[Awareness of issues in research and educational activities and expectations for young researchers' activities and supporting].

OBJECTIVES: To understand the actual situation and needs of young researchers and to provide reference for the management of Young Researchers Association (YRA) and the Japanese Society for Hygiene activities in the future. METHODS: An Internet survey was conducted on 67 members registered in YRA of the Japanese Society for Hygiene. The questions included those on basic information, research content and impressions about the activities of the society. RESULTS: Although members of YRA differ in backgrounds, research method used, and years of research experience, the respondents rated the organization as highly useful and participated continuously. In particular, they considered that participation in the planning of academic conferences and summer gatherings of YRA not only helped improve interpersonal relationships and expertise, but also provided opportunities to consult regarding educational activities and collect information. Regarding the format of conferences, it was shown that the majority of requests were for a hybrid format. It was also shown that most of the respondents expected opportunities for collaboration and joint research through participation in YRA. CONCLUSION: Through YRA, we would like to contribute to the further revitalization of young researchers and the Japanese Society for Hygiene by understanding and responding to the needs of diverse young researchers.

Silver nanoparticles induce lysosomal-autophagic defects and decreased expression of transcription factor EB in A549 human lung adenocarcinoma cells.

Although silver nanoparticles (AgNPs) are widely used in consumer and medical products, the mechanism by which AgNPs cause pulmonary damage is unclear. AgNPs are incorporated into cells and processed via the autophagy pathway. We examined the effects of AgNP exposure on autophagic flux and expression of transcription factor EB (TFEB) in A549 lung adenocarcinoma cells. In cells exposed to citrate-coated 60-nm AgNPs, confocal laser microscopic examination showed a decrease in the LysoTracker fluorescence signal and an increase in that of Cyto-ID, indicating lysosomal pH alkalization and autophagosome formation, respectively. The proteins p62 and microtubule-associated protein light chain 3B-II (LC3B-II) are both degraded by autophagy, and their levels increased depending on AgNP dose. Furthermore, AgNP-induced increase in LC3B-II was not enhanced by treatment with the autophagic inhibitor bafilomycin A1. TFEB mRNA levels, and protein levels in cytosolic and nuclear fractions, were suppressed by exposure to AgNPs, suggesting transcriptional inhibition of TFEB expression. Overexpression of TFEB did not suppress AgNP-induced LC3B-II accumulation and cellular damage, indicating that impairment of autophagic flux and cellular damage by AgNPs might not be primarily caused by reduced TFEB expression. The present study suggests that AgNP-induced lysosomal dysfunction plays a principal role in the autophagic flux defect.

Involvement of Notch1 signaling in malignant progression of A549 cells subjected to prolonged cadmium exposure.

Cadmium exposure is known to increase lung cancer risk, but the underlying molecular mechanisms in cadmium-stimulated progression of malignancy are unclear. Here, we examined the effects of prolonged cadmium exposure on the malignant progression of A549 human lung adenocarcinoma cells and the roles of Notch1, hypoxia-inducible factor 1α (HIF-1α), and insulin-like growth factor 1 receptor (IGF-1R)/Akt/extracellular signal-regulated kinase (ERK)/p70 S6 kinase 1 (S6K1) signaling pathways. Exposing A549 cells to 10 or 20 µm cadmium chloride (CdCl2) for 9-15 weeks induced a high proliferative potential, the epithelial-mesenchymal transition (EMT), stress fiber formation, high cell motility, and resistance to antitumor drugs. Of note, the CdCl2 exposure increased the levels of the Notch1 intracellular domain and of the downstream Notch1 target genes Snail and Slug. Strikingly, siRNA-mediated Notch1 silencing partially suppressed the CdCl2-induced EMT, stress fiber formation, high cell motility, and antitumor drug resistance. In addition, we found that prolonged CdCl2 exposure induced reduction of E-cadherin in BEAS-2B human bronchial epithelial cells and antitumor drug resistance in H1975 human tumor-derived non-small-cell lung cancer cells depending on Notch1 signaling. Moreover, Notch1, HIF-1α, and IGF-1R/Akt/ERK/S6K1 activated each other to induce EMT in the CdCl2-exposed A549 cells. These results suggest that Notch1, along with HIF-1α and IGF-1R/Akt/ERK/S6K1 signaling pathways, promotes malignant progression stimulated by prolonged cadmium exposure in this lung adenocarcinoma model.

