Kyu-Hyuck Chung

Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells

Cytotoxicity induced by silver nanoparticles (AgNPs) and the role that oxidative stress plays in this process were demonstrated in human hepatoma cells. Toxicity induced by silver (Ag(+)) ions was studied in parallel using AgNO(3) as the Ag(+) ion source. Using cation exchange treatment, we confirmed that the AgNP solution contained a negligible amount of free Ag(+) ions. Metal-responsive metallothionein 1b (MT1b) mRNA expression was not induced in AgNP-treated cells, while it was induced in AgNO(3)-treated cells. These results indicate that AgNP-treated cells have limited exposure to Ag(+) ions, despite the potential release of Ag(+) ions from AgNPs in cell culture. AgNPs agglomerated in the cytoplasm and nuclei of treated cells, and induced intracellular oxidative stress. AgNPs exhibited cytotoxicity with a potency comparable to that of Ag(+) ions in in vitro cytotoxicity assays. However, the toxicity of AgNPs was prevented by use of the antioxidant N-acetylcysteine, and AgNP-induced DNA damage was also prevented by N-acetylcysteine. AgNO(3) treatment induced oxidative stress-related glutathione peroxidase 1 (GPx1) and catalase expression to a greater extent than AgNP exposure, but treatment with AgNO(3) and AgNPs induced comparable superoxide dismutase 1 (SOD1) expression levels. Our findings suggest that AgNP cytotoxicity is primarily the result of oxidative stress and is independent of the toxicity of Ag(+) ions.

Oxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cells

As the applications of industrial nanoparticles are being developed, the concerns on the environmental health are increasing. Cytotoxicities of titanium dioxide nanoparticles of different concentrations (5, 10, 20 and 40 microg/ml) were evaluated in this study using a cultured human bronchial epithelial cell line, BEAS-2B. Exposure of the cultured cells to nanoparticles led to cell death, reactive oxygen species (ROS) increase, reduced glutathione (GSH) decrease, and the induction of oxidative stress-related genes such as heme oxygenase-1, thioredoxin reductase, glutathione-S-transferase, catalase, and a hypoxia inducible gene. The ROS increase by titanium dioxide nanoparticles triggered the activation of cytosolic caspase-3 and chromatin condensation, which means that titanium dioxide nanoparticles exert cytotoxicity by an apoptotic process. Furthermore, the expressions of inflammation-related genes such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), TNF-a, and C-X-C motif ligand 2 (CXCL2) were also elevated. The induction of IL-8 by titanium dioxide nanoparticles was inhibited by the pre-treatment with SB203580 and PD98059, which means that the IL-8 was induced through p38 mitogen-activated protein kinase (MAPK) pathway and/or extracellular signal (ERK) pathway. Uptake of the nanoparticles into the cultured cells was observed and titanium dioxide nanoparticles seemed to penetrate into the cytoplasm and locate in the peri-region of the nucleus as aggregated particles, which may induce direct interactions between the particles and cellular molecules, to cause adverse biological responses.

Silver nanoparticles enhance thrombus formation through increased platelet aggregation and procoagulant activity

Abstract Despite the wide use of silver nanoparticles (nano Ag), its toxicity still remains poorly understood. In this report, nano Ag induced an increase in platelet aggregation and procoagulant activation which are the key contributors to thrombotic diseases. In freshly isolated human platelets, nano Ag induced platelet aggregation and procoagulant activation evident by increased phosphatidylserine exposure and thrombin generation. Interestingly, the sub-threshold level of thrombin enhanced nano Ag-induced platelet activation significantly indicating that the prothrombotic effects of nano Ag might be further potentiated in activated platelets. An increase in intracellular calcium mediated nano Ag induced platelet activation and P-selectin expression, and serotonin release was also enhanced by nano Ag. Consistent with the in vitro results, exposure to nano Ag (0.05–0.1 mg/kg i.v. or 5–10 mg/kg intratracheal instillation) in vivo enhanced venous thrombus formation, platelet aggregation, and phosphatidylserine externalization ex vivo in rats suggesting that nano Ag, indeed, does enhance thrombus formation through platelet activation.

