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AIM: Infants with fetal growth restriction (FGR) are at an increased risk of perinatal morbidity and mortality. The long noncoding RNA H19 gene is expressed abundantly in placental villi and recent studies suggest that it regulates FGR. However, the role of H19 in the FGR placenta remains unclear. This study aimed to clarify the relationship between H19 expression and FGR using normotensive placentas after 34 weeks of gestation. METHODS: Formalin-fixed paraffin-embedded tissues from human placentas collected from pregnancies resulting in small for gestational age (SGA) and appropriate for gestational age (AGA) newborns were used. The histopathological features of placenta tissues, such as villous stromal fibrosis, the numbers of terminal villi, villous vessels and cytotrophoblasts were analyzed using hematoxylin and eosin, Masson's trichrome staining and immunostaining. The localization and expression of H19 in the placentas were demonstrated by in situ hybridization and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. Moreover, the expression levels of H19-regulated molecules such as IGF2 and decorin (DCN) were measured by RT-qPCR. RESULTS: Histopathological features of the placental villous were not different between placentas associated with SGA and AGA. H19 localized to the villous stroma, endothelial cells and cytotrophoblasts. Moreover, the expression level of H19 in SGA placentas was significantly lower than that in AGA placentas. The expression levels of IGF2 and DCN in SGA placentas tended to be lower than those in AGA placentas similarly to H19. CONCLUSION: This study highlights the potential importance of regulatory events mediated by H19 in SGA placentas without histopathological abnormalities in late pregnancy.

DOI： 10.1111/jog.14260

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DOI： 10.3892/ijo.2019.4895.

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DOI： 10.1177/0300060519873481.

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DOI： 10.1016/j.bbrc.2019.09.033.

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DOI： 10.1021/acs.chemrestox.9b00017

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Toll‑like receptor 4 (TLR4), a key regulator of the innate immune system, is expressed not only in immune cells, but also in a number of cancer cells. A biological role for TLR4 in cutaneous squamous cell carcinoma (SCC), however, is unclear. In this study, we first examined TLR4 expression and localization in cases of SCC, actinic keratosis (AK) and Bowen's disease (BD) by immunohistochemistry. TLR4 expression was significantly higher in the SCC than in the AK or BD tissues. We then determined the TLR4 expression level in vivo, in 3 histological subtypes of SCC. TLR4 expression in poorly differentiated SCC was significantly lower compared with that of the moderately and well‑differentiated type. In addition, the CD44 immunoreactivity tended to be high in the cell membrane of poorly differentiated SCC. Of note, poorly differentiated SCC is a risk factor of unfavorable outcomes in affected patients. We then assessed the biological role of TLR4 in HSC‑1 and HSC‑5 SCC cells and HaCaT human keratinocytes. TLR4 knockdown by transfection with siRNA accelerated HSC‑1 and HaCaT cell migration and invasion compared to the control siRNA‑transfected cells. TLR4 knockdown resulted in an increased CD44 expression and in an enhanced filopodia protrusion formation, particularly in HSC‑1. On the whole, these results suggest that a reduced TLR4 expression enhances the malignant features in SCC cases and cultured SCC cell lines. TLR4 may thus play an anti‑tumor role in cutaneous SCC.