Involvement of lysosomal dysfunction in silver nanoparticle-induced cellular damage in A549 human lung alveolar epithelial cells.

BACKGROUND: While silver nanoparticles (AgNPs) are widely used in consumer and medical products, the mechanism by which AgNPs cause pulmonary cytotoxicity is not clear. AgNP agglomerates are found in endo-lysosomal structures within the cytoplasm of treated cells. In this study, the functional role of lysosomes in AgNP-induced cellular damage was examined in A549 human lung alveolar epithelial cells. We evaluated the intracellular distribution of AgNPs, lysosomal pH, cellular viability, Ag dissolution, and metallothionein (MT) mRNA levels in AgNP-exposed A549 cells that were treated with bafilomycin A1, the lysosomal acidification inhibitor. FINDINGS: Exposure of A549 cells to citrate-coated AgNPs (20 nm diameter) for 24 h induced cellular damage and cell death at 100 and 200 µg Ag/ml, respectively. Confocal laser microscopic examination of LysoTracker-stained cells showed that AgNPs colocalized with lysosomes and their agglomeration increased in a dose-dependent manner (50-200 µg Ag/ml). In addition, the fluorescence signals of LysoTracker were reduced following exposure to AgNPs, suggesting the elevation of lysosomal pH. Treatment of A549 cells with 200 nM bafilomycin A1 and AgNPs (50 µg Ag/ml) further reduced the fluorescence signals of LysoTracker. AgNP-induced cell death was also increased by bafilomycin A1 treatment. Finally, treatment with bafilomycin A1 suppressed the dissolution of Ag and decreased the mRNA expression levels of MT-I and MT-II following exposure to AgNPs. CONCLUSIONS: The perturbation of lysosomal pH by AgNP exposure may play a role in AgNP agglomeration and subsequent cellular damage in A549 cells.

Difference in the toxicity mechanism between ion and nanoparticle forms of silver in the mouse lung and in macrophages.

The health effects of silver nanoparticles (AgNPs) have not been well investigated, despite AgNPs now being widely used in consumer products. We investigated the metabolic behavior and toxicity of AgNPs in comparison to silver nitrate (AgNO3) both in vivo and in vitro. AgNPs (20 nm diameter) suspended in 1% albumin solution or AgNO3 solution was injected into the mouse lung. Less than 1% of the initial dose of AgNPs and more than 7% of the initial dose of AgNO3 was recovered in the liver 4h after administration, suggesting that the ionic form of silver was absorbed by the lung tissue and entered the systemic circulation more efficiently than AgNPs. The pro-inflammatory cytokine, IL-1ß, and neutrophils in bronchoalveolar lavage fluid (BALF) increased following intratracheal instillation of AgNPs or AgNO3. AgNO3 recruited more neutrophils in the alveolar space than did AgNPs. In the in vitro study, AgNO3 was more cytotoxic than 20, 60, or 100 nm diameter AgNPs in a mouse macrophage cell line (J774.1). To investigate the intracellular distribution of Ag in detail, J774.1 cells were exposed to AgNO3 or 20 nm AgNPs and the distribution of Ag to cytosolic proteins was investigated using HPLC-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). Ag was mainly distributed to metallothioneins (MT) and to high molecular weight proteins in AgNO3- and AgNPs-exposed cells, respectively. Confocal laser microscopic examination of LysoTracker(®)-labeled cells indicated that AgNPs were colocalized with lysosomes in J774.1 cells. These results suggest that AgNPs were transported to lysosomes and only gradually dissolved in the macrophages, causing milder inflammatory stimulation in the mouse lung compared to AgNO3.

Increased expression and activation of serum- and glucocorticoid-inducible kinase-1 (SGK1) by cadmium in HK-2 renal proximal tubular epithelial cells.

