the FCHO1560-571 peptide could be a potential therapy for lung cancer.

GOLGA2 loss causes fibrosis with autophagy in the mouse lung and liver.

Autophagy is a biological recycling process via the self-digestion of organelles, proteins, and lipids for energy-consuming differentiation and homeostasis. The Golgi serves as a donor of the double-membraned phagophore for autophagosome assembly. In addition, recent studies have demonstrated that pulmonary and hepatic fibrosis is accompanied by autophagy. However, the relationships among Golgi function, autophagy, and fibrosis are unclear. Here, we show that the deletion of GOLGA2, encoding a cis-Golgi protein, induces autophagy with Golgi disruption. The induction of autophagy leads to fibrosis along with the reduction of subcellular lipid storage (lipid droplets and lamellar bodies) by autophagy in the lung and liver. GOLGA2 knockout mice clearly demonstrated fibrosis features such as autophagy-activated cells, densely packed hepatocytes, increase of alveolar macrophages, and decrease of alveolar surfactant lipids (dipalmitoylphosphatidylcholine). Therefore, we confirmed the associations among Golgi function, fibrosis, and autophagy. Moreover, GOLGA2 knockout mice may be a potentially valuable animal model for studying autophagy-induced fibrosis.

Exposure to cigarette smoke disturbs adipokines secretion causing intercellular damage and insulin resistance in high fructose diet-induced metabolic disorder mice.

A large amount of fructose intake along with smoking is associated with increased incidence of diseases linked to metabolic syndrome. More research is necessary to understand the complex mechanism that ultimately results in metabolic syndrome and the effect, if any, of high fructose dietary intake and smoking on individual health. In this study, we investigated changes in ER-Golgi network and disturbance to secretion of adipokines induced by cigarette smoking (CS) and excess fructose intake and their contribution to the disruption of metabolic homeostasis. We used high fructose-induced metabolic disorder mice model by feeding them with high fructose diet for 8 weeks. For CS exposure experiment, these mice were exposed to CS for 28 days according to OECD guideline 412. Our results clearly showed that the immune system was suppressed and ER stress was induced in mice with exposure to CS and fed with high fructose. Furthermore, their concentrations of adipokines including leptin and adiponectin were aberrant. Such alteration in secretion of adipokines could cause insulin resistance which may lead to the development of type 2 diabetes.

Dihydroceramide is a key metabolite that regulates autophagy and promotes fibrosis in hepatic steatosis model.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common chronic liver disease worldwide. Sphingolipids are a family of lipids that play essential roles as critical regulators in metabolic disorders. Some sphingolipids are known key factors in metabolic dysfunction. However, the precise effect of dihydroceramide on NAFLD remains unknown. Here, we report how dihydroceramide in autophagosome accumulation activates fibrogenesis in human liver Chang cells treated with free fatty acids (FFA). According to LC/MS lipid profiling, FFA increased the levels of sphingolipids and triacylglycerol (TG). To demonstrate the potential role of dihydroceramide metabolism in autophagy, several sphingolipid synthesis inhibitors were used. Increased dihydroceramide led to impairment of autophagic flux, resulting in increased TG storage in lipid droplets (LD) and upregulated expression of fibrosis markers. Hepatic stellate cells (HSCs, LX-2 cells) were co-cultured with Chang cells to assess the potential fibrogenic response to dihydroceramide, Treatment with rapamycin recovered autophagic flux in Chang cells and fibrogenesis in the co-culture system. Our results identified a critical function of dihydroceramide metabolism in autophagy. It could play an important role in the progression of NAFLD associated with lipid over-accumulation. Therefore, preventing autophagic flux by regulating dihydroceramide could be a potential strategic approach for providing therapy for NAFLD.

Deleterious effects in reproduction and developmental immunity elicited by pulmonary iron oxide nanoparticles.

