MicroRNAs (miRNAs) play important roles in numerous cellular processes, including development, proliferation, apoptosis, and carcinogenesis. We hypothesized that genetic variations in miRNAs are associated with bladder cancer risk and prognosis. We performed a systematic survey of single nucleotide polymorphisms (SNPs) in miRNAs and evaluated association of selected SNPs with bladder cancer risk and recurrence. The functionality of these miRNA SNPs was further examined by a series of molecular biological assays. Through bioinformatics analysis, five miRNA SNPs were selected for the association study. We found that miR-146a rs2910164 C allele was associated with a significant decreased risk of bladder cancer in both test and validation sets as well as in the combined set (P = 2.92×10^-4). Furthermore, rs2910164 GC/CC genotypes conferred a significantly reduced recurrence compared with the GG genotype (P = 0.016). Functional analysis revealed that miR-146a rs2910164 C allele inhibited cell proliferation and significantly downregulated IRAK1 and TRAF6 expressions in bladder cancer cells. Additional examination of 64 bladder tissues revealed that individuals carrying the C allele had increased expression levels of miR-146a compared with those carrying the G allele (P = 0.010). Our findings demonstrate that miR-146a rs2910164 plays important roles in the risk and recurrence of bladder cancer.

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JWA was recently demonstrated to be a novel regulator of XRCC1 in the BER protein complex to facilitate the repair of DNA SSBs. DNA repair systems have been increasingly implicated both in carcinogenesis and treatment resistance, but the expression pattern and significance of JWA and XRCC1 in cancer is unknown. In this study, expression of JWA and XRCC1 were assessed by immunohistochemistry on tissue microarray (TMA) specimens from 80 gastric carcinoma and paired adjacent non-tumoral tissues, and then validated on another two large independent TMA sets of gastric carcinoma (485 in the testing set and 662 in the validation set, respectively). Prognostic and predictive values of JWA and XRCC1 and of clinicopathologic factors, were evaluated in patients treated only with surgery and those with adjuvant chemotherapy.
JWA and XRCC1 protein levels were significantly downregulated in gastric cancer lesions compared with adjacent non-cancerous tissues. Low tumoral JWA or XRCC1 expression significantly correlated with shorter overall survival (OS) in patients without adjuvant treatment. Multivariate regression analysis showed that low JWA and XRCC1 expression, separately and together, were independent negative markers of OS. Adjuvant fluorouracil-leucovorin-oxaliplatin (FLO) significantly improved OS compared with surgery alone. However, this effect was evident only in the JWA or XRCC1 low expression group; Adjuvant fluorouracil-leucovorin-platinol (FLP) did not improve OS, except in the patients with low JWA and XRCC1expression. JWA and XRCC1 protein expression in tumor are novel candidate prognostic markers and predictive factors for benefit from adjuvant platinum-based chemotherapy (FLO or FLP) in resectable human gastric carcinoma.

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Renal tubuointerstitial fibrosis is the common process from chronic kidney disease (CKD) to end-stage renal disease. The fibrosis disorder was complex and implicated in the variant pathways including apoptosis induction, inflammation activation and endoplasmic reticulum (ER) stress. The C/EBP homologous protein (CHOP) is an important transcription factor, trans-activating infalammation or reactive oxigen species (ROS) response genes and contributed to the ER stress-induced aopoptosis. With these different fuctions, we wanted to explore the roles of CHOP in the renal tubulointerstital fibrosis process. In this poster, we used the unilateral ureteral obstruction (UUO) mouse fibrogenses model with the CHOP defencey and wild type mice. The results revealed that in CHOP knockout mice, it dramatically attenuated fibrogenesis markers incoluding α-smooth muscle actin (αSMA), fibronectin, collegen I, and plasminogen activator inhibitor-1 (PAI-1) in the UUO model on day 14. Furthermore, CHOP deficiency also reversed obstructive-induced apoptosis via JNK- related pathway, ameliorated the nutrophil marker Ly6G expression and decrease ROS injury, at last partially participated in decreasing the NOX4 generation. Taken together, our results present CHOP deficiency conspicuously attenuats obstructive-induced renal fibrosis. Furthermore, the apoptosis, inflammation (especially neutrophil infiltration) and ROS are also effectively counteracted in the UUO kidney. Base on these findings, it may have potential theaputic advantage benfits to prevent the CKD to end-stage renal disease in the future.

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CAR is a xenobiotic-responsive nuclear receptor, which regulates hepatic drug metabolism and disposition. Although CAR is also known to participate in energy metabolism, only limited amounts of information are available on this. In the present study, we have sought a new physiological function of CAR to understand the influence in the liver. DNA microarray analysis was performed using total RNAs prepared from the livers of mice treated with or without a mouse CAR activator, TCPOBOP. Possible involvements of CAR were suggested in the expression of genes associated with insulin signal and fatty acid metabolism. Among them, we focused on Hmgcs2, whose expression was down-regulated after TCPOBOP treatment. The gene encodes HMG-CoA synthase 2 (HMGCS2), a rate-limiting enzyme in ketogenesis. In reporter gene assays, CAR suppressed the Hmgcs2 expression mediated by PPARα, a critical mediator of hepatic expression of Hmgcs2. Similar suppression was observed with human HMGCS2. In DNA affinity assays and mammalian two-hybrid assays, CAR interacted with PPARα, possibly through a mechanism yet uncharacterized, on the PPAR-responsive element, which was critical for the CAR-mediated suppression of HMGCS2 in reporter assays. Finally, we found in cultured human hepatocytes that the co-treatment with human CAR activator suppressed the PPARα-mediated increase in HMGCS2 mRNA levels. Our present results suggest that CAR acts as a negative regulator for ketogenesis in the liver of mice and humans through interferring the PPARα-mediated expression of HMGCS2.