In HK-2 cells exposed to cadmium chloride (CdCl2), the level of serum- and glucocorticoid-inducible kinase-1 (SGK1) protein is increased, but the levels of SGK2 and SGK3 proteins are not. Phosphorylation of SGK1 protein is also observed. Treatment with actinomycin D abolished CdCl2-induced elevation of SGK1 mRNA level. Treatment with actinomycin D or cycloheximide suppressed SGK1 protein levels in cells exposed to CdCl2. Treatment with SGK1 inhibitor EMD638683 or knockdown of SGK1 with siRNA suppressed CdCl2-induced phosphorylation of N-Myc downstream-regulated kinase 1 (NDRG1). These results indicate that cadmium induces the transcriptional upregulation of SGK1 expression and regulates NDRG1 in HK-2 cells.

[Cellular distribution and behavior of metallothionein in mammalian cells following exposure to silver nanoparticles and silver ions].

Silver nanoparticles (AgNPs) are commercially used mainly as antibacterial reagents in wound dressing and deodorant powders. However, the mechanisms underlying Ag toxicity in mammals are not fully understood. In the present study, we assessed cellular distribution and toxicity of AgNPs and AgNO3 in mouse macrophage cell line (J774.1) and those of AgNO3 in human bronchial epithelial cell line (BEAS-2B) focusing on behavior of metallothionein (MT). J774.1 cells were exposed to 0-100 µg Ag/mL AgNPs or AgNO3 and BEAS-2B cells were exposed to 0-100 µM AgNO3 for 24 h. The cytotoxicity was assayed by a modified MTT method. The cellular concentration and distribution of Ag were evaluated by inductively coupled plasma-mass spectorometry (ICP-MS) and laser scanning microscopy. Distribution of Ag to MT and other proteins was determined using HPLC-ICP-MS. Most AgNPs were found in lysosomes in J774.1 at 3 h after post exposure. Ag was distributed to high molecular weight proteins in AgNPs-exposed cells, while most Ag was bound to MT in AgNO3-exposed cells. In AgNO3-exposed BEAS-2B cells cellular Ag concentration and Ag-bound MT (Ag-MT) were sharply increased up to 3 h and then decreased. ROS production appeared to cause relocation of MT-bound Ag to mitochondria, which evoked inhibition of electron transport chain. AgNPs were sequestered by high-molecular weight proteins rather than MT, probably because they were taken up by lysosomes before induction of MT.

Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate.

Silver (Ag) possesses antibacterial activity and has been used in wound dressings and deodorant powders worldwide. However, the metabolic behavior and biological roles of Ag in mammals have not been well characterized. In the present study, we exposed human bronchial epithelial cells (BEAS-2B) to AgNO3 and investigated uptake and intracellular distribution of Ag, expression of metallothionein (MT), generation of reactive oxygen species (ROS), and changes in mitochondrial respiration. The culture medium concentration of Ag decreased with time and stabilized at 12h. The concentration of both Ag and MT in the soluble cellular fraction increased up to 3h and then decreased, indicating that cytosolic Ag relocated to the insoluble fraction of the cells. The levels of mRNAs for the major human MT isoforms MT-I and MT-II paralleled with the protein levels of Ag-MT. The intensity of fluorescence derived from ROS was elevated in the mitochondrial region at 24h. Ag decreased mitochondrial oxygen consumption in a dose-dependent manner and the activity of mitochondrial complex I-IV enzymes was significantly inhibited following exposure to Ag. In a separate experiment, we found that hydrogen peroxide (H2O2) at concentrations as low as 0.001% (equivalent to the concentration of H2O2 in Ag-exposed cells) removed Ag from MT. These results suggest MT was decomposed by cytosolic H2O2, and then Ag released from MT relocated to insoluble cellular fractions and inhibited electron chain transfer of mitochondrial complexes, which eventually led to cell damage.

[Environmental exposure to silver and its health effects].

