Kyu Hyuck Chung

In the present study, the ecotoxicity of silver nanoparticles (AgNPs) was investigated in Caenorhabditis elegans using survival, growth, and reproduction, as the ecotoxicological endpoints, as well as stress response gene expression. Whole genome microarray was used to screen global changes in C. elegans transcription profiles after AgNPs exposure, followed by quantitative analysis of selected genes. The integration of gene expression with organism and population level endpoints was investigated using C. elegans functional genomics tools, to test the ecotoxicological relevance of AgNPs-induced gene expression. AgNPs exerted considerable toxicity in C. elegans, most clearly as dramatically decreased reproduction potential. Increased expression of the superoxide dismutases-3 (sod-3) and abnormal dauer formation protein (daf-12) genes with 0.1 and 0.5 mg/L of AgNPs exposures occurred concurrently with significant decreases in reproduction ability. Overall results of functional genomic studies using mutant analyses suggested that the sod-3 and daf-12 gene expressions may have been related to the AgNPs-induced reproductive failure in C. elegans and that oxidative stress may have been an important mechanism in AgNPs toxicity.

Abstract Fine particulate matters less than 2.5 µm (PM 2.5 ) in the ambient atmosphere are strongly associated with adverse health effects. However, it is unlikely that all fine particles are equally toxic in view of their different sizes and chemical components. Toxicity of fine particles produced from various combustion sources (diesel engine, gasoline engine, biomass burning (rice straw and pine stem burning), and coal combustion) and non-combustion sources (road dust including sea spray aerosols, ammonium sulfate, ammonium nitrate, and secondary organic aerosols (SOA)), which are known major sources of PM 2.5 , was determined. Multiple biological and chemical endpoints were integrated for various source-specific aerosols to derive toxicity scores for particles originating from different sources. The highest toxicity score was obtained for diesel engine exhaust particles, followed by gasoline engine exhaust particles, biomass burning particles, coal combustion particles, and road dust, suggesting that traffic plays the most critical role in enhancing the toxic effects of fine particles. The toxicity ranking of fine particles produced from various sources can be used to better understand the adverse health effects caused by different fine particle types in the ambient atmosphere, and to provide practical management of fine particles beyond what can be achieved only using PM mass which is the current regulation standard.

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.

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.

To elucidate the global distribution of polybrominated diphenyl ethers (PBDEs), these chemicals were determined in the muscle of skipjack tuna (Katsuwonus pelamis) collected from offshore waters of various regions in the world (Japan, Taiwan, Philippines, Indonesia, Seychelles, and Brazil, and the Japan Sea, East China Sea, South China Sea, Indian Ocean, and North Pacific Ocean). PBDEs were detected in almost all the skipjack tuna collected from the locations surveyed (from <0.1 to 53 ng/g of lipid), indicating widespread contamination by these compounds in the marine environment. Residue levels of PBDEs in these samples from the northern hemisphere seem to be higher than those from the southern hemisphere, which is plausibly due to larger usage of these compounds in the northern hemisphere. Higher concentrations of PBDEs were detected in the samples from waters around the East China Sea (up to 53 ng/g of lipid). Developing countries around the East China Sea are supposedly the “hot spots” releasing these chemicals into the marine environment. With regard to the composition of PBDE congeners, the percentage contribution by lower brominated congeners (BDE15, -28, and -47) showed an increasing trend with increasing latitude. On the other hand, higher brominated congeners (BDE153, -154, and -183) showed a reverse trend. These patterns suggest that lower brominated congeners of PBDEs (di-, tri-, and tetra-BDEs) were preferentially transported from pollution sources to northern colder regions through the atmosphere. PBDEs may have a high potency to cause global pollution like PCBs.

We used gene knockout mice to explore the role of Angiopoietin-like-4 (Angptl4) in lipid metabolism as well as to generate anti-Angptl4 mAbs with pharmacological activity. Angptl4 -/- mice had lower triglyceride (TG) levels resulting both from increased very low-density lipoprotein (VLDL) clearance and decreased VLDL production and had modestly lower cholesterol levels. Also, both Angptl4 -/- suckling mice and adult mice fed a high-fat diet showed reduced viability associated with lipogranulomatous lesions of the intestines and their draining lymphatics and mesenteric lymph nodes. Treating C57BL/6J, ApoE -/-, LDLr -/-, and db/db mice with the anti-Angptl4 mAb 14D12 recapitulated the lipid and histopathologic phenotypes noted in Angptl4 -/- mice. This demonstrates that the knockout phenotype reflects not only the physiologic function of the Angptl4 gene but also predicts the pharmacologic consequences of Angptl4 protein inhibition with a neutralizing antibody in relevant models of human disease.

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.

Rationale: Humidifier disinfectant lung injury is an acute lung disease attributed to recurrent inhalation of certain disinfectant aerosols emitted from room humidifiers. An outbreak of this toxic lung injury occurred in South Korea from 1995 until all humidifier disinfectant products were recalled from the consumer market by the government in 2011.Objectives: A nationwide study was conducted to ascertain and classify all potential cases of humidifier disinfectant lung injury in Korea and to assess dose–response relationships.Methods: By several mechanisms, clinicians and the general public were invited to report all suspected cases of humidifier disinfectant lung injury to public health officials in South Korea. A committee was convened to define diagnostic criteria based on pathologic, radiologic, and clinical findings for index cases, combined with assessment of environmental exposure to humidifier disinfectants. Clinical review and environmental assessments were performed and later combined to determine overall likelihood of disease for each study participant, classified as definite, probable, possible, or unlikely. Survival time from exposure to onset of symptoms was analyzed to assess dose–response relationships. Three broad categories of risk factors were examined: (1) biological susceptibility, (2) temporal cycle of exposure and recovery, and (3) spatial conditions and density of disinfectant.Measurements and Main Results: Of 374 possible cases identified and reviewed, 329 were unanimously classified by the diagnostic committee, as follows: 117 definite, 34 probable, 38 possible and 140 unlikely cases. A total of 62 individuals with definite or probable disease died. Risk factors examined for polyhexamethyleneguanidine phosphate exposure that were found to be significant in shortening survival included age 4 years or younger at onset, use of disinfectant for 7 days per week, airborne density of 800 μg/m3 or more of disinfectant, and daily exposure 11 or more hours in duration.Conclusions: Dose–response analysis indicated that development of humidifier disinfectant lung injury and death were associated strongly with recurrent, intense, acute exposure without sufficient recovery time between exposures, more so than long-term cumulative exposure. These findings may explain some reversible or clinically unapparent cases among coexposed family members.

The detection of high levels of microplastics in indoor and outdoor air has increased concerns regarding its toxic effects on the respiratory system. They are not easily degradable and can be deposited deep in the lungs. Although several studies have reported inhalation toxicities of microplastics, they are still controversial due to a lack of evidence. Herein, we evaluated the inhalation toxicities of three differently charged polystyrene microplastics (PS-MPs), the most abundant microplastics in the air. Cytotoxicity and ROS generation were evaluated using WST-1 and DCF-DA assays, respectively. To evaluate the toxic effects on the lung, inflammatory responses were analyzed after repeated exposure to the PS-MPs through intratracheal instillation. To explore the mechanism of toxicity, autophagy and ER stress-associated proteins were analyzed. Only the positively charged PS-MPs (NH2 -PS-MPs) showed cytotoxicity and increased ROS generation in BEAS-2B cells. Similarly, only NH2 -PS-MPs significantly increased the expression and secretion of the pro-inflammatory cytokine IL-β in the animal experiments. The expression of ER stress proteins indicated that NH2 -PS-MPs increased ER stress via PERK-EIF2α and ATF4-CHOP pathways. Moreover, accumulation of NH2 -PS-MPs in lysosomes and deformity of the nucleus were observed in BEAS-2B cells with autophagy induction. Taken together, our results demonstrated that NH2 -PS-MPs induced autophagic cell death in bronchial epithelial cells, leading to inflammatory responses in the lungs. These results suggest that repeated inhalation of microplastics can result in inflammatory responses in the lung through cellular damage of lung epithelial cells, and that inhalation microplastics should be monitored to reduce inhalation health risks.

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. Keywords: Bacterial reverse mutation test, Comet assay, Genotoxicity, Micronucleus assay, Silver nanoparticles

Particulate matter (PM) has become an important health risk factor in our society. PM can easily deposit in the bronchi and lungs, causing diverse diseases such as respiratory infections, lung cancers and cardiovascular diseases. In recent days, more and more toxicological studies have been dealing with air particles in distinctive areas including industrial areas, transportation sites, or indoors. Studies on subway PM in particular, have been recognizing PM as an important health risk factor because many people use subways as a major mode of public transportation (4 million people a day in Korea). The main aim of the present study was to evaluate the genotoxic effects of organic extract (OE) of subway PM10 and potential attribution of PAHs to these effects. Particles were collected in the subway tunnel at Kil-eum station(Line 4) for one month and then extracted with Dichloromethane (DCM). Chinese Hamster Ovary cells(CHO-K1) and human normal bronchial cells (BEAS-2B) were exposed to OE, and MN and Comet assays were conducted to analyze the genotoxicity. The results showed that OE increased DNA or chromosome damages in both cell lines. In the modified Comet assay and MN assay with free radical scavengers, we confirmed that the genotoxic effect of OE was partially due to the oxidative damage on DNA. DCFHD Aassay also indicated that OE induced ROS generation in BEAS-2B cells. PAHs [benzo(a)anthracene,benzo(k)fluoranthrene, etc.], the most well-known carcinogens in polluted air, were detected in Kil-eum PM10. In conclusion, our findings confirmed that OE of subway PM10 has genotoxic effects on normal human lung cells, and oxidative stress could be one of the major mechanisms of these genotoxic effects.In addition, some genotoxic and carcinogenic PAHs were detected in OE by GC/MS/MS, even though PAHs level was not enough to increase CYP1A1 gene. Therefore, we suggest that additive or synergistic effects by unidentified chemicals as well as PAHs contained in OE of subway PM10 may induce genotoxic effects and further researches are needed to identify the genotoxic compounds in subway PM.

Exposure to humidifier disinfectants was identified in 2011 as the potential cause of an outbreak of lung disease in Korea. It is estimated that over 8 million people have been exposed to humidifier disinfectants-chemicals added to the water used in humidifiers to prevent the growth of microorganisms-since their commercial introduction. The primary component of humidifier disinfectant products involved was polyhexamethylene guanidine phosphate (PHMG-P), a guanidine-based antimicrobial agent. Lesions observed in the lungs of patients were similar to those observed in laboratory animals exposed to PHMG-P. In this review, we outline the physicochemical and toxicological properties of PHMG-P, and introduce a putative mechanism for its lung toxicity based in large part on research findings to date.

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 11.15–0.99 mg/ml at 6 and 24 h, 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.

sObjective: The major active ingredient of humidifier disinfectant, polyhexamethylene guanidine-phosphate (PHMG-P), caused hundreds of deaths with pulmonary fibrosis. However, structurally similar guanidine-based disinfectants are still in use in various fields. Moreover, as they are precursors of excellent antimicrobial compounds, new chemicals with guanidine-based structures have been synthesized and introduced. In this study, we evaluated pulmonary fibrotic responses induced by PHMG-P, polyhexamethylene biguanide (PHMB), and oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH) and their toxicity mechanisms in type II alveolar epithelial A549 cells.Materials and methods: Cellular damage was compared by using the cytotoxicity test (WST-1 assay) and plasma membrane toxicity tests (Lactate dehydrogenase leakage detection assay and plasma membrane staining). As a measure of fibrotic response, induction of the epithelial-mesenchymal transition (EMT) was evaluated by measuring E-cadherin and α-smooth muscle actin (α-SMA) protein expression (epithelial and mesenchymal marker, respectively).Results: All tested compounds showed membrane damage; PHMG-P and PGH induced the highest and lowest damage, respectively. Moreover, they induced EMT when the test chemicals were treated with similar cytotoxic concentrations.Conclusions: Our study indicates that three guanidine-based disinfectants are potential fibrosis-inducing chemicals that induce EMT through cellular damage. Therefore, use of guanidine-based polymers should be strictly regulated by considering their potential adverse effects on the lungs.

