Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1.

ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-ß receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.

Comparative pulmonary toxicity of inhaled nickel nanoparticles; role of deposited dose and solubility.

In this pilot study, we investigated which physicochemical properties of nickel hydroxide nanoparticles (nano-NH) were mainly responsible in inducing pulmonary toxicity. First, we studied the role of nickel ions solubilized from nano-NH by comparing the toxic effects of nano-NH to those of readily soluble nickel sulfate nanoparticles (nano-NS). Additionally, to test whether there was a non-specific stress response due to particle morphology, we compared the toxicity of nano-NH with that of carbon nanoparticles (nano-C) and titanium dioxide nanoparticles (nano-Ti), both of which had similar physical properties such as particle size and shape, to nano-NH. We exposed mice to each type of nanoparticles for 4?h via a whole-body inhalation system and examined oxidative stress and inflammatory responses in the lung. We also determined the lung burden and clearance of Ni following nano-NH and nano-NS exposures. The results showed that lung deposition of nano-NH was significantly greater than that of nano-NS and nano-NH appeared to have stronger inflammogenic potential than nano-NS even when lung Ni burden taken into consideration. This suggests that the toxicity of nano-NH is not driven solely by released Ni ions from deposited nano-NH particles. However, it is unlikely that the greater toxic potential of nano-NH is attributable to a generic stress response from any nanoparticle exposure, since nano-C and nano-Ti did not elicit toxic responses similar to those of nano-NH. These results indicate that the observed pulmonary toxicity by inhaled nano-NH were chemical-specific and deposited dose and solubility are key factors to understand toxicity induced by nano-NH.

Long-Term Effects of Sirolimus on Human Skin TSC2-Null Fibroblast‒Like Cells.

Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by hamartomatous tumors of the skin, kidneys, brain, and lungs. TSC is caused by mutations in the TSC1 and TSC2 genes, which result in hyperactivation of the mTOR, leading to dysregulated cell growth and autophagy. Rapamycin (sirolimus) shrinks TSC tumors, but the clinical benefits of sirolimus are not sustained after its withdrawal. In this study, we studied the cellular processes critical for tumor formation and growth, including cell proliferation and cell size. TSC2-/- and TSC2+/- cells were isolated from TSC skin tumors and normal-appearing skin, respectively. Cells were incubated with sirolimus for 72 hours. Withdrawal of sirolimus from TSC2-/- cells resulted in a highly proliferative phenotype and caused cells to enter the S phase of the cell cycle, with persistent phosphorylation of mTOR, p70 S6 kinase, ribosomal protein S6, and 4EB-P1; decreased cyclin D kinase inhibitors; and transient hyperactivation of protein kinase B. Sirolimus modulated the estrogen- and autophagy-dependent volume of TSC2-/- cells. These results suggest that sirolimus may decrease the size of TSC tumors by reducing TSC2-/- cell volume, altering the cell cycle, and reprogramming TSC2-null cells.

Stressor identification and health assessment of fish exposed to wastewater effluents in Miho Stream, South Korea.

This study evaluated the effects of an industrial wastewater treatment plant (IWTP) and a municipal wastewater treatment plant (MWTP) effluents on a variety of bioindicators ranging from biochemical, organism, and population-level responses in pale chub (Zacco platypus) and fish community structure. The Index of Biotic Integrity (IBI) indicated that the site upstream of these wastewater treatment plant discharges is in fair-good condition and downstream of the plant is in poor condition. The EROD (ethoxyresorufin-O-deethylase) activity, condition factor, and liver somatic index were significantly increased at the downstream site compared to those of the upstream site. The most significant change observed in pale chub population in the downstream site of the Miho Stream, relative to the upstream population, was the total absence of an younger age group. Stressors impacting the downstream site were identified as mostly organic or nutrient enrichment and habitat degradation associated with wastewater treatment plants. The results of causal analysis suggest that the primary causes affecting fish population in the downstream site are through both size-selective mortality caused by ammonia toxicity and recruitment failure caused by habitat degradation and reproduction problem due to an IWTP and MWTP effluents.

Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells.

Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1alpha protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt-glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1alpha stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25 microM of FeSO(4)) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500 microM of FeSO(4)) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1alpha stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis.