Global pollution monitoring of butyltin compounds using skipjack tuna as a bioindicator

Butyltin compounds (BTs) including mono- (MBT), di- (DBT), tri-butyltin (TBT) and total tin (sigmaSn), were determined in the liver of skipjack tuna (Katsuwonus pelamis) collected from Asian offshore waters (off-Japan, the Japan Sea, off-Taiwan, the East China Sea, the South China Sea, off-Philippines, off-Indonesia, the Bay of Bengal), off-Seychelles, off-Brazil and open seas (the North Pacific). BTs were detected in all the skipjack tuna collected, suggesting widespread contamination of BTs even in offshore waters and open seas on a global scale. Considering specific accumulation, Sex-, body length- differences and migration of skipjack tuna did not seem to affect BT concentrations, indicating rapid reflection of the pollution levels in seawater where and when they were collected. Skipjack tuna is a suitable bioindicator for monitoring the global distribution of BTs in offshore waters and open seas. High concentrations of BTs were observed in skipjack tuna from offshore waters around Japan, a highly developed and industrialized region (up to 400 ng/g wet weight). Moreover skipjack tuna collected from offshore waters around Asian developing countries also revealed the levels comparable to those in Japan (up to 270 ng/g wet weight) which may be due to the recent improvement in economic status in Asian developing countries. High percentages (almost 90%) of BTs in total tin (sigmaSn: sum of inorganic tin+organic tin) were found in the liver of skipjack tuna from offshore waters around Asian developing countries. This finding suggests that the anthropogenic BTs represent the major source of Sn accumulation in skipjack tuna from these regions.

Quantitative assessment of estrogenic activity in the water environment of Korea by the E-SCREEN assay

In this study, the E-SCREEN assay was optimized and validated for the sensitive quantitative determination of the total estrogenicity in river samples. River water and sediment samples were collected and analyzed with the E-SCREEN. River water (10 l) was extracted using combined solid-phase extraction in static adsorption mode with Soxhlet extraction. Estrogenic pollutants adsorbed to the XAD-4 resin were recovered with 98.24 +/- 5.90% efficiency by elution with ethyl acetate and dichloromethane (1:9). The detection limit by 17beta-estradiol equivalent concentration (EEQ) of the E-SCREEN assay was 8.03 pg EEQ/l. Among the water samples, the estrogenic activity was observed to be higher downstream of the Kumho river (7.43 ng EEQ/l) and upstream of Kum river (2.05 ng EEQ/l) than in other samples. More than 3 mg of equivalent sediment samples from the Kumho river, Kum river and Miho stream showed partial agonistic effects, and the Mankyung river showed a partial agonistic effect with only 1.5 mg of sediment. The highest value of RPE was 83.34 downstream of the Kumho river, and the lowest value of RPE was 6.52 downstream of the Miho stream. Full estrogen agonistic activities were observed downstream of the Kumho river and upstream of the Kum river. The partial agonistic activity was observed in upstream of the Kumho river, downstream of the Mankyung river, and upstream of the Miho stream, and no agonistic action was observed downstream of the Kum river or Miho stream, or upstream of the Mankyung river. The total estrogenic activity in the river water and sediment samples was between 0.50 pg/L and 7.4 ng/L, 3.39 pg/g and 10.70 pg/g.

Environmental estrogenic effects and gonadal development in wild goldfish (Carassius auratus)

Serum vitellogenin (VTG) contents of wild goldfish (Carassius auratus) were investigated as a sensitive biomarker for artificial estrogenic compounds in aquatic environments. Goldfish was sampled from a pristine area, a river situated 5xa0km downstream from a sewage treatment works (STW), and also from the Young-San River in Korea. The female yolk precursor protein VTG was not detected when gonadosomatic index (GSI) was less than 0.85%, while VTG levels of >10xa0μg/ml were found in males whose GSI was less than 1.53%. In male goldfish sampled from STW and the Young-San River, the higher VTG corresponded to lower GSI. This study suggested a trend that gonad development was connected to VTG levels in both sexes, and the application of GSI and histological analysis provide an attractive possibility that it could be included in the panel of markers used for estrogenic activity investigation of aquatic environments.

Molecular cloning of CYP1A gene and its expression by benzo(a)pyrene from goldfish (Carassius auratus)

We cloned and sequenced the cytochrome P450 1A (CYP1A) gene from goldfish (Carassius auratus). It has a 1581 bp open reading frame that encodes a 526 amino acid protein with a theoretical molecular weight of 59.02 kDa. The CYP1A amino acid sequence clusters in a monophyletic group with other fish CYP1As, and more closely related to zebrafish CYP1A (91% identity) than to other fish CYP1As. Exposure to benzo(a)pyrene (BaP) by intraperitoneal injection increased biliary BaP metabolites and liver CYP1A gene expression. BaP exposure also increased CYP1A gene expression in extrahepatic organs, including intestine, and gill, which are sensitive to aqueous and dietary exposure to Arylhydrocarbon receptor (AhR) agonists. Therefore, goldfish CYP1A identified in this study offers basic information for further research related to biomarker use of CYP1A of goldfish.

