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The aim of this study was to determine whether membrane lipid peroxidation in mammalian cells is enhanced by X-ray irradiation at the K-shell resonance absorption peak of phosphorus. A549 and wild-type p53-transfected H1299 (H1299/wtp53) cell lines derived from human lung carcinoma were irradiated with monoenergetic X-rays at 2.153 keV, the phosphorus K-shell resonance absorption peak, or those at 2.147 or 2.160 keV, which are off peaks. Immunofluorescence staining for 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, was used as marker for protein modification. In both cell lines, the HNE production was significantly enhanced after irradiation at 2.153 keV compared to sham-irradiation. The enhancement (E) was calculated as the ratio of the fluorescence intensity of irradiated cells to that of sham-irradiated cells. In both the cell lines, E2.153 was significantly larger than E2.147 and no significant difference between E2.147 and E2.160 was observed. The extra enhancement at 2.153 keV was possibly caused by energy transition within the phosphorus K-shell resonance absorption. Our results indicate that membrane lipid peroxidation in cells is enhanced by the Auger effect after irradiation at the K-shell resonance absorption peak of phosphorus rather than by the photoelectric effect of the constituent atoms in the membrane lipid at 2.147 keV.

To clarify a possible index for long-term and low-dose irradiation, the effects of repeated low-dose X-ray irradiation on the amount of melanin-derived radicals in mouse hair and tail skin were investigated. Eight-week-old female C3H/HeSlc mice were irradiated by X-rays at a dose of 100 mGy/day 5 days/week for 12 weeks. Similarly, a 4-week irradiation experiment was carried out at 500 mGy/day for C3H/HeSlc mice, or at 10, 100, and 500 mGy/day for 8-week-old female C57BL/6NCrSlc mice. The hair sample (~10 mg) was weighed accurately and stuffed into a plastic tube. The 2-cm tip of the tail was sampled and lyophilized. Melanin-derived radicals in hair and tail samples were measured by X-band electron paramagnetic resonance spectrometry. After X-ray irradiation at 100 mGy/day for 12 weeks, no difference was found in the amount of melanin-derived radicals in the hair of the irradiated and non-irradiated groups. X-ray irradiation at 500 mGy/day for 4 weeks increased the amount of melanin-derived radicals in hair compared with the non-irradiated group, but the baseline amount of melanin-derived radicals in hair was varied. The amount of melanin-derived radicals in the tail skin dose-dependently increased. Melanin-derived radicals in skin may be an endogenous marker for long-term and low-dose irradiation.

Breast cancer is one of the most common types of cancers prevalent in women. Several types of breast cancers can easily metastasize to bone and cause disease complications such as hypercalcemia and pathologic fracture, thus compromising the quality of life of people affected by it. Bisphosphonate drugs are often used for the treatment of bone metastasis to suppress osteoclastic bone resorption. However, bisphosphonate has adverse effects on the gastrointestinal tract and kidneys and also induces osteonecrosis of the jaw. Photodynamic therapy (PDT) is an alternative cancer treatment approach with minimal invasiveness. It is a combination treatment that uses photosensitizers, which accumulate in tumor cells, followed by laser irradiation. We previously reported that the cellular incorporation of 5-aminolevulinic acid (5-ALA), which was a precursor of protoporphyrin IX (PpIX), was regulated by reactive oxygen species derived from mitochondria (mitROS). In this study, we investigated the incorporation of 5-ALA, accumulation of PpIX, and subsequent effects on cell viability after laser irradiation of two different breast cancer cell lines with different metastaticites. The highly metastatic breast cancer cell line 4T1E/M3 showed a significant increase in ROS production after treatment with indomethacin (IND). In addition, IND treatment enhanced the cellular uptake of 5-ALA via PEPT1 upregulation in 4T1E/M3, but not in the non-metastatic cell line. Overall, metastatic breast cancer is likely to be sensitive to ROS and activate signaling pathways associated with 5-ALA transportation, suggesting that ALA-PDT could be an effective treatment with low invasiveness for metastatic breast cancer.