Pulmonary fibrosis is a chronic lung disease characterized by abnormal accumulation of the extracellular matrix (ECM). Chronic damage of the alveolar epithelium leads to a process called "epithelial-mesenchymal transition" (EMT) and increases synthesis and deposition of ECM proteins. Therefore, inhibition of EMT might be a promising therapeutic approach for the treatment of pulmonary fibrosis. β-Sitosterol is one of the most abundant phytosterols in the plant kingdom and the major constituent in corn silk, which is derived from the stigma and style of maize (Zea mays). In this study, we elucidated that β-sitosterol inhibited transforming growth factor-β1 (TGF-β1)-induced EMT and consequently had an antifibrotic effect. β-Sitosterol (1-10 μg/mL) significantly downregulated the TGF-β1-induced fibrotic proteins, such as collagen, fibronectin, and α-smooth muscle actin in human alveolar epithelial cells (p < 0.01). After 24 h, relative wound density (RWD) was increased in TGF-β1 treated group (82.16 ± 5.70) compare to the control group (64.63 ± 2.21), but RWD was decreased in β-sitosterol cotreated group (10 μg/mL: 71.54 ± 7.39; 20 μg/mL: 65.69 ± 6.42). In addition, the changes of the TGF-β1-induced morphological shape and protein expression of EMT markers, N-cadherin, vimentin, and E-cadherin, were significantly blocked by β-sitosterol treatment (p < 0.01). The effects of β-sitosterol on EMT were found to be associated with the TGF-β1/Snail pathway, which is regulated by Smad and non-Smad signaling pathways. Taken together, these findings suggest that β-sitosterol can be used to attenuate pulmonary fibrosis through suppression of EMT by inhibiting the TGF-β1/Snail pathway.

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.

Citrus unshiu (Rutaceae) is an easy-peeling citrus fruit, which has been used as a traditional Korean medicine for improving skin elasticity, relieving fatigue and cough, and preventing bronchitis, flu, and various cancers. However, its active components associated with anti-inflammation and underlying mechanisms remain unknown. In this study, we investigated the active constituents from the fruits of Citrus unshiu and evaluated the anti-inflammatory activity in order to support the traditional usage of Citrus unshiu.Repeated column chromatography, together with a semi-preparative HPLC purification was used to separate the bioactive constituent from the EtOAc soluble fraction of the EtOH extract of Citrus unshiu fruits. Anti-inflammatory effects of the isolated compounds on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW264.7 macrophage cells.A new cyclic peptide, citrusin XI (1), was isolated and identified from the fruits of Citrus unshiu. The structure of compound 1 was elucidated by spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR) ((1)H, (13)C, COSY, HMQC and HMBC experiments), and high resolution (HR)-mass spectrometry, and its absolute configurations were further confirmed by the Marfey׳s method. Compound 1 decreased NO production in LPS-stimulated RAW264.7 cells in a dose-dependent manner with an IC50 value of 70μM. Compound 1 suppressed NO production by decreasing iNOS expression but COX-2 expression was slightly associated with the reduction by compound 1 in LPS-induced RAW264.7 cells. Furthermore, compound 1 inhibited NF-κB activation by blocking IκBα degradation and NF-κB phosphorylation in LPS-stimulated RAW264.7 cells.These results indicate that a new cyclic peptide, citrusin XI, from Citrus unshiu fruits has anti-inflammatory properties that inhibit the release of pro-inflammatory mediators. Compound 1 decreases NO production by decreasing iNOS expression and NF-κB activation associated with IκBα degradation and NF-κB phosphorylation in LPS-induced RAW264.7 cells. This is the first study to clarify the underlying mechanism of the anti-inflammatory effect exerted by a pure isolated compound from Citrus unshiu in LPS-stimulated RAW264.7 macrophage cells. The phytochemical, citrusin XI of Citrus unshiu may serve as lead compound in the design of new agents for preventing and treating inflammatory diseases.

Polyhexamethylene guanidine phosphate (PHMG-p) is an active ingredient of humidifier disinfectants and causes severe lung injury resulting in pulmonary fibrosis. Current evidence indicates that pulmonary fibrosis is initiated as a result of epithelial damage, which can lead to an inflammatory response and fibrotic cell infiltration; however, the toxic mechanism of PHMG-p on the epithelium is still unknown. In this study, the toxic response of PHMG-p on human lung epithelial cells was evaluated, and its mechanisms associated with reactive oxygen species (ROS), DNA damage, and its relationship with p53 activation were investigated. The toxic responses of epithelial cells were assessed by flow cytometry analysis and western blot analysis. The results revealed that PHMG-p induced G1/S arrest and apoptosis in A549 cells. Interestingly, p53 was activated by PHMG-p treatment and p53 knockdown suppressed PHMG-p-induced apoptosis and cell cycle arrest. PHMG-p promoted ROS generation and consequently increased the expression of DNA damage markers such as ATM and H2AX phosphorylation. The antioxidant N-acetylcysteine reduced the expression of phosphorylated ATM and H2AX, and the ATM inhibitor, caffeine, inhibited p53 activation. Taken together, our results demonstrate that PHMG-p triggered G1/S arrest and apoptosis through the ROS/ATM/p53 pathway in lung epithelial cells.

Polyhexamethylene guanidine phosphate (PHMG-p), used as a humidifier disinfectant, causes interstitial lung disease, obliterative bronchiolitis, and lung fibrosis; however, little is known about its effect on intercellular interactions. Extracellular vesicles (EVs), which carry diverse compounds including proteins, RNA, and DNA to mediate cell-to-cell communication through their paracrine effects, have been highlighted as novel factors in lung fibrogenesis. This study aimed to identify the effect of proteins on small EVs (sEVs) from bronchoalveolar lavage fluid (BALF) of the recipient cells after PHMG-p exposure. A week after intratracheal administration of PHMG-p, sEVs were isolated from BALF of tissue showing overexpressed inflammatory and fibrosis markers. To investigate the role of sEVs in inflammation, naïve macrophages were cultured with sEVs, which induced their activation. To identify sEV proteins that are associated with these responses, proteomics analysis was performed. In the gene ontology analysis, coagulation, fibrinolysis, and hemostasis were associated with the upregulated proteins in sEVs. The highest increase was observed in fibrinogen levels, which was also related to those gene ontologies. We validated role of exosomal fibrinogen in inflammation using recombinant fibrinogen and an inhibitor of the integrin, which is the binding receptor for fibrinogen. Overall, we elucidated that increased fibrinogen levels in the early sEVs-PHMG activated inflammatory response during early fibrosis. These results suggest that sEVs from the BALF of PHMG-p-exposed mice could aggravate fibrogenesis by activating naïve macrophages via various proteins in the sEVs, Furthermore, this finding will be broadening the spectrum of communicating mediators.

Ginkgo biloba has been used in herbal medicines for thousands of years. Although a standard G. biloba extract, EGb 761 has been used to improve cognition in breast cancer patients, its effects on breast cancer are unknown. Therefore, we investigated the antitumorigenic effects of EGb 761 using an in vitro cell model and an in vivo xenograft model. EGb 761 significantly inhibited aromatase activity in aromatase over-expressing MCF-7 cells (MCF-7 AROM). In addition, EGb 761 exposure reduced cytochrome p450 aromatase (CYP19) mRNA and protein expression; CYP19 promoter I.3 and PII expression particularly decreased. These inhibitory effects on aromatase were accompanied by reduced 17β-estradiol levels in MCF-7 AROM cells. For elucidating antitumorigenic effects, MCF-7 AROM cells were implanted in BALB/c nude mice prior to oral EGb 761 treatment for 3 weeks. EGb 761 reduced the tumor size and significantly reduced tumor CYP19 mRNA expression. Taken together, our results indicated that EGb 761 inhibited aromatase and exerted antitumor effects on breast cancer cells both in vitro and in vivo. These findings suggest that EGb761 may be a useful aromatase inhibitor for the treatment for estrogen-sensitive breast cancer.

Polyhexamethylene guanidine phosphate (PHMG-p), an antimicrobial additive, was used as a humidifier disinfectant in Korea and caused severe lung injuries, including lung fibrosis, in hundreds of victims. As PHMG-p-induced lung fibrosis is different from that induced by known fibrogenic agents such as bleomycin, it is important to understand the molecular mechanisms underlying this effect. A recent study showed that epithelial-mesenchymal transition (EMT) could play key roles in PHMG-p-induced pulmonary fibrosis. Therefore, we aimed to characterize the molecular mechanisms associated with PHMG-p-induced EMT. We observed EMT, macrophage infiltration, and fibrosis in mouse lung tissues after intratracheal instillation of PHMG-p. Furthermore, PHMG-p-induced EMT was observed in A549 cells by the evaluation of cell morphology and quantitation of mRNA and protein expression. The use of EMT inhibitors revealed that PHMG-p induced EMT through the activation of Akt and Notch signaling. Moreover, the transcription factor ZEB2 was observed in PHMG-p-treated A549 cells and mouse lungs. The results indicated that upstream regulators, including Akt and Notch 1, acted as intracellular effectors that triggered ZEB2 expression after exposure to PHMG-p. Attenuation of PHMG-p-induced EMT following inhibition or silencing of Akt and Notch signaling or ZEB2 implied that PHMG-p-induced EMT was a result of Akt, Notch, and ZEB2 activation. Our findings showed that PHMG-p induced EMT through Akt/Notch signaling pathways and that ZEB2 played an important role in PHMG-p-induced lung toxicity. This study will help to understand the mechanisms of action of PHMG-p associated with lung fibrogenesis.

To obtain the usual value of aluminum, arsenic, cadmium, chromium, copper, iron, lead, manganese, mercury, molybdenum, nickel, selenium, silicon, tin, vanadium and zinc in the normal human body, the amounts of these 16 metals were determined in 89 male and 61 female Korean cadavers, whose ages ranged from 12 to 87 years. Inductively coupled plasma atomic emission spectrometry was used for analysis of heavy metals in 9 autopsied human organs (liver, kidney, cerebrum, heart, spleen, lung, bone, hair and nail). Distribution of arsenic, nickel, selenium, lead and vanadium in the human body were almost uniform. Cadmium, mercury, manganese, molybdenum, tin and zinc were found in large quantities in the metabolic organs, whereas the concentrations of aluminum, chromium and silicon were greatest in the tissues exposed to the exterior.

We examined fate and anaerobic microbial transformation/degradation characteristics of atrazine in wetland sediment receiving wastewater from a local sugar mill. Well-known metabolites/intermediates of atrazine were not detected except hydroxyatrazine. Atrazine adsorbed on the sediment linearly with a distribution coefficient of 9.979 ml/g. Although we did not obtain a direct proof of atrazine mineralization, the values of observed yield of total NH3-N were comparable to the theoretical values suggesting the biodegradation of atrazine to its end products of NH3 and CO2. Only about 20% of atrazine was biotransformed to non-triazine species, possibly to the mineral end products, after 38 weeks of incubation in the sample reactors without organic amendments, with methanol and sodium acetate. Acetic acid appeared to enhance the biotransformation of atrazine (both in aqueous and sediment phases) if one only measured the parent atrazine compound alone. However, a comprehensive analysis of atrazine and its metabolites/intermediates showed that most of the disappeared atrazine existed as hydroxyatrazine. Glucose improved the wetland sediment's removal rate of the triazine species.

Most climacteric and postmenopausal women appear to have vasomotor symptoms as well as a high risk of osteoporosis and cardiovascular disease. Although exogenous estrogens can reduce these symptoms, women are reluctant to use hormone replacement therapy (HRT) due to its undesirable side effects, such as irregular bleeding and an increased risk of breast cancer. A previous study suggested that Ginkgo biloba extracts (GBE) have estrogenic activity and might be suitable as an alternative to HRT. However, there are no reports of the preventive effect of GBE on breast cancer, which is the side effect of classical HRT. In this study, it was confirmed that GBE exhibits estrogenic and antiestrogenic activity depending on the E2 and GBE concentration, via estrogen receptor (ER)-dependent and ER-independent pathways. In addition, GBE reduced the E2 levels by stimulating the E2 metabolism and inhibiting E2 synthesis, which indicates that GBE can induce antiestrogenic activity via the depletion of E2. Furthermore, GBE might have similar action to selective arylhydrocarbon receptor modulators (SAhRMs), which induce antiestrogenic activity through cross-talk between the arylhydrocarbon receptor (AhR) and ER. In conclusion, GBE has a biphasic effect on estrogen, and can be considered as a potential alternative to HRT with chemopreventive effects on breast cancer. However, further studies on animals and humans will be required.