With the extensive application of iron oxide nanoparticles (FeNPs), attention about their potential risks to human health is also rapidly raising, particularly in sensitive subgroups such as pregnant women and babies. In this study, we a single instilled intratracheally FeNPs (1, 2, and 4mg/kg) to the male and female parent mice, mated, then assessed reproductive toxicity according to the modified OECD TG 421. During the pre-mating period (14 days), two female parent mice died at 4mg/kg dose, and the body weight gain dose-dependently decreased in male and female parent mice exposed to FeNPs. Additionally, iron accumulation and the enhanced expression of MHC class II molecules were observed in the ovary and the testis of parent mice exposed to the highest dose of FeNPs, and the total sex ratio (male/female) of the offspring mice increased in the groups exposed to FeNPs. Following, we a single instilled intratracheally to their offspring mice with the same doses and evaluated the immunotoxic response on day 28. The increased mortality and significant hematological- and biochemical- changes were observed in offspring mice exposed at 4mg/kg dose, especially in female mice. More interestingly, balance of the immune response was shifted to a different direction in male and female offspring mice. Taken together, we conclude that the NOAEL for reproductive and developmental toxicity of FeNPs may be lower than 2mg/kg, and that female mice may show more sensitive response to FeNPs exposure than male mice. Furthermore, we suggest that further studies are necessary to identify causes of both the alteration in sex ratio of offspring mice and different immune response in male and female offspring mice.

Impact of commercial cigarette smoke condensate on brain tissue co-cultured with astrocytes and blood-brain barrier endothelial cells.

The purpose of the current study was to investigate the effect of two commercial cigarette smoke condensates (CCSC) on oxidative stress and cell cytotoxicity in human brain (T98G) or astrocytes (U-373 MG) in the presence of human brain microvascular endothelial cells (HBMEC). Cell viability of mono-culture of T98G or U-373 MG was markedly decreased in a concentration-dependent manner, and T98G was more susceptible than U-373 MG to CCSC exposure. Cytotoxicity was less prominent when T98G was co-cultured with HBMEC than when T98G was co-cultured with U-373 MG. Significant reduction in trans-epithelial electric resistance (TEER), a biomarker of cellular integrity was noted in HBMEC co-cultured with T98G (HBMEC-T98G co-culture) and U-373 MG co-cultured with T98G (U-373 MG-T98G co-culture) after 24 or 48 hr CCSC exposure, respectively. TEER value of U-373 MG co-cultured with T98G (79-84%) was higher than HBMEC co-cultured with T98G (62-63%) within 120-hr incubation with CCSC. Reactive oxygen species (ROS) generated by CCSC in mono-culture of T98G and U-373 MG reached highest levels at 4 and 16 mg/ml, respectively. ROS production by T98G fell when co-cultured with HBMEC or U-373MG. These findings suggest that adverse consequences of CCSC treatment on brain cells may be protected by blood-brain barrier or astrocytes, but with chronic exposure toxicity may be worsened due to destruction of cellular integrity.

Trifloxystrobin induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HaCaT, human keratinocyte cells.

As the outermost layer of the body, the skin plays an important role in exposure to pesticides, which could have negative impacts on human health. Trifloxystrobin is a widely used fungicide of the strobilurin class, however, there is little information regarding the skin contact-associated toxic mechanism. Therefore, the present study was performed in order to identify the skin toxicity mechanism of trifloxystrobin using HaCaT (keratinocyte of human skin) cells. Following 24 or 48 h treatment, cell viability, and subsequent Annexin V-FITC/propidium iodide assay, TUNEL assay and Western blotting were performed to investigate the cell death mechanism of trifloxystrobin. Exposure to trifloxystrobin resulted in diminished viability of HaCaT cells in both a time- and concentration-dependent manner. The cell death was derived through apoptotic pathways in the HaCaT cells. Furthermore, we explored the effect of trifloxystrobin on TRAIL-mediated extrinsic apoptosis using siRNA transfection. Knockdown of death receptor 5 suppressed trifloxystrobin-provoked apoptosis. These results indicate that trifloxystrobin induces TRAIL-mediated apoptosis and has an inhibitory effect in keratinocytes that can interfere with the barrier function and integrity of the skin. This could be proposed as a mechanism of skin toxicity by trifloxystrobin and considered in the management of pesticide exposure.

