Ha Ryong Kim

Genotoxic effects of silver nanoparticles stimulated by oxidative stress in human normal bronchial epithelial (BEAS-2B) cells.

Many classes of silver nanoparticles (Ag-NPs) have been synthesized and widely applied, but the genotoxicity of Ag-NPs and the factors leading to genotoxicity remain unknown. Therefore, the purpose of this study is to elucidate the genotoxic effects of Ag-NPs in lung and the role of oxidative stress on the genotoxic effects of Ag-NPs. For this, Ag-NPs were completely dispersed in medium by sonication and filtration. The Ag-NPs dispersed in medium were 43-260nm in size. We observed distinct uptake of Ag-NPs into BEAS-2B cells. The Ag-NPs aggregates were wrapped with an endocytic vesicle within the cytoplasm and nucleus of BEAS-2B cells. In the comet assay and micronucleus (MN) assay for BEAS-2B cells, Ag-NPs stimulated DNA breakage and MN formation in a dose-dependent manner. The genotoxic effect of Ag-NPs was partially blocked by scavengers. In particular, of the scavengers tested, superoxide dismutase most significantly blocked the genotoxic effects in both the cytokinesis-block MN assay and the comet assay. In the modified comet assay, Ag-NPs induced a significant increase in oxidative DNA damage. Furthermore, in the oxidative stress assay, Ag-NPs significantly increased the reactive oxygen radicals. These results suggest that Ag-NPs have genotoxic effects in BEAS-2B cells and that oxidative stress stimulated by Ag-NPs may be an important factor in their genotoxic effects.

Organic extracts of urban air pollution particulate matter (PM2.5)-induced genotoxicity and oxidative stress in human lung bronchial epithelial cells (BEAS-2B cells).

Traffic is a major source of particulate matter (PM), and ultrafine particulates and traffic intensity probably contribute significantly to PM-related health effects. As a strong relationship between air pollution and motor vehicle-originated pollutants has been shown to exist, air pollution genotoxicity studies of urban cities are steadily increasing. In Korea, the death rate caused by lung cancer is the most rapidly increased cancer death rate in the past 10 years. In this study, genotoxicity of PM2.5 (<2.5μm in aerodynamic diameter particles) collected from the traffic area in Suwon City, Korea, was studied using cultured human lung bronchial epithelial cells (BEAS-2B) as a model system for the potential inhalation health effects. Organic extract of PM2.5 (CE) generated significant DNA breakage and micronucleus formation in a dose-dependent manner (1μg/cm(3)-50μg/cm(3)). In the acid-base-neutral fractionation of PM2.5, neutral samples including the aliphatic (F3), aromatic (F4) and slightly polar (F5) fractions generated significant DNA breakage and micronucleus formation. These genotoxic effects were significantly blocked by scavenging agents [superoxide dismutase (SOD), sodium selenite (SS), mannitol (M), catalase (CAT)]. In addition, in the modified Comet assay using endonucleases (FPG and ENDOIII), CE and its fractions (F3, F4, and F5) increased DNA breakage compared with control groups, indicating that CE and fractions of PM2.5 induced oxidative DNA damage. These results clearly suggest that PM2.5 collected in the Suwon traffic area has genotoxic effects and that reactive oxygen species may play a distinct role in these effects. In addition, aliphatic/chlorinated hydrocarbons, PAH/alkylderivatives, and nitro-PAH/ketones/quinones may be important causative agents of the genotoxic effects.

Appropriate In Vitro Methods for Genotoxicity Testing of Silver Nanoparticles

Objectives We investigated the genotoxic effects of 40-59 nm silver nanoparticles (Ag-NPs) by bacterial reverse mutation assay (Ames test), in vitro comet assay and micronucleus (MN) assay. In particular, we directly compared the effect of cytochalasin B (cytoB) and rat liver homogenate (S9 mix) in the formation of MN by Ag-NPs. Methods Before testing, we confirmed that Ag-NPs were completely dispersed in the experimental medium by sonication (three times in 1 minute) and filtration (0.2 µm pore size filter), and then we measured their size in a zeta potential analyzer. After that the genotoxicity were measured and especially, S9 mix and with and without cytoB were compared one another in MN assay. Results Ames test using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains revealed that Ag-NPs with or without S9 mix did not display a mutagenic effect. The genotoxicity of Ag-NPs was also evaluated in a mammalian cell system using Chinese hamster ovary cells. The results revealed that Ag-NPs stimulated DNA breakage and MN formation with or without S9 mix in a dose-dependent manner (from 0.01 µg/mL to 10 µg/mL). In particular, MN induction was affected by cytoB. Conclusions All of our findings, with the exception of the Ames test results, indicate that Ag-NPs show genotoxic effects in mammalian cell system. In addition, present study suggests the potential error due to use of cytoB in genotoxic test of nanoparticles.

Identification of estrogen-like effects and biologically active compounds in river water using bioassays and chemical analysis.