Fetal age is estimated widely by body length and weight and skeletal or dental development and maturation. Regarding dental development, dental development charts reported by Schour and Massler and Ubelaker are well known. We tried to calculate the calcification rate of the deciduous teeth, mandibular cortical bone, and clavicle in fetuses utilizing postmortem computed tomography (CT) image analysis. The CT values within the circumferential area of the region of interest were automatically calculated using a software, and the calcification rate was calculated by performing single regression analysis. Our results showed that deciduous tooth calcification could be detected in over 19-week-old fetuses using CT images. The calcification of bones (mandibular cortical bone and clavicle) started earlier than the calcification of deciduous teeth. However, the calcification rate of the bones was slower compared to that of the deciduous teeth. The calcification rate of the deciduous teeth in fetuses using CT value may be effective to estimate fetal age and evaluate deciduous teeth development, suggesting that our established method is effective for age estimation in forensic dentistry.

Generation of mitochondrial reactive oxygen species (ROS), lipid peroxidation, 4-hydroxy-2-nonenal, and heat-shock protein (HSP) 47 after electron and X-ray irradiations were detected in the human neuroblastoma cell line SK-N-SH. After 10 Gy electron irradiation and 15 Gy X-ray irradiation, mitochondrial ROS production and lipid peroxidation were significantly increased. Additionally, we observed a significant increase in the levels of HSP47 after 3 and 10 Gy electron irradiation as well as 15 Gy X-ray irradiation. Furthermore, myristoylation and farnesylation were increased after 10 Gy electron and 15 Gy X-ray irradiations. We found that the level of HSP47 increased in the mitochondria after 10 Gy electron and 15 Gy X-ray irradiations. HSP47 coexisted with myristoylation and farnesylation. Furthermore, HSP47 overexpression increased mitochondrial ROS production. These results suggest that HSP47 plays an important role in mitochondria and induces mitochondrial ROS production in SK-N-SH cells.

5-Aminolevulinic acid, a precursor of heme, is utilized in a variety of applications including cancer treatment, surgery, and plant nutrition. However, 5-aminolevulinic acid itself induces oxidative stress and subsequent lipid peroxidation. Reactive oxygen species are factors in oxidative stress, not only causing cellular injury but also inducing several signal transduction pathways. Especially in cancer cells, a significant amount of signalling activation and subsequent activation of protein is caused by the enhancement of reactive oxygen species production. Reactive oxygen species levels in normal cells are low and an oxidative condition is harmful; hence, administration of 5-aminolevulinic acid to normal cells may induce oxidative stress, resulting in cell death. In this study, we investigated the effect of 5-aminolevulinic acid on normal and cancer cells with regard to oxidative stress. We used the rat normal gastric cell line RGM and its cancer-like mutant cell line RGK. 5-Aminolevulinic acid treatment of RGM cells enhanced reactive oxygen species generation and induced apoptosis associated with p53, whereas RGK cells were unaffected. In addition, RGM cell viability was recovered by application of N-acetyl-l-cysteine or p53 inhibitor. These results suggest that 5-aminolevulinic acid causes oxidative stress in normal gastric cells and induces apoptosis via the p53-dependent pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Object The aim of this study was to assess our diagnosis system for cervical node metastasis for oral squamous carcinoma (OSCC) based on the combined results of computed tomography (CT), magnetic resonance imaging (MRI), ultrasonography (US), and 18-fluorodeoxyglucose positron emission ( 18 F-FDG-PET/CT). Methods 106 patients with primary OSCC who underwent surgical treatment were divided into 3 groups. In group 1, cervical node metastasis was diagnosed by disunity combinations of diagnostic devices. In groups 2 and 3, it was assessed according to a set of diagnostic criteria that included the results of the CT/MRI, US and 18 F-FDG-PET/CT. In group 2, node status was considered negative when the results of both CT/MRI and US were negative. In group 3, node status was considered negative when the results of both CT/MRI and US were negative and the maximum standardized uptake value determined via 18 F-FDG-PET/CT was Results 67 patients were diagnosed as cN0. 50 cN0 patients adopted the wait and watch policy, 12 of whom (24.0%) developed cervical node metastasis without recurrence at the primary site. Metastasis rates after surgery improved 33.3%, 23.5%, and 13.3% in groups 1, 2, and 3, respectively. In contrast, positive predict value (PPV) were worst (50.0%, 40.0% and 33.3%). Conclusions Our diagnosis system reduces the rate of cervical lymph node metastasis after surgery.