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AIM: Cataracts were frequently observed in the rat toxicity study of a self-developed compound with stearoyl-CoA desaturase (SCD1) inhibitory activity. Swelling/degeneration of the lens epithelial cells and fibers were histopathologically observed in the eyes. SCD1 is known to convert saturated fatty acids to monounsaturated fatty acids, and then alter intracellular fatty acid composition. Although drug-induced cataracts have been reported, the relevance between cataractogenesis and SCD1 inhibitory activity is unknown. We tried to clarify the relevance by phenotypic analysis of SCD1-null mice.
METHODS: Three pairs of B6;D1Lac-Scd1ab-2J/J mice were purchased from the Jackson laboratory as SCD1-null mice, and were crossed. The offspring and parents (19 males and 19 females) were bred for 30 weeks. All animals were examined for clinical observation, body weights, and food consumption. In order to identify the cataract, the lens were ophthalmoscopically examined at regular intervals from 6 weeks of age. The eyes were removed from the animals and examined histopathologically at 10, 20, or 30 weeks of age. At the same time, blood lipids and others were also examined.
RESULTS and CONCLUSION: Cataracts with hypolipidemia were observed from 10 weeks of age in SCD1-null mice, and frequently occurred (9 of 38 rats) until 30 weeks of age. Generally, cataracts are hardly seen in C57BL/6 mice of this age. These results suggest that SCD1 the inhibitory activity might be involved in the cataractogenesis via alteration of the lipid composition of the lens.

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In this study, we focused on the periodic repeated sequence of proline residues contained in protein primary structures. As the model peptide, (Xaa-Pro) which contains a proline residue, was prepared. When a peptide containing the Lys residue for Xaa was added to the cell culture medium, formation of spheroid was induced. Here, the relationship between the length of the peptide chain and the several kinds of amino acid constituents was investigated.
All peptides were synthesized by solid phase peptide synthesis. The solutions with peptide concentration of 0.1–1.0 mg/mL were prepared. After 6hr incubation, the solution was added to cell culture medium, and cell behavior was observed (n=5).
Poly(Lys-Pro) added to the cell culture medium induced the formation of spheroid from 2 days after the addition of the peptide in both cell culture dishes for suspension culture and in tissue culture. Investigation of the chain length dependency of (Lys-Pro)_n (n=8-16) showed that spheroids were observed only for n = 10 and 12, and formation of cellular aggregates was most effective for n =10 and 12. Thus, it is considered that the peptide chain length affects the formation of spheroids. However, the formation of spheroid was not observed in both (Lys-Lys)₁₂ and (Lys-Ala)₁₂.
All spheroids obtained under the conditions had ≥80% living cells. Therefore, it is considered the formation of spheroids was not induced as a result of the aggregation of dead cells but due to the added peptides. (Lys-Pro)₁₂ peptide may find use as cell transplantation materials in vitro tissue engineering.

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CYP1A1 and CYP1A2 are involved in the both detoxification and metabolic activation of xenobiotics including procarcinogens. We have recently reported that the xenobiotic-responsive nuclear receptor CAR, in addition to Ah receptor, simultaneously transactivates both human CYP1A1 (hCYP1A1) and hCYP1A2. In this study we have investigated a possible involvement of LXRα, another xenobiotic-responsive nuclear receptor highly expressed in the liver, in the gene expression of hCYP1A1 and hCYP1A2. As these genes share an about 23 kb-5’-flanking region, reporter assays were performed with dual-reporter constructs containing the promoter sequence flanked with two different reporter genes at 5’- and 3’-ends. Results obtained from reporter assays with wild-type and deleted constructs suggested that LXRα simultaneously transactivates the expression of both genes through two regions (-554 to -511 and -510 to -461 of hCYP1A1). Gel-shift assays demonstrated that LXRα and RXRα heterodimer binds to two ER8 motifs found at around -520 and -460 of hCYP1A1. The former corresponds to the CAR-binding motif that we found previously. The critical roles of these ER8 motifs in the LXRα-mediated transcription of hCYP1A1 and hCYP1A2 was confirmed by reporter assays using reporter constructs carrying mutation(s) in these motifs. Finally, we found that LXRα synergistically transactivated the expression of hCYP1A1 and hCYP1A2 with Ah receptor but not CAR. Taken together, our present results suggest that LXRα is another transcription factor involving the expression of hCYP1A1 and hCYP1A2 and that two ER8-type responsive motifs located in the hCYP1A1 proximal promoter are important for the LXRα-mediated expression of these genes.

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The aim of this study was to assess the changes of haematological parameters and red blood cells in rainbow trout after nitrite poisoning. Experimental fish were exposed to elevated nitrite concentration of 8 mg.l^-1 for 48 hours. The changes of red blood cells were studied using light microscope and transmission electron microscope. The main haematological response of trout to nitrite exposure was significant decrease (P<0.01) in haemoglobin concentration, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration, leukocyte count and significant increase (P<0.01) in mean corpuscular volume.
No pathological changes of erythrocytes were found in the blood smears. Under transmission electron microscope, erythrocytes were observed as fusiform cells with large central, oval heterochromatic nucleus. The organelles in the cytoplasm such as mitochondria were found sporadically, exception of various types of vesicles. Four types of atypical erythrocytes were characterized in the peripheral blood of rainbow trout exposed to elevated nitrite concentration.
This research was supported by the center CENAQUA,no. CZ.1.05/2.1.00/01.0024, Project USB (GAJU), no. 047/2010/Z, Project GACR, no. P503/10/P492 and Project NAZV no. QH82117.

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The completion of a scientifically sound safety assessment of new ingredients and finished products is essential prior to their introduction into the market place. Such assessments are based on a risk assessment paradigm established by the US National Academy of Science that consists of a four-step process: hazard identification, dose-response assessment, exposure assessment, and risk characterization. Within the risk assessment process for a spray-type fabric refresher product (FR), the 2-zone model is used to assess the inhalation exposure for FR taking into account spatial variability in exposure intensity in a room where the product is used. Consumers may be exposed to aerosol particles bounced back from the surface of fabric when the product is sprayed onto fabric. The 2-zone model was formulated into a spreadsheet model and specifically parameterized for FR through a thorough understanding of consumer habits and practices (H&P) in market places. Key parameters include: 1) Far field or room volume (the recommended default is 50 m³), 2) Time period of product use (3 min), 3) Total time spent in room (480 min), 4) Number of air exchanges per hour (0.52, Shinohara et al., 2006), 5) Diameter of near field zone (1.24 m), and 6) Bounce back factor (15%), and 7) Total amount of product released (65 g). The 2-zone model is currently employed in a risk assessment-based approach to safety evaluation of FR at Procter & Gamble. This presentation will describe the source of the H&P data, the model formulation and the risk assessment for representative ingredients.

