Polystyrene microplastic particles induce autophagic cell death in BEAS-2B human bronchial epithelial cells.

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-ß 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.

Comparison of 3D airway models for the assessment of fibrogenic chemicals.

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.

Reprogrammed lung epithelial cells by decrease of miR-451a in extracellular vesicles contribute to aggravation of pulmonary fibrosis.

Extracellular vesicles (EVs) play novel roles in homeostasis through cell-to-cell communication in human airways via transferring miRNAs. However, the contribution of EV miRNAs to pulmonary phenotypic homeostasis is not clearly understood. Hence, the aim of this study was to elucidate the functional role of miRNAs obtained from epithelium-derived EVs in lung fibrogenesis. Pulmonary fibrosis was induced by exposure of polyhexamethylene guanidine phosphate (PHMG-p)-instilled mice. In histopathological changes, a clear phenotypic change was observed in bronchial epithelium. For figuring out the role of EVs derived from conditioned media of untreated cells (EV-Con) and PHMG-p-treated BEAS-2B (EV-PHMG), significant increase in EVs released from PHMG-p-treated BEAS-2B was detected. Functional analysis with targets of differentially expressed miRNAs in EVs was annotated to epithelial-mesenchymal transition (EMT). Especially, the most abundant miRNA, miR-451a, was downregulated in EV of PHMG-p-treated BEAS-2B cells. We found that odd-skipped related 1 (OSR1) was a putative target for miR-451a, which had been known as a transcription factor of several fibrosis-associated genes. Transfer of decreased miR-451a via EV-PHMG upregulated OSR1 and induced EMT compared to Con-EV-treated cells. In pulmonary fibrosis mice, miR-451a levels were significantly reduced in EV derived from bronchoalveolar lavage fluid and OSR1 expression was increased in lung tissues of mice with PHMG-p exposure. MiR-451a-transfected EVs markedly alleviated fibrogenesis in the PHMG-p-exposed lungs. Low level of miR-451a in EVs modulated EMT and fibrogenesis in recipient cells by increasing OSR1 levels in vitro and in vivo. Our results suggest that transferring EV miR-451a induces anti-fibrotic autocrine effect by downregulating its target, OSR1 maintaining pulmonary homeostasis disrupted by PHMG-p exposure, which can be a potential therapeutic target.

Effects of tobacco compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the expression of epigenetically regulated genes in lung carcinogenesis.

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.

Pre-validation of a Calu-3 epithelium cytotoxicity assay for predicting acute inhalation toxicity of chemicals.

Although in vivo inhalation toxicity tests have been widely conducted, the testing of many chemicals is limited for economic and ethical reasons. Therefore, we previously developed an in vitro acute inhalation toxicity test method. The goal of the present pre-validation study was to evaluate the transferability, reproducibility, and predictive capacity of this method. After confirming the transferability of the Calu-3 epithelium cytotoxicity assay, reproducibility was evaluated using 20 test substances at three independent institutions. Cytotoxicity data were analyzed using statistical methods, including the intra-class correlation coefficient and Bland-Altman plots for within- and between-laboratory reproducibility. The assay for the 20 test substances showed excellent agreement within and between laboratories. To evaluate the predictive capacity, 77 test substances were analyzed for acute inhalation toxicity. Accuracy was measured using a cutoff of 40%, and the relevance was analyzed as a receiver-operating characteristic (ROC) curve. An accuracy of 72.73% was obtained, and the area under the ROC curve was 0.77, indicating moderate performance. In this study, we found that the in vitro acute inhalation toxicity test method demonstrated good reliability and relevance for predicting the acute toxicity of inhalable chemicals. Hence, this assay has potential as an alternative test for screening acutely toxic inhalants.

Isolation and Characterization of Phenylpropanoid and Lignan Compounds from Peperomia pellucida [L.] Kunth with Estrogenic Activities.

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.

Tyrosine kinase inhibitor neratinib attenuates liver fibrosis by targeting activated hepatic stellate cells.

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 CCl4-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-ß)-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 CCl4. 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.

Akt and Notch pathways mediate polyhexamethylene guanidine phosphate-induced epithelial-mesenchymal transition via ZEB2.

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.

Polyhexamethylene guanidine phosphate-induced ROS-mediated DNA damage caused cell cycle arrest and apoptosis in lung epithelial cells.

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.

In vitro model for predicting acute inhalation toxicity by using a Calu-3 epithelium cytotoxicity assay.

INTRODUCTION: 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. METHOD: 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. RESULTS: 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. DISCUSSION: 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.

Polyhexamethylene guanidine phosphate-induced upregulation of MUC5AC via activation of the TLR-p38 MAPK and JNK axis.

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.

Sesquiterpenes from Cyperus rotundus and 4α,5α-oxidoeudesm-11-en-3-one as a potential selective estrogen receptor modulator.

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-ß in competitive binding assay. It was found that 2 exerted higher binding affinity to ER-ß 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.

MicroRNA regulatory networks reflective of polyhexamethylene guanidine phosphate-induced fibrosis in A549 human alveolar adenocarcinoma cells.

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.

Total Synthesis and Anti-inflammatory Evaluation of Penchinone A and Its Structural Analogues.

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.

In vitro inflammatory effects of polyhexamethylene biguanide through NF-κB activation in A549 cells.

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.

Novel approach to study the cardiovascular effects and mechanism of action of urban particulate matter using lung epithelial-endothelial tetra-culture system.

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.

Pharbilignan C induces apoptosis through a mitochondria-mediated intrinsic pathway in human breast cancer cells.

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±2.0µM). 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.

The effects of the standardized extracts of Ginkgo biloba on steroidogenesis pathways and aromatase activity in H295R human adrenocortical carcinoma cells.

OBJECTIVES: 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. METHODS: Cortisol, aldosterone, testosterone, and 17ß-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 (3ß-HSD2 and 17ß-HSD1/4). Finally, aromatase activities were measured with a tritiated water-release assay and by western blotting analysis. RESULTS: H295R cells exposed to EGb761 (10 and 100 µg/mL) showed a significant decrease in 17ß-estradiol and testosterone, but no change in aldosterone or cortisol. Genes (CYP19 and 17ß-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. CONCLUSIONS: These results suggest that EGb761 could regulate steroidogenesis-related genes such as CYP19 and 17ß-HSD1, and lead to a decrease in 17ß-estradiol and testosterone. The present study provides good information on potential therapeutic effects of EGb761 on estrogen dependent breast cancer.

Ginkgo biloba extract EGb 761-mediated inhibition of aromatase for the treatment of hormone-dependent breast cancer.

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 17ß-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.

Combined approaches using adverse outcome pathways and big data to find potential diseases associated with humidifier disinfectant.

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.