Iron is an essential nutrient for life and is involved in many important processes such as oxygen transport and DNA synthesis. However, excess amounts of iron can cause carcinogenesis by producing reactive oxygen species. Thus, the cellular transport of iron must be tightly regulated. In the human body, iron may be present as heme or non-heme iron. The mechanisms governing the cellular transport of these forms have not been clearly elucidated. We previously reported that the expression of an important heme transporter, heme carrier protein 1 was regulated by cancer-specific reactive oxygen species derived from mitochondria. In this study, we have asked if mitochondrial reactive oxygen species may also be related with non-heme iron transport. In order to address this question, we have investigated the relationship between mitochondrial reactive oxygen species and accumulation of cellular non-heme iron in a rat gastric normal, cancer and manganese superoxide dismutase-overexpressing cancer cell line, in which reactive oxygen species from mitochondria are specifically scavenged. We have also analyzed the expression of divalent metal transporter 1 and ferroprotin, involved in the incorporation and excretion of non-heme iron, respectively, as well as a hypoxia-related transcription factor HIF-1α, to elucidate the molecular mechanism of non-heme iron accumulation.

Summary The causes of pain symptoms in the temporomandibular joint (TMJ) and masticatory muscle (MM) regions may not be determined by clinical examination alone. In this review, we document that pain symptoms of the TMJ and MM regions in patients with temporomandibular disorders (TMDs) are associated with computed tomography and magnetic resonance (MR) findings of internal derangement, joint effusion, osteoarthritis, and bone marrow edema. However, it is emphasized that these imaging findings must not be regarded as the unique and dominant factors in defining TMJ pain. High signal intensity and prominent enhancement of the posterior disk attachment on fat saturation T2-weighted imaging and dynamic MR imaging with contrast material are closely correlated with the severity of TMJ pain. Magnetic transfer contrast, MR spectroscopy, diffusion tensor imaging, and ultrasonography findings have helped identify intramuscular edema and contracture as one of the causes of MM pain and fatigue. Recently, changes in brain as detected by functional MR neuroimaging have been associated with changes in the TMJ and MM regions. The thalamus, the primary somatosensory cortex, the insula, and the anterior and mid-cinglate cortices are most frequently associated with TMD pain.

Japan has entered an era of a super-aging population, and given the importance of oral nutrition, the need to evaluate swallowing function has increased. Herein, we contribute to continued developments in evaluating eating and swallowing functions by describing current videoendoscopy (VE) usage and trends to evaluate and diagnose causes of dysphagia. In all, 100 patients (58 men and 42 women; mean age: 79 years) with suspected dysphagia were enrolled; 15 of these were re-examinations. Examinations were conducted according to the Japanese Society of Dysphagia Rehabilitation VE examination guidelines for swallowing. In this study, several patients (77.8 %) with poor vocalization and a saliva reservoir were unable to eat. While evaluating the relationship between aspiration and pharyngeal or laryngeal influx, we found that when pharyngeal and laryngeal influx were present, the risk of aspiration was high. Some patients (38.9 %) were able to eat despite lacking a cough reflex; thus, the absence of a cough reflex does not necessarily equate to an inability to eat, even in patients unable to ingest nutrition orally. One case could ingest nutrition, even with no cough reflex. The 6-month survival rate after the examination of patients on nil per os status was 57.1 %, specifically in patients unable to ingest nutrition orally. These results suggest that decreased eating and swallowing functions indicate a poor prognosis for the patient's quality of life, as eating and swallowing require smooth passage in the oral phase. Therefore, actively requesting a dental intervention and oral rehabilitation is important for a patient presenting these issues.

