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Biomedical subjects

Toru Takeuchi

Publications and source records attributed to Toru Takeuchi.

At least 19 recordsLinked to original sources
PubMed2006

Elevated serum bisphenol A levels under hyperandrogenic conditions may be caused by decreased UDP-glucuronosyltransferase activity.

This study was performed to investigate the effect of androgen on the metabolism of bisphenol A (BPA), an endocrine disruptor, in order to clarify the mechanism of the higher levels of serum BPA in men and hyperandrogenemic women compared with normal women. Castrated female rats (OVX) were subcutaneously injected with testosterone propionate (TP) (0.01, 0.1, and 1 mg) every day for 2 weeks. Serum BPA concentrations in OVX rats showed a TP dose-dependent increase and were significantly higher at 0.1 and 1.0 mg of TP. The enzyme reaction of BPA glucuronidation in the rat liver microsomes showed that the ratio of glucuronide in the OVX rats was significantly reduced in a TP dose-dependent manner. Analysis of the mRNA expression of UDP-glucuronosyltransferase 2B1 (UGT2B1) by real-time quantitative RT-PCR revealed that the relative expression level of UGT2B1 mRNA showed a TP dose-dependent decrease. The results of enzyme analyses demonstrated that the ratio of BPA glucuronidation and the expression level of UGT2B1 mRNA were significantly lower under the hyperandrogenemic conditions. The clearance of BPA may be slowed in a TP dose-dependent manner, resulting in an increase of serum BPA concentration under hyperandrogenemic conditions.

Animals↗
PubMed2006

[Hirsutism].

Explore the source record for details and available documents.

Androgen Antagonists↗
PubMed2006

Detection of eight antibodies in cancer patients' sera against proteins derived from the adenocarcinoma A549 cell line using proteomics-based analysis.

To screen cancer for specific autoantibodies, we applied the approach established by Brichory et al., who reported annexins I and II as specific antigens. Solubilized proteins from a cancer cell line (A549) were separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by Western blotting (WB) analysis, in which the sera of individual patients were tested for primary antibodies. We found 11 positive spots on PVDF membrane using a WB/enhanced chemiluminescence detection Kit, and identified eight proteins, such as alpha-enolase, inosine-5'-monophosphate dehydrogenase, aldehyde dehydrogenase, 3-phosphoglycerate dehydrogenase, 3-oxoacid CoA transferase, chaperonin, peroxiredoxin 6 and triosephosphate isomerase, that reacted with these antibodies in patients' sera using MALDI-TOF/TOF. All eight antibodies were not detected in the sera derived from lung tuberculosis and healthy controls.

Adenocarcinoma↗
PubMed2006

4-Nitroquinoline 1-oxide forms 8-hydroxydeoxyguanosine in human fibroblasts through reactive oxygen species.

4-Nitroquinoline 1-oxide (4NQO) is thought to elicit its carcinogenicity by producing DNA adducts after being metabolized to 4-hydroxyaminoquinoline 1-oxide, which forms 8-hydroxydeoxyguanosine (8OHdG), oxidative damage. To determine whether reactive oxygen species (ROS) are involved in the generation of 8OHdG by 4NQO, we used high-performance liquid chromatography and immunohistochemistry to measure the levels of 8OHdG in normal human fibroblasts treated with 4NQO. The extent of ROS induced by 4NQO was determined by using fluorescent probes to detect ROS, electron paramagnetic resonance spectrometry using a cell-free system, and measurement of intracellular glutathione (GSH) levels. In fibroblasts, 4NQO dose dependently increased 8OHdG levels. Hydrogen peroxide (H2O2) and superoxide were detected in cells treated with 4NQO by using dichlorofluorescin diacetate and hydroethidine, respectively. The addition of catalase to culture medium reduced 8OHdG levels and the intensity of dichlorofluorescin fluorescence, while 4NQO generated hydroxyl radicals in the cell-free system. These findings suggest that 4NQO treatment leads to formation of superoxide, H2O2, and hydroxyl radicals, resulting in the production of a substantial amount of 8OHdG in DNA. Neither the level of 8OHdG nor that of GSH had returned to the basal level 24 h after removal of 4NQO even at a concentration as low as 1 microM. Our results suggest that generation of ROS and depletion of GSH in cells are also important factors for the generation of 8OHdG by 4NQO. This paper describes practical and sensitive ways to detect ROS and 8OHdG and discusses a new functional pathway to elicit genotoxicity.