Deregulated apoptosis has been associated with many lung diseases. Although many studies have reported the apoptotic effects exhibited by silver nanoparticles (Ag-NPs) in various circumstances, the apoptosis mechanism of Ag-NPs is unclear. We investigated oxidative stress and apoptosis in human normal bronchial epithelial (BEAS-2B) cells to elucidate the role of p53 in apoptosis by Ag-NPs. First, dispersion and stability of Ag-NPs improved using bronchial epithelial cell growth medium with 5% fetal bovine serum. Then, we observed oxidative stress in BEAS-2B cells exposed to Ag-NPs. Second, we carried out a cell death assay to measure DNA fragmentation as a biomarker of apoptosis. BEAS-2B cells were treated with p53-specific short interfering RNA (siRNA) or p53 inhibitor (pifithrin-α) to investigate whether p53 is involved in apoptosis by Ag-NPs. As a result, Ag-NPs significantly enhanced DNA fragmentation dose-dependently and treatment with p53 siRNA or pifithrin-α prevented DNA fragmentation. We also found that apoptosis-related genes (caspase-3, Bax, and Bcl-2) were regulated by Ag-NPs, which was detected by mRNA and protein levels. These results suggest that Ag-NPs induced p53-mediated apoptosis in BEAS-2B cells. Our findings may contribute to understanding the potential roles of Ag-NPs in pulmonary disease.

Mercury is a well-known environmental pollutant that can cause nephropathic diseases, including acute kidney injury (AKI). Although quercetin (QC), a natural flavonoid, has been reported to have medicinal properties, its potential protective effects against mercury-induced AKI have not been evaluated. In this study, the protective effect of QC against mercury-induced AKI was investigated using biochemical parameters, new protein-based urinary biomarkers, and a histopathological approach. A 250 mg/kg dose of QC was administered orally to Sprague-Dawley male rats for 3 days before administration of mercury chloride (HgCl2). All animals were sacrificed at 24 h after HgCl2 treatment, and biomarkers associated with nephrotoxicity were measured. Our data showed that QC absolutely prevented HgCl2-induced AKI, as indicated by biochemical parameters such as blood urea nitrogen (BUN) and serum creatinine (sCr). In particular, QC markedly decreased the accumulation of Hg in the kidney. Urinary excretion of protein-based biomarkers, including clusterin, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), monocyte chemoattractant protein-1 (MCP-1), tissue inhibitor of metalloproteinases 1 (TIMP-1), and vascular endothelial growth factor (VEGF) in response to HgCl2 administration were significantly decreased by QC pretreatment relative to that in the HgCl2-treated group. Furthermore, urinary excretion of metallothionein and Hg were significantly elevated by QC pretreatment. Histopathological examination indicated that QC protected against HgCl2-induced proximal tubular damage in the kidney. A TUNEL assay indicated that QC pretreatment significantly reduced apoptotic cell death in the kidney. The administration of QC provided significant protective effects against mercury-induced AKI.

Phytochemical investigation of an extract of the aerial part of Barleria lupulina resulted in the identification of four new iridoid glycosides (1–4), together with 14 known analogues (5–18). The structures of 1–4 were determined through 1D and 2D NMR spectroscopic data analysis, HRMS, and acid hydrolysis. This is the first report of iridoid glycosides with a formate group. The free-radical scavenging activity of compounds 9, 12, and 15–17 was assessed using the DPPH assay. Compounds 16 and 17 scavenged DPPH radicals weakly with IC50 values of 97.5 and 78.6 μg/mL, respectively.

Polyhexamethylene biguanide (PHMB) is a member of the polymeric guanidine family, which is used as a biocide and preservative in industrial, medicinal, and consumer products. Some studies reported that polyhexamethylene guanidine phosphate, which is also a member of the guanidine family, induced severe inflammation and fibrosis in the lungs. However, limited studies have evaluated the pulmonary toxicity of PHMB associated with inflammatory responses. The aim of this study was to elucidate the inflammatory responses and its mechanisms induced by PHMB in lung cells. A549 cells exposed to PHMB showed decreased viability, reactive oxygen species (ROS) generation, inflammatory cytokine secretion, and nuclear factor kappa B (NF-κB) activation. The cells showed dose-dependent cytotoxicity and slight generation of ROS. PHMB triggered inflammatory cytokine secretion and NF-κB activation by modulating the degradation of IκB-α and the accumulation of nuclear p65. TNF-α plays important roles in IL-8 expression as well as NF-κB activation. Moreover, IL-8 production induced by PHMB was completely suppressed by a NF-κB inhibitor, but not by a ROS scavenger. In conclusion, we suggest that PHMB induces the inflammatory responses via the NF-κB signaling pathway.

Hepatic fibrosis is characterized by the abnormal deposition of extracellular matrix (ECM) proteins. During hepatic fibrogenesis, hepatic stellate cell (HSC) activation followed by chronic injuries is considered a key event in fibrogenesis, and activated HSCs are known to comprise approximately 90% of ECM-producing myofibroblasts. Here, we demonstrated that (-)-catechin-7-O-β-d-apiofuranoside (C7A) significantly inhibited HSC activation via blocking the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This is the first study to show the hepatic protective effects of C7A with possible mechanisms in vitro and in vivo. In our bioactivity screening, we figured out that the EtOH extract of Ulmusdavidiana var. japonica root barks, which have been used as a Korean traditional medicine, inhibited collagen synthesis in HSCs. Four catechins isolated from the EtOAc fraction of the EtOH extract were compared with each other in terms of reduction in collagen, which is considered as a marker of hepatic protective effects, and C7A showed the strongest inhibitory effects on HSC activation in protein and qPCR analyses. As a possible mechanism, we investigated the effects of C7A on the STAT3 signaling pathway, which is known to activate HSCs. We found that C7A inhibited phosphorylation of STAT3 and translocation of STAT3 to nucleus. C7A also inhibited expressions of MMP-2 and MMP-9, which are downstream genes of STAT3 signaling. Anti-fibrotic effects of C7A were evaluated in a thioacetamide (TAA)-induced liver fibrosis model, which indicated that C7A significantly inhibited ECM deposition through inhibiting STAT3 signaling. C7A decreased serum levels of aspartate amino transferase and alanine transaminase, which were markedly increased by TAA injection. Moreover, ECM-associated proteins and mRNA expression were strongly suppressed by C7A. Our study provides the experimental evidence that C7A has inhibitory effects on HSC activation after live injury and has preventive and therapeutic potentials for the management of hepatic fibrosis.

Pubic hair is often analyzed as an alternative to scalp hair to prove previous drug use. However, urine is a potential source of external contamination. In the present study, the concentrations of methamphetamine (MA) and amphetamine (AP) in both scalp and pubic hair from illegal MA users were compared. Furthermore, in order to investigate the external contamination of pubic hair by urine, MA and AP absorbed into pubic hair that had been contaminated with authentic urine from a MA user were measured using a previously validated method. The effect of shampoo-wash on the contaminated pubic hair was also examined. However, no correlation was found in the MA and AP concentrations between scalp and pubic hair from illegal MA users. As the number of contamination events by authentic urine increased, the concentrations of MA and AP in pubic hair increased. Both MA and AP were detected in the first methanol washes of the contaminated hair samples but were not detected in the second methanol washes. As the number of shampoo-washes of the contaminated pubic hair increased, the concentrations of MA and AP gradually decreased. Even though pubic hair can be used as an alternative to scalp hair to prove previous drug use, it should be avoided when estimating drug use history. It should be also noted that higher quantitative results in pubic hair do not necessarily represent heavier drug use.

γ-Hydroxybutyrate (GHB) is a drug of abuse with a strong anesthetic effect; however, proving its ingestion through the quantification of GHB in biological specimens is not straightforward due to the endogenous presence of GHB in human blood, urine, saliva, etc. In the present study, a surrogate analyte approach was applied to accurate quantitative determination of GHB in human urine using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in order to overcome this issue. For this, 2H6-GHB and 13C2-dl-3-hydroxybutyrate were used as a surrogate standard and as an internal standard, respectively, and parallelism between the surrogate analyte approach and standard addition was investigated at the initial step. The validation results proved the method to be selective, accurate, and precise, with acceptable linearity within calibration ranges (0.1–1 μg/ml). The limit of detection and the limit of quantification of 2H6-GHB were 0.05 and 0.1 μg/ml, respectively. No significant variations were observed among urine matrices from different sources. The stability of 2H6-GHB was satisfactory under sample storage and in-process conditions. However, in vitro production of endogenous GHB was observed when the urine sample was kept under the in-process condition for 4 h and under the storage conditions of 4 and −20 °C. In order to facilitate the practical interpretation of urinary GHB, endogenous GHB was accurately measured in urine samples from 79 healthy volunteers using the surrogate analyte-based LC–MS/MS method developed in the present study. The unadjusted and creatinine-adjusted GHB concentrations in 74 urine samples with quantitative results ranged from 0.09 to 1.8 μg/ml and from 4.5 to 530 μg/mmol creatinine, respectively. No significant correlation was observed between the unadjusted and creatinine-adjusted GHB concentrations. The urinary endogenous GHB concentrations were affected by gender and age while they were not significantly influenced by habitual smoking, alcohol drinking, or caffeine-containing beverage drinking.

As several sedative-hypnotics are distributed illegally and are available domestically through media like the internet, their abuse is becoming a serious social problem. In the present study, four legal cases involving abuse of diazepam, midazolam, and/or zolpidem were proved by hair analysis using a simultaneous quantification method for the determination of diazepam (and its metabolites), lorazepam, midazolam, and zolpidem, which are often illegally abused in Korea, in hair that was developed and validated. Drugs and metabolites in hair were extracted using methanol followed by solid-phase extraction. The extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed using gas chromatography-mass spectrometry in selected ion monitoring mode. The validation parameters of the method, including selectivity, linearity, limits of detection and quantification (LOQ), recovery, intra- and interassay precision and accuracy, and processed sample stability, were satisfactory. Moreover, the developed method was successfully applied to actual cases. In case 1, which involved a pop singer who was detained for suspected drug abuse, the concentrations of diazepam and nordiazepam were 5.7 and 2.0 ng/mg in nonpigmented hair and 6.6 and 1.8 ng/mg in pigmented hair, respectively. In case 2, 0.4 ng/mg zolpidem was detected in hair from a drug abuser who purchased illegally through the internet, and 0.2 ng/mg midazolam was detected in hair from an illegal drug seller in case 3. In case 4, diazepam (lower than the LOQ), nordiazepam (0.7 ng/mg), and zolpidem (0.7 ng/mg) were detected in hair from a medical doctor who abused drugs using forged prescriptions.

Melamine-induced nephrotoxicity is closely associated with crystal formation in the kidney caused by combined exposure to melamine (Mel) and cyanuric acid (CA). However, there are few dosage-finding studies for toxicological evaluation of chronic co-exposure to Mel and CA. The objective of this study was to investigate the possible mechanism by which a Mel and CA mixture lead to renal toxicity in rats. Mel and CA were co-administered to rats via oral gavage for 50 days. Nephrotoxicity was determined by measuring blood urea nitrogen (BUN) and serum creatinine (sCr) levels. Relative kidney weights were significantly increased in rats after co-exposure to Mel+CA (63/6.3 or 630/6.3 mg/kg) mixtures. BUN and sCr levels were significantly increased after Mel and CA co-exposure. Taken together, significant increase in KIM-1, NGAL, and calbindin levels were observed in the urine of rats exposed to Mel+CA (63/6.3 or 630/6.3 mg/kg) compared with the corresponding control group. Histological analysis revealed epithelial degeneration and necrotic cell death in the proximal tubules of the kidney after co-exposure to Mel+CA (63/6.3 or 630/6.3 mg/kg). Our data suggest that Mel-mediated renal toxicity may be influenced by CA concentrations in Mel-contaminated milk or foods.