Photodynamic therapy is useful for the treatment of cancer because it is minimally invasive for patients. Certain porphyrin compounds and their derivatives have been used as the photosensitizer because they accumulate specifically in cancerous tissues. However, the detailed mechanism of this phenomenon has not been clarified. We previously reported that a proton-coupled folate transporter, HCP1, transported porphyrins and that regulation of the protein was associated with cancer-specific reactive oxygen species from mitochondria (mitROS). Therefore, over-generation of mitROS could increase HCP1 expression and the effect of photodynamic therapy. We investigated whether pretreatment with indomethacin influenced photodynamic therapy by using a rat normal gastric mucosal cell line, RGM1, its cancer-like mutated cell line, RGK1, and a manganese superoxide dismutase (MnSOD)-overexpressing RGK cell line, RGK-MnSOD. Indomethacin promotes the generation of cellular mitROS by inhibiting the electron transport chain, and MnSOD scavenges the mitROS. We elucidated that indomethacin enhanced cancer-specific mitROS generation and increased HCP1 expression. Furthermore, RGK1 cells showed higher cellular incorporation of hematoporphyrin and better therapeutic effect with indomethacin treatment whereas RGK-MnSOD cells did not show a difference. Thus, we concluded that indomethacin improved the effect of photodynamic therapy by inducing increased mitROS generation in cancer cells.

Prof. Dr. Helmut Sies is a pioneer of “Oxidative Stress”, and has published over 18 papers with the name of “Oxidative Stress” in the title. He has been Editor-in-Chief of the journal “Archives of Biochemistry and Biophysics” for many years, and is a former Editor-in-Chief of the journal “Free Radical Research”. He has clarified our understanding of the causes of chronic developing diseases, and has studied antioxidant factors. In this article, importance of “Oxidative Stress” and our mitochondrial oxidative stress studies; roles of mitochondrial ROS, effects of vitamin E and its homologues in oxidative stress-related diseases, effects of antioxidants in vivo and in vitro , and a mitochondrial superoxide theory for oxidative stress diseases and aging are introduced, and some of our interactions with Helmut are described, congratulating and appreciating his great path.

To evaluate the correlations between temporomandibular joint (TMJ) pain and both conventional and gadolinium-enhanced magnetic resonance (MR) imaging characteristics in patients with temporomandibular disorders (TMDs). T1-weighted, T2-weighted, and fat-suppressed contrast-enhanced T1-weighted MR imaging were performed in 80 TMD patients with the main complaint of TMJ pain. A kappa statistical analysis was performed to calculate the interobserver agreement between two of three radiologists. The relationships between TMJ pain and various MR findings were analyzed by multiple comparison test, Spearman’s rank correlation test, Chi-square test, Student’s t test, and multivariate logistic regression analysis. Significant correlations were observed between anterior disk displacement without reduction, joint effusion, and TMJ pain (p

Photodynamic therapy using hematoporphyrin and its derivatives is clinically useful for cancer treatments. It has been reported that cancer cells incorporate hematoporphyrin and its derivatives via heme carrier protein 1, which is a proton-coupled folate transporter. However, the mechanism of this protein expression has not been elucidated. In general, the concentration of reactive oxygen species in cancer cells is higher than that in normal cells. We previously reported that reactive oxygen species from mitochondria involved in the expression of peptide transporter 1 and accelerate the uptake of 5-aminolevulinic acid, which is a precursor of protoporphyrin IX. We suggested mitochondrial reactive oxygen species also regulated the expression of heme carrier protein 1. In this study, we used a rat gastric mucosal cell line RGM1 and its cancer-like mutated cell line RGK1. We clarified the expression of heme carrier protein 1 increased in cancer cells and it decreased in manganese superoxide dismutase expressed cancer cells. In addition, the uptake level of hematoporphyrin and photodynamic therapeutic effect were also decreased in manganese superoxide dismutase expressed cancer cells in comparison with cancer cells. Thus, we concluded that mitochondrial reactive oxygen species regulated heme carrier protein 1 expression and photodynamic therapeutic effect.

Photodynamic therapy and photodynamic diagnosis using 5-aminolevulinic acid (ALA) are clinically useful for cancer treatments. Cancer cells have been reported that 5-aminolevulinic acid is incorporated via peptide transporter 1, which is one of the membrane transport proteins, and has been reported to be significantly expressed in various gastrointestinal cancer cells such as Caco-2. However, the mechanism of this protein expression has not been elucidated. Concentration of reactive oxygen species (ROS) is higher in cancer cells in comparison with that of normal cells. We have previously reported that ROS derived from mitochondria is likely related to invasions and proliferations of cancer cells. Since 5-aminolevulinic acid is the most important precursor of heme which is necessary protein for cellular proliferations, mitochondrial ROS (mitROS) may be also related to peptide transporter 1 expressions. In this study, we used a rat gastric mucosal cell line RGM1 and its cancer-like mutated cell line RGK1, and we clarified the ALA uptake mechanism and its relations between mitROS and peptide transporter 1 expression in RGK1. We also used our self-established stable clone of cell which over-expresses manganese superoxide dismutase, a mitROS scavenger. We studied differences of the photodynamic therapy effects in these cells after ALA administrations to clear the influence of mitROS.