Silver (Ag) possesses a well-known antibacterial activity and has been used for medical treatment and cosmetics such as wound dressing and deodorant powders. Occupational Safety and Health Administration (OSHA) and Mine Safety and Health Administration (MSHA) proposed that the permissible exposure limit (PEL) for both metallic and most soluble Ag compounds should be 0.01 mg/m3. Argyria and argyrosis are known to be caused by deposition of insoluble Ag in the dermis and cornea/conjunctiva. However, the metabolic behavior and biological roles of Ag have not been well characterized in mammals. Ag can be absorbed into the systemic circulation from drinking water, and also through parenteral routes such as inhalation and dermal exposure. Experimental studies have demonstrated that Ag+ induces and binds to metallothionein I and II (MTs), which are cysteine-rich proteins, in cells. MTs are major cytoplasmic metal binding proteins and thereby reduce cellular damage caused by toxic heavy metals including Ag. Profiles of Ag distribution in MTs and other Ag-binding proteins can be determined using high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). This technique directly provides information on the intracellular behavior of Ag, which is important for elucidating the mechanism underlying Ag toxicity. Silver nanoparticles (AgNPs) are also commercially used mainly as antimicrobial agents. Despite the widespread use of AgNPs, relatively few studies have been undertaken to evaluate the health effects of AgNP exposure. In the present paper, we discuss the absorption, toxicodynamics, and metabolism of both Ag and AgNPs in mammals and their health effects.

Roles of copper chaperone for superoxide dismutase 1 and metallothionein in copper homeostasis.

Copper chaperone for SOD1 (CCS) specifically delivers copper (Cu) to copper, zinc superoxide dismutase (SOD1) in cytoplasm of mammalian cells. In the present study, small interfering RNA (siRNA) targeting CCS was introduced into metallothionein-knockout mouse fibroblasts (MT-KO cells) and their wild type cells (MT-WT cells) to reveal the interactive role of CCS with other Cu-regulating proteins, in particular, MT. CCS knockdown significantly decreased Ctr1, a Cu influx transporter, mRNA expression. On the other hand, Atp7a, a Cu efflux transporter, mRNA expression was increased 3.0 and 2.5 times higher than those of the control in MT-WT and MT-KO cells. These responses of Cu-regulating genes to the CCS knockdown reflected the presence of excess Cu in the cells. To evaluate the Atp7a function in the Cu-replete cells, siRNA of Atp7a and the other Cu transporter, Atp7b were introduced into MT-WT and MT-KO cells. The Atp7a knockdown significantly increased the intracellular Cu concentration, whereas the Atp7b knockdown had no affect. Although two MT isoforms were induced by the CCS knockdown in MT-WT cells, the expression and activity of SOD1 were maintained in both MT-WT and MT-KO cells even when CCS protein expression was reduced to 0.30-0.35 of control. This suggests that the amount of CCS protein exceeds that required to supply Cu to SOD1 in the cells. Further, the CCS knockdown induces Cu accumulation in cells, however, the Cu accumulation is ameliorated by the MT induction, the decrease of Ctr1 expression and the increase of Atp7a expression to maintain Cu homeostasis.

Roles of COMM-domain-containing 1 in stability and recruitment of the copper-transporting ATPase in a mouse hepatoma cell line.

A novel function of COMMD1 {COMM [copper metabolism MURR1 (mouse U2af1-rs1 region 1)]-domain-containing 1}, a protein relevant to canine copper toxicosis, was examined in the mouse hepatoma cell line Hepa 1-6 with multi-disciplinary techniques consisting of molecular and cellular biological techniques, speciation and elemental imaging. To clarify the function of COMMD1, COMMD1-knockdown was accomplished by introducing siRNA (small interfering RNA) into the cells. Although COMMD1-knockdown did not affect copper incorporation, it inhibited copper excretion, resulting in copper accumulation, which predominantly existed in the form bound to MT (metallothionein). It is known that the liver copper transporter Atp7b (ATP-dependent copper transporter 7beta), localizes on the trans-Golgi network membrane under basal copper conditions and translocates to cytoplasmic vesicles to excrete copper when its concentration exceeds a certain threshold, with the vesicles dispersing in the periphery of the cell. COMMD1-knockdown reduced the expression of Atp7b, and abolished the relocation of Atp7b back from the periphery to the trans-Golgi network membrane when the copper concentration was reduced by treatment with a Cu(I) chelator. The same phenomena were observed during COMMD1-knockdown when another Atp7b substrate, cis-diamminedichloroplatinum, and its sequestrator, glutathione ethylester, were applied. These results suggest that COMMD1 maintains the amount of Atp7b and facilitates recruitment of Atp7b from cytoplasmic vesicles to the trans-Golgi network membrane, i.e. COMMD1 is required to shuttle Atp7b when the intracellular copper level returns below the threshold.