4-Nitroquinoline-1-oxide↗
PubMed2006

Generation of a novel fluorescent product, monochlorofluorescein from dichlorofluorescin by photo-irradiationdagger.

Dichlorofluorescin (DCFH), a widely used fluorescent probe for reactive oxygen species (ROS) was decomposed completely and generated two distinct fluorescent products by photo-irradiation at 254 nm for 30 min. In the previous study, we had shown that one was dichlorofluorescein (DCF), a well known oxidized product of DCFH. In this study we investigated the other product and identified it as monochlorofluorescein (MCF) by 1H-NMR and fast atom bombardment/mass spectrum (FAB/MS) analyses. MCF was generated by photo-irradiation, but not by ROS. On the other hand, DCF was produced by both photo-irradiation and ROS. MCF showed similar fluorescent emission spectrum to DCF, however, its fluorescence intensity was more than that of DCF. The kinetic study suggested that MCF was not generated from DCF but from monochlorofluorescin, which might be generated from DCFH by photo-irradiation.

Chromatography, High Pressure Liquid↗
PubMed2005

Dual role of vitamin C in an oxygen-sensitive system: discrepancy between DNA damage and cell death.

Although vitamin C is considered to act both as pro-oxidant and antioxidant, the mechanisms underlying these actions are still unclear. Using the oxygen-sensitive system of a strict anaerobe, Prevotella melaninogenica, we investigated both the pro-oxidant and antioxidant mechanisms of vitamin C. In the presence of vitamin C, the 8-hydroxydeoxyguanosine (8OHdG) formation induced by oxygen exposure was enhanced, probably due to the action of vitamin C on hydrogen peroxide generated during oxygen exposure: while catalase almost completely suppressed the enhancing effect of vitamin C, 8OHdG formation induced by hydrogen peroxide was enhanced by vitamin C. By contrast, the presence of vitamin C inhibited bacterial cell death, membrane damage, and lipid peroxidation induced by oxygen exposure. Sodium azide showed similar effects to vitamin C, thus the antioxidant action of vitamin C may be due to its quenching of the singlet oxygen generated in this system. Both the pro-oxidant and antioxidant effects of vitamin C were observed only in acidic conditions.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2005

Habitual exercise induced resistance to oxidative stress.

We investigated whether habitual exercise (HE) modulates levels of oxidative DNA damage and responsiveness to oxidative stress induced by renal carcinogen Fe-nitrilotriacetic acid (Fe-NTA). During a ten week protocol, two groups of rats either remained sedentary or underwent swimming for 15--60 min per day, 5 days per week, with or without a weight equivalent to 5% of their body weight. Then we injected Fe-NTA and sacrificed the rats 1 h after the injection. We determined the activity of superoxide dismutase (SOD) in diaphragm and kidney, evaluated levels of 8-hydroxydeoxyguanosine (8OHdG), catalase, and glutathione peroxidase, and assayed OGG1 protein levels in kidney. SOD activity in the diaphragm and kidney was increased in HE rats. By itself, HE had no effect on the level of 8OHdG, but it did significantly suppress induction of 8OHdG by Fe-NTA, and the amount of suppression correlated with intensity of exercise. These results suggest that HE induces resistance to oxidative stress and, at least at the initiation stage, inhibits carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2005

Generation of 8-hydroxydeoxyguanosine from DNA using rat liver homogenates.