Tumor invasion is the most important factor to decide patient's prognosis. The relation between reactive oxygen species and tumor invasion is mainly reported that nicotinamide adenine dinucleotide phosphate oxidase in the cell membrane is a reactive oxygen species producer for formulating an invadopodia. On the other hand, mitochondrion was known as one of the most important reactive oxygen species-producer in the cell via an energy transfer system. However, the relation between mitochondrial reactive oxygen species and the tumor invasion was not well clarified. In this study, we evaluated the relation between mitochondrial reactive oxygen species and tumor invasion using a normal gastric mucosal cell-line (RGM-1) and a cancerous mutant RGM-1 cell-line (RGK-1). Manganese superoxide dismutase-expressing RGK-1 cell-lines were used for a scavenging mitochondrial reactive oxygen species. The cells have been evaluated their movement ability as follows; cellular ruffling frequencies, wound healing assay to evaluate horizontal cellular migration, and invasion assay using matrigel to analyze vertical cellular migration. All cellular movement abilities were inhibited by scavenging mitochondrial reactive oxygen species with manganese superoxide dismutase. Therefore mitochondrial reactive oxygen species was one of factors enhancing the tumor invasion in gastric cancer.

Mitochondria are a major source of intracellular energy and reactive oxygen species in cells, but are also increasingly being recognized as a controller of cell death. Here, we review evidence of signal transduction control by mitochondrial superoxide generation via the nuclear factor-κB (NF-κB) and GATA signaling pathways. We have also reviewed the effects of ROS on the activation of MMP and HIF. There is significant evidence to support the hypothesis that mitochondrial superoxide can initiate signaling pathways following transport into the cytosol. In this study, we provide evidence of TATA signal transductions by mitochondrial superoxide. Oxidative phosphorylation via the electron transfer chain, glycolysis, and generation of superoxide from mitochondria could be important factors in regulating signal transduction, cellular homeostasis, and cell death.

Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of key enzymes. This results in enhanced glucose dependency and leads to cell death under low-glucose conditions. On the other hand, the reduced requirements for oxygen and nutrients from the surrounding environment, might confer the resistance to cell death induced by hypoxia and malnutrition.

The use of hair roots as experimental samples has been a research focus for understanding the effects of spaceflight on astronauts, because it has many advantages, one of which is the fact that hair matrix cells actively divide in a hair follicle and sensitively reflect the physical conditions of the human body. In 2009, a research program focusing on the analysis of astronauts’ hairs was initiated to examine the effects of long-term spaceflight on the gene expression and mineral metabolism in the human body. Since the number of samples per astronaut is limited to 5 strands of hairs at each sampling point, due to the ethical viewpoint of astronauts or limited resources in space, it is important to develop an effective method for the molecular analysis of small amounts of hair roots. In this study, mRNA successfully extracted from 1, 5, and 10 hair follicles was amplified and subjected to the DNA microarray analysis to compare the gene expression within subjects. The results indicated that (1) it was possible to perform the genetic analysis on hair samples stored at -80℃, even without a fixation buffer and (2) the newly modified method of mRNA extraction and analysis was effective in detecting differential gene expression in samples containing only 5 hairs. In conclusion, RNA was efficiently extracted from 5 hair roots, which is the same number of hair roots used in the space experiment; therefore, this method can be applied to genetically analyze astronauts’ hair samples.

Importance The regulatory factors explaining the wide spectrum of clinical phenotypes for mitochondrial 3243A>G mutation are not known. Crosstalk between nuclear genes and mitochondrial DNA might be one factor. Observations In this case series, we compared 2 pairs of male twins with the mitochondrial 3243 A>G mutation and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome with a female control patient. One pair of monozygotic twins presented with diabetes and deafness in their 30s, stroke-like episodes in their 40s, and cardiac events and death in their 50s. Another pair of twins presented with deafness and stroke-like episodes in their 20s. The degree of heteroplasmy of 3243A>G mutation in the various tissues and organs was similar in the first pair of twins compared with the control patient. Conclusions and Relevance The clinical phenotype and segregation of mitochondrial 3243A>G mutation was similar in monozygotic twins. The onset age and distribution of the symptoms might be regulated by nuclear genes. Our findings might help to predict the clinical course of the surviving twins and afford an opportunity for therapy before the onset of mitochondrial disease, especially for monozygotic twins caused by nuclear transfer with a small amount of nuclear-donor mitochondrial DNA.