The Nackdong River is the longest river in South Korea and passes through major cities that have several industrial complexes, including chemical, electric, and petrochemical complexes, and municipal characteristics such as apartment complexes. Along the river, the Gumi region has an electric industrial complex and an apartment complex that may be possible point sources of xenoestrogens such as phenolic compounds. To identify the causative chemicals for estrogenic activity in the river water of this region, bioassay-directed chemical analysis was performed. All samples from six sampling sites (an upstream point: S1; hot spot points: S2-1, S2-2, and S2-3; and downstream points: S3, and S4) showed estrogenic activity in the E-screen assay, with bio-EEQs (17beta-E(2)-equivalent quantities) ranging from 25.35-677.15 pg/L. Samples from S2-2, the sampling point downstream of the junction of stream water, and domestic and industrial wastewater, contained the highest estrogenic activity. Since the bio-EEQ of the organic acid fraction (F2) of the S2-2 sample had the highest activity (823.25 pg-EEQ/L) and F2 may contain phenolic compounds, GC-MS analyses for phenolic xenoestrogens were conducted with the organic acid fractions of the river water samples. Six estrogenic phenolic chemicals, 4-NP, BPA, 4-t-OP, 4-t-BP, 4-n-OP, and 4-n-HTP, were detected, with the highest concentrations (I-EEQ) found in S2-2 (231.80 pg/L). Among these phenolic chemicals, 4-NP was the most potent estrogen (bio-EEF; 8.12 x1 0(-5)) and acted as a full agonist. Furthermore, 4-NP was present at levels (2.0 microg/L in S2-2) that can induce VTG induction in fish (>1 microg/L). In addition, we confirmed that river water (S2-2) significantly increased serum VTG levels in crucian carp (Carassius auratus) in a fish exposure experiment under laboratory conditions. Therefore, phenolic xenoestrogens, especially 4-NP, may be the main causative compounds responsible for the estrogenic effect on the Nackdong River.

The role of NF-κB signaling pathway in polyhexamethylene guanidine phosphate induced inflammatory response in mouse macrophage RAW264.7 cells.

Polyhexamethylene guanidine (PHMG) phosphate is a competitive disinfectant with strong antibacterial activity. However, epidemiologists revealed that inhaled PHMG-phosphate may increase the risk of pulmonary fibrosis associated with inflammation, resulting in the deaths of many people, including infants and pregnant women. In addition, in vitro and in vivo studies reported the inflammatory effects of PHMG-phosphate. Therefore, the aim of the present study was to clarify the inflammatory effects and its mechanism induced by PHMG-phosphate in murine RAW264.7 macrophages. Cell viability, inflammatory cytokine secretion, nuclear factor kappa B (NF-κB) activation, and reactive oxygen species (ROS) generation were investigated in macrophages exposed to PHMG-phosphate. PHMG-phosphate induced dose-dependent cytotoxicity, with LC50 values of 11.15-0.99mg/ml at 6 and 24h, respectively. PHMG-phosphate induced pro-inflammatory cytokines including IL-1β, IL-6, and IL-8. In particular, IL-8 expression was completely inhibited by the NF-κB inhibitor BAY11-7082. In addition, PHMG-phosphate decreased IκB-α protein expression and increased NF-κB-mediated luciferase activity, which was diminished by N-acetyl-l-cystein. However, abundant amounts of ROS were generated in the presence of PHMG-phosphate at high concentrations with a cytotoxic effect. Our results demonstrated that PHMG-phosphate triggered the activation of NF-κB signaling pathway by modulating the degradation of IκB-α. Furthermore, the NF-κB signaling pathway plays a critical role in the inflammatory responses induced by PHMG-phosphate. We assumed that ROS generated by PHMG-phosphate were associated with inflammatory responses as secondary mechanism. In conclusion, we suggest that PHMG-phosphate induces inflammatory responses via NF-κB signaling pathway.

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.

The Role of p53 in Marijuana Smoke Condensates-induced Genotoxicity and Apoptosis

Objectives Marijuana is one of the most frequently abused drug in Korea and its adverse health effects are controversial. p53 is known to be crucial in regulating the DNA damage responses, and adverse effects can occur when it is regulated by marijuana smoke. We evaluated a role of p53 on genotoxic effect and apoptosis in lung cancer cells exposed to marijuana smoke condensates (MSCs). Methods The p53-related genotoxicity and apoptosis of MSCs were evaluated using in vitro bioassay, viz., comet assay, cytokinesis-block micronucleus assay and apoptosis assay. We used two cell lines with differential p53 expression (p53-wildtype (WT) H460 and p53-null H1299). Results MSCs significantly increased DNA breakages and chromosomal changes in p53-WT H460 and p53-null H1299 cells. The genotoxicity induced by MSCs in p53-null H1299 cells showed greater sensitivity than p53-WT H460 cells. Moreover, MSCs showed a significant increase in reactive oxygen species production and apoptosis. The apoptotic responses induced by MSCs were higher in p53-WT H460 cells than in p53-null H1299 cells. Significantly increased mRNA expression or apoptosis related genes, including p53, caspase-3, and Bax/Bcl-2 ratio were observed in the p53-WT H460 cells exposed to MSCs. Conclusions These results suggest that MSCs induce DNA/chromosomal damages and apoptosis in human lung cancer cells and p53 plays an important role in the cellular response to MSCs. The present study may have border implications for our understanding of pulmonary diseases.

