Jinhee Choi

Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria-involved apoptosis.

Silver nanoparticles (AgNPs), which have well-known antimicrobial properties, are extensively used in various medical and general applications. Despite the widespread use of AgNPs, relatively few studies have been undertaken to determine the cytotoxic effects of AgNPs exposure. This study investigates possible molecular mechanisms underlying the cytotoxic effects of AgNPs. Here, we show that AgNPs-induced cytotoxicity was higher compared than that observed when AgNO(3) was used as a silver ion source. AgNPs induced reactive oxygen species (ROS) generation and suppression of reduced glutathione (GSH) in human Chang liver cells. ROS generated by AgNPs resulted in damage to various cellular components, DNA breaks, lipid membrane peroxidation, and protein carbonylation. Upon AgNPs exposure, cell viability decreased due to apoptosis, as demonstrated by the formation of apoptotic bodies, sub-G(1) hypodiploid cells, and DNA fragmentation. AgNPs induced a mitochondria-dependent apoptotic pathway via modulation of Bax and Bcl-2 expressions, resulting in the disruption of mitochondrial membrane potential (Δψ(m)). Loss of Δψ(m) was followed by cytochrome c release from the mitochondria, resulting in the activation of caspases 9 and 3. The apoptotic effect of AgNPs was exerted via the activation of c-Jun NH(2)-terminal kinase (JNK) and was abrogated by the JNK-specific inhibitor, SP600125 and siRNA targeting JNK. In summary, the results suggest that AgNPs cause cytotoxicity by oxidative stress-induced apoptosis and damage to cellular components.

p38 MAPK Activation, DNA Damage, Cell Cycle Arrest and Apoptosis As Mechanisms of Toxicity of Silver Nanoparticles in Jurkat T Cells

To identify potential harmful effects of silver nanoparticles (AgNPs) on human health, a comprehensive toxicity assay was conducted on human Jurkat T cells, using oxidative stress-related endpoint. The effect of Ag ions was also investigated and compared with that of AgNPs, as it is anticipated that Ag ions will be released from AgNPs, which may be responsible for their toxicity. Cell viability tests indicated high sensitivity of Jurkat T cells when exposed to AgNPs compared to Ag ions; however, both AgNPs and Ag ions induce similar levels of cellular reactive oxygen species during the initial exposure period and; after 24 h, they were increased on exposure to AgNPs compared to Ag ions, which suggest that oxidative stress may be an indirect cause of the observed cytotoxicity of AgNPs. AgNPs exposure activates p38 mitogen-activated protein kinase through nuclear factor-E2-related factor-2 and nuclear factor-kappaB signaling pathways, subsequently inducing DNA damage, cell cycle arrest and apoptosis. Selective toxicity of AgNPs on Jurkat T cells suggests that rigorous toxicity evaluation should be conducted using various different cell types and biological systems prior to the widespread use of AgNPs.

Ecotoxicological investigation of CeO2 and TiO2 nanoparticles on the soil nematode Caenorhabditis elegans using gene expression, growth, fertility, and survival as endpoints.

In this study, the potential harmful effect of cerium dioxide (CeO(2)), and titanium dioxide (TiO(2)) nanoparticles on the environment was investigated using Caenorhabditis elegans ecotoxicity tests. Multiple toxic endpoints, such as stress-response gene expression, growth, fertility, and survival, were analyzed in C. elegans, in response to the CeO(2) and TiO(2) exposure. To investigate relationship between sizes of nanoparticles and toxicity, C. elegans were exposed to nanoparticles to the different sizes of nanoparticles (15, 45nm for CeO(2) and 7, 20nm for TiO(2)). An increase in the expression of the cyp35a2 gene, decrease in fertility and survival parameters were observed in the 15 and 45nm of CeO(2) and in the 7nm of TiO(2) nanoparticles exposed to C. elegans. Gene knock-down experiment using RNA interference (RNAi) suggested that physiological level disturbances may be related with the cyp35a2 gene expression. Smaller sized nanoparticles (7nm of TiO(2) and 15nm of CeO(2)) seemed to be more toxic than larger sized ones (20nm of TiO(2) and 45nm of CeO(2)) on the observed toxicity. The size-dependent effect in CeO(2) and TiO(2) nanoparticles-induced toxicity needs to be investigated under more detailed experimental settings with the various sizes of nanoparticles. Further studies on the mechanism by which CeO(2) and TiO(2) nanoparticles affect cyp35a2 gene expression, fertility, and survival are warranted to better understand the CeO(2) and TiO(2) nanoparticles-induced ecotoxicity in C. elegans, as are studies with the causal relationships between these parameters. Overall results suggest that CeO(2) and TiO(2) nanoparticles have a potential for provoking ecotoxicity on C. elegans and the data obtained from this study can comprise a contribution to knowledge of the ecotoxicology of nanoparticles in C. elegans, about which little data are available.