Gemigliptin, a dipeptidyl peptidase-4 inhibitor, inhibits retinal pericyte injury in db/db mice and retinal neovascularization in mice with ischemic retinopathy.

Retinal pericyte loss and neovascularization are characteristic features of diabetic retinopathy. Gemigliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, has shown robust blood-glucose lowering effects in type 2 diabetic patients, but its effects on diabetic retinopathy have not yet been reported. We evaluated the efficacy of gemigliptin on retinal vascular leakage in db/db mice, which is an animal model for type 2 diabetes, and neovascularization in oxygen-induced retinopathy (OIR) mice, which is an animal model for ischemic proliferative retinopathy. Gemigliptin (100mg/kg/day) was orally administered to the db/db mice for 12weeks. C57BL/6 mice on postnatal day 7 (P7) were exposed to 75% hyperoxia for 5days, followed by exposure to room air from P12 to P17 to induce OIR. Gemigliptin (50mg/kg/day) was intraperitoneally injected daily from P12 to P17. Retinal neovascularization was analyzed in flat-mounted retinas on P17. We determined the efficacy and possible mechanism of gemigliptin on high glucose-induced apoptosis of primary human retinal pericytes. The oral administration of gemigliptin for 4months significantly ameliorated retinal pericyte apoptosis and vascular leakage in the db/db mice. Gemigliptin also ameliorated retinal neovascularization in the OIR mice. Gemigliptin attenuated the overexpression of plasminogen activator inhibitor-1 (PAI-1) in the retinas of diabetic and OIR mice. Gemigliptin and PAI-1 siRNA significantly inhibited pericyte apoptosis by inhibiting the overexpression of PAI-1, which is induced by high glucose. Our results suggest that gemigliptin has potent anti-angiogenic and anti-apoptotic activities via suppressing DPP-4 and PAI-1, and the results support the direct retinoprotective action of gemigliptin.

Cigarette Smoking Condensate Disrupts Endoplasmic Reticulum-Golgi Network Homeostasis Through GOLPH3 Expression in Normal Lung Epithelial Cells.

INTRODUCTION: Cigarette smoke (CS) is associated with a broad range of diseases including lung cancer. Many researchers have suggested that cigarette smoke condensate (CSC) may be more toxic compared to cigarette smoke extract (CSE) because CSC contains the lipid-soluble faction of smoke while CSE contains the hydrophilic or gas phase. The aim of this research is to investigate the effects of CSC on the disruption of endoplasmic reticulum (ER)-Golgi homeostasis in normal lung epithelial cells. METHODS: CS was generated according to the ISO 3308 method. To ascertain the mechanistic effects of CSC on lung toxicity, normal lung epithelial cells of the cell line 16HBE14o- were treated with CSC (0.1mg/mL) for 48 hours. The toxic effects of CSC on ER-Golgi homeostasis and GOLPH3 expression were observed through diverse molecular tools including transmission electron microscope analysis. RESULTS: Our results demonstrated that CSC treatment increased reactive oxygen species generation in lung cells and led to the alteration of ER-Golgi homeostasis in conjunction with increased autophagy. In particular, GOLPH3, known as an oncogene and a marker protein for the trans-Golgi network, was upregulated in CSC-treated cells. GOLPH3 protein overexpression was also confirmed in the lungs of human lung cancer patients as well as NNK-treated mice. CONCLUSION: Our study revealed that CSC caused lung damage through the disruption of ER-Golgi homeostasis and autophagy induction. The expression level of the trans-Golgi marker protein GOLPH3 could serve as a reliable bio-indicator for CS-related lung cancer. IMPLICATIONS: CS is a harmful factor in the development of many diseases including cancer. In this research, we demonstrated that CSC treatment led to malfunction of the ER-Golgi network, with the disrupted ER and Golgi causing GOLPH3 overexpression and abnormal autophagy accumulation. In addition, although the value of GOLPH3 as a predictor remains to be fully elucidated, our data suggest that GOLPH3 levels may be a novel prognostic biomarker of tobacco related lung disease.