In relation to carcinogenesis, aging and other pathologic conditions, urinary 8-hydroxydeoxyguanosine (8OHdG) is widely used as a marker for evaluating the effect of oxidative stress on DNA. Because no reports have described how 8OHdG is generated from DNA in vivo or by biological materials, and how it is excreted into urine, the authors investigated the generation of 8OHdG from DNA, using rat liver homogenate. Oxidatively damaged DNA samples containing different levels of 8OHdG were prepared using ultraviolet irradiation with three different concentrations of riboflavin. Following incubation of damaged DNA samples with rat liver homogenates, the generation of 8OHdG from the DNA was determined using high-performance liquid chromatography with electrochemical detection after ultrafiltration of the incubation mixtures. The generation of 8OHdG was also tested with an anti-8OHdG antibody. The quantity of 8OHdG generated from the DNA by rat liver homogenates was dependent on the 8OHdG levels in the DNA: almost all 8OHdG in the DNA was released as 8OHdG by rat liver homogenates. Generation of 8OHdG correlated with the degradation of DNA. Interestingly, the generated 8OHdG was stable in the presence of rat liver homogenates, whereas deoxyguanosine (dG) rapidly disappeared in the same conditions. Less than 1/10,000 of dG was converted to 8OHdG when dG was incubated with rat liver homogenate. Incubation of 8-hydroxyguanine with rat liver homogenates did not generate 8OHdG. These findings suggest that most of the 8OHdG in DNA is released as 8OHdG during DNA degradation and that, because of its stability, 8OHdG is excreted into urine, thus providing a convenient measure of oxidative damage to DNA.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2005

Radical species in DNA strand-cleavage caused by dihydropyrazines.

Dihydropyrazines (DHPs), which generate hydroxyl and carbon-centered radicals, cleaved DNA single-strand. It is new knowledge that DHPs were recently determined to produce 8-hydroxydeoxyguanosine. The remarkable increase in the DNA strand-cleavage activity upon the addition of Cu2+ suggests that the primary reactive species is carbon-centered radicals rather than the hydroxyl radical generated the initiation reaction. This proposal was in fair agreement with of the observed carbon-centered radical signal intensity, but an effect was not observed with the increase in the hydroxyl radical signal intensity.

DNA, Single-Stranded↗
PubMed2005

Differentiation, distribution, and chemical state of intracellular trace elements in LAD2 mast cell line.

Oxidation state changes of metallic ions are involved in the generation and biological defense against reactive oxygen species. The relationship between allergy and oxidative damage by metallic elements was studied by X-ray fluorescence analysis using a mast cell line. The distribution of metallic elements is changed by the induction of reactive oxygen species. In mast cells, the degranulation leading to antigen or calcium ionophore stimulation is related to excessive accumulation of iron and to its chemical state. X-ray absorption near-edge structure spectroscopy showed that the oxidation state of iron in the cells shifted from Fe(II) to Fe(III) in degranulation. This finding might have implications for understanding the mechanisms involved in IgE-mediated cell responses as seen in allergic reaction.

Calcium↗
PubMed2005

[A case of interstitial pneumonia associated with dermatomyositis effectively treated with cyclosporin-A and cyclophosphamide pulse].

A 46-year-old male demonstrated edematous fingers on both hands in November 2003, and interstitial pneumonia was noted on chest X-ray during a medical check-up in December 2003. Since muscular weakness and fever developed thereafter, and interstitial pneumonia was aggravated on chest X-ray and CT, the patient was admitted to our hospital on March 25, 2004. Heliotrope-like erythema, and Gottron's sign were noted. Laboratory findings showed the following ; LDH 876 U/l ; CK 224 U/l ; CRP 5.68 mg/dl ; and KL-6 3270 U/ml. Autoantibodies such as anti-Jo-1 antibody were all negative. Chest X-ray and CT showed ground-glass opacity in the bilateral lower dorsal regions of the lung, and reduced volume of the inferior lobe. He was diagnosed as having dermatomyositis (DM) associated with progressive interstitial pneumonia. Although a combination of steroid pulse therapy and Cyclosporin-A were administered, the pulmonary lesions became aggravated. Additional intravenous Cyclophosphamide (IVCY) was initiated on the 6th hospital day, and interstitial pneumonia was markedly improved. Cases of progressive interstitial pneumonia associated with DM that are negative for anti-Jo-1 antibody and show a low ratio of CK/LDH are resistant to various treatments. Our case suggested that combination therapy with steroid, Cyclosporin-A, and IVCY is useful for the treatment of progressive interstitial pneumonia with DM.

Cyclophosphamide↗
PubMed2004

Gender difference in serum bisphenol A levels may be caused by liver UDP-glucuronosyltransferase activity in rats.