Identification, characterization and expression profiles of Chironomus riparius glutathione S-transferase (GST) genes in response to cadmium and silver nanoparticles exposure

In this study, we report the identification and characterization of 13 cytosolic GST genes in Chironomus riparius from Expressed Sequence Tags (ESTs) database generated using pyrosequencing. Comparative and phylogenetic analyses were undertaken with Drosophila melanogaster and Anopheles gambiae GSTs and 3 Delta, 4 Sigma, 1 each in Omega, Epsilon, Theta, Zeta and 2 unclassified classes of GSTs were identified and characterized. The relative mRNA expression levels of all of the C. riparius GSTs (CrGSTs) genes under different developmental stages were varied with low expression in the larval stage. The antioxidant role of CrGSTs was studied by exposing fourth instar larvae to a known oxidative stress inducer Paraquat and the relative mRNA expression to different concentrations of cadmium (Cd) and silver nanoparticles (AgNPs) for various time intervals were also studied. All the CrGSTs showed up- or down regulation to varying levels based upon the concentration, and duration of exposure. The highest mRNA expression was noticed in Delta3, Sigma4 and Epsilon1 GST class in all treatments. These results show the role of CrGST genes in defense against oxidative stress and its potential as a biomarker to Cd and AgNPs exposure.

Silver nanoparticles down-regulate Nrf2-mediated 8-oxoguanine DNA glycosylase 1 through inactivation of extracellular regulated kinase and protein kinase B in human Chang liver cells

Recently, we reported that silver nanoparticles (AgNPs) induced reactive oxygen species (ROS) generation and the resultant oxidative stress contributes to the cell damage associated with AgNPs. 8-Oxoguanine (8-oxoG) is sensitive marker of ROS-induced DNA damage. 8-Oxoguanine DNA glycosylase 1 (OGG1) is an important DNA repair enzyme that recognizes and excises 8-oxoG. The aim of the present study was to examine the effect of AgNPs-induced oxidative stress on OGG1 and to elucidate mechanisms underlying AgNPs toxicity. AgNPs decreased OGG1 mRNA and protein expression, resulting in decreased OGG1 activity. Decreased OGG1 activity in AgNPs-treated cells led to increased 8-oxoG levels. The transcription factor NF-E2-related factor 2 (Nrf2) is an important factor in the inducible regulation of OGG1. AgNPs treatment decreased nuclear Nrf2 expression, translocation into nucleus, and transcriptional activity of Nrf2. Extracellular regulated kinase (ERK) and protein kinase B (PKB, AKT), which are upstream of Nrf2, contribute to OGG1 expression. AgNPs attenuated both active forms of ERK and AKT protein expression, resulting in suppression of Nrf2 and decrease of OGG1 expression. These studies demonstrate that down-regulation of Nrf2-mediated OGG1 in exposure to AgNPs occurs through ERK and AKT inactivation.

Effect of cadmium exposure on the globin protein expression in 4th instar larvae of Chironomus riparius Mg. (Diptera: Chironomidae): An ecotoxicoproteomics approach

In order to identify Chironomus hemoglobin (Hb) as a biomarker of ecotoxicity monitoring; herein, the effects of cadmium chloride (Cd) on Hb parameters were investigated in the 4th instar larvae of Chironomus riparius. The expressions of globin mRNA and hemolymph protein, using ecotoxicoproteomic approach, were investigated. Conventional ecotoxicity tests were also conducted to validate the ecotoxicological relevance of the response of Chironomus Hb as a biomarker. The proteomic analysis indicated that exposure to Cd lead alteration in the expression of hemolymph protein, with the total expressions of 12 hemolymph protein spots decreasing in response to treatment, with that of two increasing in response to Cd exposure. In addition, all of the spots differentially expressed in response to Cd treatment were identified as globin proteins. The decreased total Hb content observed in the hemolymph of larvae exposed to Cd suggested that the decreased expression of selected globin proteins in response to Cd exposure impacted on Hb synthesis. The overall results suggested that Hb could be a target molecule for exposure to Cd in C. riparius, with a proteomic approach appearing to be an ideal tool for the discovery of biomarkers in ecotoxicological research.

SiO(2) Nanoparticles Induced Cytotoxicity by Oxidative Stress in Human Bronchial Epithelial Cell, Beas-2B.

Objectives In this study, we investigated the potential harmful effect of the exposure to silicon dioxide (SiO2) nanoparticles through in vitro toxicity assay using human bronchial epithelial cell, Beas-2B with a focus on the involvement of oxidative stress as the toxic mechanism. Methods SiO2-induced oxidative stress was assessed by examining formation of reactive oxygen species (ROS), the induction of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1), as well as cytotoxicity effect was evaluation by cell viability. Subsequently, to understand the molecular mechanism of nanoparticle-induced oxidative stress, the involvement of oxidative stress-responding transcription factors, such as, nuclear factor-kappaB (NF-κB) and nuclear factor-E2-related factor-2 (Nrf-2), and mitogen-activated protein (MAP) kinase signal transduction pathway was also investigated. Results 5-d i phenyltera zolium bromide (MTT) assay results show that decrease 20% in cell viability and the number of cells in the subG1 phase increased. The increase in ROS formation was observed in SiO2 nanoparticle treated cells. The expression of SOD protein was not changed, whereas that of HO-1 was increased by SiO2 nanoparticle exposure. transcription factors Nrf-2 and the expression of phosphorylated form of extracellular signal-regulating kinase (ERK) was strongly induced by SiO2 nanoparticle exposure Conclusions SiO2 nanoparticles exert their toxicity through oxidative stress as they cause the significant increase ROS level. SiO2 nanoparticles induce induction of HO-1 via Nrf-2-ERK MAP kinase pathway. Our tested oxidative stress parameters are rather limited in terms of allowing the full understanding of oxidative stress and cellular response by SiO2 nanoparticle exposure.