Dihydroergotamine Tartrate Induces Lung Cancer Cell Death through Apoptosis and Mitophagy.

BACKGROUND: Mitochondria have emerged as a major target for anticancer therapy because of their critical role in cancer cell survival. Our preliminary works have suggested that dihydroergotamine tartrate (DHE), an antimigraine agent, may have effects on mitochondria. METHODS: We examined the effect of DHE on the survival of several lung cancer cells and confirmed that DHE suppressed diverse lung cancer cell growth effectively. To confirm whether such effects of DHE would be associated with mitochondria, A549 cells were employed for the evaluation of several important parameters, such as membrane potential, reactive oxygen species (ROS) generation, apoptosis, ATP production and autophagy. RESULTS: DHE decreased membrane permeability, increased ROS generation as well as apoptosis, and disturbed ATP production. Eventually, mitophagy was activated for damaged mitochondria. CONCLUSION: Taken together, our findings demonstrate that DHE induces lung cancer cell death by the induction of apoptosis and mitophagy, thus suggesting that DHE can be developed as an anti-lung cancer therapeutic agent.

Biodistribution and toxicity of spherical aluminum oxide nanoparticles.

With the rapid development of the nano-industry, concerns about their potential adverse health effects have been raised. Thus, ranking accurately their toxicity and prioritizing for in vivo testing through in vitro toxicity test is needed. In this study, we used three types of synthesized aluminum oxide nanoparticles (AlONPs): γ-aluminum oxide hydroxide nanoparticles (γ-AlOHNPs), γ- and α-AlONPs. All three AlONPs were spherical, and the surface area was the greatest for γ-AlONPs, followed by the α-AlONPs and γ-AlOHNPs. In mice, γ-AlOHNPs accumulated the most 24 h after a single oral dose. Additionally, the decreased number of white blood cells (WBC), the increased ratio of neutrophils and the enhanced secretion of interleukin (IL)-8 were observed in the blood of mice dosed with γ-AlOHNPs (10 mg kg(-1)). We also compared their toxicity using four different in vitro test methods using six cell lines, which were derived from their potential target organs, BEAS-2B (lung), Chang (liver), HACAT (skin), H9C2 (heart), T98G (brain) and HEK-293 (kidney). The results showed γ-AlOHNPs induced the greatest toxicity. Moreover, separation of particles was observed in a transmission electron microscope (TEM) image of cells treated with γ-AlOHNPs, but not γ-AlONPs or α-AlONPs. In conclusion, our results suggest that the accumulation and toxicity of AlONPs are stronger in γ-AlOHNPs compared with γ-AlONPs and α-AlONPs owing their low stability within biological system, and the presence of hydroxyl group may be an important factor in determining the distribution and toxicity of spherical AlONPs.

Recurrence-associated pathways in hepatitis B virus-positive hepatocellular carcinoma.