Gender difference in human bisphenol A (BPA) concentrations was revealed by determining serum BPA. We studied the serum concentrations and the metabolism of BPA in rats by an HPLC system. Rat serum BPA concentrations were significantly higher in males (24.9+/-7.38 ng/ml, P=0.026, n=10) than in females (8.27+/-3.11 ng/ml, n=10), as in humans. The resultant enzyme reaction products of BPA glucuronidation in the rat liver microsomes fraction were analyzed by an HPLC system. The ratio of BPA glucuronidation in the microsome reaction was significantly higher (P=0.015) in female than in male rats. The mRNA expression of UDP-glucuronosyltransferase 2B1 (UGT2B1), an isoform of UGT related to BPA glucuronidation, in the rat liver was analyzed by a real-time quantitative RT-PCR. The relative expression level of UGT2B1 mRNA was significantly higher (P<0.001) in female than in male rat livers. The gender difference in serum BPA concentrations may be explained by the difference in clearance based on the UGT activities.

Animals↗
PubMed2004

Oxidative damages in chronic inflammation of a mouse autoimmune disease model.

Reactive oxygen species are generated in many types of inflammation; it is unclear, however, if inflammation leads to oxidative damage of DNA, proteins and lipids within the inflamed tissues. In this study, we used mice that are homozygous for the alymphoplasia (aly) mutation as a model to determine if inflammation induces oxidative damage in liver and pancreas. We found that 8-hydroxy-2'-deoxyguanosine (8OHdG), which is a product of oxidative DNA damage, increases with age in livers and pancreata of C57BL/6aly/aly (aly/aly) and C57BL/6 wild type (WT) mice. The 8OHdG levels in liver, but not in pancreas, of aged aly/aly mice were significantly higher than those in age-matched WT mice. We showed that aging enhances oxidative protein damage, as measured by carbonylated protein contents, in the pancreata of WT but not aly/aly mice. In contrast, neither aging nor inflammation was associated with lipid damage, as measured by thiobarbituric acid-reactive substances (TBARS), in aly/aly or WT mice. Our results indicate that chronic inflammation in liver but not pancreas leads to increased oxidative damage to DNA, but not to lipids and proteins in aly/aly mouse model.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2004

Positive relationship between androgen and the endocrine disruptor, bisphenol A, in normal women and women with ovarian dysfunction.

This study was performed to investigate the serum levels of bisphenol A (BPA), an endocrine disruptor, in women with ovarian dysfunction and obesity. Fasting serum samples were obtained from 19 non-obese and 7 obese women with normal menstrual cycles: 7 patients with hyperprolactinemia, 21 patients with hypothalamic amenorrhea, and 13 non-obese and 6 obese patients with polycystic ovary syndrome (PCOS). BPA was measured by an enzyme-linked immunosorbent assay. BPA was detected in all human sera. Serum BPA concentrations were significantly higher in both non-obese and obese women with polycystic ovary syndrome (1.05 +/- 0.10 ng/ml, 1.17 +/- 0.16 ng/ml; p<0.05, respectively) and obese normal women (1.04 +/- 0.09 ng/ml, p<0.05) compared with those in non-obese normal women (0.71 +/- 0.09 ng/ml). There was no difference among women with hyperprolactinemia, women with hypothalamic amenorrhea, and non-obese normal women. There were significant positive correlations between serum BPA and total testosterone (r = 0.391, p<0.001), free testosterone (r = 0.504, p<0.001), androstenedione (r = 0.684, p<0.001), and DHEAS (r = 0.514, p<0.001) concentrations in all subjects. These findings show that there is a strong relationship between serum BPA and androgen concentrations, speculatively due to the effect of androgen on the metabolism of BPA.

Androgen Antagonists↗
PubMed2004

Differences in serum bisphenol a concentrations in premenopausal normal women and women with endometrial hyperplasia.