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Objective: To analyze the effect on the regulation of blood lipids and it’s mechanism of two corn pollen with different broken methods in rats.
Methods: The rats were randomly divided into three groups, a high-fat control group, a high-fat and 5.0 g / kg bw corn pollen (supercritical CO2 broken methods )and a high-fat group and 5.0 g / kg bw corn pollen (mechanical broken methods ) . All rats took foods and drunk water freely, the experimental last for 8w, the fasting level of TG, TC, HDL-C and LDL-C were determined at the end of 3rd w, 6th w end, 8th w respectively. At the end of the whole experimental, the level of serum apoB, blood leptin and various biochemical parameters were measured; then all rats were decapitated, the gross anatomy were observed, heart, liver, spleen, kidney, testis, prostate and aorta were weight and calculated the ratio of body to organ weight respectively , all above-mentioned organs were pathological histology, and measured liver fat content; 8 rats in each group were randomly selected to carry on electron microscopy of liver ultra-thin slice.
Result: The level of rats serum TG, TC, LDL-C, apoB, GPT, GOT, liver fat content, liver weight, ratio of liver to body, prostate and the ratio in two corn pollen groups were significantly lower than the control group (P <0.05 or P <0.01);and the level of HDL-C/TC, HDL-C/LDL-C were significantly higher in two corn pollen group rats (P <0.01); what’s more, the liver injury and liver cell swelling, degeneration and other diseases is significantly less in two corn pollen rats, histopathological changes in the total score was significantly lower than the control group (P <0.01).
Conclusion: Two corn pollen can significantly antagonize the increase of apoB and LDL-C and prevent or delay the level of HDL-C/LDL-C and HDL-C/TC lowering in high fat diet rat , they also can promot lipid metabolism, so they can play a important role in preventing the elevation of TG, TC and liver fat content, improving liver function, reducing liver and liver cell injury, thereby reducing risk of lipid metabolism abnormality and hyperlipidemia. Two kinds of corn pollen can significantly antagonize the increase of liver weight, liver to body ratio, prostate weight and prostate to body ratio. Two kinds of broken methods show no significant difference on lipids metabolism regulation and it’s mechanism .
Keywords :corn pollen; hyperlipidemia; lipid metabolism; morphology, ultrastructure of liver cells

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Estrogens play important roles in the growth and invasion of estrogen-dependent human neoplasms. Aromatase overexpression has been reported to be expressed in hepatitis and hepatocellular carcinoma (HCC) compared to normal liver but its details in these hepatic diseases have remained unclear. Therefore in this study, we immunolocalized aromatase using immunohistochemistry in patients with liver cirrhosis, steatosis, hepatitis, HCC, and metastasis liver carcinoma (MLC) in order to study intrahepatic aromatase in details. Aromatase immunoreactivity was predominantly expressed in non-neoplastic hepatocytes around tumor cells. We then evaluated the effects of an interaction between hepatocytes and carcinoma cells on expression of aromatase mRNA in HepG2 using a co-culture systems. Aromatase mRNA levels in HepG2 were significantly increased by co-cultivation with all carcinoma cell lines examined. The same exon 1 splicing variant was used in aromatase expression in HepG2 cells regardless of carcinoma cell lines employed in the co-culture system. These findings all indicated that carcinoma cells, whether metastatic or primary, induced aromatase expression in adjacent normal hepatocytes through the soluble aromatase inducible factors in hepatic microenvironments.

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We have developed the screening test for drug and chemical compound of food by the use of rat whole embryo culture.
The advantages of whole embryo culture are to examine the direct effects of the ARB on rat embryo.
As the testing agent, ARB was examined in this study using the rat embryo cultured from day 11 to 13 gestation.
In this whole embryo culture system,rat embryos were explanted on day 11(plug day = 0) of gestation and cultured of rat serum with 95% O₂ and 5%CO₂ using improved rotator for 48hrs.
The group of treated embryos of ARB were not exchanged in the heart beat, the crown-rump length, the embryo weight and the embryonic total somite.
On the other hand, the malformation was not observed in the embryos cultured with ARB. The blood circuration of yolk-sac or embryo was increased the control group.

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In vitro eye irritation testing, which is alternative to animal testing such as Draize eye test using rabbits, is required from an animal welfare. We have developed the reconstructed human corneal epithelium model, LabCyte CORNEA-MODEL, and investigated a test method to evaluate eye irritation effect using this model. In this presentation, we report about an optimization of eye irritation test method using LabCyte CORNEA-MODEL and about the prediction potency of an optimized LabCyte CORNEA-MODEL eye irritation test.
In evaluating the cell viability and the barrier function of LabCyte CORNEA-MODEL among inter- or intra-lots, there was little difference in each lot and showed good reproducibility. From the results of several optimization experiments of eye irritation test using LabCyte CORNEA-MODEL, the application periods of chemicals were set to 1 minute for liquid chemicals and 24 hours for solid chemicals, and the post-incubation periods were set to 24 hours for liquids and nothing for solids, respectively. If the viability was less than 50%, the chemical was judged as eye irritant. Sixty one chemical substances were applied to the optimized protocol using LabCyte CORNEA-MODEL and the correlation with in vivo class was evaluated. The prediction of the new eye irritation test methods using this model was highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%. From these results, it was suggested that the eye irritation test using LabCyte CORNEA-MODEL could be a useful alternative method to Draize eye test for variety of chemicals with irritant potency.

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The establishment of more efficient approaches for developmental neurotoxicity testing (DNT) has been an emerging issue for children’s environmental health. This study showed a systematic approach for DNT using the neuronal differentiation of mouse or human embryonic stem cells (mESCs or hESCs) as a model of fetal programming. During embryoid body (EB) formation, mESCs were exposed to 12 chemicals for 24 h and hESCs were exposed to 3 chemicals for 96 h. Then global gene expression profiling was performed using whole genome microarray analysis. Gene expression signatures for gene sets related to neuronal development and neuronal diseases were selected for further analysis. At the later stages of neuronal cell differentiation from EBs, neuronal phenotypic parameters were determined using a high-content image analyzer. Bayesian network analysis was then performed based on global gene expression and neuronal phenotypic data to generate comprehensive networks with a linkage between early events and later effects. Furthermore, the probability distribution values for the strength of the linkage between parameters in each network was calculated and then used in principal component analysis. The characterization of chemicals according to their neurotoxic potential reveals that the multi-parametric analysis based on phenotype and gene expression profiling during neuronal differentiation of mESCs or hESCs can provide a useful tool to monitor fetal programming and to predict developmentally neurotoxic compounds.

