Yiqun Mo

Comparative toxicity of standard nickel and ultrafine nickel in lung after intratracheal instillation.

Comparative Toxicity of Standard Nickel and Ultrafine Nickel in Lung after Intratracheal Instillation: Qunwei Zhang, et al.; Department of Environmental Health, School of Medicine, Fukui Medical University—A comparison was made of the bronchoalveolar lavage fluid (BALF) response to ultrafine nickel (Uf‐Ni) and standard‐sized nickel (Std‐Ni). Rats were intratracheally instilled with 0, 0.1, 0.5, 1 and 5 mg Uf‐Ni and Std‐Ni, respectively. At 3 d after instillation, the body weight and wet lung weight were determined. At the same time, BALF was analyzed for lactate dehydrogenase (LDH), total protein (TP), tumor necrosis factor‐alpha (TNF‐alpha), and total cell and differential cell counts. The results showed that indicators of lung injury and inflammation in BALF were markedly raised with increased Uf‐Ni and Std‐Ni for each from 0 to 1 mg, and there were no differences in the indices between instillation of Uf‐Ni at 1 mg and 5 mg. The results also showed that the effects of Uf‐Ni on the indices were significantly higher than those of Std‐Ni. Additional groups of rats were intratracheally instilled with 1 mg of Uf‐Ni or Std‐Ni, and wet lung weight and BALF profiles were analyzed at 1, 3, 7, 15 and 30 d later. The effect of Uf‐Ni and Std‐Ni on indices that can be presumed to reflect epithelial injury and permeability (LDH or TP), and release of proinflammatory cytokine (TNF‐alpha) were increased throughout the 30 d post‐exposure and the effects of Uf‐Ni on these indices were significantly higher than those of Std‐Ni from 1 to 30 d after instillation. Moreover, the number of neutrophils and LDH activity in BALF of rats after exposure to Uf‐Ni were significantly greater than those of Std‐Ni‐exposed rats up to 30 d after instillation. Our findings suggest that Uf‐Ni has a much more toxic effect on the lung than St‐Ni, but the mechanism remains to be elucidated.

Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes: The role of oxidative stress and protein tyrosine kinase activation.

Recently, many studies have shown that nanoparticles can translocate from the lungs to the circulatory system. As a particulate foreign body, nanoparticles could induce host responses such as reactive oxygen species (ROS) generation, inflammatory cytokine and matrix metalloproteinase (MMP) release which play a major role in tissue destruction and remodeling. However, the direct effects of nanoparticles on leukocytes, especially monocytes, are still unclear. The objective of the present study was to compare the ability of Nano-Co and Nano-TiO(2) to cause alteration of transcription and activity of MMPs and to explore possible mechanisms. We hypothesized that non-toxic doses of some transition metal nanoparticles stimulate an imbalance of MMP/TIMP that cause MMP production that may contribute to their health effects. To test this hypothesis, U937 cells were treated with Nano-Co and Nano-TiO(2) and cytotoxic effects and ROS generation were measured. The alteration of MMP-2 and MMP-9 expression and activity of MMP-2 and MMP-9 after exposure to these metal nanoparticles were subsequently determined. To investigate the potential signaling pathways involved in the Nano-Co-induced MMP activation, the ROS scavengers or inhibitors, AP-1 inhibitor, and protein tyrosine kinase (PTK) inhibitors were also used to pre-treat U937 cells. Our results demonstrated that exposure of U937 cells to Nano-Co, but not to Nano-TiO(2), at a dose that does not cause cytotoxicity, resulted in ROS generation and up-regulation of MMP-2 and MMP-9 mRNA expression(..) Our results also showed dose- and time-related increases in pro-MMP-2 and pro-MMP-9 gelatinolytic activities in conditioned media after exposure of U937 cells to Nano-Co, but not to Nano-TiO(2). Nano-Co-induced pro-MMP-2 and pro-MMP-9 activity increases were inhibited by pre-treatment with ROS scavengers or inhibitors. We also demonstrated dose- and time-related decreases in tissue inhibitors of metalloproteinases 2 (TIMP-2) in U937 cells after exposure to Nano-Co, but not to Nano-TiO(2). However, neither Nano-Co nor Nano-TiO(2) exposure led to any transcriptional change of TIMP-1. The decrease of TIMP-2 after exposure to Nano-Co was also inhibited by pre-treatment with ROS scavengers or inhibitors. Our results also showed that pre-treatment of U937 cells with AP-1 inhibitor, curcumin, or the PTK specific inhibitor, herbimycin A or genistein, prior to exposure to Nano-Co, significantly abolished Nano-Co-induced pro-MMP-2 and-9 activity. Our results suggest that Nano-Co causes an imbalance between the expression and activity of MMPs and their inhibitors which is mediated by the AP-1 and tyrosine kinase pathways due to oxidative stress.