HLE, a human hepatocellular carcinoma cell line was transiently transfected with normal human MnSOD and MnSOD without a mitochondrial target signal (MTS). Mitochondrial reactive oxygen species (ROS), lipid peroxidation and apoptosis were examined as a function of time following 18.8 Gy X-ray irradiation. Our results showed that the level of mitochondrial ROS increased and reached a maximum level 2 hours after X-ray irradiation. Authentic MnSOD, but not MnSOD lacking MTS, protected against mitochondrial ROS, lipid peroxidation and apoptosis. In addition, the levels of mitochondrial ROS were consistently found to always correlate with the levels of authentic MnSOD in mitochondria. These results suggest that only when MnSOD is located in mitochondria is it efficient in protecting against cellular injuries by X-ray irradiation and that mitochondria are the critical sites of X-ray-induced cellular oxidative injuries.

著者人数: 12名, Accepted: 2011-12-09, 資料番号: SA1004105000

Summary Up to now, we have performed scintigraphy with 201-thallium chloride (201-TlCl) and 99m-Tc-hexakis-2-methoxy-isobutyl-isonitrile (99m-Tc-MIBI) for malignant tumors and lymphoscintigraphy with 99m-Tc-rhenium-colloid (99m-Tc-Re) and 99m-Tc-human-serum-albumin-diethylene-triamine-penta-acetic-acid (99m-Tc-HSA-D) for lymph node metastasis. In this article, we re-evaluated scintigraphic images retrospectively with a hope that the results might be a clue, even if it is small, for dentists to try to improve the accuracy of diagnosis of malignant tumors. From scintigraphy, we obtained the tumor retention index as a factor to estimate the uptake of radioactive agents in tumor cells. Moreover, we estimated transport proteins of Na + /K + -ATPase and permeability-glycoprotein (P-gp) expressed on the cell membrane that might regulate the kinetic condition of radioactive agents. Among the tumor retention index, the transport protein and the histopathologic finding of tumors, there were relatively well correlations. The tumor retention index showed a difference clearly between malignant tumor and benign tumor. The transport protein revealed a distinct expression in accordance with the malignancy of tumor, and the uptake clearly depended upon the expression of transport protein. Moreover, the lymph node metastasis was detected well by lymphoscintigraphy with 99m-Tc-Re and 99m-Tc-HSA-D.

Gastric hydrochloric acid (HCl) has been regarded as an inciting factor in gastric mucosal injuries and has been reported to induce lipid peroxidation in vitro. However, because HCl is not an oxidant per se, the exact mechanism by which the acid induces lipid peroxidation is unknown. We hypothesized that gastric acid may disrupt mitochondrial transmembrane potential and induce the production of superoxide in mitochondria, which subsequently may induce lipid peroxidation and apoptosis in gastric mucosal cells. Firstly we treated gastric epithelial RGM1 cells with solutions containing various concentrations of HCl (i.e., of varying pH), and examined cellular injury, lipid peroxidation, and apoptosis with specific fluorescent dyes. Secondly, we performed electron paramagnetic resonance (EPR) spectroscopy of isolated, acid-exposed mitochondria from the cells, using a spin-trapping reagent for superoxide, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). Finally, we established novel RGM1 cells that overexpressed manganese superoxide dismutase (MnSOD), which removes superoxide from mitochondria, and examined the effect of acid treatment on cellular membrane lipid peroxidation. The results indicated that the exposure to acid indeed induced cellular injury, cellular lipid peroxidation, apoptosis, and the demonstration of the exact superoxide spectra on EPR spectroscopy in gastric epithelial cells, and that overexpression of MnSOD decreased superoxide production and prevented cellular lipid peroxidation. These results suggested that gastric acid, like nonsteroidal anti-inflammatory drugs (NSAIDs), induces mitochondrial superoxide production, which induces gastric cellular injury by triggering cellular lipid peroxidation and apoptosis.