DOI： 10.3892/ijo.2019.4790

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DOI： 10.3892/or.2019.6999

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Protein disulfide-isomerase A3 (PDIA3) is a chaperone protein that modulates folding of newly synthesized glycoproteins and responds to endoplasmic reticulum (ER) stress. Previous studies reported that increased expression of PDIA3 in hepatocellular carcinoma (HCC) is a marker for poor prognosis. However, the mechanism remains poorly understood. The aim of the present study, therefore, was to understand the role of PDIA3 in HCC development. First, immunohistochemical staining of tissues from 53 HCC cases revealed that HCC tissues with high PDIA3 expression exhibited a higher proliferation index and contained fewer apoptotic cells than those with low expression. In addition, the knockdown of PDIA3 significantly inhibited cell proliferation and induced apoptosis in HCC cell lines. These results suggest that PDIA3 regulates cell proliferation and apoptosis in HCC. An examination of whether PDIA3 knockdown induced apoptosis through ER stress revealed that PDIA3 knockdown did not increase ER stress marker, 78 kDa glucose-regulated protein, in HCC cell lines. Furthermore, the association between PDIA3 and the signal transducer and activator of transcription 3 (STAT3) signaling pathway were investigated in vitro and in vivo. Immunofluorescence staining and co-immunoprecipitation experiments revealed colocalization and binding, respectively, of PDIA3 and STAT3 in HCC cell lines. The knockdown of PDIA3 decreased the levels of phosphorylated STAT3 (P-STAT3; Tyr705) and downstream proteins of the STAT3 signaling pathway: The anti-apoptotic proteins (Bcl-2-like protein 1, induced myeloid leukemia cell differentiation protein Mcl-1, survivin and X-linked inhibitor of apoptosis protein). In addition, PDIA3 knockdown provided little inhibitory effect on cell proliferation in HCC cell lines treated with AG490, a tyrosine-protein kinase JAK/STAT3 signaling inhibitor. Finally, an association was demonstrated between PDIA3 and P-STAT3 expression following immunostaining of 35 HCC samples. Together, the present data suggest that PDIA3 promotes HCC progression through the STAT3 signaling pathway.

DOI： 10.3892/ijo.2019.4710

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DOI： 10.4172/2157-7099.1000527

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

The glycolytic inhibitor 2-deoxy-D-glucose (2DG) causes energy starvation, affecting cell viability in a wide range of cancer cell lines. To determine the action of 2DG in pancreatic cancer, we performed proteomic analysis of pancreatic cancer cell line after 2DG treatment. Eighty proteins showed differential expression and among these, proteins involved in phosphohexose metabolism were upregulated. Up-regulation of glutamine: fructose 6-phosphate aminotransferase 1 (GFAT1), which belongs to the hexosamine biosynthesis pathway (HBP) that produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to maintain glycoprotein, was validated by evaluation of mRNA and protein levels. Therefore, we assessed the amounts of total N-glycoproteins. Unexpectedly, we found a reduction of total N-glycoproteins and phosphorylation of GFAT1 by AMP-activated protein kinase (AMPK). These data may shed light on HBP dysfunction. Furthermore, we found endoplasmic reticulum (ER) stress accompanied by increased expression of ER stress markers, such as glucose response protein 78 (GRP78) and C/EBP-homologous protein (CHOP), in 2DG-treated cells. Moreover, the additive activation of AMPK by metformin (Met) synergistically enhanced the reduction of protein N-glycosylation and cell growth inhibition in the presence of 2DG. These results suggest that 2DG reduces N-glycosylation of proteins following the increase of phosphorylation of GFAT1 and results in the inhibition of cell growth mediated by ER stress in pancreatic cancer cells.

DOI： 10.1016/j.bbrc.2018.05.041

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DOI： 10.3892/ijo.2018.4238

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

The prognosis of hepatocellular carcinoma (HCC) is unfavorable following complete tumor resection. The aim of the present study was to identify a molecule able to predict HCC prognosis through comprehensive protein profiling and to elucidate its clinicopathological significance. Comprehensive protein profiling of HCC was performed by liquid chromatography-tandem mass spectrometry. Through the bioinformatic analysis of proteins expressed differentially in HCC and non-HCC tissues, protein disulfide-isomerase A3 (PDIA3) was identified as a candidate for the prediction of prognosis. PDIA3 expression was subsequently examined in 86 cases of HCC by immunostaining and associations between PDIA3 expression levels and clinicopathological characteristics were evaluated. The Ki-67 index and apoptotic cell death of carcinoma cells were examined by immunostaining and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay in 24 cases. The results demonstrated that PDIA3 was expressed in all 86 HCC cases; 56 HCC cases (65%) exhibited high expression of PDIA3 and 30 (35%) exhibited low expression. The disease-free and overall survival times of HCC patients with high PDIA3 expression were significantly shorter than in HCC patients with low expression. Furthermore, increased expression of PDIA3 was associated with an elevated Ki-67 index, indicating increased cancer cell proliferation and a reduction in apoptotic cell death. Taken together, these results suggest that PDIA3 expression is associated with tumor proliferation and decreased apoptosis in HCC, and that increased expression of PDIA3 predicts poor prognosis. PDIA3 may therefore be a key molecule in the development of novel targeting therapies for patients with HCC.