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We present a non-invasive technique for the characterization of contractile behaviors of cardiomyocytes, based on the high-speed video imaging and the analysis using motion vector prediction (MVP) algorithm (1). With this method, we could detect contraction and relaxation motions of cardiomyocytes separately, and could obtain quantitative information on contractile behaviors of cardiomyocytes, such as the beating rate, contraction/relaxation velocity, orientation of contraction, beating cooperativity/homogeneity in the monolayer, and propagation of contraction wave in the monolayers. Furthermore, the effects of drugs that will alter inotropism and chronotropism of cardiomyocytes (e.g., autonomic agents, gap junction inhibitors, ion channel blockers) were clearly detected. Since this method could sensitively evaluate the contractile behavior of cardiomyocytes without using any labeling or specialized culture dishes, it may be used to study and/or monitor cardiomyocyte during prolonged culture periods and in screens for drugs that may alter the contraction of cardiomyocytes. At the meeting, we will show the details of this method and discuss the correlation between the motion pattern of contraction/relaxation and extracellular field potential parameters, evaluated for human-induced pluripotent stem cell-derived cardiomyocytes.
(1) Hayakawa, T. et al., “Non-invasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion vector analysis.” Tissue Eng Part C Methods, 2012, 18, 21 - 32.

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Most of infectious diseases are intricately linked to the global environment. Alterations of environment like global warming have a significant potential to intensify selected infectious diseases. Climatic effects are predicted to include crowding, famine, water contamination, human migration, and alterations in vector ecology, all of which increase infectious diseases.
The fist line to protect body from the attack of pathogens by innate immune system.
The innate immune system plays a crucial part in the initiation and subsequent direction of adaptive immune responses. Moreover, because there is a delay of 4–7 days before the initial adaptive immune response takes effect, the innate immune response has a critical role in controlling infections during this period.
The present study was carried out to investigate the immunmodulating activity of WK4 on the expression of nitric oxide (NO), tumor necrosis factor alpha (TNF-a), interferon stimulated gene factor 3 (IRF3), cyclooxygenase-2 (COX-2), interferon gamma(IFN-r) and Nuclear factor kaffa B (NF-kB). The phagocytosis of peritoneal macrophage by uptaking E.coli FITC between WK4-untreated and WK4-treated group was compared. Significant augmentation in the phagocytosis of WK4-treated peritoneal macrophage was seen compare to untreated group. It also enhanced the survival rate of E. coli infected mice by augmenting the phagocytosis activity of macrophages. These results support that WK4 effectively prevented infection by E. coli by the activation of peritoneal macrophage. WK4 would be useful to prevent pathogenic microbial infection in human and farm animals.

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Constitutive androstane receptor (CAR) is highly expressed in the liver and plays central roles in the xenobiotic-induced expression of drug-metabolizing enzymes. CAR is also involved in hepatocyte proliferation and hepatocarcinogenesis, at least in rodents, and it remains unknown if CAR shows similar influences in human became clear species differences are often observed on the action of CAR activators. CAR is retained in cytoplasm and CAR activators induce its nuclear translocation to enhance target gene expression. As CAR is barely expressed in culture cells such as human hepatoma HepG2 cells, it is necessary to overexpress CAR in these cells through transfecting its expression plasmid, in order to analyze CAR functions and/or assess CAR activators. However, it is known that overexpressed CAR tends to stimulate the gene transcription even in the absence of CAR activators because of its automatic translocation into nucleus. In this study, we have sought to establish a new cell-based reporter assay system for the identification of species-selective activators of hCAR using tag-fused CAR proteins. We found that in reporter assays with HepG2 cells hCAR fused with V5 and His tag showed low basal activity and considerable response to a hCAR agonist CITCO treatment but not to phenobarbital. One hundred and seventy-six industrial chemicals were thus subjected to this reporter assay system to assess their ability to activate hCAR. As results, four compounds activated hCAR were identified as agonistic hCAR activators. In conclusion, our reporter assay system may be a promising tool to assess chemicals’ agonistic activities toward hCAR.

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Intracellular levels of redox molecules, such as hydrogen peroxide (H2O2), glutathione (GSH), and NADPH etc., and their electron transfer are controlled by redox network with nuclear factor-E2 p45-related factor (Nrf) 2 regulation. Electron transfer fluxes of redox molecules were numerically calculated using our constructed model of redox network by biochemically-based modeling using literature data under H2O2 stress. The reaction rate of glutathione peroxidase (GPx) - glutathione reductase (GR) - glucose-6-phosphate dehydrogenase (G6PD) pathways was greatly increased in oxidative condition. Core regulatory mechanism was considered as follows: cytosolic H2O2 was elevated by huge influx of extracellular H2O2, and showed superlinear threshold increase with GSH depletion caused by saturation of negative feedback loop via GR reaction. NADPH regeneration simultaneously increased for keeping GSH homeostasis by negative feedback loop via G6PD reaction, but it could not prevent GSH depletion. Then subsequent adaptive response rescued GSH depletion by induction of GR, glutamate-cysteine ligase (GCL) and G6PD through Nrf2-dependent gene network to prevent H2O2 elevation. This model provided basic regulatory mechanism of redox network and allowed long-term simulation under wide range of H2O2 stress.

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A stable human cell line, termed HPL-A3, has newly established by co-transfection of a human pregnane X receptor (hPXR) expression vector and a reporter plasmid (p3A4-hPXRE-Luc) containing a luciferase gene and a promoter/enhancer region of the human CYP3A4 gene into a human hepatoma-derived cell line, HepG2. Then, the usefulness of HPL-A3 for chemically activated luciferase expression (CALUX) assay for the screening of human CYP3A inducers was assessed. The induction of CALUX in APL-A3 cell line was observed by hPXR activators, including rifampicin, but not by rat/mouse PXR activators, such as pregnenolone-16α-carbonitrile and dexamethasone. The hPXR activator-mediated induction of CYP3As, including CYP3A4, was also observed at both levels of mRNA and enzyme activity. There were positive correlations between chemical-mediated inductions of CALUX and CYP3A4 mRNA. Interestingly, expression levels of not only hPXR but also vitamin D receptor (VDR), one of positive transcription factors for CYP3A subfamily genes, in HPL-A3 cells were significantly increased as compared with those in a parent cell line HepG2, and consequently, VDR ligand (1,25-dihydroxyvitamin D₃)-mediated inductions of CALUX and CYP3A4 mRNA were observed in the cells. These findings verified the usefulness of HPL-A3 for the screening of the CYP3A inducers, which can activate the hPXR and/or hVDR.