Lansoprazole is effective in healing non-steroidal anti-inflammatory drugs induced ulcers, and antioxidant properties have been thought to play a key role in healing ulcers. We hypothesize that lansoprazole exerts a cytoprotective effect by inhibiting reactive oxygen species leakage from mitochondria and lipid peroxidation. We pretreated gastric epithelial RGM1 cells with lansoprazole and then treated them with indomethacin in vitro. We found that the lansoprazole pretreatment significantly reduced cellular injury, maintained mitochondrial transmembrane potential, and decreased lipid peroxidation. Furthermore, the signal intensity of the electron spin resonance spectrum of the indomethacin-treated mitochondria which were pretreated with lansoprazole showed considerable reduction compared to those without the lansoprazole pretreatment. These results suggest that lansoprazole reduced superoxide production in the mitochondria of indomethacin treated cells, and subsequently inhibited lipid peroxide and cellular injury in gastric epithelial cells.

Iron is an essential substance for human and play many important roles such as transportation of oxygen and synthesis of DNA. However, the concentration of iron in body is strictly regulated because excess iron causes reactive oxygen species (ROS) and damages cells. Most of iron in body classified as follows: heme iron and non-heme iron. We previously reported that high level ROS derived from mitochondria (mitROS) in cancer cells enhanced the expression of heme iron transporter. In this study, we examined the relationship between non-heme iron transportation and mitROS. We used a rat gastric normal, cancer and manganese superoxide dismutase (MnSOD)-overexpressing cancer cell line, in which mitROS are specifically scavenged. Among these cell lines, non-heme iron incorporation was compared using radioactive iron compound. The incorporation in cancer cells was greater than in normal cells and decreased by overexpressing MnSOD. We also studied the expression of transporters which was related to incorporation and excretion of non-heme iron. The expression of incorporation protein DMT1 in cancer cells was greater than in normal and MnSOD-overexpressing cancer cells. On the other hand, excretion protein ferroportin decreased in cancer compared to MnSOD-overexpressing cancer cells. Thus, we concluded that cancer cellular ROS from mitochondria regulated the incorporation and excretion of non-heme iron via expression of iron transport protein.

In this study, the DNA array, quantitative PCR, 8-hydroxydeoxyguanosine (8OHdG detection), relative mtDNA copy number compared to the nuclear DNA, and mitochondrial mutation rate were analyzed using blood samples kindly donated by 135 persons, who live in Amami island district, highest centenarian rate (the number of centenarian in 100,000 people) in Japan. The results showed that three genes significantly decreased in over 90-aged population. The genes are, HSPD1, heat shock 60 kDa protein 1 (chaperonin), GADD45G, GADD45gamma, growth arrest and DNA-damage-inducible, and LTBP-3, latent transforming growth factor beta binding protein 3. The results of 8OHdG detection showed that gradually and significantly increase of 8OHdG as a function of the age. The detection copy number of mtDNA resulted in increase in the copy number dependent on the age. The mtDNA mutation resulted in no difference among the age. The results showed that the Amami population over 90 years old did not show substantial change in the copy number and the frequency of mutation in mtDNA, suggesting maintaining good health in Amami region.

OBJECTIVE: To evaluate inflammatory changes in masticatory muscles by magnetization transfer contrast (MTC) imaging. METHODS: Gradient-recalled echo (GRE) and MTC-GRE images of relaxed masticatory muscles in 28 healthy volunteers were obtained before and after exercise. At the same time, muscle stiffness and pain in the masseter muscles were also measured. Magnetization transfer ratios (MTRs) of the muscles were calculated from the GRE and MTC-GRE images. The MTRs of the masticatory muscles in 50 patients with temporomandibular disorder were compared with those in the volunteers. RESULTS: Immediately after the exercise, the MTRs of the masseter muscles significantly decreased (P < 0.05), whereas muscle stiffness and pain increased in the healthy volunteers. In patients with masseter muscle pain, the MTRs of the masseter muscles were significantly lower than in the healthy volunteers (P < 0.05). CONCLUSIONS: Magnetization transfer contrast imaging strongly reflects the masticatory muscle edematous changes, possibly leading to masseter muscle pain.