DOI： 10.3892/ol.2016.5304

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

In the histopathological diagnosis of cutaneous tumors, the differential diagnosis of squamous cell carcinoma (SCC) with crateriform architecture and keratoacanthoma (KA) is often difficult so an accurate understanding of the biological features and the identification of reliable markers of SCC and KA are crucial issues. Insulin-like growth factor 2 mRNA-binding protein-3 (IGF2BP3, also known as IMP3) is thought of as a bona fide oncofetal protein, which is overexpressed and is involved in cell proliferation, migration, and invasion in several kinds of tumors. However, the role of IMP3 in cutaneous SCC and KA has not been well studied. Therefore, we focused on studying the biological functions of IMP3 in SCC and KA. In human skin SCC cell lines, HSC-1 and HSC-5, and the human keratinocyte cell line, HaCaT, IMP3 mRNA levels were significantly higher than that of normal human skin. The knockdown of IMP3 expression reduced the proliferation of HSC-1, and significantly reduced invasion by HSC-1 and HSC-5. In contrast, the knockdown of IMP3 did not significantly affect invasion by HaCaT cells. In immunohistochemical studies of SCC and KA tissues, the Ki-67 labeling index (LI) of the suprabasal cell layer was significantly higher in SCC, compared with KA tissues and the tumor-free margin (TFM) adjacent to SCC and KA. Most SCC tissues stained strongly positive for IMP3, but KA tissues and TFM were mostly negative for IMP3. The Ki-67 LI of the IMP3-positive group was significantly higher than that of the IMP3-negative group in the suprabasal cell layer of SCC. These results suggest that IMP3 plays an important role in proliferation and, more significantly, in the invasion of SCC, and may be a suitable marker for the histopathological diagnosis of SCC with a crateriform architecture and KA. Furthermore, IMP3 may potentially be a new therapeutic target for SCC.

DOI： 10.3892/ijo.2016.3323

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Obesity is a risk factor for colorectal cancer. The accumulation of abdominal fat tissue causes abundant reactive oxygen species production through the activation of NADPH oxidase due to excessive insulin stimulation. The enzyme NADPH oxidase catalyzes the production of reactive oxygen species and evokes the initiation and progression of tumorigenesis. Apocynin is an NADPH oxidase inhibitor that blocks the formation of the NADPH oxidase complex (active form). In this study, we investigated the effects of apocynin on the development of azoxymethane-induced colonic aberrant crypt foci in obese KK-A(y) mice and on the development of intestinal polyps in Apc mutant Min mice. Six-week-old KK-A(y) mice were injected with azoxymethane (200 μg/mouse once per week for 3 weeks) and given 250 mg/L apocynin or 500 mg/L apocynin in their drinking water for 7 weeks. Six-week-old Min mice were also treated with 500 mg/L apocynin for 6 weeks. Treatment with apocynin reduced the number of colorectal aberrant crypt foci in KK-A(y) mice by 21% and the number of intestinal polyps in Min mice by 40% compared with untreated mice. Both groups of mice tended to show improved oxidation of serum low-density lipoprotein and 8-oxo-2'-deoxyguanosine adducts in their adipose tissues. In addition, the inducible nitric oxide synthase mRNA levels in polyp tissues decreased. Moreover, apocynin was shown to suppress nuclear factor-κB transcriptional activity in vitro. These results suggest that apocynin and other NADPH oxidase inhibitors may be effective colorectal cancer chemopreventive agents.

DOI： 10.1111/cas.12801

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We report a case of Peutz-Jeghers syndrome (PJS) in a 33-year-old female patient with synchronous uterine cervical minimal deviation adenocarcinoma (MDA) and gastric type adenocarcinoma (GTA). The patient was diagnosed with PJS at the age of 10. At the time of consultation, she complained of watery discharge. Magnetic resonance imaging of the pelvis showed a poorly circumscribed mass in the uterine cervix. Histologically, both MDA and GTA components, as well as their transitional area, were observed. Both components were diffusely positive for MUC6, CK7 and, robustly, for p16. Moreover, the components were negative for ER, PgR and CEA, while HIK1083 and CK20 positive cells were found focally. Ki-67 labeling index in the MDA component was 5% while that in the GTA component was 50%. This case of GTA accompanied by MDA in a patient with PJS is distinct from the single previously-reported comparable case of which we are aware, with respect to the overexpression of p16 protein, an event considered rare in these tumors, and the continuity between the MDA and GTA components. This continuity favors the hypothesis that GTA arises from the dedifferentiation of MDA.