Effect of glutathione depletion on removal of copper from LEC rat livers by tetrathiomolybdate.

Tetrathiomolybdate (TTM) is a powerful and selective copper (Cu) chelator that is used as a therapeutic agent for Wilson disease. TTM is the sole agent that can remove Cu bound to metallothionein (MT) in the livers of Long-Evans rats with a cinnamon-like coat color (LEC rats). However, the administration of excess TTM causes the deposition of Cu and molybdenum (Mo) in the liver. In the present study, the effect of hepatic glutathione (GSH) depletion on the removal of Cu from the livers of LEC rats was evaluated to establish an effective therapy by TTM. Pretreatment with l-buthionine sulfoximine (BSO), a depletor of GSH in vivo, reduced the amounts of Cu and Mo excreted into both the bile and the bloodstream, and increased the amounts of Cu and Mo deposited in the livers of LEC rats in the form of an insoluble complex 4h after the TTM injection. The results suggest that GSH depletion creates an oxidative environment in the livers of LEC rats, and the oxidative environment facilitates the insolubilization of Cu and Mo in the livers of LEC rats after the TTM injection. Therefore, the effect of TTM on the removal of Cu from the liver was reduced in the oxidized condition. Wilson disease patients and LEC rats develop liver injury caused by oxidative damage. From a clinical viewpoint, increasing in the GSH concentration is expected to enhance the effect of TTM.

Copper accumulation and compartmentalization in mouse fibroblast lacking metallothionein and copper chaperone, Atox1.

Copper (Cu) is the active center of some enzymes because of its redox-active property, although that property could have harmful effects. Because of this, cells have strict regulation/detoxification systems for this metal. In this study, multi-disciplinary approaches, such as speciation and elemental imaging of Cu, were applied to reveal the detoxification mechanisms for Cu in cells bearing a defect in Cu-regulating genes. Although Cu concentration in metallothionein (MT)-knockout cells was increased by the knockdown of the Cu chaperone, Atox1, the concentrations of the Cu influx pump, Ctr1, and another Cu chaperone, Ccs, were paradoxically increased; namely, the cells responded to the Cu deficiency despite the fact that cellular Cu concentration was actually increased. Cu imaging showed that the elevated Cu was compartmentalized in cytoplasmic vesicles. Together, the results point to the novel roles of MT and cytoplasmic vesicles in the detoxification of Cu in mammalian cells.

Narrow-bore HPLC-ICP-MS for speciation of copper in mutant mouse neonates bearing a defect in Cu metabolism.

Minute amounts of tissue supernatants from mouse neonates bearing a mutation in the copper (Cu)-transporter gene, Atp7a, were injected into narrow-bore HPLC coupled with an inductively coupled plasma-mass spectrometer (ICP-MS) to examine Cu metabolism. In the 14-day-old mutant neonates, Cu accumulated in the intestine in the metallothionein (MT)-bound form, and mRNA expression of the two MT isoforms was increased. Meanwhile, Cu in the MT-bound form (Cu-MT) was depleted in the liver and mRNA expression decreased in comparison with wild-type mice. These results suggest that Cu is not secreted by intestinal microvillus cells into bloodstream due to the defect of Atp7a, and systemic depletion of Cu occurred. On the other hand, in the kidneys of mutant mice, Cu accumulated in the MT-bound form despite the fact that mRNA expression of the two MT isoforms was low. Part of Cu-MT in microvillus cells may be released into bloodstream at turnover and be preferably taken up by the kidneys. Consequently, the mRNA expression of MT isoforms was not always coincident with the amounts of MT proteins binding Cu, and narrow bore HPLC-ICP-MS used for MT protein determination is a complementary technique to real-time RT-PCR used for MT mRNA determination in Cu speciation.