Inhalation exposure to nickel hydroxide nanoparticles induces systemic acute phase response in mice.

It has been proposed that acute phase response can be a mechanism by which inhaled particles exert adverse effects on the cardiovascular system. Although some of the human acute phase proteins have been widely studied as biomarkers of systemic inflammation or cardiovascular diseases, there are only a few studies that investigated the role of serum amyloid P (SAP) , a major acute phase protein in mice. In this study, we investigated the changes in SAP, following inhalation exposure to nickel hydroxide nanoparticles (nano-NH) . We conducted 1) acute (4 h) exposure to nano-NH at 100, 500, and 1000 µg/m(3) and 2) sub-acute (4h/d for 3d) exposure at 1000 µg/m(3), then measured serum SAP protein levels along with hepatic Sap mRNA levels. The results show that inhaled nano-NH can induce systemic acute phase response indicated by increased serum SAP levels and hepatic Sap mRNA levels. To the best of our knowledge, this is the first study showing induction of SAP in response to repeated particle exposure, and the results suggest that SAP can be used as a biomarker for systemic inflammation induced by inhaled particles.

Long-term inhalation exposure to nickel nanoparticles exacerbated atherosclerosis in a susceptible mouse model.

BACKGROUND: Because associations have been reported between inhaled ambient ultrafine particles and increased risk of cardiopulmonary disease, it has been suggested that inhaled engineered nanoparticles (NPs) may also induce adverse effects on the cardiovascular system. OBJECTIVE: We examined the long-term cardiovascular effects of inhaled nickel hydroxide NPs (nano-NH) using a sensitive mouse model. METHODS: Hyperlipidemic, apoprotein E-deficient (ApoE-/-) mice were exposed to nano-NH at either 0 or 79 µg Ni/m3, via a whole-body inhalation system, for 5 hr/day, 5 days/week, for either 1 week or 5 months. We measured various indicators of oxidative stress and inflammation in the lung and cardiovascular tissue, and we determined plaque formation on the ascending aorta. RESULTS: Inhaled nano-NH induced significant oxidative stress and inflammation in the pulmonary and extrapulmonary organs, indicated by up-regulated mRNA levels of certain antioxidant enzyme and inflammatory cytokine genes; increased mitochondrial DNA damage in the aorta; significant signs of inflammation in bronchoalveolar lavage fluid; changes in lung histopathology; and induction of acute-phase response. In addition, after 5-month exposures, nano-NH exacerbated the progression of atherosclerosis in ApoE-/- mice. CONCLUSIONS: This is the first study to report long-term cardiovascular toxicity of an inhaled nanomaterial. Our results clearly demonstrate that long-term exposure to inhaled nano-NH can induce oxidative stress and inflammation, not only in the lung but also in the cardiovascular system, and that this stress and inflammation can ultimately contribute to progression of atherosclerosis in ApoE-/- mice.

Pulmonary response after exposure to inhaled nickel hydroxide nanoparticles: short and long-term studies in mice.

Short and long-term pulmonary response to inhaled nickel hydroxide nanoparticles (nano-Ni(OH)(2), CMD = 40 nm) in C57BL/6 mice was assessed using a whole body exposure system. For short-term studies mice were exposed for 4 h to nominal concentrations of 100, 500, and 1000 mg/m(3). For long-term studies mice were exposed for 5 h/d, 5 d/w, for up to 5 months (m) to a nominal concentration of 100 mg/m(3). Particle morphology, size distribution, chemical composition, solubility, and intrinsic oxidative capacity were determined. Markers of lung injury and inflammation in bronchoalveolar lavage fluid (BALF); histopathology; and lung tissue elemental nickel content and mRNA changes in macrophage inflammatory protein-2 (Mip-2), chemokine ligand 2 (Ccl2), interleukin 1-alpha (Il-1α), and tumor necrosis factor-alpha (Tnf-α) were assessed. Dose-related changes in BALF analyses were observed 24 h after short-term studies while significant changes were noted after 3 m and/or 5 m of exposure (24 h). Nickel content was detected in lung tissue, Ccl2 was most pronouncedly expressed, and histological changes were noted after 5 m of exposure. Collectively, data illustrates nano-Ni(OH)(2) can induce inflammatory responses in C57BL/6 mice.