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DOI： 10.3892/ijo.2015.2858

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Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields; however, its toxicological properties are not well documented. In our previous report, MGT showed genotoxicity in both in vitro and in vivo assay systems, and it was suggested that inflammatory responses exist behind the genotoxicity. To further clarify mechanisms underlying the genotoxicity, a comprehensive DNA adduct (DNA adductome) analysis was conducted using DNA samples derived from the lungs of mice exposed to MGT. In total, 30 and 42 types of DNA adducts were detected in the vehicle control and MGT-treated groups, respectively. Principal component analysis (PCA) against a subset of DNA adducts was applied and several adducts, which are deduced to be formed by inflammation or oxidative stress, as the case of etheno-deoxycytidine (εdC), revealed higher contributions to MGT exposure. By quantitative-LC-MS/MS analysis, εdC levels were significantly higher in MGT-treated mice than those of the vehicle control. Taken together with our previous data, it is suggested that inflammatory responses might be involved in the genotoxicity induced by MGT in the lungs of mice.

DOI： 10.3390/ijms16023474

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Lipid peroxidation directly reacts with DNA and produces various exocyclic etheno-base DNA adducts, some of which are considered to contribute to carcinogenesis. However, the system for repairing them in humans is largely unknown. We hypothesized that etheno-DNA adducts are repaired by base excision repair initiated by DNA glycosylase. To test this hypothesis, we examined the activities of the DNA glycosylase proteins OGG1, SMUG1, TDG, NEIL1, MUTYH, NTH1, MPG, and UNG2 against double-stranded oligonucleotides containing 1,N-6-ethenoadenine (epsilon A), 3,N-4-ethenocytosine (epsilon C), butanone-ethenocytosine (B epsilon C), butanone-ethenoguanine (B epsilon G), heptanone-ethenocytosine (H epsilon C), or heptanone-ethenoguanine (H epsilon G) using a DNA cleavage assay. We found that TDG is capable of removing thymine that has mispaired with epsilon C, B epsilon C, B epsilon G, H epsilon C, or H epsilon G in vitro. We next examined the effect of TDG against etheno-DNA adducts in human cells. TDG-knockdown cells exhibited the following characteristics: (a) higher resistance to cell death caused by the induction of etheno-DNA adducts; (b) lower repair activity for epsilon C; and (c) a modest acceleration of mutations caused by epsilon C, compared with the rate in control cells. All these characteristics suggest that TDG exerts a repair activity against etheno-DNA adducts in human cells. These results suggest that TDG has novel repair activities toward etheno-DNA adducts. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2014.07.044

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Nanomaterials are useful for their characteristic properties and are commonly used in various fields. Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields, whereas their toxicological properties are not well documented. A safety assessment is thus urgently required for MGT, and genotoxicity is one of the most serious concerns. In the present study, we examined genotoxic effects of MGT using mice and revealed that DNA damage analyzed by a comet assay in the lungs of imprinting control region (ICR) mice intratracheally instilled with a single dose of 0.05 or 0.2 mg/animal of MGT was approximately two-to three-fold higher than that of vehicle-control animals. Furthermore, in gpt delta transgenic mice, gpt mutant frequency (MF) in the lungs of the group exposed to four consecutive doses of 0.2 mg MGT was significantly higher than in the control group. Mutation spectrum analysis showed that base substitutions were predominantly induced by MGT, among which G:C to A:T transition and G:C to T:A transversion were the most significant. To clarify the mechanism of mutation caused by MGT, we analyzed the formation of DNA adducts in the lungs of mice exposed to MGT. DNA was extracted from lungs of mice 3, 24, 72 and 168 h after intratracheal instillation of 0.2 mg/body of MGT, and digested enzymatically. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and lipid peroxide-related DNA adducts were quantified by stable isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS). Compared with vehicle control, these DNA adduct levels were significantly increased in the MGT-treated mice. In addition to oxidative stress-and inflammation related-DNA adduct formations, inflammatory cell infiltration and focal granulomatous formations were also observed in the lungs of MGT-treated mice. Based on these findings, it is suggested that inflammatory responses are probably involved in the genotoxicity induced by MGT in the lungs of mice.