BACKGROUND: Despite the recent identification of several prognostic gene signatures, the lack of common genes among experimental cohorts has posed a considerable challenge in uncovering the molecular basis underlying hepatocellular carcinoma (HCC) recurrence for application in clinical purposes. To overcome the limitations of individual gene-based analysis, we applied a pathway-based approach for analysis of HCC recurrence. RESULTS: By implementing a permutation-based semi-supervised principal component analysis algorithm using the optimal principal component, we selected sixty-four pathways associated with hepatitis B virus (HBV)-positive HCC recurrence (p < 0.01), from our microarray dataset composed of 142 HBV-positive HCCs. In relation to the public HBV- and public hepatitis C virus (HCV)-positive HCC datasets, we detected 46 (71.9%) and 18 (28.1%) common recurrence-associated pathways, respectively. However, overlap of recurrence-associated genes between datasets was rare, further supporting the utility of the pathway-based approach for recurrence analysis between different HCC datasets. Non-supervised clustering of the 64 recurrence-associated pathways facilitated the classification of HCC patients into high- and low-risk subgroups, based on risk of recurrence (p < 0.0001). The pathways identified were additionally successfully applied to discriminate subgroups depending on recurrence risk within the public HCC datasets. Through multivariate analysis, these recurrence-associated pathways were identified as an independent prognostic factor (p < 0.0001) along with tumor number, tumor size and Edmondson's grade. Moreover, the pathway-based approach had a clinical advantage in terms of discriminating the high-risk subgroup (N = 12) among patients (N = 26) with small HCC (<3 cm). CONCLUSIONS: Using pathway-based analysis, we successfully identified the pathways involved in recurrence of HBV-positive HCC that may be effectively used as prognostic markers.

Hepatic differentiation of human adipose tissue-derived mesenchymal stem cells and adverse effects of arsanilic acid and acetaminophen during in vitro hepatic developmental stage.

In the present study, we differentiated hepatocyte-like cells (HLCs) from human adipose tissue-derived mesenchymal stem cells (AT-MSCs). The hepatic differentiation was confirmed by increases in hepatic proteins or genes, the cytochrome P450 (CYP) activities, albumin secretion, and glycogen storage. To determine the developmental toxic effect of arsanilic acid (Ars) and acetaminophen (AAP) on the hepatic development, the differentiating cells were treated with the test chemicals (below IC12.5) from day 4 to day 13. The enzymatic activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) did not significantly differ in response to Ars treatment. AAP treatment increased the activities of all enzymes in a dose-dependent manner, significantly at concentrations of 2.5 and 5 mM of AAP. On the expressions of hepatic genes for Ars, the expressions were significantly inhibited by more than 0.5 mM for Albumin (ALB), but only 2.5 mM for α-feto protein (AFP). In the AAP-treated group, the expressions of ALB and AFP were significantly decreased at the concentrations exceeding 0.625 mM. The activities of CYP3A4 were not changed by both treatments. The activities of CYP1A2 were increased by AAP, whereas it was decreased by Ars treatment. In conclusion, AAP could cause serious adverse effects during the hepatic development as compared to Ars.

Comparison of the toxicity of aluminum oxide nanorods with different aspect ratio.

Aluminum oxide nanoparticles are listed among 14 high-priority nanomaterials published by the Organization for Economic Co-operation and Development, but limited information is available on their potential hazards. In this study, we compared the toxicity of two different aluminum oxide nanorods (AlNRs) commercially available in vivo and in vitro. Considering aspect ratio, one was 6.2 ± 0.6 (long-AlNRs) and the other was 2.1 ± 0.4 (short-AlNRs). In mice, long-AlNRs induced longer and stronger inflammatory responses than short-AlNRs, and the degree reached the maximum on day 7 for both types and decreased with time. In addition, in vitro tests were performed on six cell lines derived from potential target organs for AlNPs, HEK-293 (kidney), HACAT (skin), Chang (liver), BEAS-2B (lung), T98G (brain), and H9C2 (heart), using MTT assay, ATP assay, LDH release, and xCELLigence system. Long-AlNRs generally produced stronger toxicity than short-AlNRs, and HEK-293 cells were the most sensitive for both AlNRs, followed by BEAS-2B cells, although results from 4 kinds of toxicity tests conflicted among the cell lines. Based on these results, we suggest that toxicity of AlNRs may be related to aspect ratio (and resultant surface area). Furthermore, novel in vitro toxicity testing methods are needed to resolve questionable results caused by the unique properties of nanoparticles.

A 13-week repeated-dose oral toxicity and bioaccumulation of aluminum oxide nanoparticles in mice.