Characterization and expression analysis of phospholipid hydroperoxide glutathione peroxidase cDNA from Chironomus riparius on exposure to cadmium.

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) is an antioxidant enzyme in the glutathione peroxidases (GPx) family that reduces hydroperoxides of phospholipids and maintains the integrity of biomembranes. Here, we report the identification and characterization of a full length cDNA of PHGPx from the ecotoxicologically important aquatic midge Chironomus riparius (CrPHGPx1) from the Expressed Sequence Tags (ESTs) database generated through pyrosequencing. The 837 base pair (bp) cDNA contained an open reading frame of 597 bp, and a 75 bp 5 and a 159 bp 3untranslated region. The theoretical molecular mass of the deduced amino acid (aa) sequence (197 aa) was 22.40 kDa with an estimated pI of 8.77. The Cys-codon was present at residue 74 and also the active site residues Gln(91) and Trp(164). The active-site motifs and GPx family signature motifs LAFPCNQF(101-108) and WNFTK(163-168) were also found. Phylogenetic analysis showed that CrPHGPx1 is grouped with PHGPx1 from other species and is more closely related to insects belonging to the dipteran order. The mRNA of CrPHGPx1 was detected in larvae, pupae and adults. The expression of CrPHGPx1 is induced by cadmium exposure indicating that the mRNA expression of CrPHGPx1 is differently regulated in response to oxidative stress caused by environmental stressors.

Cyp35a2 gene expression is involved in toxicity of fenitrothion in the soil nematode Caenorhabditis elegans.

In this study, the effect of organophosphorous (OP) pesticide, fenitrothion (FT), on the non-target organism was investigated using the soil nematode, Caenorhabditis elegans. Toxicity was investigated on multiple biological levels, from organism to molecular levels, such as, immoblity, growth, fertility, development, acetyl cholinesterase (AChE) activity and stress-response gene expressions. FT may provoke serious consequences on the C. elegans population, as it induced significant developmental disturbance. As expected, FT exposure inhibits AChE activity of C. elegans. The increased expression of the cytochrome p450 family protein 35A2 (cyp35a2) gene was also observed in FT exposed worms. To experimentally demonstrate the relationships between organism-level effects and the cyp35a2 gene expression in FT-exposed C. elegans, the integration of the gene expression with biochemical-, and organism level endpoints were attempted using a C. elegans cyp35a2 RNA interference (RNAi) and cyp35a2 mutant (gk317). The 24 h-EC50s of C. elegans on FT exposure were in the order of cyp35a2 RNAi in cyp35a2 mutant (gk317)>cyp35a2 mutant (gk317)>cyp35a2 RNAi in wildtype (N2)>wildtype (N2). The higher EC50 values of cyp35a2 RNAi and cyp35a2 mutant (gk317) compared to that of wildtype C. elegans strongly supported that cyp35a2 gene plays an important role in the toxicity of FT towards C. elegans. The experiments with cyp35a2 RNAi also indicated that the development disturbance and decreased AChE activity, which were observed in FT exposed wildtype C. elegans were significantly rescued in the cyp35a2 RNAi C. elegans. Overall results suggest that the cyp35a2 may be an important gene for exerting FT toxicity in C. elegans.

Cytoprotective Effects of Triphlorethol-A Against Formaldehyde-Induced Oxidative Damage and Apoptosis: Role of Mitochondria-Mediated Caspase-Dependent Pathway

The toxicity of formaldehyde (HCHO) has been attributed to its ability to form adducts with DNA and proteins. Triphlorethol-A, derived from Ecklonia cava, was reported to exert a cytoprotective effect against oxidative stress damage via an antioxidant mechanism. The aim of this study was to examine the mechanisms underlying the triphlorethol-A ability to protect Chinese hamster lung fibroblast (V79-4) cells against HCHO-induced damage. Triphlorethol-A significantly decreased the HCHO-induced intracellular reactive oxygen species (ROS) production. Triphlorethol-A prevented increased cell damage induced by HCHO via inhibition of mitochondria-mediated caspase-dependent apoptosis pathway. Triphlorethol-A diminished HCHO-induced mitochondrial dysfunction, including loss of mitochondrial membrane action potential (Δψ) and adenosine triphosphate (ATP) depletion. Furthermore, the anti-apoptotic effect of triphlorethol-A was exerted through inhibition of c-Jun NH2-terminal kinase (JNK), which was enhanced by HCHO. Our data indicate that triphlorethol-A exerts a cytoprotective effect in V79-4 cells against HCHO-induced oxidative stress by inhibiting the mitochondria-mediated caspase-dependent apoptotic pathway.