DOI： 10.3390/nano4010175

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Language：English   Publisher：ELSEVIER IRELAND LTD  

Metabolic syndrome (MS) and related disorders, including cancer, are steadily increasing in most countries of the world. However, mechanisms underlying the link between MS and colon carcinogenesis have yet to be fully elucidated. In this review article we focus on the relationships between various individual associated conditions (obesity, dyslipidemia, diabetes mellitus type 2 and hypertension) and colon cancer development, and demonstrate probable related factors revealed by in vivo and in vitro studies. Furthermore, molecules suggested to be involved in cancer promotion are addressed, and the potential for cancer prevention by targeting these molecules is discussed. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.canlet.2012.10.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

The genotoxic effects of multi-walled carbon nanotubes (MWCNTs) were examined by using in vitro and in vivo assays. MWCNTs significantly induced micronuclei in A549 cells and enhanced the frequency of sister chromatid exchange (SCE) in CHO AA8 cells. When ICR mice were intratracheally instilled with a single dose (0.05 or 0.2 mg/animal) of MWCNTs, DNA damage of the lungs, analysed by comet assay, increased in a dose-dependent manner. Moreover, DNA oxidative damage, indicated by 8-oxo-7,8-dihydro-2'-deoxyguanosine and heptanone etheno-deoxyribonucleosides, occurred in the lungs of MWCNT-exposed mice. The gpt mutation frequencies significantly increased in the lungs of MWCNT-treated gpt delta transgenic mice. Transversions were predominant, and G: C to C: G was clearly increased by MWCNTs. Moreover, many regions immunohistochemically stained for inducible NO synthase and nitrotyrosine were observed in the lungs of MWCNT-exposed mice. Overall, MWCNTs were shown to be genotoxic both in in vitro and in vivo tests; the mechanisms probably involve oxidative stress and inflammatory responses.

DOI： 10.3109/17435390.2012.674571

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

We examined the genotoxicity of magnetite nanoparticles (primary particle size: 10 nm) on human A549 and Chinese hamster ovary (CHO) AA8 cells. Six hours' treatment with the particles dose-dependently increased the frequency of micronuclei (MN) in the A549 and CHO AA8 cells up to 5.2% and 5.0% at a dose of 200 mu g/ml (34 mu g/cm(2)), respectively. In A549 cells, treatment with the nanoparticles (2 mu g/ml) for 1 hr induced H2AX phosphorylation, which is suggestive of DNA double strand breaks (DSB). Treating CHO AA8 cells with 2 mu g/ml (0.34 mu g/cm(2)) magnetite for 1 hour resulted in a five times higher frequency of sister chromatid exchange (SCE) than the control level. We detected reactive oxygen species (ROS) in CHO cells treated with the particles. These findings indicate that magnetite nanoparticles induce ROS in mammalian cells, leading to the direct or indirect induction of DSB, followed by clastogenic events including MN and SCE.

DOI： 10.2131/jts.38.503

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Language：English   Publisher：The Japanese Environmental Mutagen Society  

Nanomaterials are being utilized for many kinds of industrial products, and the assessment of genotoxicity and safety of nanomaterials is therefore of concern. In the present study, we examined the genotoxic effects of fullerene (C₆₀) and kaolin using in vitro and in vivo genotoxicity systems. Both nanomaterials significantly induced micronuclei and enhanced frequency of sister chromatid exchange (SCE) in cultured mammalian cells. When ICR mice were intratracheally instilled with these nanomaterials, DNA damage of the lungs increased significantly that of the vehicle control. Formation of DNA adducts in the lungs of mice exposed to nanomaterials were also analyzed by stable isotope dilution LC-MS/MS. 8-Oxodeoxyguanosine and other lipid peroxide related adducts were increased by 2- to 5-fold in the nanomaterial-exposed mice. Moreover, multiple (four consecutive doses of 0.2 mg per animal per week) instillations of C₆₀ or kaolin, increased gpt mutant frequencies in the lungs of gpt delta transgenic mice. As the result of mutation spectrum analysis, G:C to C:G transversions were commonly increased in the lungs of mice exposed to both nanomaterials. In addition, G:C to A:T was increased in kaolin-exposed mice. In immunohistochemical analysis, many regions of the lungs that stained positively for nitrotyrosine (NT) were observed in mice exposed to nanomaterials. From these observations, it is suggested that oxidative stress and inflammatory responses are probably involved in the genotoxicity induced by C₆₀ and kaolin.<br>