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.

Welders working in a confined space, as in the shipbuilding industry, are at risk of being exposed to high concentrations of welding fumes and developing pneumoconiosis or other welding-fume exposure related diseases. Among such diseases, manganism resulting from welding-fume exposure remains a controversial issue, as the movement of manganese into specific brain regions has not yet been clearly established. Accordingly, to investigate the distribution of manganese in the brain after welding-fume exposure, male Sprague–Dawley rats were exposed to welding fumes generated from manual metal arc-stainless steel (MMA-SS) at concentrations of 63.6 ± 4.1 mg/m3 (low dose, containing 1.6 mg/m3 Mn) and 107.1 ± 6.3 mg/m3 (high dose, containing 3.5 mg/m3 Mn) total suspended particulate (TSP) for 2 h per day in an inhalation chamber over a 60-day period. Blood, brain, lung, and liver samples were collected after 2 h, 15, 30, and 60 days of exposure and the tissues analyzed for their manganese concentrations using an atomic absorption spectrophotometer. Although dose- and time-dependent increases in the manganese concentrations were found in the lungs and livers of the rats exposed for 60 days, only slight manganese increases were observed in the blood during this period. Major statistically significant increases in the brain manganese concentrations were detected in the cerebellum after 15 days of exposure and up until 60 days. Slight increases in the manganese concentrations were also found in the substantia nigra, basal ganglia (caudate nucleus, putamen, and globus pallidus), temporal cortex, and frontal cortex, thereby indicating that the pharmacokinetics and distribution of the manganese inhaled from the welding fumes were different from those resulting from manganese-only exposure.

Opium poppy products are often illegally used for both recreational and medicinal purposes. In order to demonstrate the ingestion of opium poppy substances, morphine, codeine and their metabolites have been identified. However, morphine and codeine also originate from the ingestion of therapeutic drugs. Therefore, thebaine, one of the main opium alkaloids, in hair was suggested as a marker for chronic use of illegal opium poppy substances in the present study. First, thebaine was included in the analyte list of our routine analytical method for the simultaneous quantification of codeine, morphine, norcodeine, normorphine and 6-acetylmorphine (6-AM) in hair, which was fully validated previously. Then, the incorporation of thebaine and other opiates into hair and the effect of hair pigmentation were examined using lean Zucker rats with both dark grey and white hair on the same body. Thebaine was also measured in hair samples from actual cases of opium poppy substance use. Consequently, thebaine in hair was demonstrated as a marker of chronic use of illegal opium poppy substances using an animal study and actual cases. Thebaine and other opiates were successfully measured in pigmented hair from rats that ingested raw opium suspension. Moreover, thebaine identified in hair excluded possibility of ingestion of pharmaceutical opiates in actual cases.

Polyhexamethylene guanidine phosphate (PHMG-phosphate), an active component of humidifier disinfectant, is suspected to be a major cause of pulmonary fibrosis. Fibrosis, induced by recurrent epithelial damage, is significantly affected by epigenetic regulation, including microRNAs (miRNAs). The aim of this study was to investigate the fibrogenic mechanisms of PHMG-phosphate through the profiling of miRNAs and their target genes. A549 cells were treated with 0.75 μg/mL PHMG-phosphate for 24 and 48 h and miRNA microarray expression analysis was conducted. The putative mRNA targets of the miRNAs were identified and subjected to Gene Ontology analysis. After exposure to PHMG-phosphate for 24 and 48 h, 46 and 33 miRNAs, respectively, showed a significant change in expression over 1.5-fold compared with the control. The integrated analysis of miRNA and mRNA microarray results revealed the putative targets that were prominently enriched were associated with the epithelial-mesenchymal transition (EMT), cell cycle changes, and apoptosis. The dose-dependent induction of EMT by PHMG-phosphate exposure was confirmed by western blot. We identified 13 putative EMT-related targets that may play a role in PHMG-phosphate-induced fibrosis according to the Comparative Toxicogenomic Database. Our findings contribute to the comprehension of the fibrogenic mechanism of PHMG-phosphate and will aid further study on PHMG-phosphate-induced toxicity.

Welder’s pneumoconiosis has generally been determined to be benign and unassociated with respiratory symptoms based on the absence of pulmonary-function abnormalities in welders with marked radiographic abnormalities. In previous studies, the current authors suggested a three-phase lung fibrosis process to study the pathological process of lung fibrosis and found that the critical point for recovery was after 30 days of welding-fume exposure at a high dose, at which point early and delicate fibrosis was observed in the perivascular and peribronchiolar regions. Accordingly, the current study investigated the inflammatory and genotoxic responses during a 30-day period of welding-fume exposure to elucidate the process of fibrosis. As such, rats were exposed to manual metal arc-stainless steel (MMA-SS) welding fumes at concentrations of 65.6 ± 2.9 (low dose) and 116.8 ± 3.9 mg/m3 (high dose) total suspended particulate for 2 h per day in an inhalation chamber for 30 days. Animals were sacrificed after the initial 2 h exposure, and after 15 and 30 days of exposure. The rats exposed to the welding fumes exhibited a statistically significant (P < 0.05) decrease in body weight when compared to the control during the 30-day exposure period, yet an elevated cellular differential count and higher levels of albumin, LDH, and β-NAG, but not elevated TNF-α, and IL-1β in the acellular bronchoalveolar lavage fluid. In addition, the DNA damage resulting from 30 days of welding-fume exposure was confirmed by a comet assay and the inmmunohistochemistry for 8-hydroxydeoxyguanine (8-OH-dG). Consequently, the elevated inflammatory and genotoxic indicators confirmed the lung injury and inflammation caused by the MMA-SS welding-fume exposure.

The abuse of sildenafil and its analogous, accelerated by their inappropriate or illegal distribution, is a serious social issue globally. However, no studies have been conducted to monitor these drugs simultaneously in hair, which can provide valuable information on chronic drug use. In the present study, an LC–MS/MS method was developed for the simultaneous determination in hair of five erectile dysfunction drugs having a high risk for abuse (mirodenafil, sildenafil, tadalafil, udenafil and vardenafil) and their selected metabolites (SK3541, desmethylsildenafil, DA8164 and desethylvardenafil). The novel method was fully validated after optimizing matrix effects and extraction efficiency. The optimized sample preparation included acidic methanol extraction followed by solid phase extraction using C18 mixed mode strong cation exchange polymeric cartridges. The prepared samples were analyzed by LC–MS/MS with electrospray ion source in the positive ionization mode. The validation results proved the method to be selective, sensitive, accurate and precise, with acceptable linearity within calibration ranges. LODs ranged from 0.05 (DA8164) to 1 ng/10 mg hair (tadalafil). LOQs were 1 ng/10 mg hair except for DA8164 and vardenafil, of which they were 2.5 ng/10 mg hair. No significant variations were observed by different sources of matrices in both human and rat hair, except for tadalafil, for which a stable isotope-labeled internal standard was effective. The animal study suggested hair pigmentation was a major factor for the incorporation of the drugs and metabolites into hair. However, a wide variation of the sildenafil-to-desmethylsildenafil ratios was observed in human hair samples. The developed method will be very useful for monitoring the abuse of erectile dysfunction drugs for both legal and public health aspects.

Several studies showed that the inflammatory and fibrotic responses induced by polyhexamethylene guanidine phosphate (PHMG-p) were similar to those observed for idiopathic pulmonary fibrosis in South Korea in 2011. "Omic" technologies can be used to understand the mechanisms underlying chemical-induced diseases. Studies to determine the toxicity of chemicals may facilitate understanding of the mechanisms underlying the development of pulmonary fibrosis at a molecular level; thus, such studies may provide information about the toxic characteristics of various substances. In this review, we have outlined the cellular and molecular mechanisms underlying idiopathic pulmonary fibrosis and described pulmonary fibrosis induced by various chemicals, including bleomycin, paraquat, and PHMG-p, based on the results of studies performed to date.

Acute kidney injury (AKI) occurs in a half of cisplatin (CDDP)-treated patients. Traditional biomarkers including blood urea nitrogen (BUN) and serum creatinine (SCr) are still used for detection of CDDP-induced AKI, but these biomarkers are not specific or sensitive. The aim of this study was to identify the specific and sensitive biomarkers against CDDP-induced renal injury between young (3-week-old) and old (20-week-old) rats. All animals were intraperitoneally injected once with CDDP (6 mg/kg). After 3 days, all animals were sacrificed and serum, urine, and kidney tissues were collected. Urinary and serum biomarkers as well as histological changes were measured. CDDP-induced proximal tubular damage was apparent from histopathological examination, being more severe in 3-week-old rats accompanied by increased number of TUNEL-positive apoptotic cells. This was associated with elevated urinary kidney injury molecule-1 (KIM-1), glutathione-S-transferase alpha (GST-α), vascular endothelial growth factor (VEGF), and tissue inhibitor of metalloproteinases-1 (TIMP-1). In contrast, the levels of neutrophil gelatinase-associated lipocalin (NGAL) and osteopontin were significantly increased in 20-week-old rats after CDDP treatment. These results indicate that the use of age-specific urinary biomarkers is necessary to diagnosis of CDDP-induced AKI. Especially, urinary KIM-1, GST-α, TIMP-1, and VEGF levels may help in the early diagnosis of young patients with CDDP-induced AKI.

Pharbitidis Semen, the seed of Morning glory (Pharbitis nil), is a medicinal agent that has traditionally been used as a purgative in Korea. Pharbilignan C (PLC) is a dihydro[b]-benzofuran-type neolignan from Pharbitidis Semen, which reportedly exhibited the most potent cytotoxicity against human tumor cells. To further study the antiproliferative activity of PLC, its molecular mechanisms of action in two breast adenocarcinoma cells, MCF-7 and MDA-MB 231 cells were investigated. PLC inhibited the proliferation of MDA-MB 231 and MCF-7 cells, in order of sensitivity (IC50 of MDA-MB 231 cells: 7.0 ± 2.0 μM). PLC induced apoptosis in MDA-MB 231 cells with down regulation of Bcl-2 and up-regulation of Bax expression. It also decreased mitochondrial membrane potential accompanied with increasing initiator caspase, caspase-9 activation and executioner caspase, caspase-3 activation. This study demonstrates that PLC inhibited proliferation of MDA-MB 231 cells by inducing apoptosis via the mitochondria-mediated intrinsic pathway.

Estrogenic activity-oriented fractionation and purification of methanol extract from the rhizome of Cyperus rotundus, a well-known traditional herbal medicine, led to the isolation of six sesquiterpenes. 4α,5α-Oxidoeudesm-11-en-3-one (2) and cyper-11-ene-3,4-dione (3) together with four known sesquiterpenes, cyperotundone (1), caryophyllene α-oxide (4), α-cyperone (5), and isocyperol (6) were obtained from the hexane and dichloromethane fractions. Compounds 2 and 3 were newly isolated from natural resources in particular. To identify the possible use of isolated compounds as an alternative to hormone replacement therapy (HRT), estrogenic activity was evaluated by E-screen assay on MCF-7 BUS cells. Among the all isolated compounds from the rhizome of Cyperus rotundus, newly isolated from natural resource, 2 exhibited the most potent estrogenic activity. In an estrogen sensitive reporter gene assay, 2 significantly increased transcriptional activities. As a phytoestrogen, 2 was assessed by investigating dual action on ER-α and ER-β in competitive binding assay. It was found that 2 exerted higher binding affinity to ER-β than ER-α and it also showed both estrogenic and antiestrogenic effects depending on the E2 concentration. Our results indicate that newly isolated from Cyperus rotundus, 2 has biphasic activities on estrogen receptors which could be useful as an alternative HRT.