DNA damage caused by metal nanoparticles: involvement of oxidative stress and activation of ATM.

Nanotechnology is a fast growing emerging field, the benefits of which are widely publicized. Our current knowledge of the health effects of metal nanoparticles such as nanosized cobalt (Nano-Co) and titanium dioxide (Nano-TiO(2)) is limited but suggests that metal nanoparticles may exert more adverse pulmonary effects as compared with standard-sized particles. To investigate metal nanoparticle-induced genotoxic effects and the potential underlying mechanisms, human lung epithelial A549 cells were exposed to Nano-Co and Nano-TiO(2). Our results showed that exposure of A549 cells to Nano-Co caused reactive oxygen species (ROS) generation that was abolished by pretreatment of cells with ROS inhibitors or scavengers, such as catalase and N-acetyl-L(+)-cysteine (NAC). However, exposure of A549 cells to Nano-TiO(2) did not cause ROS generation. Nano-Co caused DNA damage in A549 cells, which was reflected by an increase in length, width, and DNA content of the comet tail by the Comet assay. Exposure of A549 cells to Nano-Co also caused a dose- and a time-response increased expression of phosphorylated histone H2AX (γ-H2AX), Rad51, and phosphorylated p53. These effects were significantly attenuated when A549 cells were pretreated with catalase or NAC. Nano-TiO(2) did not show these effects. These results suggest that oxidative stress may be involved in Nano-Co-induced DNA damage. To further investigate the pathways involved in the Nano-Co-induced DNA damage, we measured the phosphorylation of ataxia telangiectasia mutant (ATM). Our results showed that phosphorylation of ATM was increased when A549 cells were exposed to Nano-Co, and this effect was attenuated when cells were pretreated with catalase or NAC. Pretreatment of A549 cells with an ATM specific inhibitor, KU55933, significantly abolished Nano-Co-induced DNA damage. Furthermore, pretreatment of A549 cells with ROS scavengers, such as catalase and NAC, significantly abolished Nano-Co-induced increased expression of phosphorylated ATM. Taken together, oxidative stress and ATM activation are involved in Nano-Co-induced DNA damage. These findings have important implications for understanding the potential health effects of metal nanoparticle exposure.

Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O(2)(-) generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67(phox) siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91(phox) knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47(phox), p67(phox) and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67(phox) siRNA. Exposure of MPMVEC obtained from gp91(phox) knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to activation of MAPKs through induced phosphorylation of p38 and ERK1/2 MAPKs that may further result in endothelial dysfunction through production of cytokines such as IL-6. Our results suggest that endothelial oxidative stress may be an important mechanism for PM-induced cardiovascular effects.

Endoplasmic reticulum stress and oxidative stress are involved in ZnO nanoparticle-induced hepatotoxicity

Zinc oxide nanoparticles (Nano-ZnO) are widely used in sunscreens, clothes, medicine and electronic devices. However, the potential risks of human exposure and the potential for adverse health impacts are not well understood. Previous studies have demonstrated that exposure to Nano-ZnO caused liver damage and hepatocyte apoptosis through oxidative stress, but the molecular mechanisms that are involved in Nano-ZnO-induced hepatotoxicity are still unclear. Endoplasmic reticulum (ER) is sensitive to oxidative stress, and also plays a crucial role in oxidative stress-induced damage. Previous studies showed that ER stress was involved in many chemical-induced liver injuries. We hypothesized that exposure to Nano-ZnO caused oxidative stress and ER stress that were involved in Nano-ZnO-induced liver injury. To test our hypothesis, mice were gavaged with 200 mg/kg or 400 mg/kg of Nano-ZnO once a day for a period of 90 days, and blood and liver tissues were obtained for study. Our results showed that exposure to Nano-ZnO caused liver injury that was reflected by focal hepatocellular necrosis, congestive dilation of central veins, and significantly increased alanine transaminase (ALT) and aspartate transaminase (AST) levels. Exposure to Nano-ZnO also caused depletion of glutathione (GSH) in the liver tissues. In addition, our electron microscope results showed that ER swelling and ribosomal degranulation were observed in the liver tissues from mice treated with Nano-ZnO. The mRNA expression levels of ER stress-associated genes (grp78, grp94, pdi-3, xbp-1) were also up-regulated in Nano-ZnO-treated mice. Nano-ZnO caused increased phosphorylation of RNA-dependent protein kinase-like ER kinase (PERK) and eukaryotic initiation factor 2α (eIF2α). Finally, we found that exposure to Nano-ZnO caused increased ER stress-associated apoptotic protein levels, such as caspase-3, caspase-9, caspase-12, phosphorylation of JNK, and CHOP/GADD153, and up-regulation of pro-apoptotic genes (chop and bax). These results suggest that oxidative stress and ER stress-induced apoptosis are involved in Nano-ZnO-induced hepatotoxicity in mice.