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Endothelial nitric oxide synthase (eNOS) is the enzyme that endogenously biosynthesize nitric oxide (NO) from L-arginine. Because NO serves important functions to maintain vascular homeostasis, it must be controlled tightly. If not, endothelial dysfunction occurs. The activity of eNOS is regulated by multiple interdependent control mechanisms and signaling pathways, and one of that is phosphorylation at Ser1177 residue of the enzyme. In this study, we figured out that orotic acid, an intermediate in pyrimidine synthesis, inhibits activation of Akt/eNOS signaling pathway by insulin. In ECV304 human endothelial cells, orotic acid inhibits insulin activation of eNOS. In ECV304 cells incubated with orotic acid, phosphorylation at Ser1177 of eNOS by insulin is decreased in dose-dependent manner. Orotic acid also reduces NO production by insulin. However, orotic acid didn’t affect eNOS expression or eNOS phosphorylation without insulin signaling. To investigate the effect of orotic acid on upstream signaling pathway, we also checked Akt, a well-known kinase which phosphorylates eNOS. Insulin activates Akt through PI3K/Akt signaling pathway, and Akt phosphorylates eNOS directly. Orotic acid also inhibits Akt phosphorylation by insulin. In conclusion, orotic acid inhibits insulin activation of Akt/eNOS signaling pathway and decreases NO production. Therefore, orotic acid breaks vascular homeostasis and induces endothelial dysfunction.

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[Purpose] Recently we have reported that mitochondrial membrane bound glutathione transferase (MtMGST1) contributes to the mitochondrial permeability transition (MPT) (Lee et al., Toxicol Appl Pharmacol 2008) and MtMGST1 in the inner membrane (IM) could form disulfide-linked high molecular weight protein (HMP) with MPT regulator proteins, adenine nucleotide translocator (ANT) and cyclophilin D by the oxidant peroxynitrite (PON) (Imaizumi and Aniya, Arch. Biochem. Biophys. 2011). In the present study, we purified IM-MGST1 and isoforms of ANT and examined whether IM-MGST1 directly interacts with ANT followed by HMP formation by PON treatment. [Method] IM-MGST1 and ANT were purified from IM as described previously. IM-MGST1 and ANT were incubated with PON in the presence or absence of phospholipids, and then GST activity and HMP formation were measured by the method of Habig and immunoblot analysis, respectively. [Results and Discussion] IM-MGST1 and two types of ANT (30 kDa and 48 kDa) were purified from IM. IM-MGST1 activity was increased by incubation with ANTs, cardiolipin and PON. When IM-MGST1 was incubated with ANTs in the presence of PON, HMP was observed dominantly by incubation with ANT fraction with 48 kDa. Thus it was confirmed that IM-MGST1 activity is modulated by cardiolipin and ANTs and that IM-MGST1 directly interacts with ANT and can form disulfide-linked HMP in the presence of PON. These results suggest that IM-MGST1 attributes to a MPT pore formation with ANT in oxidative stress.

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The concept of ‘epigenetics’ was established by Waddington (1942) to explain the development and cellular differentiation of animals. Several processes, such as DNA methylation, histone modification, non-coding RNA transcription, and finally chromatin modification, control the cellular molecular mechanisms underlying epigenetics. Epigenetics was originally defined as the interaction between the genome and various environmental conditions. This scientific field is known as ‘Environmental Epigenetics’ and refers to the disruption of normal epigenetics by various environmental factors. Epigenetics comprises biochemical processes; therefore, epigenetics is easily disrupted by a variety of chemical substances in the environment. ‘Environmental Epigenetics’ is strongly correlated with toxicology, clinical medicine, and the psychosocial behaviour of humans. Furthermore, ‘The Developmental Origin of Health and Disease (DOHaD) Theory (Barker, 1968)’ has been accepted in various fields of clinical medicine. The main causes of DOHaD are attributable to epigenetics. Therefore, as in toxicology, the embryonic stages are very important from the standpoint of epigenetics as well.
We have been studying mutagenesis and epimutagenesis induced by chemical substances in mouse somatic and germ cells. We believe that ‘Epigenetic Toxicology’ is especially important during the embryonic stages. Herein, we propose the concept of ‘Embryonic Cells-originated Epigenetic Toxicology’. The test substance will be administered at the embryonic stage. The treated male mice will be mated with untreated female mice for 2 generations. We will test for somatic epigenetic toxicity in the F1 generation and for germinal epigenetic toxicity in the F2 generation . The various toxicological phenomena observed during this experiment could be considered from the viewpoint of ‘Environmental Epigenetics’.

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Increasing evidence showed that hyperhomocysteinemia plays an important role in the development of steatosis. In this study we evaluated the effects of L-serine (L-ser) on the ethanol-induced steatosis and elucidated the possible mechanisms of action in terms of the inhibition of the maturation of SREBP-1c through suppression of hyperhomocysteinemia. In vitro studies showed that treatment of AML-12 murine hepatocytes with methionine and homocysteine increased the intracellular homocysteine levels and triglyceride contents, whereas cotreatment with L-ser decreased the homocysteine and triglyceride accumulation. L-ser treatment increased the expression of insulin-induced gene-1 (INSIG-1) and inhibited the maturation and translocation of SREBP-1c to the nucleus. Transfection of the cells with siRNA for betaine homocysteine S-methyltransferase (BHMT) or cystathionine beta-synthase (CBS) increased cellular homocysteine and triglyceride level possibly due to the increase in the activation of SREBP-1. L-ser treatment reversed the SREBP-1 activation and triglyceride accumulation in BHMT siRNA-transfected cells, but not in CBS siRNA transfected cells. Male C57BL/6 mice received ethanol (5g/kg body weight) by gavage every 12 hours for a total 3 times. L-ser (200mg/kg body weight) was administrated by gavage for the last two ethanol treatment. L-Ser treatment significantly attenuated hyperhomocysteinemia. Ethanol-induced activation of SREBP-1 and accumulation of triglyceride in the liver were reversed by L-ser. These results demonstrate that L-ser inhibits hyperhomocysteinemia by accelerating the conversion of homocysteine to cystathionine which results in the amelioration of SREBP-1 activation and fatty liver.