Chronic exposure to marijuana produces adverse effects on the endocrine and reproductive systems in humans; however, the experimental evidence for this presented thus far has not been without controversy. In this study, the estrogenic effect of marijuana smoke condensate (MSC) was evaluated using in vitro bioassays, viz., the cell proliferation assay, the reporter gene assay, and the ER competitive binding assay. The results of these assays were compared with those of three major cannabinoids, i.e., THC, CBD, and CBN. The estrogenic effect of MSC was further confirmed by the immature female rat uterotrophic assay. MSC stimulated the estrogenicity related to the ER-mediated pathway, while neither THC, CBD, nor CBN did. Moreover, treatment with 10 and 25 mg/kg MSC induced significant uterine response, and 10 mg/kg MSC resulted in an obvious change in the uterine epithelial cell appearance. MSC also enhanced the IGFBP-1 gene expression in a dose-dependent manner. To identify the constituents of MSC responsible for its estrogenicity, the MSC fractionated samples were examined using another cell proliferation assay, and the estrogenic active fraction was analyzed using GC-MS. In the organic acid fraction that showed the strongest estrogenic activity among the seven fractions of MSC, phenols were identified. Our results suggest that marijuana abuse is considered an endocrine-disrupting factor. Furthermore, these results suggest that the phenolic compounds contained in MSC play a role in its estrogenic effect.

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 measurement uncertainty (MU) of methamphetamine (MA) and amphetamine (AP) was estimated in an authentic urine sample with a relatively low concentration of MA and AP using the bottom-up approach. A cause and effect diagram was deduced; the amount of MA or AP in the sample, the volume of the sample, method precision, and sample effect were considered uncertainty sources. The concentrations of MA and AP in the urine sample with their expanded uncertainties were 340.5 +/- 33.2 ng/mL and 113.4 +/- 15.4 ng/mL, respectively, which means 9.7% and 13.6% of the concentration gave an estimated expanded uncertainty, respectively. The largest uncertainty originated from sample effect and method precision in MA and AP, respectively, but the uncertainty of the volume of the sample was minimal in both. The MU needs to be determined during the method validation process to assess test reliability. Moreover, the identification of the largest and/or smallest uncertainty source can help improve experimental protocols.

The abuse or misuse of forged erectile-dysfunction drugs, containing phosphodiesterase type 5 inhibitors (e.g. sildenafil), is a serious issue globally. Therefore, the detection of sildenafil and related active analogues in counterfeit pharmaceuticals or the differentiation between counterfeit and authentic drugs has been performed with a variety of analytical techniques. Recently, a liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS)-based in-house library, consisting of accurate mass ion fragmentation information and retention times, was effectively applied to screen a large number of compounds in field of forensic toxicology. However, a comprehensive LC-QTOF-MS spectral library of sildenafil and related active analogues has not yet been reported. In the present study, a spectral library of 40 compounds of sildenafil and related analogues was developed with accurate mass spectra and retention times using LC-QTOF-MS, and applied to screen nine marketed counterfeit products. The in-house library successfully identified sildenafil, dimethylsildenafil, hydroxyhomosildenafil, demethylhongdenafil, pseudovardenafil and vardenafil in the samples. Our LC-QTOF-MS-based spectral library search is considered a powerful approach for identifying sildenafil and related active analogues in counterfeit pharmaceuticals.

The first total synthesis and biological evaluation of penchinone A and its structural analogues are described. The key steps for the preparation of penchinone A derivatives involve the oxime-directed palladium(II)-catalyzed oxidative acylation, Claisen rearrangement, and base-mediated olefin migration. This transformation efficiently provides a range of allyl-substituted biaryl ketones with site-selectivity and functional group compatibility. In addition, all synthetic compounds were screened for anti-inflammatory activity against nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) with lipopolysaccharide (LPS)-induced RAW264.7 cells. Generally, a range of penchinone A derivatives potently inhibited NO, TNF-α, and IL-6 productions, compared to dexamethasone as a positive control. Notably, penchinone A (8g) and its derivatives (8e and 8f) were found to exhibit anti-inflammatory activity stronger than that of dexamethasone.

Hepatic fibrosis is a chronic liver disease characterized by the accumulation of extracellular matrix (ECM). Activation of hepatic stellate cells (HSCs) after repetitive liver damage is a key event in hepatic fibrogenesis. As part of ongoing research projects to identify pharmacologically effective natural products, the phytochemical investigation of a MeOH extract of Centipeda minima led to the isolation of a sesquiterpene lactone, brevilin A, which was explored to elucidate potential anti-fibrotic effects by reversing HSC activation. First, we observed that transforming growth factor (TGF)-β1 treatment significantly increased the expression levels of HSC activation marker, α-smooth muscle actin (α-SMA), and ECM protein such as collagen and fibronectin. Then, we demonstrated that brevilin A reversed the TGF-β1-induced increase in protein and mRNA expression levels of α-SMA and collagen. To investigate the underlying molecular mechanism of brevilin A, we evaluated the effects of brevilin A on the STAT3 signaling pathway. STAT3 phosphorylation, increased by TGF-β1 treatment, was strongly inhibited by brevilin A; the expression levels of fibronectin and connective tissue growth factor were also significantly decreased by brevilin A. The present study indicated that brevilin A has a preventive and therapeutic potential against hepatic fibrosis.

Lung epithelial cells and fibroblasts play key roles in pulmonary fibrosis and are involved in fibrotic signaling and production of the extracellular matrix (ECM), respectively. Recently, 3D airway models consisting of both cell types have been developed to evaluate the fibrotic responses while facilitating cell-cell crosstalk. This study aimed to evaluate the fibrotic responses in these models using different fibrogenic agents, which are known as key events in adverse outcome pathways of pulmonary fibrosis. We quantified cell injury and several sequential steps in fibrogenesis, including inflammation, the epithelial-mesenchymal transition (EMT), fibroblast activation, and ECM accumulation, using two different 3D airway models, the EpiAirway™-full thickness (Epi/FT) and MucilAir™-human fibroblast (Mucil/HF) models. In the Epi/FT model, fibrogenic agents induced the expression of inflammation and EMT-associated markers, while in the Mucil/HF model, they induced fibroblast activation and ECM accumulation. Using this information, we conducted gene ontology term network analysis. In the Epi/FT model, the terms associated with cell migration and response to stimulus made up a large part of the network. In the Mucil/HF model, the terms associated with ECM organization and cell differentiation and proliferation constituted a great part of the network. Collectively, our data suggest that polyhexamethyleneguanidine phosphate and bleomycin induce different responses in the two 3D airway models. While Epi/FT was associated with inflammatory/EMT-associated responses, Mucil/HF was associated with fibroblast-associated responses. This study will provide an important basis for selecting proper 3D airway models and fibrogenic agents to further research or screen chemicals causing inhalation toxicity.

Welders with radiographic pneumoconiosis abnormalities have exhibited a gradual clearing of the X-ray identified effects following removal from exposure. In some cases, the pulmonary fibrosis associated with welding fumes appears in a more severe form in welders. Accordingly, to investigate the disease and recovery process of pneumoconiosis induced by welding-fume exposure, rats were exposed to welding fumes with concentrations of 63.6+/-4.1 mg/m(3) (low dose) and 107.1+/-6.3 mg/m(3) (high dose) of total suspended particulate for 2 h per day in an inhalation chamber for a total of 2 h or 15, 30, 60 or 90 days. Thereafter, the rats were no longer exposed and allowed to recover from the welding fume-induced lung fibrosis for 90 days. When compared to the unexposed control group, the lung weights significantly increased in both the low- and high-dose rats from day 15 to 90. A histopathological examination combined with fibrosis-specific staining revealed that the lungs from the low-dose rats did not exhibit any significant progressive fibrotic changes. Whereas, the lungs from the high-dose rats exhibited early delicate fibrosis from day 15, which progressed into the perivascular and peribronchiolar regions by day 30. Interstitial fibrosis appeared at day 60 and became prominent by day 90, along with the additional appearance of pleural fibrosis. Recovery, evaluated based on the body and lung weights and a histopathological examination, was observed in both the high and low-dose rats that were exposed up to 30 days. The rats exposed for 60-90 days at the low dose also recovered from the fibrosis, yet the rats exposed for 60-90 days at the high dose did not fully recover. Consequently, recovery from pneumoconiosis induced by welding-fume exposure was observed when the degree of exposure was short-term and moderate.

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. Keywords: Apoptosis, Genotoxicity, Lung neoplasms, Marijuana smoke condensates, Tumor suppressor protein p53

Plants of the genus Taraxacum, commonly known as dandelions, are used to treat breast cancer in traditional folk medicine. However, their use has mainly been based on empirical findings without sufficient scientific evidence. Therefore, we hypothesized that dandelions would behave as a Selective estrogen receptor modulator (SERM) and be effective as hormone replacement therapy (HRT) in the postmenopausal women. In the present study, in vitro assay systems, including cell proliferation assay, reporter gene assay, and RT-PCR to evaluate the mRNA expression of estrogen-related genes (pS2 and progesterone receptor, PR), were performed in human breast cancer cells. Dandelion ethanol extract (DEE) significantly increased cell proliferation and estrogen response element (ERE)-driven luciferase activity. DEE significantly induced the expression of estrogen related genes such as pS2 and PR, which was inhibited by tamoxifen at 1 μmol·L(-1). These results indicated that DEE could induce estrogenic activities mediated by a classical estrogen receptor pathway. In addition, immature rat uterotrophic assay was carried out to identify estrogenic activity of DEE in vivo. The lowest concentration of DEE slightly increased the uterine wet weight, but there was no significant effect with the highest concentration of DEE. The results demonstrate the potential estrogenic activities of DEE, providing scientific evidence supporting their use in traditional medicine.

A series of azaisoflavone analogs were designed and synthesized and their transactivation activities and binding affinities for ERα and ERβ were investigated. Among these compounds, 2b and 3a were the most potent with 6.5 and 1.1 μM of EC50, respectively. Molecular modeling study showed putative binding modes of the compound 3a in the active site of ERα and ERβ, which were similar with that of genistein and provided insight of the effect of N-alkyl substitution of azaisoflavones on ERβ activity. Also, a biphasic effect of azaisoflavone analogs on MCF-7 cell growth depending on their concentrations was investigated.

Rhubarb is a well-known herb worldwide and includes approximately 60 species of the Rheum genus. One of the representative plants is Rheum palmatum, which is prescribed as official rhubarb due to its pharmacological potential in the Korean and Chinese pharmacopoeia. In our bioactive screening, we found out that the EtOH extract of R. palmatum inhibited hepatic stellate cell (HSC) activation by transforming growth factor β1 (TGF-β1). Chemical investigation of the EtOH extract led to the isolation of chrysophanol 8-O-glucoside, which was determined by structural analysis using NMR spectroscopic techniques and electrospray ionization mass spectrometry (ESIMS). To elucidate the effects of chrysophanol 8-O-glucoside on HSC activation, activated LX-2 cells were treated for 48 h with chrysophanol 8-O-glucoside, and α-SMA and collagen, HSC activation markers, were measured by comparative quantitative real-time PCR (qPCR) and western blotting analysis. Chrysophanol 8-O-glucoside significantly inhibited the protein and mRNA expression of α-SMA and collagen compared with that in TGF-β1-treated LX-2 cells. Next, the expression of phosphorylated SMAD2 (p-SMAD2) and p-STAT3 was measured and the translocation of p-STAT3 to the nucleus was analyzed by western blotting analysis. The expression of p-SMAD2 and p-STAT3 showed that chrysophanol 8-O-glucoside strongly downregulated STAT3 phosphorylation by inhibiting the nuclear translocation of p-STAT3, which is an important mechanism in HSC activation. Moreover, chrysophanol 8-O-glucoside suppressed the expression of p-p38, not that of p-JNK or p-Erk, which can activate STAT3 phosphorylation and inhibit MMP2 expression, the downstream target of STAT3 signaling. These findings provided experimental evidence concerning the hepatoprotective effects of chrysophanol 8-O-glucoside against liver damage and revealed the molecular basis underlying its anti-fibrotic effects through the blocking of HSC activation.