DOI： 10.3123/jemsge.33.14

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Other Link： https://jlc.jst.go.jp/DN/JALC/00363915935?from=CiNii

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Nanomaterials are being utilized for many kinds of industrial products, and the assessment of genotoxicity and safety of nanomaterials is therefore of concern. In the present study, we examined the genotoxic effects of fullerene (C 60) and kaolin using in vitro and in vivo genotoxicity systems. Both nanomaterials significantly induced micronuclei and enhanced frequency of sister chromatid exchange (SCE) in cultured mammalian cells. When ICR mice were intratracheally instilled with these nanomaterials, DNA damage of the lungs increased significantly that of the vehicle control. Formation of DNA adducts in the lungs of mice exposed to nanomaterials were also analyzed by stable isotope dilution LC-MS/MS. 8-Oxodeoxyguanosine and other lipid peroxide related adducts were increased by 2- to 5-fold in the nanomaterial-exposed mice. Moreover, multiple (four consecutive doses of 0.2 mg per animal per week) instillations of C 60 or kaolin, increased gpt mutant frequencies in the lungs of gpt delta transgenic mice. As the result of mutation spectrum analysis, G:C to C:G transversions were commonly increased in the lungs of mice exposed to both nanomaterials. In addition, G:C to A:T was increased in kaolin-exposed mice. In immunohistochemical analysis, many regions of the lungs that stained positively for nitrotyrosine (NT) were observed in mice exposed to nanomaterials. From these observations, it is suggested that oxidative stress and inflammatory responses are probably involved in the genotoxicity induced by C 60 and kaolin. © The Japanese Environmental Mutagen Society.

DOI： 10.3123/jemsge.33.14

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-N(epsilon)-3-[(hept-1-enyl)-4-hexyl-pyridinium]lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenal-lysine adducts were indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.

DOI： 10.1074/jbc.M109.068023

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Various lines of evidence indicate that the oxidative modification of protein and the subsequent accumulation of the degenerated proteins have been found in cells and tissues during aging, oxidative stress, and in a variety of pathological states. The critical agents that give rise to this protein degeneration may be represented by aldehydes. Although the covalent modification of proteins by aldehydes alone has been well-studied, the effect of reactive oxygen species, such as H2O2, upon aldehyde modification of the protein has received little attention. We have now established a unique protein modification in which H2O2 and, to a lesser extent, alkyl hydroperoxides mediate the binding of alkanals to the lysine residues of protein to generate structurally unusual N-acylation products. Upon the reaction of a lysine-containing peptide, N(alpha)-benzoylglycyl-lysine, with hexanal in the presence of H2O2, a product containing one molecule of hexanal per peptide was detected. On the basis of the chemical and spectroscopic evidence, the product was identified to be the acylation product, N(epsilon)-hexanoyllysine. H2O2 mediated the N-acylation of the lysine derivative by the saturated aldehydes of 1-6 carbons in length. The H2O2-mediated acylation of the protein was immunochemically confirmed by reaction of the proteins with hexanal in the presence of H2O2. Furthermore, the enhanced N-acylations (N-acetylation and N-hexanoylation) were also observed in the kidney of rats exposed to ferric nitrilotriacetate, a well-characterized inducer of oxidative stress. Mechanistic studies using a phosphonium lysine derivative suggest a Baeyer-Villiger-like reaction proceeding through peroxide addition to the aldehyde Schiff base. These data suggest that the hydroperoxides, including H2O2, might be involved not only in the oxidative modification of protein but also in the covalent binding of the saturated aldehydes to proteins under oxidative stress.

