Qunwei Zhang

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.

Molecular mechanisms of nickel carcinogenesis

Nickel treatment of intact cultured cells oxidized dichlorofluorescin to a fluorescent product indicating that nickel elevated the level of oxidants in cells. Nickel also caused an increase in crosslinking of amino acids to DNA and these complexes did not appear to involve the direct participation of Ni2+. Histidine, cysteine and tyrosine were prominent among the amino acids crosslinked to DNA. Nickel selectively damaged heterochromatin and this resulted in deletions of heterochromatic regions during nickel carcinogenesis. Thrombospondin was one of the genes expressed in normal cells that was not expressed in nickel-transformed cells. Other aspects of the molecular mechanism of nickel carcinogenesis are discussed.

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.

1-Cys peroxiredoxin knock-out mice express mRNA but not protein for a highly related intronless gene

1‐Cys peroxiredoxin (1‐cysPrx), a member of the peroxiredoxin family with a single conserved cysteine, is a unique antioxidant enzyme. We have generated mice in which the 1‐cysPrx gene has been inactivated; they are viable and fertile. Mice have a highly related intronless gene (1‐cysPrx‐P1, GenBank accession number AF085220) with the same length of open reading frame (224 aa) as 1‐cysPrx but located on a different chromosome. Since the product of this gene possibly could mimic 1‐cysPrx function, we compared expression of 1‐cysPrx and 1‐cysPrx‐P1 in mouse tissues by real‐time polymerase chain reaction and Western blot. 1‐cysPrx mRNA and protein were expressed in all mouse tissues that were examined with the highest expression level in lung. 1‐cysPrx‐P1 mRNA was expressed only in testis. In the 1‐cysPrx ‘knock‐out’ mouse, 1‐cysPrx‐P1 mRNA expression level was similar to the wild type but protein expression was not detected. Thus, mouse 1‐cysPrx‐P1 is an mRNA‐expressed pseudogene that does not result in detectable protein in vivo.

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.

INVOLVEMENT OF ERKS ACTIVATION IN CADMIUM-INDUCED AP-1 TRANSACTIVATION IN VITRO AND IN VIVO

Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category 1 carcinogen. Cadmium-induced up‐regulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP‐1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP‐1 activity by cadmium appears to involve activation of Erks, since the induction of AP‐1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP‐1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP‐1 in AP‐1‐luciferase report transgenic mice. Considering the role of AP‐1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmium‐induced carcinogenesis.

The histone deacetylase inhibitor trichostatin A reduces nickel-induced gene silencing in yeast and mammalian cells

We have previously reported that nickel (Ni)-silenced expression of the URA3 gene in yeast (Saccharomyces cerevisiae) and gpt transgene in G12 Chinese hamster cells. In both cases, close proximity to a heterochromatic region was required for gene silencing. Yeast exposed to Ni exhibited reduced acetylation of the lysine residues in the N-terminal tail of histone H4. Ni-induced silencing of the gpt gene in mammalian cells involved hypermethylation of promoter region DNA. Yeast do not employ DNA methylation to silence gene expression. To determine if histone deacetylation participates in Ni-induced silencing of the URA3 and gpt genes, we exposed yeast and G12 hamster cells to the histone deacetylase inhibitor trichostatin A (TSA) prior to and concurrently with Ni. Treatment of yeast cells with 0.2-0.6mM NiCl(2) resulted in reduced expression of the URA3 gene as assessed by increased resistance to 1g/l 5-fluorotic acid (5-FOA). This effect was lessened when yeast were pre-treated with 50 microg TSA/ml. Similarly, treatment of G12 cells with 5 ng/ml TSA during and after exposure to 0.3 microg Ni(3)S(2)/cm(2) reduced silencing of the gpt gene as gauged by resistance to 10 microg/ml 6-thioguanine (6-TG). The ability of TSA alone and in combination with the DNA-demethylating agent (5-AzaC) to reactivate the gpt gene in Ni-silenced variants was also assessed. Although treatment with 100 ng/ml TSA for 48 h was partially effective in reactivating the gpt gene, treatment with 5 microM 5-AzaC was more efficacious. The greatest gpt gene reversion frequencies were observed following a sequential 5-AzaC/TSA treatment. Taken all together, our data from mammalian cells suggests that both DNA methylation and histone deacetylation participate in Ni-induced silencing of the gpt gene with DNA hypermethylation playing the more dominant role in maintaining the silenced state.

Comparative Pulmonary Responses Caused by Exposure to Standard Cobalt and Ultrafine Cobalt.

Comparative Pulmonary Responses Caused by Exposure to Standard Cobalt and Ultrafine Cobalt: Qunwei Zhang, et al. Department of Environmental Health, School of Medicine, Fukui Medical University—The aim of this study was to compare the pulmonary toxicity after exposure to standard cobalt (Std‐Co) and ultrafine cobalt (Uf‐Co). Rats were intratracheally instilled with 1 mg of Std‐Co and Uf‐Co, and wet lung weight and bronchoalveolar lavage fluid (BALF) profiles were analysed 1,3,7,15 and 30 d later. The effects of Std‐Co and Uf‐Co on indices that can be presumed to reflect epithelial injury and permeability (lactate dehydrogenase (LDH) and total protein (TP)), and release of proinflammatory cytokine (tumor necrosis factor‐alpha (TNF‐alpha)) were increased throughout the 30 d post‐exposure period. The results showed that the effects of Std‐Co and Uf‐Co on these indices were significantly higher than those of control. The results also showed that the effects of Uf‐Co on indices were significantly higher than those of Std‐Co from 1 to 15 d after instillation. Moreover, the number of neutrophils and LDH activity in BALF in rats after exposure to Uf‐Co were significantly greater than those of Std‐Co‐exposed rats up to 30 d after instillation. Our findings suggest that Uf‐Co has a much more toxic effect on the lungs than Std‐Co, but the mechanism remains to be elucidated.