In the past a few decades, particular interest has been focused on the distribution and interaction between toxic and essential elements in animals and humans, since such interactions might have adaptive implications to environmental pollution. The current study was performed to assess the correlation of elemental concentration with age and the correlation between toxic and essential elements in Koreans. Toxic elements, such as Cd, Pb, Hg, and essential elements such as Se and Zn, were analyzed in internal organs (liver, kidney cortex, kidney medulla, heart, lung, spleen, cerebrum, testis and bone) of 162 Korean cadavers. The tissues were digested with a microwave digestion system and the 5 elements were determined by inductively coupled plasma atomic emission spectrometry. Positive correlation with age was observed in the following cases: Cd in the liver, kidney cortex, kidney medulla, heart and testis; Pb in testis and bone. The concentration of Hg, Se and Zn was not correlated with age in any of the tissues tested. A significantly high correlation between Hg and Se, Pb and Se was observed in liver, kidney cortex, kidney medulla, heart, lung, spleen, cerebrum, testis and bone. The correlation between Cd and Zn was significant in the liver, kidney cortex, kidney medulla, heart, lung, cerebrum, testis and bone. These results indicate that the distribution of toxic elements is similar to that of essential elements in all tissues.

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-chlorotriazine), one of the most widely used herbicides, is classified as a possible human carcinogen by the US Environmental Protection Agency and is a known endocrine disruptor. Recent research has indicated that the estrogenic and antiestrogenic effects elicited by this chemical are not mediated by the estrogen receptor. In this study, we also observed that not only atrazine but also its metabolites did not affect basal or 17β-estradiol induced MCF7-BUS cell proliferation. Therefore, to explain the apparent endocrine disrupting effects of atrazine in vivo, an estrogen-independent mechanism and its metabolites were studied. We examined the alteration in aromatase (CYP19) activity in human choriocarcinoma JEG-3 cells by atrazine and its major metabolites (hydroxyatrazine, diaminochlorotriazine), the conversion of estradiol to hydroxylated metabolites by these compounds associated with cytochrome P4501A. The common metabolite of atrazine, diaminochlorotriazine inhibited the aromatase activity responsible for estrogen synthesis in JEG-3 cells. Atrazine and its metabolites induced 7-ethoxyresorufin-O-deethylase activity and catalyzed estrogen metabolism in MCF-7 cells. These results suggest that the antiestrogenic effects of atrazine may relate to a decrement in estrogen levels caused by aromatase inhibition and estrogen metabolism stimulation by atrazine and its metabolites.

In the preparation of a reference material (RM) for quality assurance, both homogeneity and stability studies are integral parts. In the present study, both homogeneity and stability of a candidate RM for the determination of methamphetamine and amphetamine in hair were examined by an isotope dilution gas chromatography/mass spectrometry (GC/MS) method, which is not only one of the analytical methods validated in our previous study but also one of the primary methods for the preparation of a certified reference material (CRM). Additionally, homogeneity was monitored using a different method: micropulverized extraction followed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), which was fully validated in the previous study. In order to demonstrate the suitability of the method as an isotope dilution with mass spectrometry (IDMS), the extraction efficiency was also determined according to time. Our results showed that the current method, i.e., agitating hair with isotope internal standards in the extraction solvent for 20 h followed by GC-MS, was accepted as an IDMS. No significant difference was observed between bottles of the candidate CRM. The statistical results also showed no significant trends in stability for 92 days at room temperature and 4 degrees C. An inter-laboratory quality assurance program was also performed successfully using this material. The candidate CRM developed in the present study demonstrated its suitability for quality assurance in hair drug analysis. Even though a RM is necessity as a quality control tool, it is not always easy to have an authentic RM containing target drugs and metabolites. Even when an in-house quality control material is used, both homogeneity and stability should be investigated.

Phentermine (PT) has been widely used as an anti-obesity drug. This drug has to be used with caution due to its close resemblance with amphetamines in its structure and toxicity profile. Recently, PT is in distribution by illegal modes and is found to be available through sources such as the internet, thus their misuse and/or abuse is threatening to be a serious social issue. In the present study, 32 cases of drug suspects were observed for PT abuse, detected using hair samples for drug analysis. PT and other amphetamines, such as methamphetamine (MA), amphetamine (AP), 3,4-methylenedioxyamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA), were extracted using 1% HCl in methanol for 20 h at 38°C. The extracts were derivatized with trifluoroacetic anhydride (TFAA) and analyzed using gas chromatography/mass spectrometry (GC/MS). Among the 32 cases of PT abuse, MA and its main metabolite, AP were identified in seven cases and MDMA and its main metabolite, MDA were detected in two other cases.

Medical costs in South Korea have risen, in part due to increased demand and consumption of pharmaceutical products by an aging population and also because of the introduction of newer, more expensive drugs. In an effort to stabilize the financing of health insurance and alleviate the financial burden on individuals, the government implemented a policy changing the national health insurance drug-listing system from a negative list system to a positive list system (PLS).The goal of this study was to compare differences in drug-listing rates for new chemical entities (NCEs) and incrementally modified drugs (IMDs) after South Korea introduced the PLS in December 2006. Parameters significantly affecting NCE and IMD listings were also identified.New drug-listing data for 2007 and 2008 were obtained from the databases of the Health Insurance Review Agency and the Ministry of Health and Welfare. Descriptive analyses on the reimbursement rate and logistic regression analysis were conducted. Statistical significance was tested for all results, and P < 0.05 was considered statistically significant.A total of 150 reimbursement applications (79 for NCEs, 71 for IMDs) were examined for this study. The overall drug-listing rate was lower than before the introduction of the PLS. Drug reimbursement rates for NCEs (50.6%) were lower than those for IMDs (74.6%) (P = 0.0025). However, the price negotiation rate was 85.0% for NCEs compared with 73.6% for IMDs (P = 0.1847). The time required for both reimbursement and drug pricing was significantly longer for NCE than for IMD listings (P < 0.05). Cost-effectiveness and budget impact were 2 significant variables affecting the listing of NCEs. However, no significant variable was identified for IMDs.The PLS challenges the drug-listing system by decreasing the drug-listing rate and lengthening the period for reimbursement determinations. These effects were more pronounced for NCE listings than for IMD listings.

As the current methods to predict the inhalation toxicity of chemicals using animal models are limited, alternative methods are required. We present a new in vitro prediction method for acute inhalation toxicity using the Calu-3 epithelial cytotoxicity assay applicable for water-soluble inhalable chemicals. To confirm the characteristics of the optimal Calu-3 epithelium, tight-junction formation, morphology, and mucus secretion were verified using scanning electron microscopy, transepithelial electrical resistance analysis, and immunofluorescence after growth in an air-liquid interface (ALI). Sixty chemicals, including 38 positive and 22 negative for acute inhalation toxicity, were selected from the European Chemical Agency chemical database. The cell viability of the exposed cells was assessed using an MTT assay to predict the acute inhalation toxicity by calculating the area under the receiver operating characteristic (ROC) curve and accuracy. When cultivated in an ALI, the epithelium was thicker and secreted more mucin than that under submerged cultivation, characteristic of the in vivo respiratory epithelium. The areas under the ROC curve were 0.75 and 0.78 when exposed to chemicals at concentrations of 2.5 and 5%, respectively. The highest accuracy of the methods was 68 and 78% at cut-off values of 85 and 40% cell viability, respectively. The in vitro model was moderately accurate with good prediction. It is replicable because of its advantages, i.e., the use of cultured cells and the simplicity of the method. Overall, the Calu-3 epithelial cytotoxicity assay may be a useful and simple approach to identify substances that cause acute inhalation toxicity.

Abstract Liver fibrosis, a common outcome of chronic liver disease characterized by excessive accumulation of extracellular matrix (ECM), is a leading cause of mortality worldwide. The tyrosine kinase inhibitor neratinib is a human epidermal growth factor receptor 2 (HER2) inhibitor approved by the FDA for HER2-positive breast cancer treatment; however, it has not yet been evaluated for liver fibrosis treatment. We elucidated the anti-fibrotic effects of neratinib in hepatic stellate cells (HSCs) and in vivo models of CCl 4 -induced liver fibrosis. HSC activation is a key step in liver fibrogenesis and has a crucial role in collagen deposition, as it is primarily responsible for excessive ECM production. The effect of neratinib on HSC was evaluated in transforming growth factor (TGF-β)-incubated LX-2 cells and culture-activated primary human HSCs. In vivo study results indicated that neratinib inhibited the inflammatory response, HSC differentiation, and collagen accumulation induced by CCl 4 . Moreover, the anti-fibrotic effects of neratinib were not associated with the HER2 signaling pathways. Neratinib inhibited FGF2 expression in activated HSCs and serum FGF2 level in the model, suggesting that neratinib possessed therapeutic potency against liver fibrosis and the potential for application against other fibrotic diseases.

Extracts of Peperomia pellucida [L.] Kunth have previously been demonstrated to have in vivo estrogenic-like effects, thereby functioning as an anti-osteoporotic agent. However, the compounds responsible for these effects have not yet been determined. Therefore, the aim of this study is to isolate and elucidate potential compounds with estrogenic activity. The structures of the isolated compounds were identified using 1D 1H and 13C-NMR and confirmed by 2D FT-NMR. The estrogenic activity was evaluated using the E-SCREEN assay, and a molecular docking study was performed to predict the binding affinity of the isolated compounds to estrogen receptors. In this experiment, we successfully isolated three phenylpropanoids and two lignan derivatives, namely, 6-allyl-5-methoxy-1,3-benzodioxol-4-ol (1), pachypostaudin B (2), pellucidin A (3), dillapiole (4), and apiol (5). Among these compounds, the isolation of 1 and 2 from P. pellucida is reported for the first time in this study. Activity assays clearly showed that the ethyl acetate extract and its fractions, subfractions, and isolated compounds exerted estrogenic activity. Methanol fraction of the ethyl acetate extract produced the highest estrogenic activity, while 1 and 2 had partial agonist activity. Some compounds (derivates of dillapiole and pellucidin A) also had, in addition, anti-estrogenic activity. In the docking study, the estrogenic activities of 1–5 appeared to be mediated by a classical ligand-dependent mechanism as suggested by the binding interaction between the compounds and estrogen receptors; binding occurred on Arg 394 and His 524 of the alpha receptor and Arg 346 and His 475 of the beta receptor. In summary, we reveal that P. pellucida is a promising anti-osteoporotic agent due to its estrogenic activity, and the compounds responsible for this activity were found to be lignan and phenylpropanoid derivatives. The presence of other compounds in either the extract or fraction may contribute to a synergistic effect, as suggested by the higher estrogenic activity of the methanol fraction. Hence, we suggest further research on the osteoporotic activity and safety of the identified compounds, especially regarding their effects on estrogen-responsive organs.

In vitro models have become increasingly sophisticated, and their usefulness in supporting toxicity testing is well established. The present study was designed to establish a novel in vitro model that mimics the cellular network surrounding airways and pulmonary blood vessels, to study the cardiovascular toxic effects of particulate matter (PM). Transwell culture method was used to develop a novel tetra-culture system consisting of tri-cultures (one lung epithelial and two immune cell lines) in the apical chamber and endothelial cells in the basolateral chamber. Tri-cultures were exposed to standard reference material (SRM) 1648a, an urban PM. SRM 1648a did not show cytotoxic effects; however, it increased IL-6 level in apical and basolateral chambers. The cells in the basolateral chamber showed increased monocyte adhesion. Furthermore, exposure of tri-cultured cells to SRM 1648a in the apical chamber induced ICAM-1 expression in endothelial cells in the basolateral chamber by activating the IL-6/STAT3 pathway. In conclusion, a tetra-culture system was established to facilitate the identification of cellular adhesion molecule expression induced by the interaction between pulmonary epithelial and endothelial cells. The tetra-culture system will contribute to elucidation of the relationships between inhalable PM and cardiovascular diseases.