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[Background and aim]
In wild bird species, the metabolic activity of warfarin by cytochrome P450 (CYP) was reported to be much lower compared to chicken. However, even the important CYP isoforms in avian xenobiotic metabolism have not been clarified due to the lack of information about the expression level and the function of each isoforms. Thus, the reported species difference of metabolic ability cannot be attributed to any isoform. In this study, we aimed to identify the isoform dominant in avian xenobiotic metabolism and to characterize the species difference of the isoform.
[Method and result]
We compared the mRNA copy number among CYP isoforms in chicken liver. CYP1A5, 2C23 and 2C45 were highly expressed in liver, while CYP3A37, which has been well studied in poultry species, showed lower expression level. Therefore, we focused on CYP2C23 of avian species. Partial sequences of CYP2C23 from 8 avian species were cloned and the anti-peptide antibody was synthesized against the conserved amino acid sequence among them. In immunoblot with the antibody and the microsomes of the avian species, we could detect CYP2C23s from all the avian species.
[Discussion]
With mRNA expression, CYP2C subfamily was assumed to be important isoforms in chicken xenobiotics metabolism. The expression levels of CYP2C23 proteins showed not as much difference among bird species as the metabolic activity. With all results above, the species difference of metabolic ability among bird species cannot be attributed to the protein expression levels, but the functional difference.

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Purpose: Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is transcription factor that regulates expression of antioxidant and phase II detoxifying genes and that promotes tumor growth and chemoresistance. Nuclear import and export of Nrf2 controls the cellular oxidoreduction homeostasis. In the current study, we explored whether expression of Nrf2 in the cytoplasm could promote tumor progression and subsequent poor survival in colorectal cancer patients.
Experimental Design: One hundred and fifty tumors from colorectal cancer patients were enrolled to evaluate Nrf2 expression by immunohistochemistry. The prognostic value of Nrf2 was analyzed by Kaplan-Meier and multivariate Cox regression models. The nuclear localization sequence (NLS) of Nrf2 was mutated by site-directed mutagenesis for transfection into Nrf2-knockdown HCT116 stable clones. HCT116 and HCT116 p53 null cells were also treated with a NO scavenger (carboxy-PTIO) and donor (GSNO), respectively. The effects of cytoplasmic Nrf2 on cell growth and migration capability were evaluated by colony formation and Boyden chamber assays.
Results: Fifty percent of the tumors displayed only cytoplasmic Nrf2, 32% of the tumors displayed cytoplasmic/nuclear Nrf2, and 18% of the tumors showed no Nrf2 expression by immunohistochemistry. Kaplan-Meier and Cox regression models showed that cytoplasmic Nrf2 may independently predict poor survival in colorectal cancer patients. Mechanistically, the elevation of cytoplasmic Nrf2 expression in the HCT116 shNrf2 stable clone by NLS-mutated Nrf2 transfection or NO scavenger treatment was responsible for the increase in colony formation and migration capability in these colon cells.
Conclusion: Cytoplasmic Nrf2 promotes tumor progression and subsequent poor survival in colorectal cancer patients.

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Chromosome aberrations (CA) are associated with several genetic heredity diseases. Of concerns, prenatal diagnosis or cytogenetic screening was administered to determine if there are chromosome abnormalities and genetic diseases in a fetus or embryo. However, conventional G-banding technique which is generally applied in CA detection has limit on exploit of copy number variants (CNVs) due to low resolution and tedious multiple processes. Instead, array comparative genomic hybridization (aCGH) technology enabling incremented high-resolution can be considered as an alternative approach for improving prenatal diagnosis. In this study, our aCGH (coverage of SNP tag and CNV regions) was used to identify copy number variations from genomic DNA in human amniotic cell specimens. The aCGH results revealed various karyotypes of CNVs including loss, homozygous loss, gain, high copy gain, and copy neutral LOH whereas using conventional G-banding only one benign cytogenetic CNV was observed in one case study. The aCGH was compatible to define small-scale chromosomal imbalances segment that were undetectable risk segment by G-banding. In contrast, abnormal G-banded karyotypes as balanced rearrangements were hidden from detection by aCGH analysis. The aCGH also provides detailed database of copy number variant regions (CNVRs) in each chromosome. Interestingly, CNVRs containing important genes (ACADM, PPM1B, UGT2B17 and ZDHHC11) were discovered from our data, in which their defects or mutations have been reportedly to be involved in certain genetic heritable diseases and/or syndromes. Gathering together, our detailed CNVs and CNVR via aCGH of amniotic cells might be meaningful database to improve strategy in disease-specific genotoxicity researches.

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The disruption of androgen receptor (AR) mediated androgen signaling played very important roles in several androgen related diseases and symptoms. In order to screen the androgen modulating potency of chemicals, we developed stable AR-GreenS cell lines to access AR mediated transcriptional activation and optimized the protocol. Stable AR-GreenS cell line was stably transfected with pGL4-MMTV/Hygro, which is a firefly luciferase reporter vector bearing androgen responsive element (ARE), in 22RV1 cell line which is a human prostate cancer cells contained functional AR. AR-GreenS cell line was characterized the expression of hormone receptors. In this stable cell line, 5α-Dihydrotestosterone (DHT) was dose-dependently induced the luciferase activity and the activity was significantly increased at 1.0 x 10^-10M and stated to reach to plateu 10^-8M with maximum about 15 folds compared with vehicle control. This DHT induced luciferase activity was inhibited by the treatment of AR antagonist, bicalutamide. AR-GreenS cells have maintained their growth rate, morphology and responsiveness to DHT until now 85 passages cultured for over 13 months. The inter variation of assay was relatively small with about 5.1± 1.3 mean value of CV (the coefficient of variation). Using AR-GreenS cells, we established the 3 days test protocol and optimized the testing condition for AR transcriptional activation assay. We validated the stable cell line using 20 compounds among 78 substances which are recommended substances for validation of in vitro androgen receptor transcriptional activation assays by ICCVAM.
*This research was support by a grant (12162KFDA737) from Korea Food & Drug Administration in 2012

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Sirtuins (SIRTs), NAD⁺-dependent class III histone deacetylases (HDACs), play an important role in the regulation of cell division, survival, and senescence. Although several effective SIRT inhibitors have been developed, little is known about the specific mechanisms of their anti-cancer activity. Here, we investigate the anti-cancer effects of Sirtinol, a SIRT inhibitor, on human breast cancer MCF-7 cells. In the present study, apoptotic and autophagic cell death were measured. Sirtinol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. The IC₅₀ values of Sirtinol were 48.6 µM (24 h) and 43.5 µM (48 h) in MCF-7 cells. As expected, Sirtinol significantly increased acetylation of p53, which has been reported to be a target of SIRT1/2. Flow cytometry analysis revealed that Sirtinol significantly increased the G1 phase of the cell cycle. Up-regulation of Bax, down-regulation of Bcl-2, and cytochrome c release into the cytoplasm were observed in MCF-7 cells, these are involved in the mechanism of apoptotic cell death. The annexin V-FITC assay was used to confirm Sirtinol-induced apoptotic cell death. Furthermore, expression of LC3-II, an autophagy-related molecule, was significantly increased in MCF-7 cells after Sirtinol treatment. Autophagic cell deaths were confirmed by acridine orange and dansylcadaverine (MDC) staining. Interestingly, pretreatment of 3-methyladenine (3-MA) increased the Sirtinol-induced MCF-7 cells cytotoxicity, which is associated with blocking autophagic cell death and increasing apoptotic cell death. Based on our results, down-regulation of SIRT1/2 expression may play an important role in the regulation of breast cancer cell death; thus SIRT1/2 could be a novel molecular target for cancer therapy and these finding provide a molecular basis for targeting SIRT1/2 for future cancer therapy.