Regulation of plasminogen activator inhibitor-1 expression in endothelial cells with exposure to metal nanoparticles.

Recent studies demonstrated that exposure to nanoparticles could enhance the adhesion of endothelial cells and modify the membrane structure of vascular endothelium. The endothelium plays an important role in the regulation of fibrinolysis, and imbalance of the fibrinolysis system potential contributes to the development of thrombosis. Plasminogen activator inhibitor-1 (PAI-1) is the most potent endogenous inhibitor of fibrinolysis and is involved in the pathogenesis of several cardiovascular diseases. The aim of this study was to investigate the alteration of PAI-1 expression in mouse pulmonary microvascular endothelial cells (MPMVEC) exposed to the metal nanoparticles that are known to be reactive, and the potential underlying mechanisms. We compared the alteration of PAI-1 expression in MPMVEC exposed to non-toxic doses of nano-size copper (II) oxide (Nano-CuO) and nano-size titanium dioxide (Nano-TiO(2)). Our results showed that Nano-CuO caused a dose- and time-dependent increase in PAI-1 expression. Moreover, exposure of MPMVEC to Nano-CuO caused reactive oxygen species (ROS) generation that was abolished by pre-treatment of cells with ROS scavengers or inhibitors, DPI, NAC and catalase. Exposure of MPMVEC to Nano-CuO also caused a dose- and time-dependent increase in p38 phosphorylation by Western blot. These effects were significantly attenuated when MPMVEC were pre-treated with DPI, NAC and catalase. To further investigate the role of p38 phosphorylation in Nano-CuO-induced PAI-1 overexpression, the p38 inhibitor, SB203580, was used to pre-treat cells prior to Nano-CuO exposure. We found that Nano-CuO-induced overexpression of PAI-1 was attenuated by p38 inhibitor pre-treatment. However, Nano-TiO(2) did not show the same results. Our results suggest that Nano-CuO caused up-regulation of PAI-1 in endothelial cells is mediated by p38 phosphorylation due to oxidative stress. These findings have important implications for understanding the potential health effects of metal nanoparticle exposure.

Differential susceptibility of inbred mouse strains to chlorine-induced airway fibrosis

Chlorine is a reactive gas that is considered a chemical threat agent. Humans who develop acute lung injury from chlorine inhalation typically recover normal lung function; however, a subset can experience chronic airway disease. To examine pathological changes following chlorine-induced lung injury, mice were exposed to a single high dose of chlorine, and repair of the lung was analyzed at multiple times after exposure. In FVB/NJ mice, chlorine inhalation caused pronounced fibrosis of larger airways that developed by day 7 after exposure and was associated with airway hyperreactivity. In contrast, A/J mice had little or no airway fibrosis and had normal lung function at day 7. Unexposed FVB/NJ mice had less keratin 5 staining (basal cell marker) than A/J mice in large intrapulmonary airways where epithelial repair was poor and fibrosis developed after chlorine exposure. FVB/NJ mice had large areas devoid of epithelium on day 1 after exposure leading to fibroproliferative lesions on days 4 and 7. A/J mice had airways covered by squamous keratin 5-stained cells on day 1 that transitioned to a highly proliferative reparative epithelium by day 4 followed by the reappearance of ciliated and Clara cells by day 7. The data suggest that lack of basal cells in the large intrapulmonary airways and failure to effect epithelial repair at these sites are factors contributing to the development of airway fibrosis in FVB/NJ mice. The observed differences in susceptibility to chlorine-induced airway disease provide a model in which mechanisms and treatment of airway fibrosis can be investigated.