Breast cancer is the most common cancer in women worldwide. There are many endocrine adjuvant therapies for breast cancer patients that are categorized according to their mechanisms. Among them, aromatase inhibitors (AIs) that block the synthesis of estrogens have proven superiority compared with tamoxifen and have replaced it as a first‐line hormonal therapy. However, AIs also have limitations due to their side effects – increased rate of bone loss and musculoskeletal complaints. We therefore need new candidate AIs with fewer side effects. The extracts of Ginkgo biloba (EGb), which contain phytochemicals from the tree, had biphasic effects for estrogens and osteoporosis‐inhibiting activities in our previous experiments. In this study, we explored the possibility of EGb as an AI and their mechanisms. Aromatase activities were inhibited by EGb both in JEG‐3 cells and in recombinant CYP19 microsomes. The results of polymerase chain reaction for aromatase from a coding sequence and specific promoter sequences (exon I.a, exon I.c) in JEG‐3 cells as well as the results of reporter gene assays showed that EGb dose‐dependently decreased the aromatase gene expression. The decreased protein levels were demonstrated by Western blotting. From these results, we concluded that EGb could act as an AI at both the enzyme and transcriptional levels. Copyright © 2013 John Wiley &amp; Sons, Ltd.

Cigarette smoking is associated with carcinogenesis owing to the mutagenic and genotoxic effects of cigarette smoke. The aim of this study was to evaluate the mutagenic and genotoxic effects of Korean cigarettes using in vitro assays.We selected 2 types of cigarettes (TL and TW) as benchmark Korean cigarettes for this study, because they represent the greatest level of nicotine and tar contents among Korean cigarettes. Mutagenic potency was expressed as the number of revertants per μg of cigarette smoke condensate (CSC) total particulate matter whereas genotoxic potency was expressed as a concentration-dependent induction factor. The CSC was prepared by the International Organization for Standardization 3308 smoking method. CHO-K1 cells were used in vitro micronucleus (MNvit) and comet assays. Two strains of Salmonella typhimurium (Salmonella enterica subsp.enterica; TA98 and TA1537) were employed in Ames tests.All CSCs showed mutagenicity in the TA98 and TA1537 strains. In addition, DNA damage and micronuclei formation were observed in the comet and MNvit assays owing to CSC exposure. The CSC from the 3R4F Kentucky reference (3R4F) cigarette produced the most severe mutagenic and genotoxic potencies, followed by the CSC from the TL cigarette, whereas the CSC from the TW cigarette produced the least severe mutagenic and genotoxic potencies.The results of this study suggest that the mutagenic and genotoxic potencies of the TL and TW cigarettes were weaker than those of the 3R4F cigarette. Further study on standardized concepts of toxic equivalents for cigarettes needs to be conducted for more extensive use of in vitro tests.

Epidemiological and toxicological studies indicate that polyhexamethylene guanidine phosphate (PHMG-p) is a guanidine-based cationic disinfectant strongly associated with interstitial lung diseases. As individuals exposed to aerosolized PHMG-p complain of respiratory problems (asthma and rhinitis), whether PHMG-p can cause respiratory diseases other than interstitial fibrosis should be investigated. MUC5AC, the predominant mucin gene expressed in airways, is associated with obstructive respiratory disease pathogenesis. Therefore, in this study, we elucidated the relationship between PHMG-p and MUC5AC overexpression. First, in immunofluorescence studies, the bronchial epithelia of mice intratracheally administrated PHMG-p appeared to be sloughing and tethered by MUC5AC. Second, Calu-3 cells exposed to PHMG-p showed concentration-dependent increases in MUC5AC mRNA and protein expression. c-Jun N-terminal kinase (JNK), p38, and c-jun were phosphorylated in cells exposed to PHMG-p. SP600125 and SB203580, JNK and p38 inhibitors, respectively, reduced the upregulation of MUC5AC by PHMG-p in Calu-3 cells. When toll-like receptor (TLR)2, 4, and 6 were silenced, PHMG-p-induced JNK and p38 phosphorylation decreased. Furthermore, TLR2-, 4-, and 6-silenced cells showed reduced levels of MUC5AC mRNA and protein induced by PHMG-p, with TLR6 knockdown showing the greatest effect. In conclusion, PHMG-p induced MUC5AC overexpression via activation of the TLR-p38 MAPK and JNK axis.

Epithelial-to-mesenchymal transition (EMT) is increasingly recognized as contributing to the pathogenesis of idiopathic pulmonary fibrosis. Therefore, novel plant-based natural, active compounds have been sought for the treatment of fibrotic EMT. The aim of the present study was to investigate the inhibitory effects of Astilbe rubra on TGF-β1-induced EMT in lung alveolar epithelial cells (A549). A. rubra was subjected to extraction using 70% ethanol (ARE), and ethanol extracts of the aerial part and that of the rhizome were further partitioned using various solvents. Protein expression and cell motility were investigated to evaluate the inhibitory effects of ARE on EMT. EMT occurred in A549 cells treated with TGF-β1, but was prevented by co-treatment with ARE. The dichloromethane fractions showed the strongest inhibitory effect on TGF-β1-induced EMT. β-Peltoboykinolic acid was isolated from the dichloromethane fractions of A. rubra by activity-oriented isolation. β-Peltoboykinolic acid not only attenuated TGF-β1-induced EMT, but also the overproduction of extracellular matrix components including type I collagen and fibronectin. The Smad pathway activated by TGF-β1 was inhibited by co-treatment with β-peltoboykinolic acid. Taken together, these results indicate that β-peltoboykinolic acid from A. rubra and dichloromethane fractions shows potential as an antifibrotic agent in A549 cells treated with TGF-β1.

According to previous survey, about two million of people were expected to suffer from toxic effects due to humidifier disinfectant (HD), regardless of healing or not. Extremely small group are recognized as HDs' victims. Up to now, previous research tried to focus on interstitial fibrosis on terminal bronchiole because it is specific finding, compared with other diseases. To figure out overall effects from HDs, we recommend adverse outcome pathways (AOPs) as new approach. Reactive oxygen species (ROS) generation, decreased T-cell and pro-inflammatory cytokine release from macrophage could be key events between the exposure to HDs and diseases. ROS generation, decreased cell and pro-inflammatory cytokine release from macrophage could be cause of interstitial fibrosis, pneumonia and many other diseases such as asthma, allergic rhinitis, allergic dermatitis, fetal death, premature baby, autoimmune disease, hepatic toxicity, renal toxicity, cancer, and so on. We predict potential disease candidate by AOPs. We can validate the real risk of the adverse outcome by epidemiologic and toxicologic study using big data such as National Health Insurance data and AOPs knowledge base. Application of these kinds of new methods can find the potential disease list from the exposure to HD.

Benzylpiperazine (BZP) and trifluoromethylphenylpiperazine (TFMPP) are commonly used constituents of party pill drugs. They are reported to induce psychoactive effects such as euphoria and provide effects similar with other illicit drugs such as methylenedioxymethamphetamine (MDMA). A great deal of evidence has proven that party pills, as alternatives for MDMA, exert harmful effects on users. However, their toxicological effects have not been fully understood and endocrine disruptive effects are still unknown. In this study, we identified estrogenic effects of BZP and TFMPP by using in vitro and in vivo assays. BZP and TFMPP stimulated cell proliferation in a dose-dependent manner, while co-treatment with tamoxifen and BZP or TFMPP showed a decrease of E(2)-induced cell proliferation. In an estrogen sensitive reporter gene assay, BZP and TFMPP significantly increased transcriptional activities of party pill drugs. In addition, ER-related genes, PR and pS2, were significantly stimulated by BZP and TFMPP. These results indicated that BZP and TFMPP could have estrogenic activities related to the ER-mediated pathway. Unlike the in vitro assay results, BZP and TFMPP did not show significant effects on weight increase in a rodent uterotrophic assay. However, further studies would be necessary to verify the estrogenic activities of BZP and TFMPP by a chronic exposure animal study.

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.

Since the largely abused worldwide used of marijuana, there have been many ongoing debates regarding the adverse health effects of marijuana smoking. Marijuana smoking was recently proved to cause pulmonary toxicity by inducing genotoxic effects or generating reactive oxygen species. Because p53, a tumor suppressor gene, has an important pathophysiologic role in the regulation of lung epithelial cell DNA damage responses, we hypothesized that p53 may be involved in the oxidative stress-mediated apoptosis induced by marijuana smoking. First, we confirmed that marijuana smoke condensate (MSC) induces oxidative stress in BEAS-2B cells. We observed that reactive oxygen species (ROS) generation was increased by MSC in the DCFH-DA assay. Also, antioxidant enzyme (superoxide dismutase, catalase) activity and their mRNA expressions were up-regulated by MSC. Second, we investigated p53 involvement in the MSC-induced apoptotic pathway in BEAS-2B cells. The results showed that MSC increased caspase-3 activation and DNA fragmentation as markers of apoptosis. In addition, the mRNA levels of apoptosis-related genes (p53 and Bax) were increased by MSC and phospho-p53, along with the increase of Bax protein expression by MSC. Apoptosis and apoptosis-related gene expression were partially blocked by an inhibitor of p53-dependent transcriptional activation (pifithrin-α). The results indicate that p53 plays a role in MSC-induced apoptosis. Taken together, the findings of the present study suggest that MSC partially induces p53-mediated apoptosis through ROS generation in human lung epithelial cells and this may have broader implications for our understanding of pulmonary diseases.

Indigenous aquatic population such as fish could be used as a successful test species for evaluating the ecological effects in aquatic environment. In the present study, vitellogenin (Vtg) from slender bitterling (Acheilognathus yamatsutae), an indigenous aquatic species in Korea, was cloned and sequenced to determine if the Vtg gene possesses an important characteristic so as to act as a sensitive biomarker for estrogenic endocrine disrupting chemicals (EEDCs). The sbVtg cDNA is 5010 bp in length, containing a 4653 bp open reading frame, which encodes 1550 amino acid residues. The sbVtg cDNA was divided into lipovitellin heavy chain (LvH), phosvitin (Pv), lipovitellin light chain (LvL) as well as a beta'-component (beta'-c) domain, and belongs to VtgAo2. SbVtg has conserved important sequences for Vtg functions such as signal peptide, VtgR-binding region, and disulfide bond formation, all of which are consistent with those of other teleosts. In addition, the male slender bitterling aqueous exposed to 17 alpha-ethinylestradiol (EE2, 12.5, 25, and 50 ng/L) produced a statistically significant and concentration-dependent increase in hepatic Vtg mRNA expression, which showed a similar pattern to biliary estrogenic activity, measured by ERE-reporter gene assay. Thus, this study clearly indicates that the induction of Vtg in slender bitterling might be a suitable biomarker in toxicological research of EEDCs.

Aromatase inhibitors that block estrogen synthesis are a proven first-line hormonal therapy for postmenopausal breast cancer. Although it is known that standardized extract of Ginkgo biloba (EGb761) induces anti-carcinogenic effects like the aromatase inhibitors, the effects of EGb761 on steroidogenesis have not been studied yet. Therefore, the effects of EGb761 on steroidogenesis and aromatase activity was studied using a H295R cell model, which was a good in vitro model to predict effects on human adrenal steroidogenesis.Cortisol, aldosterone, testosterone, and 17β-estradiol were evaluated in the H295R cells by competitive enzyme-linked immunospecific assay after exposure to EGb761. Real-time polymerase chain reaction were performed to evaluate effects on critical genes in steroid hormone production, specifically cytochrome P450 (CYP11/ 17/19/21) and the hydroxysteroid dehydrogenases (3β-HSD2 and 17β-HSD1/4). Finally, aromatase activities were measured with a tritiated water-release assay and by western blotting analysis.H295R cells exposed to EGb761 (10 and 100 μg/mL) showed a significant decrease in 17β-estradiol and testosterone, but no change in aldosterone or cortisol. Genes (CYP19 and 17β-HSD1) related to the estrogen steroidogenesis were significantly decreased by EGb761. EGb761 treatment of H295R cells resulted in a significant decrease of aromatase activity as measured by the direct and indirect assays. The coding sequence/ Exon PII of CYP19 gene transcript and protein level of CYP19 were significantly decreased by EGb761.These results suggest that EGb761 could regulate steroidogenesis-related genes such as CYP19 and 17β-HSD1, and lead to a decrease in 17β-estradiol and testosterone. The present study provides good information on potential therapeutic effects of EGb761 on estrogen dependent breast cancer.