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We have found that a yeast major peroxiredoxin Tsa1 forms transient disulfide bond with pyruvate kinase (Pyk1), and regulates Pyk1 enzyme activity in redox-dependent manner. Because Pyk1 catalyzes a rate-limiting step in glycolysis, we thought metabolic changes might affect cellular redox status.
Here, we investigated contributions of Tsa1-dependent redox regulation of Pyk1 on cellular antioxidant response. We generated a PYK1 mutant yeast strain carrying mutations in cysteine residues responsible for interaction with Tsa1 (PYK1C/A). Wild Type (WT) and PYK1C/A cells were cultured in a glucose medium from a log phase to a stationary phase and spotted on the ager medium containing hydrogen peroxide (H₂O₂). H₂O₂ sensitivity of PYK1C/A was equivalent to WT at the log phase. In contrast, PYK1C/A was more sensitive to H₂O₂ than WT at the stationary phase. Since glucose was depleted from the medium at the stationary phase, NADPH might be supplied from a gluconeogenesis-induced pentose phosphate pathway. NADPH is required for detoxification of H₂O₂. Therefore, we examined requirement of fuructose-1,6-bisphosphatase (fbp1Δ), a key enzyme on gluconeogenesis for the phenotype. As we anticipated, the H₂O₂ sensitivity of PYK1C/A was equivalent to that of WT in fbp1Δ genetic background. Furthermore, TSA1 was also required for the difference of the H₂O₂ sensitivity. Taken together, these results suggested that Tsa1-mediated Pyk1 downregulation might upregulate gluconeogenesis to enhance resistance to H₂O₂ after depletion of glucose at the stationary phase. Our results suggest a novel mechanism on controlling metabolism in response to oxidative stress.

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Many studies have indicated that abnormal protein degradation is related to Parkinson’s disease (PD). Autophagy is intracellular degradation system that delivers cytoplasmic constituents surrounded by isolation membrane to the lysosome and can degrade protein aggregates and damaged organelles. It has been reported that brain specific autophagy-deficient mice exhibit the neuronal cell death accompanied by accumulation of protein aggregates and neurodegenerative disorder-like symptoms. 1-methyl-4-phenylpyridinium (MPP⁺) has been widely used in preparation of cell model of PD, and several mechanisms of MPP⁺-induced cytotoxicity at high concentrations (a few hundred µM-a few mM) have been reported. But high concentration-induced intracellular changes do not seem to reflect PD pathogenesis, because MPP⁺ is not present at such high concentrations in the brain of MPTP-induced animal model of PD. In this study, we investigated the effects of low concentration of MPP⁺ (10 µM) on autophagy and its related proteins, on the basis of morphological changes in the human neuroblastoma cell line SH-SY5Y. We found that 10 µM MPP⁺ induces an increase in the number of vacuoles by about 2.5 times compared with control. We revealed that these vacuoles are autophagic vacuoles by means of immunocytochemistry and western blotting for an autophagy marker LC3. These results suggest that low concentrations of MPP⁺ induce an abnormality of autophagy.

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Nephrotoxicity is one of critical toxicological manifestations in clinical and preclinical investigation. Conventionally, serum creatinine and blood nitrogen urea (BUN) have been used for screening nephrotoxicity status. Advanced research in nephrotoxicity biomarkers was able to demonstrate promising biological molecules produced in biological fluids and tissues for the prediction of nephropathy. Of numerous nephrotoxicity biomarkers, some of them are comparatively evaluated for their potential usefulness. The biomarkers proposed are as follows: kidney injury molecule-1 (KIM-1), cystatin C (Cys), neutrophil gelatinase associated lipocalin (NGAL), N-acetyl-β-Dglucosaminidase (NAG) interleukin-18 (IL-18), and liver-type fatty acid binding protein (L-FABP). NGAL has been shown to detect kidney injury earlier than changes in serum creatinine (Scr) in kidney patients. Scr value at the time of diagnosis of acute kidney injury (AKI) or other types of kidney disorders may not provide a reliable diagnostic information about prognosis of kidney injury because it could not reflect the severity or the stage specificity of nephropathy. NGAL levels in urine may be better to predict nephrotoxicity due to its high sensitivity and specificity. Other metabolomic biomarker of citrate was also proposed as a potential useful target metabolite for nephrotoxicity induced by xenobiotics.
(Acknowledgement: This research was support by a grant (10182KFDA992-1203) from Korea Food & Drug Administration in 2010)

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A wide variety of chemicals have been used for manufacturing consumer products and humans are exposed to them in our daily life. To protect consumers from exposure to hazardous chemicals, risk management system needs to be prepared. Developed countries such as United States (U.S.) and Canada have applied their own risk management systems for regulating the chemicals. However, the risk management systems prepared by developed countries may not be readily applicable to developing or underdeveloped countries because economic, political, or social situations are diverse in each country. Generally, risk management framework includes risk assessment, risk confrontation, risk intervention, risk communication, and risk management evaluation, but it may differ in the process, contents, risk intervention of stakeholders, etc. European Commission (EC) requires a social- economy analysis for formulating an option on restrictions suggested by European Chemicals Agency and EC has early warning system for safety management system, Rapid Alert System (RAPEX). In the comparison of merits or demerits of the risk management system, the five parts of monitoring, risk assessment, risk communication, risk management and decision-making part can be considered. The monitoring part is the collection of information and evaluation in monitoring agencies. The risk assessment process includes the scientific evaluation of potential adverse health effects. The risk communication tasks are to identify stakeholders, develop stakeholder analysis, assess stakeholder acceptability, consult with stakeholders, inform stakeholders about the options, evaluate control options and monitor changing issues. The risk management process is weighting of policy options and selecting regulatory options. The decision-making step is related to the determination of governmental or voluntary actions. This paper reviews the critical points of risk management systems to effectively control hazardous chemicals for human health.