Intracellular delivery of adenosine triphosphate enhanced healing process in full-thickness skin wounds in diabetic rabbits

BACKGROUND Small unilamellar lipid vesicles were used to encapsulate adenosine triphosphate (ATP-vesicles) for intracellular energy delivery and were tested for diabetic skin wounds in rabbits. METHODS Diabetes was induced by alloxan. The mean peak blood glucose concentration was 505 mg/dL. One ear was made ischemic and 80 full-thickness wounds were created in 10 animals. ATP-vesicles or saline was used and healing was compared. RESULTS On the non-ischemic ears, mean closure time for ATP-vesicles-treated wounds was 13.7 days versus 16.4 days for saline-treated wounds (P < .05). On the ischemic ears, mean closure time for ATP-vesicles-treated wounds was 15.3 days versus 19.3 days for saline-treated wounds (P < .01). Histological study indicated better healing and re-epithelialization in the ATP-vesicles-treated wounds. CONCLUSIONS Intracellular delivery of ATP accelerated the healing process of diabetic skin wounds on ischemic and non-ischemic rabbit ears. The mechanisms deserve further study but may be related to improved cellular energy supplies.

Cytokine and NO release from peripheral blood neutrophils after exposure to metal nanoparticles: in vitro and ex vivo studies

We have observed that nano-size metal particles such as nickel (Nano-Ni), cobalt (Nano-Co), and titanium dioxide (Nano-TiO2) have much more toxic effects on rat lungs than standard-size Ni, Co, and TiO2 particles. Recent evidence indicates that inhaled nanoparticles can penetrate alveolar membranes and enter the circulation, which may result in adverse effects in extra-pulmonary organs. We have proposed that metal nanoparticles may directly or indirectly activate peripheral blood neutrophils after they have translocated from the lungs to the circulation. The neutrophils are the most numerous phagocytes in the body, whose function is not only to defense against bacterial and viral infection, but also to be capable of ingesting particles. Neutrophils contain enzymes that may cause tissue damage if they are excessive or inappropriate activated. In this study, we determined the release of cytokines and nitrite from neutrophils after exposure to metal nanoparticles in vitro and ex vivo. The results showed dose-related increases of TNF-α, MIP-2, and nitrite levels in the supernatants of neutrophils treated with various doses of Nano-Ni and Nano-Co. Neutrophils treated with Nano-Ni and Nano-Co released significantly higher levels of TNF-α, MIP-2, and nitrite than those treated with Nano-TiO2 and the control. The above results were further confirmed by ex vivo experiments that showed dose-related increases in TNF-α, MIP-2, and nitrite release from neutrophils from rats exposed to Nano-Ni and Nano-Co. The level of TNF-α and MIP-2 reached their highest level after 24 h exposure, while the level of nitrite continued to increase, reaching its highest level after 48 h exposure. The levels of TNF-α, MIP-2, and nitrite released by neutrophils from rats intratracheally instilled with Nano-Ni and Nano-Co were consistently significantly higher than those from rats instilled with physiological saline and Nano-TiO2. These results indicate that Ni, Co, and TiO2 nanoparticles of the similar diameter are dramatically different in their ability to activate neutrophils to release cytokines and nitrite, and each metal exerts a different effect.

Creating a long-term diabetic rabbit model.

This study was to create a long-term rabbit model of diabetes mellitus for medical studies of up to one year or longer and to evaluate the effects of chronic hyperglycemia on damage of major organs. A single dose of alloxan monohydrate (100 mg/kg) was given intravenously to 20 young New Zealand White rabbits. Another 12 age-matched normal rabbits were used as controls. Hyperglycemia developed within 48 hours after treatment with alloxan. Insulin was given daily after diabetes developed. All animals gained some body weight, but the gain was much less than the age-matched nondiabetic rabbits. Hyperlipidemia, higher blood urea nitrogen and creatinine were found in the diabetic animals. Histologically, the pancreas showed marked beta cell damage. The kidneys showed significantly thickened afferent glomerular arterioles with narrowed lumens along with glomerular atrophy. Lipid accumulation in the cytoplasm of hepatocytes appeared as vacuoles. Full-thickness skin wound healing was delayed. In summary, with careful management, alloxan-induced diabetic rabbits can be maintained for one year or longer in reasonably good health for diabetic studies.