Pulmonary fibrosis (PF) is a chronic and fatal lung disease with few treatment options. Although the pathogenesis of PF is not clear, a chronic inflammatory response to continuous damage is considered the cause of pulmonary fibrosis. PF is characterized by excessive accumulation of extracellular matrix (ECM), therefore, inhibition of myofibroblast differentiation is a good therapeutic target for PF. As part of our continuing endeavor to explore biologically active metabolites from insect-associated microbes, we found that the MeOH extract of the culture broth from the entomopathogenic fungus Beauveria bassiana inhibited collagen induction and E-cadherin down-regulation. In order to identify active compounds, we carried out chemical analysis of the MeOH extract with the assistance of LC/MS-guided isolation approach, which led to the successful identification of four cyclodepsipeptides 1–4. Among the isolates, compound 2 showed inhibitory effects on myofibroblast differentiation induced by TGF-β1. Compound 2 inhibited induction of α-SMA and N-cadherin, which are myofibroblast markers, and blocked the accumulation of ECM proteins such as collagen and fibronectin. Overall these findings demonstrate that compound 2 can be used to attenuate pulmonary fibrosis by targeting myo- fibroblast differentiation.

&lt;p&gt;&lt;em&gt;Sphagnum palustre&lt;/em&gt; (a moss) has been traditionally used in Korea for the cure of several diseases such as cardiac pain and stroke. In this research, the inhibitory effect of &lt;em&gt;S. palustre&lt;/em&gt; on aromatase (cytochrome P&lt;sub&gt;450&lt;/sub&gt; 19, CYP19) activity was studied. [1β-&lt;sup&gt;3&lt;/sup&gt;H] androstenedione was used as a substrate and incubated with &lt;em&gt;S. palustre&lt;/em&gt; extract and recombinant human CYP19 in the presence of NADPH. &lt;em&gt;S. palustre&lt;/em&gt; extract inhibited aromatase in a concentration-dependent manner (IC&lt;sub&gt;50&lt;/sub&gt; value: 36.4 ± 8.1 µg/mL). To elucidate the major compounds responsible for the aromatase inhibitory effects of &lt;em&gt;S. palustre&lt;/em&gt; extract, nine compounds were isolated from the extract and tested for their inhibition of aromatase activity. Compounds &lt;strong&gt;1&lt;/strong&gt;, &lt;strong&gt;6&lt;/strong&gt;, and &lt;strong&gt;7&lt;/strong&gt; displayed aromatase inhibition, while the inhibition by the other compounds was negligible.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Video Clip&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;a href="https://youtube.com/v/n6xeo3RXJVY"&gt;Aromatase enzyme activity:&lt;/a&gt; 4 min 16 sec &lt;/p&gt;

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.

Cigarette smoking is a major cause of lung cancer. Although tobacco smoking-induced genotoxicity has been well established, there is apparent lack of abundance functional epigenetic effects reported On cigarette smoke-induced lung carcinogenesis. The aim of this study was to determine effects of intratracheal administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) utilizing target gene expression DNA methylation patterns in lung tissues of mice following twice weekly for 8 weeks treatment. An unbiased approach where genomic regions was undertaken to assess early methylation changes within mouse pulmonary tissues. A methylated-CpG island recovery assay (MIRA) was performed to map the DNA methylome in lung tissues, with the position of methylated DNA determined using a Genome Analyzer (MIRA-SEQ). Alterations in epigenetic-regulated target genes were confirmed with quantitative reverse transcription-PCR, which revealed 35 differentially hypermethylated genes including Cdkn1C, Hsf4, Hnf1a, Cdx1, and Hoxa5 and 30 differentially hypomethylated genes including Ddx4, Piwi1, Mdm2, and Pce1 in NNK-exposed lung tissue compared with controls. The main pathway of these genes for mediating biological information was analyzed using the Kyoto Encyclopedia of Genes and Genomes database. Among them, Rssf1 and Mdm2 were closely associated with NNK-induced lung carcinogenesis. Taken together, our data provide valuable resources for detecting cigarette smoke-induced lung carcinogenesis.

Abstract We investigated the chemical transformation of atrazine with sodium azide. Sodium azide reduced the atrazine level in the control reactors at a rate much faster than the natural anaerobic biotransformation rate in wetland sediment. The chemical transformation of sodium azide with atrazine was observed in the D.I. water sample reactors. It was evident from the study that higher the sodium azide concentration, the faster the dissipation of atrazine. Atrazine disappeared faster in anaerobic conditions than aerobic conditions. Based on limited mass spectral analysis, 3‐ethylamino, 5‐isopropylamino‐s‐triazyl azide and 3‐ethylamino, 5‐isopropylamino‐s‐triazinone appeared to be the final transformation products from the chemical interactions between atrazine and sodium azide. This study clearly demonstrated that sodium azide must not be used to prepare control reactors in atrazine degradation studies.

The response of environmental pollutants can be detected bioanalytically focusing on the source and matrices of concern. Cell culture bioassays are rapid and inexpensive and thus have great potential for routine monitoring of aquatic resources. Such novel in vitro assays are a new tool to investigate lipophilic and low volatile compounds and are a powerful complement to instrumental analysis. The ethoxyresorufin O-deethylase (EROD) microbioassay was conducted to determine cytochrome P4501A (CYP1A) activity in environmental samples, and the calculated 3-methylchloranthrene (3-MC) equivalent concentration (MEQ) was introduced as a new quantitative water quality parameter. The chemical MEQ was calculated by multiplying induction equivalency factor (IEF) to GC-MS analysis data. And biological (bio-) MEQ was calculated by comparing the concentration response curve of the sample with those of the 3-MC calibration curve. Therefore, chemical MEQ is an estimation of the toxic effects of polycyclic aromatic hydrocarbons (PAHs) and bio-MEQ is the total toxic effects of various CYP1A-inducing chemicals in water samples. In this study, bio-MEQ values of water samples were higher than chemical MEQ values and total PAH concentrations, indicating that there must be other compounds in the water sample effective as inducers of EROD and that the biological activities of mixture compounds are mainly due to additive effects. There was a good correlation between bio-MEQ and total PAH concentration. The difference between bio-MEQ and total PAH concentration was high in downstream areas, i.e., polluted sites. Bio-MEQ calculated based on the enzyme-inducing effect of water samples could give information about the biological potency of water samples caused by PAH-like compounds.

Abstract Background Hydrocodone is a semi-synthetic derivative of codeine and produces opioid-like effects similar to morphine. Hydrocodone (Trade names: Vicodin, Lortab, Hycodan, Vicoprofen) is one of the most frequently prescribed opioids in the U.S. as an antitussive (cough suppressant) and narcotic analgesic agent for the treatment of moderate to severe pain. Its ease of prescription and opioid-like effects has led to widespread drug diversion and abuse. The two commercially available homogeneous immunoassays for the detection of hydrocodone in urine at a cutoff of 300 ng/mL suffer undesirable cross-reactivity to oxycodone and morphine at concentrations below 25 000 ng/mL. ARK Diagnostics has developed the ARK Hydrocodone Assay to detect hydrocodone at a cutoff of 300 ng/mL with no cross-reactivity to oxycodone, morphine, and codeine at concentrations below 100 000 ng/mL. Methods The ARK™ Hydrocodone Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff of 300 ng/mL and semi-quantitative range up to 800 ng/mL. The performance of this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, spiked recovery, specificity, Histogram Overlap Analysis of ±25% controls and the cutoff, and method comparison with LC-MS/MS were evaluated. Results In semi-quantitative mode, total precision ranged from 3.9% to 9.9%CV. Spiked recovery of hydrocodone ranged from 86.2% (720 ng/mL) to 102.9% (240 ng/mL). The active metabolite, hydromorphone, showed an approximate equivalence to the 300 ng/mL hydrocodone cutoff at 299 ng/mL (100.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 96.9% (98 positives and 128 negatives). Conclusion The ARKTM Hydrocodone Assay measures hydrocodone and its metabolite hydromorphone in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.

OBJECTIVES: Inhalation exposure to humidifier disinfectants has resulted to various types of health damages in Korea. To determine the epidemiological correlation necessary for presuming the legal causation, we aimed to develop a method to synthesize the entire evidence.METHODS: Epidemiological and toxicological studies are systematically reviewed. Target health problems are selected by criteria such as frequent complaints of claimants. Relevant epidemiologic studies are reviewed and the risk of bias and confidence level of the total evidence are evaluated. Toxicological literature reviews are conducted on three lines of evidence including hazard information, animal studies, and mechanistic studies, considering the source-to-exposure-to-outcome continuum. The confidence level of the body of evidence is then translated into the toxicological evidence levels for the causality between humidifier disinfectant exposure and health effects. Finally, the levels of epidemiological and toxicological evidence are synthesized.RESULTS: Under the Special Act revised in 2020, if the history of exposure and the disease occurred/worsened after exposure were approved, and the epidemiological correlation between the exposure and disease was verified, the legal causation is presumed unless the company proves the evidence against it. The epidemiological correlation can be verified through epidemiological investigations, health monitoring, cohort investigations and/or toxicological studies. It is not simply as statistical association as understood in judicial precedents, but a general causation established by the evidence as a whole, i.e., through weight-of-the-evidence approach.CONCLUSIONS: The weight-of-the-evidence approach differs from the conclusive single study approach and this systematic evidence integration can be used in presumption of causation.

Abstract Anaerobic biotransformation of atrazine under three different nutrient conditions, i.e., D.I water, wetland water, and basal salt media (BSM), was investigated with the sediment from a wetland receiving sugar mill wastewa‐ter in Louisiana. None of the well‐known metabolites and their hydroxy analogs, except hydroxyatrazine were detected in all sample reactors. About 50% of 10 mg/L of atrazine disappeared after 38 weeks of incubation from liquid and sediment phases of the sample reactors with D.I. water and wetland water. However, more than 25% of the disappeared atrazine still existed as hydroxyatrazine in those sample reactors. BSM substantially increased the removal rate of atrazine. Atrazine concentrations dropped to less than detectable level after 38 weeks of incubation in the sample reactors with BSM. However, the hydroxyatrazine level in liquid and sediment phases of the BSM reactors was still about 2.5 mg/L. BSM without (NH4)2SO4 decreased the atrazine transformation rate.

Objectives In order to identify the possibility of striped bitterling (SB) (Acheilognathus yamatsutae) being used as a test species for estrogenic endocrine disrupting chemicals (EEDCs), we carried out the cloning and sequence characterization of the estrogen receptor (ER). Methods The ER from a striped bitterling was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), 5'- and 3'-rapid amplification of cDNA ends (5'-RACE and 3'-RACE) and T-vector cloning. The expression of ER mRNA was also analyzed in six tissues (brain, liver, kidney, gill, gonad, and intestines) by real-time PCR. Results We obtained an ER from the striped bitterling. The SB ER cDNA was 2189 base pairs (bp) in length and contained a 1707 bp open reading frame that encoded 568 amino acid residues. The SB ER amino acid sequence clustered in a monophyletic group with the ERα of other fish, and was more closely related to zebrafish ERα (88% identity) than to the ERα of other fish. The SB ER cDNA was divided into A/B, C, D, E and F domains. The SB ER has conserved important sequences for ER functions, such as the DNA binding domain (D domain), which are consistent with those of other teleosts. Conclusions The ER of the striped bitterling could provide basic information in toxicological studies of EEDCs in the striped bitterling. Keywords: Estrogen receptor, Estrogenic endocrine disrupting effects, Gene cloning, Striped bitterling

Abstract We investigated the aerobic biotransformation of atrazine under three different nutrient conditions, i.e., wetland water, D.I. water, and basal salt media (BSM), using the wetland sediment receiving wastewater from a local sugar mill in Louisiana. None of the well‐known metabolites and their hydroxy analogs were detected, nor prevailing hydroxyatrazine. The atrazine biotransformation rate was the highest in the sample reactors with indigenous wetland water having some of necessary nutrients for the sediment microorganisms. The concentration of atrazine dropped from 10 mg/L to less than 10 μg/L within three weeks of incubation at room temperature once the sediment microorganisms were adjusted to new environments. Contrary to our expectation, BSM significantly hindered the aerobic biotransformation rate, removing only 25 % of atrazine during 18 weeks of incubation. The high level of nitrogen source in the BSM was suspected for the cause of the inhibition.