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Leptin, a representative adipokine secreted from the white adipose tissue, is considered as a potential linker between obesity and cancer. SIRT1 is an NAD⁺-dependent histone/protein deacetylase speculated to function as an oncogene. This prompted us to investigate the role of SIRT1 in leptin-induced colon carcinogenesis. In the present study, we found that leptin signaling-defective ob/ob and db/db mice showed lower expression of SIRT1 in colon tissues compared with leptin signaling-intact C57BL/6J mice. Moreover, leptin induced upregulation of SIRT1 in human colon cancer (HCT-116) cells. Leptin promoted migration and invasion of HCT-116 cells and tumor growth in the xenograft assay, which was abrogated by treating with a SIRT1 inhibitor sirtinol, suggesting that SIRT1 accounts for leptin-induced colon carcinogenesis. Notably, leptin-induced SIRT1 expression was regulated by the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2). Leptin stimulated nuclear accumulation of Nrf2 as well as its direct interaction with antioxidant response elements located in the SIRT1 promoter. We also found that siRNA knockdown of Nrf2 abrogated leptin-induced SIRT1 expression. Moreover, SIRT1 was significantly reduced in colon tissues of Nrf2-null mice, lending further support to Nrf2-dependent SIRT1 expression. Based on the observations from using N-acetylcysteine and AG490, the ROS-JAK2-STAT3 axis may be involved in leptin-induced Nrf2 activation. The expression of leptin, Nrf2 and SIRT1 was coordinately increased in human colon tumor tissues, providing substantial evidence. In conclusion, the obese protein leptin might stimulate colon tumor promotion as well as progression via the Nrf2-dependent SIRT1 overexpression.

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Phagocytosis of apoptotic neutrophils by macrophages, called efferocytosis, is critical to resolution of inflammation as this process prevents the exposure of surrounding tissues at the inflammatory site to the toxic contents of lytic cells. Docosahexaenoic acid-derived resolvin D1 (RvD1), endogenously generated during resolution of inflammation, is known to stimulate efferocytosis. However, little is known about the mechanism of RvD1-mediated enhancement of efferocytosis. In the present study, we found that murine macrophage-like RAW264.7 cells treated with lipopolysaccharide (LPS) had markedly decreased efferocytic activity, but the incubation with RvD1 restored the efferocytic ability of the LPS-treated RAW264.7 cells. RvD1 restored the efferocytic activity by down-regulating the LPS-induced upregulation of TNF-α. The inhibitory effect of RvD1 on LPS-induced TNF-α expression was associated with enhanced nuclear localization of p50/p50 homodimer and concomitant reduction of p65/p50 heterodimer in the nucleus. RvD1 triggered extracellular signal-regulated kinase (ERK)-mediated degradation of nuclear factor κB1 (NF-κB1) p105 to generate p50, which was subsequently translocated to the nucleus as p50/p50 homodimer. Knockdown of NF-κB p50 abolished the ability of RvD1 to suppress TNF-α expression and also to restore efferocytosis, suggesting that the replacement of p65/p50 with p50/p50 homodimer in the nucleus is an essential event for RvD1-mediated stimulation of efferocytosis. In a murine peritonitis model, intraperitoneal administration of RvD1 abrogated the zymosan A-induced TNF-α production, thereby stimulating efferocytosis. Taken together, these findings indicate that RvD1 expedites the resolution of inflammation through induction of efferocytosis by p50/p50 homodimer-mediated repression of TNF-α.

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Neuroinflammation has been known to play a critical role in the pathogenesis of Alzheimer’s disease (AD) through amyloidogenesis. However, it is not yet clear whether intraperitoneal (i.p.) injection of lipopolysacchride (LPS) is sufficient for inducing AD model. In this present study, we investigated the amyloidogenic and memorial dysfunctional effects by differential dosages and duration of injected LPS in different aged mice, and acceleration effect of LPS on the amyloidogenesis and memory dysfunction in Tg2576 mice. We found that i.p. injection of LPS at both doses (0.25 and 0.75 mg/kg/day, 7 times) could induce memory dysfunction and amyloidogenesis, but did not show significant difference between two doses. In addition, LPS-injection to three ages (4, 6 and 10 weeks old) of mice revealed memory decline and amyloidogenic effect in the age-dependent manner. To define LPS-induced elevation on memory and amyloidogenesis, we injected LPS (0.25 mg/kg/day, 7 times) to APP over-expressing Tg2576 mice and found that systemic injection of LPS also accelerated memory decline and amyloidogenesis in AD model mice. Therefore, this study showed that LPS-induced memory dysfunction and amyloidogenesis was sufficient through 7 times injection of 0.25 mg/kg/day, and 10 or more weeks old mice were sufficient. In addition, we confirmed LPS-induced acceleration of amyloidogenesis in APP-overexpressing mice, suggests that systemic injection of LPS might be a useful method for neuroinflammation-associated AD.

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The tumor suppressor gene phosphatase and tensin homolog deleted on chromosome 10 gene (PTEN) encodes a 403-aminao acid protein with an evolutionarily conserved dual lipid and protein phosphatase domain at the NH2 terminus. Germ line mutations in PTEN confer elevated risks in the development of many cancers. PTEN is well documented in antagonizing the phosphoinosital-3-kinase (PI3K)/AKT signaling in the cytoplasm. Here, we reveal novel functions for PTEN in the DNA-damage response. We show phosphorylated PTEN-Ser380 recruitments to nuclear in a sprinkle manner and colocations with ATM-pSer-1981 and γ-H2AX at the DNA damage sites. Using large-scale chromatin relaxation mammalian cell model, we find PTEN induces large-scale chromatin decondensation, the protein phosphatase activity and phosphorylation statue of the PTEN-Ser380 are essential for chromatin unfolding. In addition, the lipid and protein phosphatase domain significantly increases the extent of chromatin unfolding as compared with wild type PTEN. Inhibition of ATM significantly down-regulates the phosphorylation level of PTEN and impairs the chromatin remodeling activity. Our results, showing that PTEN couples to DNA damage response in chromatin remodeling way, have important implications for better understanding of PTEN’s tumor-suppressor function and also reveal the potential target for drug design.

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