DOI： 10.1021/tx800080x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Several lines of evidence indicate that the nonenzymatic oxidative modification of proteins and the subsequent accumulation of the modified proteins have been found in cells during aging and oxidative stress and in various pathological states, including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis. Our previous work suggested the existence of molecular mimicry between antibodies raised against hydroxy-2-nonenal (HNE)-modified protein and anti DNA autoantibodies, a serologic hallmark of systemic lupus erythematosus (SLE). In the present study, we investigated the possible involvement of HNE-modified proteins as the endogenous source of the anti-DNA antibodies. Accumulation of the antigen recognized by the antibody against the HNE-modified protein was observed in the nucleus of almost all of the epidermal cells from patients with autoimmune diseases, including SLE. The SLE patients also showed significantly higher serum levels of the anti- HNE titer than healthy individuals. To determine if a specific anti- DNA response could be initiated by the HNE-derived epitopes, we immunized BALB/c mice with the HNE-modified protein and observed a progressive increase in the anti- DNA response. Moreover, we generated the monoclonal antibodies, showing recognition specificity toward DNA, and found that they can bind to two structurally distinct antigens (i. e. the native DNA and protein-bound 4-oxo-2-nonenal). The findings in this study provide evidence to suspect an etiologic role for lipid peroxidation in autoimmune diseases.

DOI： 10.1074/jbc.M703039200

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DOI： 10.1016/j.bbapap.2007.02.006

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DOI： 10.1107/S1744309106016630

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

4-Hydroxy-2-nonenal (HNE), a racemic mixture of 4R- and 4S-enantiomers, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress. HNE reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. We have previously raised the mAbs, anti-R mAb 310 and anti-S mAb S412, that enantioselectively recognized the R-HNE-histidine and R-HNE-histidine adducts, respectively, and demonstrated the presence of both epitopes in vivo. In the present study, to further investigate the anti-HNE immune response, we analyzed the variable genes and primary structure of these Abs and found that the sequence of R310 was highly homologous to anti-DNA autoantibodies, the hallmark of systemic lupus erythematosus. An x-ray crystallographic analysis of the R310 Fab fragment showed that the R-HNE-histidine adduct binds to a hydrophobic pocket in the antigen-binding site. Despite the structural identity to the anti-DNA autoantibodies, however, R310 showed only a slight crossreactivity with the native double-stranded DNA, whereas the Ab immunoreactivity was dramatically enhanced by the treatment of the DNA with 4-oxo-2-nonenal (ONE), an analog of HNE. Moreover, the 7-(2-oxo-heptyl)-substituted 1,N-2-etheno-type ONE-2'-deoxynucleoside adducts were identified as alternative epitopes of R310. Molecular mimicry between the R-HNE-histidine configurational isomers and the ONE-DNA base adducts is proposed for the dual crossreactivity.

DOI： 10.1073/pnas.0600865103

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Language：Japanese   Publisher：(一社)日本消化器外科学会  

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Language：Japanese   Publisher：(一社)日本内分泌外科学会  

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J-GLOBAL

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Language：Japanese   Publisher：(一社)日本消化器外科学会  

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J-GLOBAL

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Language：English   Publisher：日本癌学会  

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Language：Japanese  

J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SPANDIDOS PUBL LTD  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SPANDIDOS PUBL LTD  

Web of Science

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Language：Japanese   Publisher：日本プロテオーム学会（日本ヒトプロテオーム機構）  

DOI： 10.14889/jhupo.2014.0.84.0

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Language：English   Publisher：日本癌学会  

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：The Environmental Mutagen Society of Japan  

J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：The Environmental Mutagen Society of Japan  

CiNii Books

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J-GLOBAL

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J-GLOBAL

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Presentation type：Poster presentation  

Venue：Tokyo  

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Venue：Tokyo  

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Venue：Sapporo  

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Venue：Tokyo  

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Venue：Sendai  

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Venue：Tokyo  

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Venue：Tokyo  

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Venue：Tokyo  

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Venue：Tokyo  

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Venue：大阪  

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Venue：京都  

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Venue：名古屋  

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Presentation type：Poster presentation  

Venue：神戸   Country：Japan  

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Presentation type：Poster presentation  

Venue：神戸  

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Venue：京都  

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Venue：東京  

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Venue：東京  

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Venue：東京  

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Venue：東京  

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Venue：京都  

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Venue：東京  

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Venue：品川  

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Venue：東京  

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Venue：東京  

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Venue：神戸  

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Venue：神戸  

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Venue：神戸  

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Presentation type：Poster presentation  

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Presentation type：Oral presentation (general)  

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Presentation type：Poster presentation  

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Presentation type：Poster presentation  

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