Exposure to endocrine disrupting chemicals (EDCs) has been raised in relation to its potential for adverse health outcomes. Bisphenol A (BPA) is an estrogenic EDC widely found in plastic products. We determined BPA concentrations in premenopausal women by an enzyme-linked immunosorbent assay and evaluated possible linkage between its contamination levels and endometrial hyperplasia, an estrogen-related disorder of the uterus. It has been implied that higher levels of BPA, which binds to estrogen receptor and plays estrogenic roles may, enhance endometrial hyperplasia. Serum BPA was detectable in all subjects and its concentrations in healthy controls with normal endometrium were 2.5 +/- 1.5 ng/ml (mean +/- SD). BPA levels in patients with simple endometrial hyperplasia with benign nature were 2.9 +/- 2.0 ng/ml and were not significantly different from the controls. Unexpectedly, BPA levels in patients with complex endometrial hyperplasia with malignant potential were 1.4 +/- 0.4 ng/ml and significantly lower compared to both control and simple endometrial hyperplasia groups. In addition, we measured the serum BPA levels in postmenopausal endometrial cancer patient (1.4 +/- 0.5 ng/ml), which were also significantly lower than control and simple endometrial hyperplasia groups. These findings suggest the presence of associations between BPA exposure and complex endometrial hyperplasia and endometrial cancer. The mode of action of BPA may be more complex than expected and the contradictory results may serve as a clue to addressing the mechanisms of linkage between occurrence of estrogen-dependent diseases and endocrine disruption.

Benzhydryl Compounds↗
PubMed2003

Method to overcome photoreaction, a serious drawback to the use of dichlorofluorescin in evaluation of reactive oxygen species.

Non-fluorescent dichlorofluorescin (DCFH) was converted to fluorescent products by photo-irradiation during observations with spectrofluorometer and fluorescence microscopy. Photo-irradiation of DCFH at 250, 300, 330, 400, 500, or 600 nm generated fluorescent dichlorofluorescein (DCF), an oxidation product of DCFH, and an unrecognized fluorescent product. The ratio of the unknown product to DCF varied from 0.15 to 8.21 depending on wavelength. Although reactive oxygen species scavengers, such as catalase, superoxide dismutase, and sodium azide, did not suppress the increase in non-specified fluorescence, reagents such as ascorbic acid, mercaptopropionyl glycine, and methoxycinnamic acid, in a cell-free system, almost completely suppressed it with little effect on the fluorescence of DCF. Meanwhile, ascorbic acid also suppressed non-specified fluorescence in cells, but not completely. At low concentrations of DCFH, the speed of increasing fluorescence was considerably retarded, to such a degree that the fluorescence increase in cells during fluorescence microscopic observation was negligible. The addition, at the time of evaluation, of the above reagents to cell-free systems and, in cell systems, reducing the concentration of DCFH, effectively suppressed the photoreaction of DCFH.

Antioxidants↗
PubMed2003

Role of Fas/Fas ligand-mediated apoptosis in murine contact hypersensitivity.

Apoptosis plays an important role in immune responses, but little is known about its involvement in contact hypersensitivity (CH). In this study, we have investigated the role of Fas/Fas ligand (FasL)-mediated apoptosis in the pathogenesis of CH. Mice were sensitized by one topical application of 100 microl of 3% oxazolone to shaved skin of the abdomen. Six days later, CH was provoked by challenging both sides of sensitized mouse right ear with 15 microl of 1% oxazolone. Using a DNA ladder assay, we found that apoptosis was induced in the skin of oxazolone-sensitized mice 24-96 h after allergen challenge. Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) apoptosis flow cytometric assay showed that early apoptotic CD4(+) T cells (annexin V-FITC(+)PI(-)), but not late apoptotic CD4(+) T cells (annexin V-FITC(+)PI(+)), increased in the inflamed skin of mice with CH. Moreover, the expressions of mRNAs for T helper (Th2) cytokine (interleukin (IL)-4), Th1 cytokine (interferon (IFN)-gamma) and proapoptotic molecules (Bax, Fas, FasL and IL-1beta-converting enzyme (ICE)/caspase-1) were significantly elevated in the oxazolone-sensitized mouse skin 6-72 h after allergen challenge. Dramatic increase in IL-10 mRNA was only observed in the sensitized mouse skin 6 and 12 h after allergen challenge. Furthermore, CH was significantly inhibited with decreased apoptosis and early apoptotic CD4(+) T cells in inflamed skin in Fas mutant lpr/lpr mice compared to wild-type mice, whereas there were no significant differences in IL-4, IFN-gamma, IL-10, Bax and ICE mRNAs in the inflamed skin of CH between lpr/lpr and wild-type mice. Our results thus suggest that Fas/FasL pathway partially contributes to apoptosis in murine CH and that Fas/FasL-mediated apoptosis plays a partial role in the development of CH. The contribution of Fas/FasL-mediated apoptosis to CH appears independent of Th1 and Th2 cytokines.

Adjuvants, Immunologic↗