Inhibitory Aromatase Effects of Flavonoids from Ginkgo Biloba Extracts on Estrogen Biosynthesis

Ginkgo biloba extract (GBE) is a popular phytomedicine and has been used for disorders of the central nervous system, cardiovascular, renal, respiratory, and circulatory diseases. Although GBE is a complex mixture of over 300 compounds, its major components are 24% flavonoids and 6% terpene lactones. In this study, we tested the inhibitory effects of the three major flavonoids (kaempferol, quercetin, and isorhamnetin) from GBE, independently and as mixtures, on aromatase activity using JEG-3 cells (human placental cells) and recombinant proteins (human placental microsome). In both systems, kaempferol showed the strongest inhibitory effects among the three flavonoids; the flavanoid mixtures exerted increased inhibitory effects. The results of exon I.1-driven luciferase reporter gene assays supported the increased inhibitory effects of flavonoid mixtures, accompanied by suppression of estrogen biosynthesis. In the RT-PCR analysis, decreased patterns of aromatase promoter I.1 mRNA expressions were observed, which were similar to the aromatase inhibition patterns of flavonoids and their mixtures. The present study demonstrated that three flavonoids synergistically inhibit estrogen biosynthesis through aromatase inhibition, decrease CYP19 mRNA, and induce transcriptional suppression. Our results support the usefulness of flavonoids in adjuvant therapy for breast cancer by reducing estrogen levels with reduced adverse effects due to estrogen depletion.

Developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in artificially fertilized crucian carp (Carassius auratus) embryo.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent bioaccumulative environmental contaminant that is an endocrine disruptor. Embryos of various fish species are responsive to TCDD and have been used as an alternative method to the acute toxicity test with juvenile and adult fish. The TCDD test has similar endpoints of developmental toxicity. However, their sensitivity and signs of TCDD-induced toxicity are different depending on fish species and its habit. Crucian carp (Carassius auratus) - the sentinel species for persistent organic pollutants and a common foodfish in China, Japan, and Korea - was used to identify the developmental toxicity of TCDD. We obtained the fertilized eggs from the artificial fertilization of crucian carp (97.45% success rate). Embryos at 3h post fertilization (hpf) were exposed to no vehicle, vehicle (dimethylsulfoxide, 0.1% v/v) or TCDD (0.128, 0.32, 0.8, 2 and 5 μg/L) for 1h and then fresh water was changed and aerated. Embryonic development and toxicity were monitored until 150 hpf. TCDD-exposed group showed no effects on embryo mortality and hatching rate from 6 to 126 hpf. On the other hand, the post-hatching mortality rate in TCDD-exposed group was increased in a dose-dependent manner, especially at high doses (0.8, 2 and 5 μg/L). The LD50 for larval mortality was calculated to 0.24 ng TCDD/g embryo. Pericardial edema was continuously observed in larvae of TCDD-exposed groups from hatching complete time (78 hpf), followed by the onset of yolk sac edema. Hemorrhage and edema showed a significant increase depending on exposure concentration and time. Expression of TCDD-related CYP1A genes was evaluated quantitatively. Embryo and larvae in TCDD-exposed groups displayed a significant increase of CYP1A gene expression. Overall, we defined TCDD-induced toxicity in artificially fertilized crucian carp embryo and these results suggest that crucian carp can be applied as an early life stage model of TCDD-induced toxicity.

In vitro estrogenic and antiestrogenic potential of chlorostyrenes.

Chlorostyrenes (CSs) are primarily derived from industrial by-products and are persistent and accumulative in the environment. In this study, the estrogenic and antiestrogenic activities of CSs (o-CS, m-CS, p-CS, DiCS, octa-CS) were evaluated using in vitro bioassays. o-CS and octa-CS have both estrogenic and antiestrogenic activity in the E-SCREEN assay and the ERE-reporter gene assay, indicating effects on a classical ER-mediated pathway. m-CS showed estrogenic activity in E-SCREEN but not in ERE-reporter gene assays, indicating that it may work through a non-classical ER-mediated pathway. Finally, DiCS only showed antiestrogenic activity via an ER-independent pathway, which can be induced by depletion of endogenous E(2) level by the inhibition of aromatase activity and the stimulation of E(2) metabolism. Although CSs have structural similarities to dioxins/furans, they did not have AhR agonist effects. This study is the first to show the estrogenic and antiestrogenic activity of several CSs using in vitro bioassay systems, including whether the compounds work via ER-mediated or/and non-ER-mediated pathways.