Distribution of organochlorines and PCB congeners in Korean human tissues

In order to investigate the residual amounts of organochlorines and polychlorinated biphenyls (PCBs) in Korean human tissues (blood, adipose tissue, liver, kidney cortex, and lung), the samples were collected from the autopsied cadavers of 40 men and 40 women (from teens to seventies of age) α-BHC, β-BHC, γ-BHC, σ-BHC,p,p′-DDT,p,p′-DDD,p,p′-DDE, endrin, dieldein, aldrin, and 7 marker PCBs (28, 52, 101, 118, 138, 153, and 180) were determined in human tissues. The levels of organochlorines and PCB congeners indicated that they have been widely distributed in Korean human body. Positive correlations in terms of age were observed for the following cases:p,p′-DDE,p,p′-DDT, θ-DDT, PCB 118, PCB 138, PCB 153, and θ-PCB in the adipose tissue, andp,p′-DDE in the lung. Concentration of these compounds showed a significant age-related increase. Accumulation of these compounds in aged people revealed that these compounds were more slowly eliminated in our environment and risk assessment was necessary for further proper action. Significant differences in the levels of PCBs between genders were found for PCB 118 in the adipose tissue and PCB 138 in the liver. Positive correlation coefficients between tissues were detected withp,p′-DDE and β-BHC.

Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutants

The sequence of cytochrome P4501A (CYP1A) cDNA of medaka (Oryzias latipes) was determined, and its messenger ribonucleic acid (mRNA) regulation by beta-naphthoflavone (betaNF) was evaluated. The determined cDNA sequence contained 2,349 base pairs (bp), and the open reading frame contained a total of 1,563 bp encoding 521 predicted amino acids. The induction of CYP1A mRNA in medaka was evaluated using reverse transcription-polymerase chain reaction. The concentration-dependent induction of CYP1A mRNA in the liver was observed after exposure to betaNF at nominal concentrations of 20, 100, and 500 microg/L for 2 d. Time-dependent changes of CYP1A mRNA levels were also observed in the liver, gill, gut, and caudal fin tissues of medaka exposed to 100 microg/L of betaNF for 7 d. Our results showed that the degree of CYP1A mRNA induction in the gill, gut, and caudal fin after exposure to betaNF was relatively higher than that in the liver, possibly because of low basal levels of CYP1A mRNA in the gill, gut, and caudal fin of nonexposed fish. The induction of medaka CYP1A mRNA was also observed after exposure to an environmental sample, landfill leachate. The CYP1A mRNA inductions in the gill, gut, and caudal fin were also higher than that in the liver as shown in the betaNF-treated groups. These results show that CYP1A mRNA determination in the gill, gut, and caudal fin, which are in direct contact with the polluted water, may become a useful method for monitoring CYP1A-inducible chemicals.

Molecular characterization of the aryl hydrocarbon receptor (AhR) pathway in goldfish (Carassius auratus) exposure to TCDD: the mRNA and protein levels.

In bony fish or other aquatic vertebrates, the aryl hydrocarbon receptor (AhR) signaling pathway is initiated by exposure to polycyclic (or/and halogenated) aromatic hydrocarbons (PAHs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), which subsequently induces the up-regulated expression of a series of related genes (such as cytochrome P4501A (CYP1A)). However, a lack of applicable protein reagents hinders our further understanding of the AhR signaling pathway, which focuses only on gene-based investigations. The goldfish (Carassius auratus) is an ideal model for a study of environmental pollution in whole-Asian fresh water. Here, three sensitive and specific polyclonal antisera against goldfish AhR1, AhR2, and CYP1A proteins were developed. These antisera not only bound the in-vitro synthesized target proteins, but recognized the real proteins expressed in goldfish tissues, with minimal cross-reactivity to non-specific proteins. Together with the analysis of semi-quantitative RT-PCR and polyclonal-antibody-based sandwich ELISA, we confirmed that goldfish AhRs differed in the expression (mRNA and protein levels) patterns among test tissues. Importantly, the relative abundance of each AhR mRNA levels from the different tissues showed no obvious consistency with their protein levels. After exposure to TCDD, goldfish AhR2 showed a more sensitivity than AhR1, and stimulated CYP1A expression directly, similar with the other reported fish models. Overall, development of these antibodies in this study will allow valuable and versatile investigations to further understand the AhR signaling pathway, and different expression (mRNA and protein) patterns represent the first step in determining the regulatory mechanisms underlying the TCDD-exposed aquatic environment.