Because of an increase in the commercial applications of manufactured nanoparticles, the issue of potential adverse health effects of nanoparticles following intended or unintended exposure is rapidly gaining attention. In this study, we evaluated the toxicity of aluminum oxide nanoparticles (AlNPs, rod-type, 1.5, 3, and 6 mg/kg) after oral administration to mice for 13 weeks. Compared with the control group, the consumption of diet and drinking water and body weight gain decreased in the group treated with AlNPs. The group treated with 6 mg/kg AlNPs also showed a marked elevation in the count of white blood cells that associated with a significant decrease and increase to the proportion of eosinophils and lymphocytes, respectively. In addition, the secretion of IL-6 and monocyte chemotactic protein-1 increased in a dose-dependent manner in the treated groups. Furthermore, AlNPs showed the highest accumulation in the liver and kidneys compared with the control group, increased the lactate dehydrogenase level in the blood, and induced the development of a pathological lesion in the liver and kidneys. Taken together, we suggest that the target organs of rod-type AlNPs may be the liver, kidneys and the immune system, and the not-observed adverse effect level may be lower than 6 mg/kg.

Toxic response of graphene nanoplatelets in vivo and in vitro.

With the development of nanotechnology, myriad types of novel materials have been discovered at the nanoscale, among which the most interesting material is graphene. However, the toxicity data available on graphene are extremely limited. In this study, we explored toxic response of commercially available graphene nanoplatelets (GNPs) in vivo and in vitro. The GNPs used in this study had a high surface area and feature considerably few defects. In mice, GNPs (2.5 and 5 mg/kg) remained in the lung until 28 days after a single instillation, and the secretion of inflammatory cytokines reached the maximal level at Day 14 and then decreased over time. In vitro study using BEAS-2B cells, a human bronchial epithelial cell line, GNPs located within autophagosome-like vacuoles 24 h after exposure. The GNPs (2.5, 5, 10, and 20 µg/mL) also dose-dependently reduced cell viability, which was accompanied by an increase in the portion of cells in the subG1 and S phases. Moreover, the GNPs down-regulated the generation of reactive oxygen species, suppressed ATP production, caused mitochondria damage, and elevated the levels of autophagy-related proteins. Based on these results, we suggest that GNPs provoked a subchronic inflammatory response in mice and that GNPs induced autophagy accompanying apoptosis via mitochondria damage in vitro.

Oxidative Stress Induced by Cigarette Smoke Extracts in Human Brain Cells (T98G) and Human Brain Microvascular Endothelial Cells (HBMEC) in Mono- and Co-Culture.

The objective of the current study was to examine oxidative stress induced by cigarette smoke extract (CSE) or cigarette smoke condensate (CSC) in human brain cells (T98G) and human brain microvascular endothelial cells (HBMEC) in mono- and co-culture systems. Cell viability of T98G cells exposed to CSC (0.05-4 mg/ml) was significantly decreased compared to CSE (0.025-20%). There were no marked differences between quantities of reactive oxygen species (ROS) generation by either CSE (2, 4, and 10%) or CSC (0.2, 0.4, and 0.8 mg/ml) treatment compared to control. However, a significant effect was noted in ROS generation following CSC incubation at 4mg/ml. Cellular integrity of HBMEC decreased to 74 and 64% within 120 h of exposure at the IC50 value of CSE and CSC, respectively. This study suggests that chronic exposure to cigarette smoking might initiate damage to the blood-brain barrier.

Physicochemical determinants of multiwalled carbon nanotubes on cellular toxicity: influence of a synthetic method and post-treatment.

Since the discovery of carbon nanotubes (CNTs), scientists have performed extensive studies on nanotubes in the fields of materials science, physics, and electronic engineering. Because multiwalled CNTs (MWCNTs) are not homogeneous materials, and because it is not feasible to test every newly synthesized MWCNT, this study was aimed at investigating the physicochemical properties that primarily determine the cellular toxicity of MWCNTs. This study analyzed the relationship between cell viability and physicochemical characteristics following exposure to eight different MWCNTs. We generated eight different MWCNTs using various synthetic methods and post-treatments. From this analysis, we sought to identify the major physicochemical determinants that could predict the cellular toxicity of MWCNTs, regardless of the synthetic method and post-treatment conditions. Creation of binding sites on the tube walls by breaking C-C bonds played a pivotal role in increasing toxicity and was most clearly demonstrated by a Raman G peak shift and the ID/IG ratio. In addition, several factors were found to be strongly related to cellular toxicity: surface charge in the case of MWCNTs created by the chemical vapor deposition method and surface area and EPR intensity in the case of MWCNTs created by the arc discharge based method. The methods developed in this study could be applied to the prediction of the toxicity of newly synthesized MWCNTs.

ERK pathway is activated in bare-FeNPs-induced autophagy.

Iron oxide nanoparticles (FeNPs) are known to be one of the most biocompatible and safe nanoparticles. However, their long-term persistence remains a problem, and macrophages play as an important mediator in continuous stimulation of the immune system due to biopersistence of nanoparticles. In the present study, we identified the mechanisms underlying the uptake and toxicity of bare-FeNPs using RAW264.7 cells, a mouse peritoneal macrophage cell line. The bare-FeNPs penetrated the cell membrane through electrostatic interactions together with the general phagocytic pathway. At 24 h after exposure, they distributed freely in the cytosol or within autophagosome-like vacuoles. Bare-FeNPs induced decrease in the cell viability along with the cell cycle arrest in G1 phase. In addition, they increased the generation of ROS and the secretion of NO and TNF alpha as well as the expression of SOD-1 and SOD-2 proteins, which are an antioxidant. While the mitochondrial calcium level, the intensity of labeled mitochondria, and ATP production decreased, the levels of autophagy-related proteins such as p62, beclin 1, ATG5, and LC3B increased in a dose-dependent manner together with the levels of ATF 3, p-EGFR, and p-ERK proteins. However, the level of p-JNK protein clearly decreased. TEM images also showed that damaged organelle exist within autophagosome-like vacuoles with bare-FeNPs. On the basis of these results, we suggest that bare-FeNPs induce autophagy by initiating oxidative stress in RAW264.7 cells. Furthermore, ERK, but not JNK, pathway is activated in bare-FeNPs-induced autophagy.

Magnetite- and maghemite-induced different toxicity in murine alveolar macrophage cells.

The unique properties of nanoparticles and biological systems are important factors affecting the biological response following nanoparticle exposure. Iron oxide nanoparticles are classified mainly as magnetite (M-FeNPs) and maghemite (NM-FeNPs). In our previous study, NM-FeNPs induced autophagic cell death in RAW264.7, a murine peritoneal macrophage cell line, which has excellent lysosomal activity. In this study, we compared the toxicity of M-FeNPs and NM-FeNPs in MH-S, a murine alveolar macrophage cell line, which has relatively low lysosomal activity. At 24 h post-exposure, M-FeNPs decreased cell viability and ATP production, and elevated the levels of reactive oxygen species, nitric oxide, and pro-inflammatory cytokines to a higher extent than NM-FeNPs. Damage of mitochondria and the endoplasmic reticulum and the down-regulation of mitochondrial function and transcription-related genes were also higher in cells exposed to M-FeNPs than in cells exposed to NM-FeNPs (50 µg/ml). In addition, cells exposed to M-FeNPs (50 µg/ml) showed an increase in the number of autophagosome-like vacuoles, whereas cells exposed to NM-FeNPs formed large vacuoles in the cytosol. However, an autophagy-related molecular response was not induced by exposure to either FeNPs, unlike the results seen in our previous study with RAW264.7 cells. We suggest that M-FeNPs induced higher toxicity compared to NM-FeNPs in MH-S cells, and lysosomal activity plays an important role in determining cell death pathway.